145. Betz, EC; Punja, ZK. (2021) First report of Verticillium isaacii causing wilting and vascular blackening on wasabi (Wasabia japonica) plants.Can. J. Plant Pathol. 43: 658-669 First report of Verticillium isaacii causing wilting and vascular blackening on wasabi (Wasabia japonica) plants
Eutrema japonicum; vascular blackening; Verticillium isaacii; verticillium wilt; wasabi; Wasabia japonica
Wasabi (Wasabia japonica (Miq.) Matsum) plants are grown over a period of 12-18 months for their valuable rhizomes, and pathogens infecting the rhizome are of economic importance for producers. During 2016 and 2017, symptoms of wilting and internal vascular blackening of rhizomes were observed in two greenhouses in British Columbia, with disease incidence in the range of 5-10%. Isolates recovered from diseased tissues were examined morphologically and a majority (70%) resembled a Verticillium species. Molecular analysis using PCR of the ITS1-ITS2 region of rDNA and a portion of the actin gene identified the isolates as V. isaacii Inderb. R.M. Bostock, R.M. Davis & Subbarao. The optimal temperature for growth of V. isaacii on potato dextrose agar was 20 degrees C and a distinct yellow pigment was produced, which is characteristic of species in the clade Flavexudans. Inoculation of detached wounded wasabi tissues with V. isaacii produced blackening symptoms and chlorosis on leaves. Black streaks developed on petioles and internal blackening of rhizomes was also observed. Plants inoculated in the greenhouse by immersing wounded roots in a spore suspension of V. isaacii developed a reduced root system compared with control plants. Blackening of the rhizome tissue was observed after three months. Differences were observed in pathogenicity between V. isaacii isolates. We demonstrate for the first time that V. isaacii is the cause of wilting and vascular blackening on wasabi plants. It is considered a weak pathogen, only affecting wounded tissues with symptoms developing after an extended time following inoculation. DOI
143. MacDonald, JL; Punja, ZK; Xiang, Y; Bouthillier, MJ; Reade, R; DeYoung, RM; Bhagwat, B; Betz, EC; Li, YQ; Chen, XB. (2021) First report of wasabi mottle virus causing ringspot and vein-clearing symptoms on wasabi (Wasabia japonica) in North America.Can. J. Plant Pathol. 43: 311-322 First report of wasabi mottle virus causing ringspot and vein-clearing symptoms on wasabi (Wasabia japonica) in North America
Eutrema japonicum; genome; ringspot; Tobamovirus; wasabi; Wasabi mottle virus
Symptoms of ringspots and vein-clearing were observed on wasabi (Wasabia japonica(Miq) Matsum) plants in three greenhouses in British Columbia during 2017. Ten indicator plant species, including fourNicotianaspecies, were inoculated with sap extracts from symptomatic leaves; after 4-11 days, necrotic lesions developed on all plants. Transmission electron microscopy revealed rod-shaped virions, 250-300 nm in length, in leaves ofN. occidentalisandN. clevelandii. Total RNA from symptomatic wasabi tissues was used in RT-PCR with universal primers corresponding to five virus genera and specific primers for turnip ringspot virus and alfalfa mosaic virus. Amplicons similar to 400 bp in size were obtained with tobamovirus primer set TobN up3/TobN do4 and amplicons of similar to 300 and similar to 600 bp were obtained with ilarvirus primer set Ilar1F5/Ilar1R7. Sequencing and MegaBLAST (NCBI) query of the Ilar amplicons showed 99% identity to wasabi mottle virus (WMoV), a member of the genusTobamovirus. Next-generation Sequencing confirmed WMoV as the only virus present in diseased plants. BC isolates (GenBank accession no. MK431779) showed 99.43% sequence identity to isolate 'Alishan' (GenBank accession no. KJ207375.1) and 98.62% identity to 'Tochigi' strain (GenBank accession no. AB017504.1). Mechanical inoculation of wasabi cultivars 'Green Thumb' and 'Daruma' produced ringspots and vein-clearing symptoms after 22-23 days on the former while the latter was asymptomatic, but WMoV was detected in all plants by RT-PCR. WMoV may have been introduced into Canada on imported infected 'Green Thumb' plants and subsequently spread through commercial vegetative propagation. The effects on yield or rhizome quality are yet unknown. DOI
142.Punja, ZK. (2021) Emerging diseases of Cannabis sativa and sustainable management.Pest Manag. Sci. 77: 3857-3870 Emerging diseases of Cannabis sativa and sustainable management
biocontrol; disease management; fungal pathogens; hemp; marijuana; plant pathology
Cultivation of cannabis plants (Cannabis sativa L., marijuana) has taken place worldwide for centuries. In Canada, legalization of cannabis in October 2018 for the medicinal and recreational markets has spurned interest in large-scale growing. This increased production has seen a rise in the incidence and severity of plant pathogens, causing a range of previously unreported diseases. The objective of this review is to highlight the important diseases currently affecting the cannabis and hemp industries in North America and to discuss various mitigation strategies. Progress in molecular diagnostics for pathogen identification and determining inoculum sources and methods of pathogen spread have provided useful insights. Sustainable disease management approaches include establishing clean planting stock, modifying environmental conditions to reduce pathogen development, implementing sanitation measures, and applying fungal and bacterial biological control agents. Fungicides are not currently registered for use and hence there are no published data on their efficacy. The greatest challenge remains in reducing microbial loads (colony-forming units) on harvested inflorescences (buds). Contaminating microbes may be introduced during the cultivation and postharvest phases, or constitute resident endophytes. Failure to achieve a minimum threshold of microbes deemed to be safe for utilization of cannabis products can arise from conventional and organic cultivation methods, or following applications of beneficial biocontrol agents. The current regulatory process for approval of cannabis products presents a challenge to producers utilizing biological control agents for disease management. (c) 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. DOI PubMed
141.Punja, ZK. (2021) First report of the powdery mildew pathogen of hops, Podosphaeria macularis, naturally infecting cannabis (Cannabis sativa L., marijuana) plants under field conditions.Can. J. Plant Pathol.First report of the powdery mildew pathogen of hops, Podosphaeria macularis, naturally infecting cannabis (Cannabis sativa L., marijuana) plants under field conditions
cannabis; cross-infectivity; Golovinomyces; hops; Humulus; Podosphaeria; powdery mildew
Plants of the cannabis (Cannabis sativa L., marijuana) strain 'Chronic Ryder', grown outdoors in the Fraser Valley of British Columbia (BC), displayed symptoms of powdery mildew in late July 2019. The disease progressed rapidly under cool, wet conditions to infect leaves, stems, shoot tips and inflorescences by early September. To identify the pathogen, DNA was extracted from healthy and diseased leaves and subjected to PCR using eukaryotic universal primers that amplified the ITS1-5.8S-ITS2 region of rDNA. A 650 bp band present only in the diseased plants, showed 99.45% sequence homology to Podosphaeria macularis, the powdery mildew pathogen that infects hop plants. In greenhouse-grown cannabis infected with the powdery mildew pathogen Golovinomyces ambrosiae, a 700 bp size band was present. Conidial morphology also distinguished the two pathogens - P. macularis produced ovoid-oval conidia with fibrosin bodies, while G. ambrosiae produced cylindrical-oblong conidia with no fibrosin bodies. A survey of powdery mildew-infected hop fields and indoor cannabis growing facilities in different geographical locations in BC, showed that plants were infected exclusively by either P. macularis or G. ambrosiae, respectively. Inoculations using P. macularis-infected leaf segments placed on cannabis 'Pink Kush' leaves under high humidity at 22-26 degrees C gave rise to small mildew colonies after 24-35 days, confirmed to be P. macularis by PCR. Development of P. macularis in cannabis plants was slower compared with G. ambrosiae. To date, only one outdoor location for cannabis has been confirmed to have P. macularis; the potential for spread to other sites or to indoor cultivation facilities is unknown. DOI
140.Punja, ZK; Ni, L. (2021) The bud rot pathogens infecting cannabis (Cannabis sativa L., marijuana) inflorescences: symptomology, species identification, pathogenicity and biological control.Can. J. Plant Pathol.The bud rot pathogens infecting cannabis (Cannabis sativa L., marijuana) inflorescences: symptomology, species identification, pathogenicity and biological control
biological control; Botrytis cinerea; Botrytis pseudocinerea; Diaporthe eres; Fusarium graminearum; grey mould; Sclerotinia sclerotiorum
Bud rot pathogens cause diseases on Cannabis sativa L. (cannabis, hemp) worldwide through pre- and post-harvest infections of the inflorescence. Seven indoor or outdoor cannabis production sites and three hemp fields were sampled for bud rot and stem canker presence during 2019-2020. Among 178 isolates recovered from diseased tissues, sequences of the ITS1-5.8S-ITS2 region of rDNA, the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene and the heat shock 60 (HSP) gene identified the following: Botrytis cinerea (162 isolates), B. pseudocinerea (2), B. porri (1), Sclerotinia sclerotiorum (5), Diaporthe eres (3) and Fusarium graminearum (5). Pathogenicity studies conducted on fresh detached cannabis buds inoculated with spore suspensions or mycelial plugs showed that B. cinerea, S. sclerotiorum and F. graminearum were the most virulent, while B. pseudocinerea, B. porri and D. eres caused significantly less bud rot. Optimal growth of Botrytis species occurred at 15-25 degrees C. In vitro antagonism tests showed that Bacillus spp., Trichoderma asperellum and Gliocladium catenulatum inhibited B. cinerea and S. sclerotiorum colony growth. When applied as a spray 48 h prior to B. cinerea inoculation, all biocontrol agents significantly (P < 0.01) reduced disease development on detached inflorescences. Prolific growth and sporulation of T. asperellum and G. catenulatum were observed on bud tissues. The pathogens B. porri, S. sclerotiorum, D. eres and F. graminearum are described for the first time as cannabis bud rot pathogens. Inoculum from neighbouring fields of diseased garlic, cabbage, blueberry and hay pasture, respectively, likely initiated infection of inflorescences. Several biological control agents show potential for disease reduction through competitive exclusion. DOI
139.Punja, ZK; Ni, L; Roberts, A. (2021) The Fusarium solani species complex infecting cannabis (Cannabis sativa L., marijuana) plants and a first report of Fusarium (Cylindrocarpon) lichenicola causing root and crown rot.Can. J. Plant Pathol. 43: 567-581 The Fusarium solani species complex infecting cannabis (Cannabis sativa L., marijuana) plants and a first report of Fusarium (Cylindrocarpon) lichenicola causing root and crown rot
brown root rot; Cylindrocarpon root rot; Fusarium solani; Neocosmaspora lichenicola; onychomycoses; opportunistic pathogen
Greenhouse-grown cannabis (Cannabis sativa L., marijuana) plants with yellowing, crown rot and root-browning symptoms were sampled from six production facilities during 2019-2020. Among 34 fungal isolates recovered, 28 were identified as Fusarium solani and six isolates were provisionally identified as Cylindrocarpon sp. based on morphology. These latter isolates produced slow-growing colonies with grey-white aerial mycelium and a chestnut-brown colour below. Cylindrical 1-3 septate conidia without a distinctive foot cell were produced. Microconidia were absent and chlamydospores were produced in culture. Phylogenetic analysis of three isolates based on the elongation factor (TEF-1 alpha) and ITS1-5.8S-ITS2 regions identified them as Fusarium lichenicola (formerly Cylindrocarpon lichenicola), a member of F. solani species complex (FSSC) subclade 16. Pathogenicity tests using a mycelial and spore suspension were performed on cannabis cuttings and rooted plants. Isolates of F. solani originating from diseased crowns caused symptoms in 10-14 days, while those of F. lichenicola caused yellowing and wilting after 3 weeks, suggesting that F. lichenicola is less virulent. Inoculum of F. lichenicola was detected in coco coir samples used for plant propagation. Previous reports of F. lichenicola are from tropical climates, where the fungus has been associated with dermal and ocular infections of human tissues, with a few reports of it causing diseases on pomelo fruits, taro corms and tea plants. This study demonstrates the first occurrence worldwide of F. lichenicola on cannabis plants, on which it is considered a weak introduced tropical pathogen, likely to have originated from coco coir imported into Canada. DOI
138.Punja, ZK; Scott, C; Lung, S. (2021) Several Pythium species cause crown and root rot on cannabis (Cannabis sativa L., marijuana) plants grown under commercial greenhouse conditions.Can. J. Plant Pathol.Several Pythium species cause crown and root rot on cannabis (Cannabis sativa L., marijuana) plants grown under commercial greenhouse conditions
crown rot; damping-off; disease complex; Pythium root rot; wilting
Cannabis (Cannabis sativa L., marijuana) plants with symptoms of crown rot, root decay, wilting and plant death were sampled during 2018 and 2019 from seven licensed production greenhouses. Affected tissues from 140 diseased plants were surface-sterilized and plated onto potato dextrose agar. Ninety-five isolates morphologically resembling Pythium species were subcultured and subjected to PCR of the ITS1-5.8-ITS2 region of ribosomal DNA. The following species were identified based on >99% sequence identity to reference isolates in GenBank: P. myriotylum (43 isolates), P. dissotocum (35 isolates), P. aphanidermatum (3 isolates) and Globisporangium ultimum (syn. P. ultimum) (2 isolates). A fifth species - P. catenulatum (12 isolates), was distinguished from P. rhizo-oryzae using the cytochrome oxidase c subunit I (COI) sequence. Cannabis licensed production facilities in British Columbia had all five species present, while P. dissotocum was found in two facilities in Ontario, and P. myriotylum was present in one facility in northern California. Isolates selected to represent each Pythium species were grown on potato dextrose agar at 25 degrees C and they all showed comparable colony growth after 6 days. The same isolates caused root browning, decay and stunting of cannabis plants grown in a coco: perlite potting medium. Plant mortality was similar after 21 days but rates of disease progression varied depending on the isolate tested. Wounding of roots and prolonged periods of saturation enhanced disease development. These results demonstrate for the first time that crown and root rot on greenhouse-grown cannabis plants can be caused by up to five Pythium species. DOI
137. Scott, C; Punja, ZK. (2021) Evaluation of disease management approaches for powdery mildew on Cannabis sativa L. (marijuana) plants.Can. J. Plant Pathol. 43: 394-412 Evaluation of disease management approaches for powdery mildew on Cannabis sativa L. (marijuana) plants
biocontrol agents; disease control; marijuana; powdery mildew; UV-C
Powdery mildew on cannabis (Cannabis sativa L., marijuana), caused by Golovinomyces cichoracearum, reduces plant growth and overall quality. To investigate disease management options, biological, chemical and physical approaches were assessed. A mildew-susceptible strain, 'Copenhagen Kush', was grown indoors with continual exposure to mildew inoculum. Treatments were applied weekly over a four-week period to groups of four plants once mildew infection had established itself. Trials were repeated thrice under varying initial disease pressures. Disease assessments were made weekly and the percentage of area infected on 30 leaflets per plant was used to calculate a disease rating score for treated and control plants. Disease progress curves were plotted and AUDPC values were determined for each treatment. To test the effect of UV-C light on mildew development, plants were exposed daily for 3-5 s over 28 days to UV-C light. The response of 12 cannabis strains to powdery mildew infection was assessed after exposing them to inoculum over a period of two weeks. The most effective treatments that significantly (P < 0.05) reduced disease in three trials were Luna Privilege SC (fluopyram), Regalia(R) Maxx, MilStop(R), Rhapsody ASO(TM), neem oil, and Stargus(R). Treatments that were less effective included ZeroTol(R), boric acid, and Actinovate(R) SP. Daily exposure of plants to UV-C light significantly reduced disease (by 45.2%, P < 0.05). Seven of 12 cannabis strains had significantly lower disease severity compared with the other five strains. The disease management strategies evaluated in this study have potential for reducing powdery mildew development on cannabis. DOI
136. Betz, EC; Punja, ZK. (2020) Management of powdery mildew, caused byErysiphe cruciferarum, on wasabi (Wasabia japonica) plants in British Columbia.Can. J. Plant Pathol.Management of powdery mildew, caused byErysiphe cruciferarum, on wasabi (Wasabia japonica) plants in British Columbia
Erysiphe cruciferarum; Eutrema japonicum; powdery mildew; wasabi; Wasabia japonica
Powdery mildew on wasabi (Wasabia japonica(Miq.) Matsumura)plants reduces photosynthesis and severe infections can result in chlorosis and defoliation, resulting in reduced yields.Erysiphe cruciferarumOpiz ex L. Junell was confirmed as the causal agent using sequence analysis of the ITS1-5.8 S-ITS2 region, along with conidial and conidiophore morphology. To evaluate reduced-risk management options, the efficacy of four commercially available products was assessed over three separate trials conducted in a commercial wasabi greenhouse in British Columbia. The products tested included two biological pesticides,Bacillus subtilisstrain QST 713 (formulated as Rhapsody ASO), andStreptomyces lydicusstrain WYEC 108 (formulated as Actinovate SP), as well as a copper-based fungicide, Cueva, and a plant formulated extract, Regalia Maxx (fromReynoutria sachalinensis). Treatments were applied at two-week intervals over 10-12 weeks starting with onset of disease symptoms. Natural infection byE. cruciferarumwas allowed to progress on the plants during the trials. Leaves were assessed for powdery mildew biweekly based on percentage of leaf surface infected, which was then converted to area under the disease progress curve (AUDPC) for each treatment. Five to six applications of Cueva and Regalia significantly (p < 0.05) reduced the progression of powdery mildew in all trials. Rhapsody also reduced disease progression in some trials, but to a lesser extent. Actinovate was not effective. These results provide producers of wasabi with reduced-risk options for management of powdery mildew. DOI
133. Ni, L; Punja, ZK. (2020) Management of powdery mildew on greenhouse cucumber (Cucumis sativus L.) plants using biological and chemical approaches.Can. J. Plant Pathol.Management of powdery mildew on greenhouse cucumber (Cucumis sativus L.) plants using biological and chemical approaches
Bacillus subtilis; biological control; chemical control; cucumber; powdery mildew
Powdery mildew, caused by Podosphaera xanthii (U. Braun & Shishkoff), is a widespread disease on greenhouse cucumber in many countries, including Canada. The efficacy of seven biological or chemical products to manage powdery mildew was evaluated. These included Rhapsody (R) (Bacillus subtilis Ehrenberg strain QST 713), Microflora PROTM (B. subtilis, Bacillus amyloliquefaciens Priest et al., Bacillus pumilus Meyer & Gottheil), Prestop (R) (Gliocladium catenulatum J.C. Gilman & E.V. Abbott strain J1446), Double Nickel LC (B. amyloliquefaciens strain D747), Active Flower (TM) (fertilizer containing N:P:K of 8:4:12 plus 3% boron), Luna (R) (fluopyram and pyrimethanil) and Pristine (R) (pyraclostrobin and boscalid). Products were applied as preventative or eradicative treatments at weekly intervals to the moderately susceptible cucumber cultivars 'Picowell RZ' or 'Tasty Green'. Disease severity was rated after 3-4 applications as the number of colonies that developed on leaves, or as a percentage of leaf area infected calculated using image analysis. Rhapsody at a rate of 1.5% provided the most significant disease reduction compared with the other three biological products, although rates of 2% and 4% also resulted in high disease suppression in a second experiment. The fungicides Luna (0.5 mL L-1) and Pristine (0.66 g L-1) provided a level of control comparable to Rhapsody. Application of ActiveFlower at 0.5 and 0.3 mL 100 mL(-1) significantly reduced powdery mildew after three applications, but some phytotoxicity was observed at the higher rate. The products tested in this study can be part of an integrated powdery mildew management programme for greenhouse cucumber. DOI
132.Punja, ZK. (2020) First report ofFusarium proliferatumcausing crown and stem rot, and pith necrosis, in cannabis (Cannabis sativaL., marijuana) plants.Can. J. Plant Pathol.First report ofFusarium proliferatumcausing crown and stem rot, and pith necrosis, in cannabis (Cannabis sativaL., marijuana) plants
crown rot; damping-off; Gibberella fujikuroi; marijuana; pith necrosis
Cannabis (Cannabis sativaL., marijuana) plants grown under greenhouse or controlled environments with symptoms of leaf yellowing, leaf necrosis and defoliation were observed during 2018-2019. Additional symptoms included crown rot and internal browning or blackening of the pith tissues. Stock (mother) plants as well as plants in the vegetative and flowering stages of 15 cannabis strains (genotypes) were affected. In addition, damping-off symptoms were observed on rooted cuttings in propagation rooms. Isolations from diseased tissues yielded predominantlyFusarium proliferatum, with someF. oxysporumalso recovered. Phylogenetic analysis of sequences from the translation elongation factor 1 alpha (TEF-1 alpha) region of 29 isolates ofF. proliferatumfrom eight licenced production facilities in three provinces in Canada (British Columbia, Ontario and New Brunswick), and one cannabis production site in northern California, grouped isolates from cannabis with a large clade of isolates from a wide range of other hosts in different geographic regions. Pathogenicity studies confirmed the ability ofF. proliferatumto cause symptoms of wilting, leaf and pith necrosis, and plant death on cuttings, rooted plants and stock plants. Inoculated tomato and cucumber plants developed similar symptoms. Stem colonization was more extensive byF. proliferatumcompared toF. oxysporumon cannabis cuttings. Both grew optimally at 25 degrees C on agar media althoughF. oxysporumgrew faster thanF. proliferatumat all temperatures tested. The occurrence ofF. proliferatumon cannabis plants has not been previously reported, adding to recent reports ofF. oxysporumandF. solanithat cause similar symptoms on cannabis plants. DOI
131.Punja, ZK. (2020) Epidemiology ofFusarium oxysporumcausing root and crown rot of cannabis (Cannabis sativaL., marijuana) plants in commercial greenhouse production.Can. J. Plant Pathol.Epidemiology ofFusarium oxysporumcausing root and crown rot of cannabis (Cannabis sativaL., marijuana) plants in commercial greenhouse production
bud rot; cannabis; Cannabis sativa; crown rot; damping-off; epidemiology; fusarium root rot
Fusarium oxysporum causes root browning and crown infection on marijuana (Cannabis sativaL.) plants, resulting in stunted growth, yellowing of leaves, and plant death. Pathogen presence and diversity were assessed in samples of diseased crowns, stems, pith tissues and roots from five commercial production facilities in British Columbia and Ontario. PCR of the elongation factor (EF-1 alpha) region and sequence analysis confirmed the identity and phylogenetic relationships among 33 representative isolates from over 200 isolates collected. Stock (mother) plants of eight cannabis strains (genotypes) with symptoms of yellowing, crown rot and internal stem decay yieldedF. oxysporumat a frequency of 70-100%. Cuttings obtained from asymptomatic plants showed pathogen recovery rates of 1-2% on potato dextrose agar at distances of 90-150 cm from the crowns. Symptoms of damping-off were observed on cuttings in a rooting and propagation facility. Pathogenicity tests confirmed the development of characteristic symptoms on cuttings and rooted plants inoculated withF. oxysporum. Phylogenetic analysis indicated that isolates ofF. oxysporumfrom BC belonged to a separate clade from ON isolates and that clonal spread was occurring within licenced facilities. Air sampling conducted in a greenhouse facility revealedF. oxysporumwas present in areas used for rooting, vegetative propagation, and where stock plants were grown. The pathogen was also recovered from inflorescences. These findings indicate thatF. oxysporumis present in several commercial production facilities in Canada and reduces root development and establishment of rooted cuttings, and causes yellowing and stunting on flowering plants of many cannabis strains. DOI
130.Punja, ZK. (2020) The diverse mycoflora present on dried cannabis (Cannabis sativaL., marijuana) inflorescences in commercial production.Can. J. Plant Pathol.The diverse mycoflora present on dried cannabis (Cannabis sativaL., marijuana) inflorescences in commercial production
Contaminants; medical marijuana; moulds; pathogens; quality assurance
The objective of this study was to assess harvested dried inflorescences (buds) of cannabis (Cannabis sativaL., marijuana) for fungal presence and diversity. Samples from drying rooms of three licenced facilities in British Columbia were tested repeatedly during 2017-2019. A swab method was used, wherein sterile cotton swabs were gently swabbed over bud surfaces and directly streaked onto potato dextrose agar containing 140 mg L(-1)streptomycin sulphate. Petri dishes were incubated at 21-24 degrees C for 5-6 days and the fungal colonies that developed were recorded. The testing was repeated to provide >40 cumulative sampling times over a 2-year period. Representative colonies of each unique morphological type were identified to genus and species by PCR of the ITS1-5.8-ITS2 region of rDNA and sequence analysis. Among 34 different fungal species identified, the most prevalent werePenicillium(comprising 17 different species), followed by species ofCladosporium, Botrytis, Aspergillus, Fusarium, TalaromycesandAlternaria. All samples had several fungal species present and the number and composition varied at different sampling times and within different facilities. The swab method provided a qualitative assessment of viable mould contaminants on cannabis buds and reflected the diversity of mycoflora present, many of which are previously unreported. Fungi on cannabis buds may originate from spores released from diseased or decomposing plant materials, from growing substrates used in cannabis production, or as airborne contaminants in post-harvest trimming and drying rooms. Samples of dried buds exposed to electrobeam (e-beam) radiation treatment had no detectable fungal contamination when assessed using the swab method. DOI
129.Punja, ZK; Holmes, JE. (2020) Hermaphroditism in Marijuana (Cannabis sativaL.) Inflorescences - Impact on Floral Morphology, Seed Formation, Progeny Sex Ratios, and Genetic Variation.Front. Plant Sci. 11 Hermaphroditism in Marijuana (Cannabis sativaL.) Inflorescences - Impact on Floral Morphology, Seed Formation, Progeny Sex Ratios, and Genetic Variation
self-fertilization; feminized seeds; hermaphroditic flowers; genetic diversity; transposable elements; sexual reproduction; Cannabis sativaL; marijuana
Cannabis sativaL. (hemp, marijuana) produces male and female inflorescences on different plants (dioecious) and therefore the plants are obligatory out-crossers. In commercial production, marijuana plants are all genetically female; male plants are destroyed as seed formation reduces flower quality. Spontaneously occurring hermaphroditic inflorescences, in which pistillate flowers are accompanied by formation of anthers, leads to undesired seed formation; the mechanism for this is poorly understood. We studied hermaphroditism in several marijuana strains with three objectives: (i) to compare the morphological features of this unique phenotype with normal male flowers; (ii) to assess pollen and seed viability from hermaphroditic flowers; and (iii) to assess the effect of hermaphroditism on progeny male:female (sex) ratios and on genetic variation using molecular methods. The morphological features of anthers, pollen production and germination in hermaphroditic flowers and in staminate inflorescences on male plants were compared using light and scanning electron microscopy. Seeds produced on hermaphroditic plants and seeds derived from cross-fertilization were germinated and seedlings were compared for gender ratios using a PCR-based assay as well as for the extent of genetic variation using six ISSR primers. Nei's index of gene diversity and Shannon's Information index were compared for these two populations. The morphology of anthers and pollen formation in hermaphroditic inflorescences was similar to that in staminate flowers. Seedlings from hermaphroditic seeds, and anther tissues, showed a female genetic composition while seedlings derived from cross-fertilized seeds showed a 1:1 male:female sex expression ratio. Uniquely, hermaphroditic inflorescences produced seeds which gave rise only to genetically female plants. In PCR assays, a 540 bp size fragment was present in male and female plants, while a 390 bp band was uniquely associated with male plants. Sequence analysis of these fragments revealed the presence ofCopia-like retrotransposons within theC. sativagenome which may be associated with the expression of male or female phenotype. In ISSR analysis, the percentage of polymorphic loci ranged from 44 to 72% in hermaphroditic and cross-fertilized populations. Nei's index of gene diversity and Shannon's Information index were not statistically different for both populations. The extent of genetic variation after one generation of selfing in the progeny from hermaphroditic seed is similar to that in progeny from cross-fertilized seeds. DOI PubMed
128. Betz, EC; Roberts, AJ; Punja, ZK. (2019) 2017 AND 2018 SURVEYS OF MICROBES ASSOCIATED WITH WASABI DISEASES IN BRITISH COLUMBIA GREENHOUSES.Can. J. Plant Pathol. 41: 190-191 2017 AND 2018 SURVEYS OF MICROBES ASSOCIATED WITH WASABI DISEASES IN BRITISH COLUMBIA GREENHOUSES
Six wasabi greenhouses in the Lower Mainland and Vancouver Island areas of British Columbia were surveyed for disease symptoms during the summers of 2017 and 2018. Disease symptoms were observed at low to moderate levels, depending on the greenhouse sampled. Over the two years, 86 symptomatic plants were collected and a total of 16 potential pathogens were identified, including 12 fungi, 1 oomycete, 2 bacteria, and 1 virus.
127. Ni, L; Punja, ZK. (2019) Effects of a foliar fertilizer containing boron on the development of Sclerotinia stem rot (Sclerotinia sclerotiorum) on canola (Brassica napus L.) leaves.J. Phytopathol.Effects of a foliar fertilizer containing boron on the development of Sclerotinia stem rot (Sclerotinia sclerotiorum) on canola (Brassica napus L.) leaves
boron; canola; foliar fertilizer; fungal disease; phenolic compounds; Sclerotinia stem rot
Sclerotinia stem rot (Sclerotinia sclerotiorum Lib. De Bary) is one of the most destructive fungal diseases on canola (Brassica napus L.). The effect of a foliar fertilizer containing 3% boron (Active Flower (TM) [AF]) in reducing disease severity was evaluated. AF at 0.1, 0.3 and 0.5 ml/100 ml was first tested for growth inhibition of S. sclerotiorum in potato dextrose broth. Growth was reduced at 0.5 ml/100 ml by around 90%. Boric acid (BA), an important component of AF, was tested against fungal growth at 10 ml/L, and no significant effect (p = .05) was found. Foliar applications of AF and AF formulation that did not contain boron at 0.1, 0.3 and 0.5 ml/100 ml were made weekly to canola 'Westar' grown under greenhouse conditions. Treatments were also made with BA at 10 ml/L to canola plants. After four applications, AF at 0.5 ml/100 ml and BA at 10 ml/L enhanced boron levels in leaves by fivefold and threefold, respectively, compared with the control. Lesion size of S. sclerotiorum on detached leaves was significantly (p < .05) reduced by AF at 0.5 ml/100 ml, but lesion size was not reduced on AFWB-treated leaves. Experiments were repeated twice with the same results. Levels of phenolic compounds in leaves treated with 0.5 ml/100 ml AF were enhanced by twofold compared with the control. There were no significant differences in lignin, peroxidase (POD) or polyphenoloxidase (PPO) between the control and AF treatments. These results suggest that enhanced boron levels in canola leaves were associated with a suppressive effect on disease due to S. sclerotiorum. DOI
126.Punja, ZK; Collyer, D; Scott, C; Lung, S; Holmes, J; Sutton, D. (2019) Pathogens and Molds Affecting Production and Quality of Cannabis sativa L.Front. Plant Sci. 10 Pathogens and Molds Affecting Production and Quality of Cannabis sativa L.
diseases; plant pathogens; epidemiology; post-harvest molds; fungi; root infection; endophytes
Plant pathogens infecting marijuana (Cannabis sativa L.) plants reduce growth of the crop by affecting the roots, crown, and foliage. In addition, fungi (molds) that colonize the inflorescences (buds) during development or after harvest, and which colonize internal tissues as endophytes, can reduce product quality. The pathogens and molds that affect C. sativa grown hydroponically indoors (in environmentally controlled growth rooms and greenhouses) and field-grown plants were studied over multiple years of sampling. A PCR-based assay using primers for the internal transcribed spacer region (ITS) of ribosomal DNA confirmed identity of the cultures. Root-infecting pathogens included Fusarium oxysporum, Fusarium solani, Fusarium brachygibbosum, Pythium dissotocum, Pythium myriotylum, and Pythium aphanidermatum, which caused root browning, discoloration of the crown and pith tissues, stunting and yellowing of plants, and in some instances, plant death. On the foliage, powdery mildew, caused by Golovinomyces cichoracearum, was the major pathogen observed. On inflorescences, Penicillium bud rot (caused by Penicillium olsonii and Penicillium copticola), Botrytis bud rot (Botrytis cinerea), and Fusarium bud rot (F. solani, F. oxysporum) were present to varying extents. Endophytic fungi present in crown, stem, and petiole tissues included soil-colonizing and cellulolytic fungi, such as species of Chaetomium, Trametes, Trichoderma, Penicillium, and Fusarium. Analysis of air samples in indoor growing environments revealed that species of Penicillium, Cladosporium, Aspergillus, Fusarium, Beauveria, and Trichoderma were present. The latter two species were the result of the application of biocontrol products for control of insects and diseases, respectively. Fungal communities present in unpasteurized coconut (coco) fiber growing medium are potential sources of mold contamination on cannabis plants. Swabs taken from greenhouse-grown and indoor buds pre- and post-harvest revealed the presence of Cladosporium and up to five species of Penicillium, as well as low levels of Alternaria species. Mechanical trimming of buds caused an increase in the frequency of Penicillium species, presumably by providing entry points through wounds or spreading endophytes from pith tissues. Aerial distribution of pathogen inoculum and mold spores and dissemination through vegetative propagation are important methods of spread, and entry through wound sites on roots, stems, and bud tissues facilitates pathogen establishment on cannabis plants. DOI PubMed
124.Punja, ZK; Tirajoh, A; Collyer, D; Ni, L. (2019) Efficacy of Bacillus subtilis strain QST 713 (Rhapsody) against four major diseases of greenhouse cucumbers.Crop Prot. 124 Efficacy of Bacillus subtilis strain QST 713 (Rhapsody) against four major diseases of greenhouse cucumbers
Biological control; Pythium root rot; Pythium aphanidermatum; Fusarium root and stem rot; Fusarium oxysporum; Gummy stem blight; Didymella bryoniae; Powdery mildew; Podosphaera xanthii
Diseases of cucumber (Cucumis sativus L.) are a major limiting factor during commercial greenhouse production of the crop. The biological control agent Bacillus subtilis strain QST 713 (Rhapsody, containing 1 x 10(9) colony-forming units per g) was evaluated for efficacy against four major diseases affecting greenhouse cucumbers grown in British Columbia, Canada. These diseases included powdery mildew, Fusarium root and stem rot, Pythium crown and root rot, and gummy stem blight. Rhapsody was applied as a root drench or spray to leaves or flowers at a rate of 1.0 L product/100 L water, or in some experiments at 1.5 L product/100 L water. Applications were made 1 and 4 days prior to, as well as after, pathogen inoculation. Weekly foliar applications of Rhapsody significantly (P <= 0.05) reduced powdery mildew colony development and showed eradicative effects on established leaf infections. Leaf area infected on untreated plants was 80-100% compared to 15-20% on Rhapsody-treated plants. Two drenches of Rhapsody made 1 and 4 days prior to Fusarium inoculation significantly (P <= 0.05) improved plant growth (height and dry weight) after five weeks, but growth was lower compared to the noninoculated control plants. Treatments made 1 and 4 days post-inoculation were much less effective in preventing disease development. Application of Rhapsody to Pythium-inoculated plants, prior to or after inoculation, did not significantly increase plant height or dry weight compared to the pathogen-only treatment. Developing cucumber fruit inoculated with a mycelial suspension of the gummy stem blight pathogen caused 24-30% post-harvest decay after fruit were incubated at 20-22 degrees C for 7-10 days. Fruit infection was reduced by almost 55% when Rhapsody was applied 24 h prior to pathogen inoculation while an application made 24 h after pathogen inoculation had no effect. Preventative applications of Rhapsody were effective in reducing development of powdery mildew, Fusarium root and stem rot, and gummy stem blight on cucumber, while eradicative applications were effective against powdery mildew only. There was no effect observed against Pythium root rot. DOI
122.Punja, ZK. (2018) Flower and foliage-infecting pathogens of marijuana (Cannabis sativa L.) plants.Can. J. Plant Pathol. 40 Flower and foliage-infecting pathogens of marijuana (Cannabis sativa L.) plants
bud rot; cannabis; disease management; marijuana; pathogen detection; powdery mildew
Flower buds of Cannabis sativa develop as inflorescences (buds) which are harvested and dried prior to sale. The extent to which fungal plant pathogens can colonize the buds prior to harvest has not been previously studied. Flower buds were sampled at various pre-harvest and harvest time periods during 2015-2017 at locations in British Columbia and Alberta to determine the range of fungi present. Isolated fungi were inoculated onto developing buds to determine the extent of tissue colonization. A pre- and post-harvest internal rot was associated with Botrytis cinerea, causing botrytis bud rot. In addition, two species of Penicillium - P. olsonii and P. copticola - were recovered from pre-harvest flower buds, as well as dried buds, and shown to cause penicillium bud rot. Scanning electron microscopy studies revealed colonization and sporulation on bracts and stigmas of the flower buds by P. olsonii. Several Fusarium species, which were identified using ITS rDNA sequences as F. solani, F. oxysporum and F. equiseti, were isolated from pre-harvest flower buds. These fungi colonized the flower buds following artificial inoculation and caused visible rot symptoms. The most severe symptoms were caused by F. solani, followed by F. oxysporum and, to a much lesser extent, F. equiseti. Powdery mildew infection of the foliage and flower buds was caused by Golovinomyces(Erysiphe) cichoracearum. The pathogen was detected on young vegetatively propagated cuttings and sporulation was abundant on older plants and on flower buds. The various fungi recovered from cannabis flower buds may be present as contaminants from aerially dispersed spores and have the potential to cause various types of pre- and post-harvest bud rot under conducive environmental conditions. Powdery mildew may be spread through aerially disseminated spores and infected propagation materials. Management of these pathogens will require monitoring of the growth environment for spore levels and implementation of sanitization methods to reduce inoculum sources. DOI
120.Punja, ZK; Rodriguez, G. (2018) Fusarium and Pythium species infecting roots of hydroponically grown marijuana (Cannabis sativa L.) plants.Can. J. Plant Pathol. 40 Fusarium and Pythium species infecting roots of hydroponically grown marijuana (Cannabis sativa L.) plants
disease management; hydroponics; marijuana; pathogen detection; root rot; wilt
An increase in the cultivation of Cannabis sativa (cannabis or marijuana) plants in Canada is becoming associated with increased incidence and severity of various diseases, many of which have not been previously reported. In this study, hydroponically grown C. sativa plants were sampled over a 3-year period (2014-2017) to determine the prevalence of root pathogens. Following isolation, pathogenicity studies were conducted to establish the extent of disease symptoms caused by the recovered microbes. Root rot was found to be caused by two Pythium species - Pythium dissotocum Drechsler and P. myriotylum Drechsler. As well, two Fusarium species were recovered from diseased plants - Fusarium oxysporum Schlecht. emend. Snyder & Hansen and F. solani (Mart.) Sacc. Upon inoculation onto healthy plants, all isolates of Pythium spp. caused browning and a reduction in root mass, accompanied by stunting. Inoculation of plants with F. oxysporum caused browning of roots and crown rot infection, accompanied by pith and vascular discolouration, and in some cases wilting of plants, while root and crown infection was observed with F. solani. Phylogenetic analysis of internal transcribed spacer (ITS) and elongation factor 1 (EF-1) sequences revealed that the Fusarium species affecting cannabis plants shared 99-100% sequence homology with isolates causing stem rot and wilt in other hosts, including cumin and tomato, suggesting they were not uniquely adapted to cannabis. The potential for spread of F. oxysporum through the hydroponic system was confirmed by its detection in the recirculating nutrient solution. Furthermore, rooted cuttings obtained from commercial propagators were found to harbour Fusarium root infection that resulted in subsequent stunting, yellowing and occasional death of plants. This demonstrates the potential for long-distance spread of the pathogen. The two Pythium species recovered from cannabis plants have an extremely broad host range and are not unique to this host. An additional species, P. aphanidermatum (Edson) Fitzp., was recovered from diseased plants grown under greenhouse conditions in 2018. The management of these root pathogens on C. sativa will require the evaluation and implementation of sanitization methods, biological control agents, and chemical products adapted from greenhouse vegetable production practices. The use of pathogen-free propagation materials and identification of potential sources of disease resistance should also become a priority. DOI
119.Punja, ZK; Scott, C; Chen, S. (2018) Root and crown rot pathogens causing wilt symptoms on field-grown marijuana (Cannabis sativa L.) plants.Can. J. Plant Pathol. 40 Root and crown rot pathogens causing wilt symptoms on field-grown marijuana (Cannabis sativa L.) plants
crown rot; fusarium root rot; pythium root rot; stem colonization; wilting
Yellowing and wilting symptoms on field-grown Cannabis sativa (cannabis) plants followed by total plant collapse under conditions of extreme hot weather were observed in northern California in 2017. The crown regions of affected plants were dark and sunken and internal tissue discolouration extended 10-15 cm above the soil surface. Isolations made from the pith, vascular and cortical tissues in the crown region yielded Fusarium oxysporum (40% frequency), F. brachygibbosum (28% frequency), Pythium aphanidermatum (22% frequency), Fusarium solani and F. equiseti (5% frequency each). Pathogenicity tests were conducted on rooted plantlets to establish the extent of root and crown decay, as well as on mature stems to determine the extent of stem tissue colonization caused by these species. Extensive reduction in root length was caused by F. solani, F. oxysporum, F. brachygibbosum and P. aphanidermatum and wounding significantly enhanced disease development. Stem tissue colonization by these pathogens at wound sites was similarly extensive. Isolates of F. equiseti were non-pathogenic. Both F. solani and P. aphanidermatum caused plant mortality within 6-10weeks following inoculation. In phylogenetic analyses using the internal transcribed spacer (ITS) rDNA region and the elongation factor 1 (EF-1) region, F. oxysporum isolates from cannabis plants in northern California were grouped separately from all other formae speciales and from isolates previously recovered from British Columbia. Two isolates of F. brachygibbosum were identical to an isolate previously reported to infect almond stems in cold storage and field-grown seedlings in northern California. These findings indicate that a complex of pathogens potentially can cause root and crown rot under field conditions, resulting in wilt symptoms and collapse of cannabis plants. DOI
117. Macdonald, JL; Punja, ZK. (2017) Occurrence of botrytis leaf blight, anthracnose leaf spot, and white blister rust on Wasabia japonica in British Columbia.Canadian Journal of Plant Pathology 39: 60-71 Occurrence of botrytis leaf blight, anthracnose leaf spot, and white blister rust on Wasabia japonica in British Columbia
Albugo candida; Botrytis cinerea; Colletotrichum higginsianum; leaf blight; leaf spot; wasabi; white rust; Wasabi; brulure helminthosporienne; tache des feuilles; rouille blanche; Colletotrichum higginsianum; Botrytis cinerea; Albugo candida
Diseases of wasabi (Wasabia japonica) are the most important reason for crop failure in commercial greenhouses, and expanding disease issues highlight the importance of identifying the causal agents. Diseased wasabi leaves were collected during 2013-2015 from greenhouses in the Fraser Valley of British Columbia. Isolations from plants showing symptoms of leaf spot and blight yielded a Botrytis sp. and a Colletotrichum sp. when plated onto PDA. In addition, pustules containing sporangiospores of an Albugo sp. were observed on naturally infected leaves. Molecular identification using the ITS1-ITS4 region of rDNA revealed the pathogens isolated from wasabi leaves were B. cinerea and C. higginsianum, while the Albugo species was A. candida. The B. cinerea isolates were shown to be weakly pathogenic, infecting only succulent and senescing or wounded leaves. Inoculation studies with C. higginsianum showed that it caused lesions on Brassica juncea (mustard) leaves and on wasabi, but not on Medicago sativa (alfalfa). In culture, fastest growth occurred at 25 and 30 degrees C, and the highest conidial production after 7days occurred under continuous darkness. Isolates of A. candida collected from naturally infected Capsella bursa-pastoris (shepherd's purse) plants were identical to those from wasabi plants. These previously unreported pathogens on wasabi in Canada will continue to provide challenges to commercial producers and further research into disease control methods is warranted. ResumeLes maladies du wasabi (Wasabi japonica) sont les principales causes des recoltes deficitaires dans les serres commerciales, et les problemes relatifs a l'expansion de ces maladies mettent en evidence l'importance d'en identifier les agents causaux. De 2013 a 2015, des feuilles infectees de wasabi ont ete collectees dans des serres de la vallee du Fraser, en Colombie-Britannique. Lorsque places sur de la gelose dextrosee a la pomme de terre, les tissus de plants affichant des symptomes de la tache des feuilles et de la brulure helminthosporienne ont produit une espece Botrytis et une de Colletotrichum. De plus, des pustules contenant des sporangiospores d'une espece d'Albugo ont ete observees sur des feuilles naturellement infectees. L'identification moleculaire basee sur la region de l'ITS (amorces ITS1-ITS4) de l'ADNr a permis de conclure que les agents pathogenes isoles a partir des feuilles de wasabi etaient B. cinerea et C. higginsianum, tandis que l'espece d'Albugo etait A. candida. Les isolats de B. cinerea se sont averes faiblement pathogenes, infectant seulement les feuilles succulentes et senescentes ou encore les feuilles blessees. Des etudes d'inoculation realisees avec C. higginsianum ont montre qu'il causait des lesions sur les feuilles de Brassica juncea (moutarde) et le wasabi, mais pas sur Medicago sativa (luzerne). En culture, la croissance la plus rapide s'est produite a 25 et 30 degrees C, et la production conidienne la plus elevee s'est manifestee au bout de sept jours d'obscurite totale. Les isolats d'A. candida collectes sur des plants de Capsella bursa-pastoris (bourse-a-pasteur) naturellement infectes etaient identiques a ceux provenant des plants de wasabi. Ces agents pathogenes non rapportes anterieurement sur le wasabi au Canada continueront de constituer un defi pour les producteurs commerciaux et, en consequence, des recherches plus poussees concernant les methodes de lutte contre cette maladie sont justifiees. DOI
114.Punja, ZK; Chandanie, WA; Chen, X; Rodriguez, G. (2017) Phoma leaf spot of wasabi (Wasabia japonica) caused by Leptosphaeria biglobosa.Plant Pathology 66: 480-489 Phoma leaf spot of wasabi (Wasabia japonica) caused by Leptosphaeria biglobosa
black streak; blackleg; Brassica napus; Leptosphaeria biglobosa 'occiaustralensis'; Leptosphaeria maculans 'brassicae'
A leaf spot disease on wasabi plants grown in commercial greenhouses in the Fraser Valley of British Columbia was characterized. Mycelial growth and pycnidial formation were observed within lesions when leaves were incubated under conditions of high humidity. Isolation from diseased tissues consistently yielded colonies of a Phoma species. Sequence analysis of the rDNA internal transcribed spacer (ITS1-5.8S-ITS2) region of eight isolates showed 100% nucleotide sequence identity with Phoma wasabiae and Leptosphaeria biglobosa subspecies 'occiaustralensis' and 99.2% identity with L. biglobosa 'canadensis'. Pathogenicity studies on wasabi leaves showed that wounding greatly facilitated infection and enhanced lesion development for most isolates but was not required for all isolates. Chlorotic areas appeared around the inoculation sites within 4 days, followed by necrosis. Isolates displayed a range of virulence, from weakly to highly virulent, on wasabi leaves. Similar results were observed on leaves of canola cultivar Westar, i.e. wounding significantly increased lesion size and isolates displayed a range of virulence. An isolate of Leptosphaeria maculans 'brassicae' from canola was highly virulent on wasabi and canola leaves, causing lesions similar to those of L. biglobosa 'occiaustralensis' while an isolate of L. biglobosa 'canadensis' from canola was weakly virulent on both hosts and required wounds to infect. These results demonstrate that isolates of L. biglobosa 'occiaustralensis' from wasabi are as virulent as L. biglobosa 'canadensis' on wasabi and canola leaves but in some cases were comparable in virulence to L. maculans 'brassicae'. DOI
112. Sutton, DB; Punja, ZK. (2017) Investigating biospeckle laser analysis as a diagnostic method to assess sprouting damage in wheat seeds.Comput. Electron. Agric. 141: 238-247 Investigating biospeckle laser analysis as a diagnostic method to assess sprouting damage in wheat seeds
Biospeckle; Pre-harvest sprouting; Machine vision; Germination; Wheat
Sprouting Damage is a persistent quality control concern in the cereals industry, as sprouting damaged kernels (SDK) contain enzymes that have a detrimental effect on flour quality. Furthermore, the severity of sprouting damage is difficult to detect using standard visual grading methods. In this work, we present Biospeclde Laser Analysis (BLA) as a diagnostic tool to measure the germination progress and the simulated SDK severity of Canadian Western Red Spring wheat seeds. We first analysed dissected seeds and found that high frequency biospeckle activity in the germ correlated with germination progress. Following this, a novel whole seed grading protocol was developed using qualitative and quantitative data provided by the biospeclde measurement. Using our whole seed grading protocol, seeds subjected to simulated SDK treatments at two levels (10 and 20 h pre-trial water exposure) could be differentiated from healthy seeds and from each respective treatment (p < 0.05). Our results indicate that BLA has the ability to detect latent SDK and may have further applications such as characterizing the dormancy traits of wheat cultivars and studying the seed germination process. (C) 2017 Elsevier B.V. All rights reserved. DOI
109.Punja, ZK; Rodriguez, G; Tirajoh, A. (2016) Effects of Bacillus subtilis strain QST 713 and storage temperatures on post-harvest disease development on greenhouse tomatoes.Crop Protection 84: 98-104 Effects of Bacillus subtilis strain QST 713 and storage temperatures on post-harvest disease development on greenhouse tomatoes
Bacterial populations; Biological control; Fruit rot; Fresh market tomatoes; Rhapsody; Penicillium
Tomato plants in two commercial greenhouses were treated with Rhapsody (Bacillus subtilis strain QST 713, rate of 1.45%) once every 4 weeks during 2012-2013 to determine effects on post-harvest fruit infection. Populations of Bacillus and disease incidence were monitored weekly from harvested fruit over an 18-week period. Population levels of Bacillus ranged from 75 to 110 x 10(4) colony forming units (cfu) cm(-2) of fruit surface area one week after application to 25-30 x 10(4) cfu cm(-2) of fruit surface area 4 weeks after application. Disease incidence on harvested fruit incubated at 21 degrees C for 7-10 days was variable, due to variation in inoculum levels within the greenhouse as well as variable environmental conditions. Both disease incidence and severity were significantly reduced on Rhapsody-treated fruit, especially in the 1-2 week period following application. Post-harvest storage temperature (13 degrees C vs. 21 degrees C) and incubation time (12 vs. 16 days) had a significant effect on final disease severity. Rhapsody treated fruit incubated at 13 degrees C had an average of 1-2% fruit infection compared to up to 20% infection on untreated fruit at 21 degrees C. The most frequent pathogens affecting fruit quality were Penicillium sp. and Rhizopus stolonifer. Rhapsody applications made every 4 weeks maintained sufficiently high populations of Bacillus on the fruit surface to prevent spread of these fungi onto the fruit, resulting in significant post-harvest disease control on fresh market tomatoes. When combined with storage at 13 degrees C for no more than 12 days, disease was reduced to negligible levels. (C) 2016 Elsevier Ltd. All rights reserved. DOI
108.Punja, ZK; Rodriguez, G; Tirajoh, A; Formby, S. (2016) Role of fruit surface mycoflora, wounding and storage conditions on post-harvest disease development on greenhouse tomatoes.Canadian Journal of Plant Pathology 38: 448-459 Role of fruit surface mycoflora, wounding and storage conditions on post-harvest disease development on greenhouse tomatoes
fruit decay; fungal pathogens; post-harvest storage; Solanum lycopersicum
Fungi causing post-harvest decay of greenhouse-grown tomato fruits in British Columbia were recovered from diseased samples collected during 2010 and 2011. The most frequently isolated fungi were Penicillium olsonii, Botrytis cinerea, Rhizopus stolonifer and Alternaria alternata, and to a lesser extent Galactomyces geotrichum. Pathogenicity tests showed that R. stolonifer caused the greatest fruit decay, followed by P. olsonii. The remaining fungi also caused some fruit rot, and lesion development by all fungi was enhanced by wounding of the fruits. A post-inoculation incubation temperature of 21 degrees C promoted much greater disease development compared with 13 degrees C. To assess the composition of the mycoflora on the surface of ripening tomato fruits, swab samples were collected weekly over a 6-18 week period from two commercial greenhouses during 2011 and 2012 and streaked onto potato dextrose agar. The most commonly recovered fungi from the fruit surfaces were species of Penicillium, Cladosporium, Aspergillus, Rhizopus and Alternaria. The composition of fungal populations fluctuated from one week to the next in both greenhouses and over both years. Samples of leaf litter consisting of discarded leaf prunings harboured all of these fungi that were present on the fruit surface. Harvested fruit samples were subsequently incubated at 21 degrees C to assess the level of disease development. The resulting fruit decay was caused primarily by species of Penicillium, Alternaria and Rhizopus, and a major source of inoculum originated from the stem and calyx tissues, which led to fruit infection near the stem end. Shipments of tomato fruits in refrigerated trucks were monitored over a 6-10 day period after harvest and showed variable temperature and humidity levels during transportation. Post-harvest decay of tomatoes is influenced by the composition of the fruit surface mycoflora, presence of fruit injury, storage temperatures and environmental conditions during shipping. DOI
107.Punja, ZK; Wally, O; Jayaraj, J. (2016) Transgenic approaches to enhance disease resistance in carrot plants to fungal pathogens.International Symposium On Biotechnology and Other Omics in Vegetable Science 1145: 143-152 Transgenic approaches to enhance disease resistance in carrot plants to fungal pathogens
transgenic plants; Daucus carotae; genetic engineering; pathogenesis-related proteins; thaumatin; chitinase; glucanase; promoters; necrotrophic fungi
Diseases caused by fungi and bacteria are among the most important constraints to carrot production worldwide. These disease afflict the carrots not only during the growing season but also during postharvest storage. In this study, transgenic expression of antifungal genes was investigated to enhance tolerance in carrots to two necrotrophic fungi - Botrytis cinerea and Sclerotinia sclerotiorum. Promoters were evaluated for suitable transgene expression in carrot tissues. The Arabidopsis ubiquitin promoter and Cauliflower mosaic virus 35S promoter showed high-level p-glucuronidase (uidA) gene expression in root tips, leaves and tap roots. Using an Agrobacterium-mediated transformation system, a thaumatin-like protein, chitinase, glucanase and peroxidase genes were expressed in carrot tissues. Transgene expression was confirmed by PCR and by protein expression analysis. Leaf tissues were challenged with the fungal pathogens in vitro. The most significant reduction was observed in the peroxidase-expressing lines, with 75-90% disease reduction. One chitinase-expressing line and 2 TLP-expressing lines had a 15-50% reduction in disease but no glucanase-expressing lines showed significant disease reduction. This is the first report of peroxidase over-expression leading to a significant reduction in disease caused by necrotrophic fungal pathogens. DOI
104. Chatterton, S; Punja, ZK. (2012) Colonization of geranium foliage by Clonostachys rosea f. catenulata, a biological control agent of botrytis grey mould.Botany-Botanique 90: 1-10 Colonization of geranium foliage by Clonostachys rosea f. catenulata, a biological control agent of botrytis grey mould
biological control; geranium; leaf colonization; Gliocladium catenulatum
The ecological requirements for the colonization of geranium leaves by the biocontrol agent Clonostachys rosea f. catenulata strain J1446 were investigated. Although this biocontrol agent is a soil-inhabiting fungus, treatment of geranium foliage with the agent can reduce grey mould caused by Botrytis cinerea in the greenhouse. To characterize the extent of foliar colonization, a GUS-transformed isolate of C. rosea f. catenulata was applied to foliage of two geranium cultivars, Pelargonium x hortorum and Pelargonium x domesticum. Population levels of C. rosea f. catenulata were found to be highest on senescent leaves and stems, followed by fully expanded leaves, and lowest on newly emerged leaves of both cultivars. Optimum temperature for leaf and petiole colonization was 20-25 degrees C for both cultivars. The biocontrol agent required at least 12 h of continuous leaf wetness to achieve maximum population densities on the leaves and stems of both cultivars. On whole plants, colonization was significantly higher on wounded leaves, stems, and senescing leaves compared with that on nonwounded leaves, stems, and mature leaves, respectively. GUS staining indicated that the fungus preferentially colonized the wound sites of leaves and the cut portions of stems. Results indicate that this biocontrol agent can successfully colonize the foliage of geraniums, thus demonstrating the endophytic ability of C. rosea f. catenulata in both root and foliar tissues. DOI
103. Elmhirst, JF; Haselhan, C; Punja, ZK. (2011) Evaluation of biological control agents for control of botrytis blight of geranium and powdery mildew of rose.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 33: 499-505 Evaluation of biological control agents for control of botrytis blight of geranium and powdery mildew of rose
biological control; botrytis; geranium; ornamental disease management; powdery mildew; rose
Four commercially formulated biological control products, containing Gliocladium catenulatum (Prestop (R) WP), Trichoderma harzianum (PlantShield (R)) and Bacillus subtilis [Serenade (R) MAX (TM) (wettable powder) and Rhapsody (R) ASO (TM) (liquid)] were evaluated for control of rose powdery mildew (Podosphaera pannosa) on outdoor, container-grown roses and botrytis blight (Botrytis cinerea) on greenhouse-grown zonal geraniums in 2006 and 2007. The products were applied every 7-14 days and disease incidence and severity were compared to the fungicides captan for botrytis blight control and myclobutanil for powdery mildew control. Gliocladium catenulatum provided the best control of botrytis blight of geranium in both years, and disease incidence was significantly lower compared with plants treated with captan. Both G. catenulatum and B. subtilis (Serenade MAX (TM) applied every 14 days or Rhapsody ASO (TM) every 7 days) provided significant control of rose powdery mildew, which was comparable to that provided by myclobutanil. These preventative disease-suppressive biological control products may have a useful role in commercial nursery crop production. DOI
102. Jayaraman, J; Norrie, J; Punja, ZK. (2011) Commercial extract from the brown seaweed Ascophyllum nodosum reduces fungal diseases in greenhouse cucumber.Journal of Applied Phycology 23: 353-361 Commercial extract from the brown seaweed Ascophyllum nodosum reduces fungal diseases in greenhouse cucumber
Ascophyllum; Cucumber; Fungal diseases; Resistance; Mechanism
This study examined the effects of Stimplex (TM), a marine plant extract formulation from Ascophyllum nodosum, on some common cucumber fungal pathogens. Greenhouse cucumber plants were sprayed and/or root drenched using Stimplex (TM) at 0.5% or 1% concentration twice at 10-day intervals. Treatments also included application of fungicide (chlorothalonil, 2 g L(-1)) alternating with Stimplex (TM) application. Treated plants were inoculated with four cucumber fungal pathogens including Alternaria cucumerinum, Didymella applanata, Fusarium oxysporum, and Botrytis cinerea. Stimplex (TM) application resulted in a significant reduction in disease incidence of all the pathogens tested. The disease control effect was greater for Alternaria and Fusarium infection, followed by Didymella and Botrytis. Combined spray and root drenching with Stimplex (TM) was more effective than either spray or root drenching alone. The alternation of one fungicide application, alternated with Stimplex (TM) application, was highly effective and found to be the best treatment in reducing the disease ratings. Plants treated with Stimplex T showed enhanced activities of various defense-related enzymes including chitinase, beta-1,3-glucanase, peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, and lipoxygenase. Altered transcript levels of various defense genes, including chitinase, lipoxygenase, glucanase, peroxidase, and phenylalanine ammonia lyase were observed in treated plants. Cucumber plants treated with Stimplex (TM) also accumulated higher level of phenolics compared to water controls. These results suggest that seaweed extracts enhance disease resistance in cucumber probably through induction of defense genes or enzymes. DOI
101.Punja, ZK. (2011) American Ginseng: Research Developments, Opportunities, and Challenges.Journal of Ginseng Research 35: 368-374 American Ginseng: Research Developments, Opportunities, and Challenges
Panax quinquefolius; Genetic variation; Disease; Transgenic research
American ginseng (Panax quinquefolius L.) is grown in some regions of the USA and Canada and marketed for its health promoting attributes. While cultivation of this plant species has taken place in North America for over 100 years, there are many challenges that need to be addressed. In this article, the current production method used by growers is described and the challenges and opportunities for research on this valuable plant are discussed. These include studies on pharmacological activity, genetic diversity within the species, genetic improvement of currently grown plants, molecular characterization of gene expression, and management of diseases affecting plant productivity. The current research developments in these areas are reviewed and areas requiring further work are summarized. Additional research should shed light on the nature of the bioactive compounds and their clinical effects, and the molecular basis of active ingredient biosynthesis, and provide more uniform genetic material as well as improved plant growth, and potentially reduce losses due to pathogens. DOI
100. Bradley, GG; Punja, ZK. (2010) Composts containing fluorescent pseudomonads suppress fusarium root and stem rot development on greenhouse cucumber.Canadian Journal of Microbiology 56: 896-905 Composts containing fluorescent pseudomonads suppress fusarium root and stem rot development on greenhouse cucumber
biological control; Pseudomonas; Fusarium oxysporum; diacetylphloroglucinol; compost
Three composts (Ball, dairy, and greenhouse) were tested for the ability to suppress the development of Fusarium root and stem rot (caused by Fusarium oxysporum f. sp. radicis-cucumerinum) on greenhouse cucumber. Dairy and greenhouse composts significantly reduced disease severity (P = 0.05), while Ball compost had no effect. Assessment of total culturable microbes in the composts showed a positive relationship between disease suppressive ability and total population levels of pseudomonads. In vitro antagonism assays between compost-isolated bacterial strains and the pathogen showed that strains of Pseudomonas aeruginosa exhibited the greatest antagonism. In growth room trials, strains of P. aeruginosa and nonantagonistic Pseudomonas maculicola, plus 2 biocontrol strains of Pseudomonas fluorescens, were tested for their ability to reduce (i) survival of F. oxysporum, (ii) colonization of plants by the pathogen, and (iii) disease severity. Cucumber seedlings grown in compost receiving P. aeruginosa and P. fluorescens had reduced disease severity index scores after 8 weeks compared with control plants without bacteria. Internal stem colonization by F. oxysporum was significantly reduced by P. aeruginosa. The bacteria colonized plant roots at 1.9 x 10(6) +/- 0.73 x 10(6) CFU.(g root tissue)(-1) and survival was >10(7) CFU.(g compost)(-1) after 6 weeks. The locus for 2,4-diacetylphloroglucinol production was detected by Southern blot analysis and confirmed by PCR. The production of the antibiotic 2,4-diacetylphloroglucinol in liquid culture by P. aeruginosa was confirmed by thin layer chromatography. These results demonstrate that composts containing antibiotic-producing P. aeruginosa have the potential to suppress diseases caused by Fusarium species. DOI
99. Chatterton, S; Punja, ZK. (2010) Factors influencing colonization of cucumber roots by Clonostachys rosea f. catenulata, a biological disease control agent.Biocontrol Science and Technology 20: 37-55 Factors influencing colonization of cucumber roots by Clonostachys rosea f. catenulata, a biological disease control agent
MONITORING TRICHODERMA-HARZIANUM; SP RADICIS-CUCUMERINUM; FUSARIUM-OXYSPORUM; DAMPING-OFF; STEM ROT; NONPATHOGENIC FUSARIUM; GREENHOUSE CUCUMBER; POPULATION-DYNAMICS; GUS TRANSFORMANT; TOMATO ROOT
The biocontrol fungus Clonostachys rosea f. catenulata (Gliocladium catenulatum) strain J1446, commercially available as Prestop(R) (Verdera Oy, Finland), is an effective antagonist against several root and foliar greenhouse pathogens. The biocontrol agent forms dense networks of hyphae on plant roots, grows internally in root epidermal cells, and produces hydrolytic enzymes, all of which lead to a reduction in pathogen propagules. An understanding of the environmental and host factors that influence root colonization by C. rosea f. catenulata is important to maximize disease control efficacy. Cucumber roots grown in nutrient solution in containers were inoculated with conidia of a GUS-transformed strain of C. rosea f. catenulata. Population levels associated with roots over time were assessed by colony-plate counts, GUS staining and enzymatic assays to determine GUS activity. Variables such as pH, temperature and growing medium were major factors that influenced population levels, while cucumber cultivar, addition of nutrients, and wounding of roots did not appear to significantly affect colonization. Population density of C. rosea f. catenulata on roots was highest when the nutrient solution was maintained at pH 5, 6, or 7, and at temperatures of 18-22 degrees C. Lowest colonization levels were observed on roots of plants grown in potting mix or in field soil. Measurement of GUS activity provided a slightly more accurate assessment of root colonization levels compared to colony-plate counts. These results illustrate the optimal environmental conditions which can ensure maximum root colonization by C. rosea f. catenulata and enhance disease control by the biocontrol agent. DOI
98. Wally, O; Punja, ZK. (2010) Enhanced disease resistance in transgenic carrot (Daucus carota L.) plants over-expressing a rice cationic peroxidase.Planta 232: 1229-1239 Enhanced disease resistance in transgenic carrot (Daucus carota L.) plants over-expressing a rice cationic peroxidase
Peroxidase; Lignin; Hydrogen peroxide; Pathogen resistance; Oxidative burst
Plant class III peroxidases are involved in numerous responses related to pathogen resistance including controlling hydrogen peroxide (H2O2) levels and lignin formation. Peroxidases catalyze the oxidation of organic compounds using H2O2 as an oxidant. We examined the mechanisms of disease resistance in a transgenic carrot line (P23) which constitutively over-expresses the rice cationic peroxidase OsPrx114 (previously known as PO-C1) and which exhibits enhanced resistance to necrotrophic foliar pathogens. OsPrx114 over-expression led to a slight enhancement of constitutive transcript levels of pathogenesis-related (PR) genes. These transcript levels were dramatically increased in line P23 compared to controls [GUS construct under the control of 35S promoter (35S::GUS)] when tissues were treated with cell wall fragments of the fungal pathogen Sclerotinia sclerotiorum (SS-walls), and to a lesser extent with 2,6-dichloroisonicotinic acid. There was no basal increase in basal H2O2 levels in tissues of the line P23. However, during an oxidative burst response elicited by SS-walls, H2O2 accumulation was reduced in line P23 despite, typical media alkalinization associated with oxidative burst responses was observed, suggesting that OsPrx114 was involved in rapid H2O2 consumption during the oxidative burst response. Tap roots of line P23 had increased lignin formation in the outer periderm tissues, which was further increased during challenge inoculation with Alternaria radicina. Plant susceptibility to a biotrophic pathogen, Erysiphe heraclei, was not affected. Disease resistance to necrotrophic pathogens in carrot as a result of OsPrx114 over-expression is manifested through increased PR transcript accumulation, rapid removal of H2O2 during oxidative burst response and enhanced lignin formation. DOI
97. Chatterton, S; Punja, ZK. (2009) Chitinase and beta-1,3-glucanase enzyme production by the mycoparasite Clonostachys rosea f. catenulata against fungal plant pathogens.Canadian Journal of Microbiology 55: 356-367 Chitinase and beta-1,3-glucanase enzyme production by the mycoparasite Clonostachys rosea f. catenulata against fungal plant pathogens
TRICHODERMA-HARZIANUM; BIOLOGICAL-CONTROL; GLIOCLADIUM-CATENULATUM; DAMPING-OFF; ANTIFUNGAL ACTIVITY; RHIZOCTONIA-SOLANI; BOTRYTIS-CINEREA; BIOCONTROL; EXPRESSION; CUCUMBER
Clonostachys rosea f. catenulata (syn. Gliocladium catenulatum) is an effective fungal biological agent against Fusarium root and stem rot and Pythium damping-off diseases on cucumber plants. Both chitinase and beta-1,3-glucanase enzymes were produced when C. rosea was grown on a synthetic medium containing chitin or laminarin as a sole carbon source, respectively. Chitinase production was also induced by Fusarium cell walls, while beta-1,3-glucanase activity was induced by both Fusarium and Pythium cell walls, as well as by growth on homogenized cucumber roots and on low-carbon media. Mycelial growth of Fusarium and Pythium, when exposed to C. rosea culture filtrates that contain glucanase activity, was significantly reduced compared with the controls, and cell walls of both pathogens were degraded. On excised cucumber roots, hyphae of C. rosea formed appressorium-like structures and coiled around hyphae of Pythium. In culture, C. rosea caused localized degradation of Fusarium hyphae. Cucumber root tissues colonized by C. rosea showed higher levels of beta-1,3-glucanase activity at 7 days post-application compared with untreated controls. To determine if this activity was derived from C. rosea, glucanase isoforms were separated on activity gels. Fungal culture filtrates and root extracts contained the same predominant 20 kDa isoform. Reverse-transcription polymerase chain reaction (RT-PCR) using primers designed to amplify a beta-1,3-glucanase gene in C. rosea confirmed glucanase expression on roots. These results show that C. rosea produces beta-1,3-glucanase in situ, which can degrade hyphae of Fusarium and Pythium and contribute to biological control efficacy. DOI
96. Chatterton, S; Punja, ZK. (2009) Interactions Between Clonostachys rosea f. catenulata, Fusarium oxysporum and Cucumber Roots Leading to Biological Control of Fusarium Root and Stem Rot.Recent Developments in Management of Plant Diseases 1: 93-106 Interactions Between Clonostachys rosea f. catenulata, Fusarium oxysporum and Cucumber Roots Leading to Biological Control of Fusarium Root and Stem Rot
Clonostachys rosea f. catenulata; Gliocladium catenulatum; Fusarium oxysporum f. sp radicis-cucumerinum; Cucumis sativus; Biological control; GUS transformation; Colonization; Competition; Mycoparasitism
Clonostachys rosea f. catenulata (Gliocladium catenulatum) strain J1446 (formulated as Prestop WP) suppressed Fusarium root and stem rot caused by Fusarium oxysporum f. sp. radicis-cucumerinum on greenhouse cucumber plants. In culture, C. rosea produced chitinase and beta-1,3-glucanase enzymes on chitin or laminarin as a sole carbon source, respectively, and caused localized degradation of Fusarium hyphae. These enzymes were also induced by growth on Fusarium mycelial fragments and homogenized cucumber roots in vitro. Cucumber root extracts from C. rosea-colonized plants had significantly higher levels of glucanase at 7 days post-application compared to untreated controls. Reverse-transcription polymerase chain reaction using primers designed to amplify a beta-1,3-glucanase gene confirmed C. rosea glucanase expression on roots. Following transformation of the biocontrol agent with Agrobacterium tumefaciens strain AGL-1 containing the hygromycin resistance (hph) and beta-glucuronidase (uidA) genes, blue-stained mycelia could be seen growing on the surface and within epidermal and cortical cells of roots, stems and shoots 3 weeks after treatment. Application of C. rosea preceding inoculation with Fusarium significantly reduced pathogen populations on roots compared to plants inoculated with Fusarium alone, while densities of the biocontrol agent appeared to increase in the presence of the pathogen. Colonization of infection sites by C. rosea in the root zone is one of the mechanisms by which pathogen development and disease incidence is reduced. DOI
92. Jayaraj, J; Rahman, M; Wan, A; Punja, ZK. (2009) Enhanced resistance to foliar fungal pathogens in carrot by application of elicitors.Annals of Applied Biology 155: 71-80 Enhanced resistance to foliar fungal pathogens in carrot by application of elicitors
ACIBENZOLAR-S-METHYL; BOTRYTIS-CINEREA; SYSTEMIC RESISTANCE; SALICYLIC-ACID; TOMATO PLANTS; CHITINASE; INDUCTION; CHITOSAN; LEAVES; EXPRESSION
Treatment of greenhouse-grown carrot plants with salicylic acid (SA) (100 mu m), chitosan (0.02%) and the nutrient-chelate product Alexin (1%) followed 10 h later by inoculation with the necrotrophic fungal pathogens Alternaria radicina and Botrytis cinerea significantly reduced disease development 10 days after inoculation (d.a.i.) compared with control plants sprayed with water. The most effective treatment was chitosan, followed by Alexin and SA. Additional sprays of elicitors resulted in significantly lower disease development 25 d.a.i. Treated plants had elevated transcript levels of pathogenesis-related protein 1 (PR1), chitinase, lipid transfer protein (LTP), chalcone synthase, nonexpressor of PR1 and pathogenesis-related protein 5 (PR5) genes compared with control plants when assayed 10-70 h after treatment. The activity of peroxidase, polyphenoloxidase, phenylalanine ammonia-lyase, chitinase, beta-1,3-glucanase and lipoxygenase was significantly increased in elicitor-treated plants compared with control plants 12-72 h after treatment. Microscopic examination of treated leaves revealed reduced fungal growth and colonisation, 48 h after treatment, accompanied by fewer lesions at 120 h, compared with the control. Protein extracts from elicitor-treated plants reduced spore germination and germ tube elongation of the pathogens in vitro by 30-45%. Elicitor-treated plants accumulated higher amounts of total phenolics, 6-methoxymellin and H2O2 compared with the control. Both chitosan and Alexin induced responses similar to that of SA, suggesting that these elicitors may activate the salicylate pathway, leading to induction of defence genes, enzymes, phytoalexin and phenolics, which collectively reduced fungal colonisation. DOI
89. Rodriguez, G; Punja, ZK. (2009) Vascular blackening of wasabi rhizomes caused by Pectobacterium carotovorum subsp carotovorum.European Journal of Plant Pathology 124: 483-493 Vascular blackening of wasabi rhizomes caused by Pectobacterium carotovorum subsp carotovorum
RAPID IDENTIFICATION; ERWINIAS; PLANTS; CELLS; WILT; PCR
Wasabi (Wasabia japonica) is grown for its highly-valued rhizome which is used as a condiment in Japanese food. Symptoms of vascular blackening in the rhizome were first observed in 2005 in plants grown in British Columbia, Canada. Microscopic observations and microbial isolation from infected tissues revealed that most of the xylem tracheid cells were blackened and bacteria were consistently associated with symptomatic plants. The bacterium most frequently recovered was identified as Pectobacterium carotovorum subsp. carotovorum (Pcc) using BioLog (TM) and sequencing of a specific similar to 510 bp IGS region. Pathogen-free plants obtained using meristem-tip micropropagation were inoculated with a wasabi isolate of Pcc. Vascular blackening symptoms developed in the rhizome after 8 weeks when the rhizome was first wounded by stabbing or cutting, or if the roots were pre-inoculated with Pythium species isolated from rhizome epidermal tissues, followed by inoculation with Pcc at 1 x 10(8) cells ml(-1). Xylem tracheid cells were blackened and Pcc was reisolated from all diseased tissues. The highest frequency of rhizome vascular blackening occurred at 22A degrees C and 27A degrees C and these tissues occasionally succumbed to soft rot at higher temperatures, but not when inoculated tissues were incubated at 10A degrees C. The rooting medium used by growers for vegetative propagation of wasabi was shown to contain Pcc but the pathogen was not recovered from the irrigation water. Entry of Pcc through wounds on wasabi rhizomes and the host tissue response result in symptoms of vascular blackening. DOI
88. Wally, O; Jayaraj, J; Punja, Z. (2009) Comparative resistance to foliar fungal pathogens in transgenic carrot plants expressing genes encoding for chitinase, beta-1,3-glucanase and peroxidise.European Journal of Plant Pathology 123: 331-342 Comparative resistance to foliar fungal pathogens in transgenic carrot plants expressing genes encoding for chitinase, beta-1,3-glucanase and peroxidise
THAUMATIN-LIKE PROTEIN; FUSARIUM-GRAMINEARUM; ENHANCES RESISTANCE; DISEASE-RESISTANCE; WHEAT; TOBACCO; TRANSFORMATION; GLUCANASE; CUCUMBER; LIGNIN
Genes encoding an acidic wheat class IV chitinase (383), an acidic wheat beta 1,3-glucanase (638) and a rice cationic peroxidase (POC1) were introduced into 'Nantes Coreless' carrot (Daucus carota) by Agrobacterium-mediated transformation. The genes were introduced singly or in various combinations followed by selection imposed by the herbicide phosphinothricin. Regenerated plantlets were screened for presence and expression of the three transgenes using PCR, Southern and Northern hybridisations. Eighteen transgenic lines expressing a single transgene and 2 lines each co-expressing 638/383 and 383/POC1 were assessed for resistance to the necrotrophic fungal pathogens Botrytis cinerea and Sclerotinia sclerotiorum. Percentage leaf area diseased was measured 4 and 7 days after inoculation (dai) and compared to non-transformed control plants. Six lines expressing beta-1,3-glucanase 638 alone had no enhanced resistance to B. cinerea at 4 dai and only slight resistance to S. sclerotiorum; there was no effect at 7 dai. Two out of the six lines expressing 383 alone had enhanced tolerance to both pathogens with a 20-50% reduction in disease development at 7 dai. Two lines co-expressing 638/383 had slight reductions in disease by (10-20%) similar to that of the lines expressing chitinase 383 alone. Highest levels of disease resistance were seen in transgenic lines expressing POC1, alone or in combination with chitinase 383. Disease symptoms were slower to develop and symptoms were reduced by up to 90% for B. cinerea and 70% for S. sclerotiorum. The 383/POC1 co-expressing plants developed disease at levels similar to that of POC1 alone. Petioles of plants over-expressing POC1 had higher levels of lignin accumulation constitutively compared to control plants, which was greatly enhanced following inoculation with S. sclerotiorum. These results indicate that peroxidase over-expression can lead to significant disease reduction against necrotrophic pathogens in transgenic carrot plants. DOI
87. Wally, O; Jayaraj, J; Punja, ZK. (2009) Broad-spectrum disease resistance to necrotrophic and biotrophic pathogens in transgenic carrots (Daucus carota L.) expressing an Arabidopsis NPR1 gene.Planta 231: 131-141 Broad-spectrum disease resistance to necrotrophic and biotrophic pathogens in transgenic carrots (Daucus carota L.) expressing an Arabidopsis NPR1 gene
SYSTEMIC ACQUIRED-RESISTANCE; FOLIAR FUNGAL PATHOGENS; DISPLAY ENHANCED RESISTANCE; SALICYLIC-ACID; DEFENSE RESPONSES; SCLEROTINIA-SCLEROTIORUM; SIGNALING PATHWAY; BOTRYTIS-CINEREA; NPR1-LIKE GENES; RICE
The development of transgenic plants highly resistant to a range of pathogens using traditional signal gene expression strategies has been largely ineffective. Modification of systemic acquired resistance (SAR) through the overexpression of a controlling gene such as NPR1 (non-expressor of PR genes) offers an attractive alternative for augmenting the plants innate defense system. The Arabidopsis (At) NPR1 gene was successfully introduced into 'Nantes Coreless' carrot under control of a CaMV 35S promoter and two independent transgenic lines (NPR1-I and NPR1-XI) were identified by Southern and Northern blot hybridization. Both lines were phenotypically normal compared with non-transformed carrots. Northern analysis did not indicate constitutive or spontaneous induction in carrot cultures of SAR-related genes (DcPR-1, 2, 4, 5 or DcPAL). The duration and intensity of expression of DcPR-1, 2 and 5 genes were greatly increased compared with controls when the lines were treated with purified cell wall fragments of Sclerotinia sclerotiorum as well as with 2,6-dichloroisonicotinic acid. The two lines were challenged with the necrotrophic pathogens Botrytis cinerea, Alternaria radicina and S. sclerotiorum on the foliage and A. radicina on the taproots. Both lines exhibited 35-50% reduction in disease symptoms on the foliage and roots when compared with non-transgenic controls. Leaves challenged with the biotrophic pathogen Erysiphe heraclei or the bacterial pathogen Xanthomonas hortorum exhibited 90 and 80% reduction in disease development on the transgenic lines, respectively. The overexpression of the SAR controlling master switch in carrot tissues offers the ability to control a wide range of different pathogens, for which there is currently little genetic resistance available. DOI
85. Chatterton, S; Jayaraman, J; Punja, ZK. (2008) Colonization of cucumber plants by the biocontrol fungus Clonostachys rosea f. catenulata.Biological Control 46: 267-278 Colonization of cucumber plants by the biocontrol fungus Clonostachys rosea f. catenulata
Clonostachys rosea f. catenulata; Gliocladium catenulatum; Fusarium oxysporum f. sp radicis-cucumerinum; Cucumis sativus; biological control; GUS transformation; Agrobacterium tumefaciens; colonization; competition
Clonostachys rosea f. catenulata (Gliocladium catenulatum) strain J1446 (formulated as Prestop WP) suppressed Fusarium root and stem rot caused by Fusarium oxysporum f. sp. radicis-cucumerinum (Forc) on cucumber plants grown hydroponically in rockwool medium. Sixty days following application at seeding, the biocontrol agent had proliferated through the rockwool blocks and was present on cucumber roots and the crown region of the stem at populations >1 X 10(5) CFU/g fresh weight. Scanning electron micrographs showed that C rosea had rapidly colonized the root surface and was associated with root hairs and epidermal cell junctions. Following transformation of the fungus with Agrobacterium tumefaciens strain AGL-1 containing the hygromycin resistance (hph) and P-glucuronidase (uidA) genes, blue-stained mycelia could be seen growing on the surface and within epidermal and cortical cells of roots, stems and shoots 3 weeks after treatment. Quantification of GUS activity by fluorometric assays showed that fungal biomass was highest in the roots and crown area, while the extent of colonization of upper stems and true leaves was variable. Higher population levels resulted following application to rockwool blocks compared to seed treatment. Application of C rosea preceding inoculation with Forc significantly reduced pathogen populations on roots compared to plants inoculated with Forc alone. Colonization of infection sites in the root zone reduced pathogen development and disease incidence. Densities of the biocontrol agent appeared to increase in the presence of the pathogen. (C) 2008 Elsevier Inc. All rights reserved. DOI
83. Goswami, RS; Dong, YH; Punja, ZK. (2008) Host range and mycotoxin production by Fusarium equiseti isolates originating from ginseng fields.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 30: 155-160 Host range and mycotoxin production by Fusarium equiseti isolates originating from ginseng fields
root rot; trichothecene; zearalenone
Fusarium equiseti is prevalent in soil and straw mulch in ginseng (Panax ginseng) fields in British Columbia and causes a reddish brown discolouration on ginseng roots. The pathogenicity of two isolates of F equiseti from ginseng fields to other plant species belonging to different families was evaluated. Mycelial plugs and spore suspensions were used to inoculate seeds and roots in laboratory, growth room, and greenhouse experiments. Seed decay and reddish brown to black lesions were observed on hypocotyls and roots of kidney bean (Phaseolus vulgaris), bush bean (Phaseolus lunatus), broad bean (Vicia faba), chickpea (Cicer arietinum), and pea (Pisum sativum). A brownish discolouration and water-soaking symptoms developed on roots of alfalfa (Medicago sativa), canola (Brassica napus), wheat (Triticum aestivum), barley (Hordeum vulgare), and oat (Avena sativa) seedlings. Tomato (Lycopersicon esculentum), pepper (Capsicum annum), carrot (Daucus carota), and cucumber (Cucumis sativus) plants did not exhibit any visible symptoms. Diseased tissues from several affected plant species contained the mycotoxins nivalenol and (or) zearalenone at concentrations ranging from 0.3 to 11 ppm. This study shows that the host range of F. equiseti includes several members of the Leguminoseae, in addition to some cereals. The fungus may be one of the potential causes of damping-off and root rot on these plant species.
82. Goswami, RS; Punja, ZK. (2008) Molecular and biochemical characterization of defense responses in ginseng (Panax quinquefolius) roots challenged with Fusarium equiseti.Physiological and Molecular Plant Pathology 72: 10-20 Molecular and biochemical characterization of defense responses in ginseng (Panax quinquefolius) roots challenged with Fusarium equiseti
EXPRESSED SEQUENCE TAGS; PLANT DEFENSE; PHENOLIC-COMPOUNDS; DISEASE RESISTANCE; LIGNIN BIOSYNTHESIS; OXIDATIVE STRESS; MESSENGER-RNA; GENE FAMILY; ARABIDOPSIS; PATHOGENS
Fusarium equiseti causes a discoloration on ginseng roots that significantly affects their marketability. The cellular and biochemical changes in affected roots that lead to this symptom, as well as differential gene expression following pathogen inoculation were studied. Accumulation of phenolics, cell disruption, and development of a zone of lignified cells were observed in affected tissues. A number of genes involved in host defense responses, particularly those induced by jasmonic acid and genes mediating phenolic production and detoxification, were up-regulated. The defense reactions in the perennial roots of ginseng are highlighted and compared to those of other plant species. Crown Copyright (C) 2008 Published by Elsevier Ltd. All rights reserved. DOI
81. Jayaraj, J; Devlin, R; Punja, Z. (2008) Metabolic engineering of novel ketocarotenoid production in carrot plants.Transgenic Research 17: 489-501 Metabolic engineering of novel ketocarotenoid production in carrot plants
beta-carotene ketolase; carotenoid pathway engineering; ketocarotenoids; astaxanthin; root pigmentation; biopharming
Carotenoids constitute a vast group of pigments that are ubiquitous throughout nature. Carrot (Daucus carota L.) roots provide an important source of dietary beta-carotene (provitamin A), alpha-carotene and lutein. Ketocarotenoids, such as canthaxanthin and astaxanthin, are produced by some algae and cyanobacteria but are rare in plants. Ketocarotenoids are strong antioxidants that are chemically synthesized and used as dietary supplements and pigments in the aquaculture and neutraceutical industries. We engineered the ketocarotenoid biosynthetic pathway in carrot tissues by introducing a beta-carotene ketolase gene isolated from the alga Haematococcus pluvialis. Gene constructs were made with three promoters (double CaMV 35S, Arabidopsis-ubiquitin, and RolD from Agrobacterium rhizogenes). The pea Rubisco small sub-unit transit peptide was used to target the enzyme to plastids in leaf and root tissues. The phosphinothricin acetyl transferase (bar) gene was used as a selectable marker. Following Agrobacterium-mediated transformation, 150 plants were regenerated and grown in a glasshouse. All three promoters provided strong root expression, while the double CaMV 35S and Ubiquitin promoters also had strong leaf expression. The recombinant ketolase protein was successfully targeted to the chloroplasts and chromoplasts. Endogenous expression of carrot beta-carotene hydroxylases was up-regulated in transgenic leaves and roots, and up to 70% of total carotenoids was converted to novel ketocarotenoids, with accumulation up to 2,400 mu g/g root dry weight. Astaxanthin, adonirubin, and canthaxanthin were most prevalent, followed by echinenone, adonixanthin and beta-cryptoxanthin. Our results show that carrots are suitable for biopharming ketocarotenoid production for applications to the functional food, neutraceutical and aquaculture industries. DOI
80. Jayaraj, J; Liang, GH; Muthukrishnan, S; Punja, ZK. (2008) Generation of low copy number and stably expressing transgenic creeping bentgrass plants using minimal gene cassette bombardment.Biologia Plantarum 52: 215-221 Generation of low copy number and stably expressing transgenic creeping bentgrass plants using minimal gene cassette bombardment
gene integration; green fluorescent protein expression; plant transformation
A minimal gene cassette comprised of the ubiquitin (Ubi) promoter + green fluorescent protein (Gfp) gene + Nos terminator DNA sequences, derived from the plasmid vector pPZP201-Gfp was utilized for transformation of creeping bentgrass using particle bombardment. Bentgrass calli bombarded individually with equivalent amounts of the cassette or whole plasmid DNA were compared for Gfp expression and the GFP-positive calli were subsequently regenerated into plants. Percentage of GFP expressing calli and the number of GFP spots/calli were significantly higher in calli that were bombarded with the minimal gene cassette when compared to the whole plasmid. The Gfp expression was stable up to the T-2 generation in minimal gene cassette transformants and there was a lower degree of gene silencing. Southern blot analysis of transgenic plants derived from minimum gene cassette bombardment revealed the presence of single or few copy of the transgene and fairly simple integration patterns. In comparison, whole plasmid transformants had multiple copies and complex integration patterns of the transgene. These results illustrate the advantages of using simple gene cassette for stable plant transformation in bentgrass with possible applications to other plant species.
79. Jayaraj, J; Punja, ZK. (2008) Transgenic carrot plants accumulating ketocarotenoids show tolerance to UV and oxidative stresses.Plant Physiology and Biochemistry 46: 875-883 Transgenic carrot plants accumulating ketocarotenoids show tolerance to UV and oxidative stresses
beta-Carotene ketolase; Oxidative stress tolerance; UV-B tolerance; Daucus carota
Ketocarotenoids are strong antioxidant compounds which accumulate in salmon, shrimp, crustaceans and algae, but are rarely found naturally in higher plants. In this study, we engineered constitutive expression of an algal beta-carotene ketolase gene (bkt) in carrot plants to produce a number of ketocarotenoids from beta-carotene. These included astaxanthin, adonirubin, canthaxanthin, echinenone, adonixanthin and beta-cryptoxanthin. Leaves accumulated up to 56 mu g/g total ketocarotenoids and contained higher beta-carotene levels but lower levels of alpha-carotene and lutein. The photosynthetic capacity of transgenic plants was not significantly altered by these changes. However, when high-expressing transgenic plants were exposed to UV-B irradiation, they grew significantly better than the wild-type controls. Similarly, leaf tissues exposed to various oxidative stresses including treatment with H2O2 and methyl viologen showed less injury and retained higher levels of chlorophyll a + b. Total carotenoid extracts from transgenic leaves had higher antioxidant and free-radical scavenging activity in vitro compared to control leaves. Transgenic tissues also accumulated lower amounts of H2O2 following exposure to oxidative stresses, suggesting that free radical and reactive oxygen species were quenched by the ketocarotenoids. (C) 2008 Elsevier Masson SAS. All rights reserved. DOI
77. Jayaraj, J; Wan, A; Rahman, M; Punja, ZK. (2008) Seaweed extract reduces foliar fungal diseases on carrot.Crop Protection 27: 1360-1366 Seaweed extract reduces foliar fungal diseases on carrot
disease resistance; elicitors; fungal pathogens; Daucus carota; Alternaria radicina; Botrytis cinerea
Greenhouse-grown carrot plants were sprayed with an extract (0.2%) of the seaweed Ascophyllum nodosum (SW) and then inoculated 6 h later with the fungal pathogens Alternaria radicina and Botrytis cinerea. Additional applications of SW were made 10 and 20 d after inoculation. Treated plants showed significantly reduced disease severity at 10 and 25 d after inoculation compared to control plants sprayed with water. SW was more effective than salicylic acid (SA) (100 mu M) in reducing infection. Activity of certain defence-related enzymes, including peroxidase (PO), polyphenoloxidase, phenylalanine ammonia lyase, chitinase and beta-1,3-glucanase, were significantly increased in plants treated with SW and SA compared to the control 12 h after treatment. The treated plants also had higher transcript levels of pathogenesis-related protein I (PR-1), chitinase, lipid transfer protein (Ltp), phenylalanine ammonia lyase (Pal), chalcone synthase, non-expressing pathogenesis-related protein (NPR-1) and pathogenesis-related protein 5 (PR-5) genes compared to control plants. These results show that SW enhances disease resistance in carrot, likely through induction of defence genes or proteins. (C) 2008 Elsevier Ltd. All rights reserved. DOI
76.Punja, ZK; Wan, A; Goswami, RS. (2008) Root rot and distortion of ginseng seedling roots caused by Fusarium oxysporum.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 30: 565-574 Root rot and distortion of ginseng seedling roots caused by Fusarium oxysporum
SP RADICIS-LYCOPERSICI; BRITISH-COLUMBIA; DIVERSITY
Stunted ginseng (Panax quinquefolius) seedlings with distorted roots as a result of girdling of the taproot and rotting of the root tips were observed in British Columbia and Ontario in 2005 in four fields of 1- or 2-year-old plants. Isolation from affected root tissues yielded > 90% recovery of Fusarium oxysporum. Population levels of the fungus were up to 40-fold higher in the rhizosphere of affected plants compared with that of healthy plants. Inoculation of 1-year-old seedlings with four isolates of F. oxysporum under greenhouse conditions resulted in rotting of root tips, brown discolouration and death of lateral roots, followed by distortion of the taproots at a frequency of 52%-70%. DNA of F. oxysporum was detected in flowers, green berries, ripe berries, and seeds of ginseng using a DNA hybridization array. Stratified seeds showed up to an 80% incidence of F oxysporum both in the seed coat and endosperm. Isolates of F. oxysporum from ginseng grew best under laboratory conditions at 25-30 degrees C and PH 5.7-6.8. Mycelial growth was significantly reduced by 10 mu g/mL of the fungicides benomyl, propiconazole, fludioxonil, and thiophanate-methyl. Phylogenetic comparisons of the sequence a portion of the elongation factor-1 alpha gene grouped all 13 isolates of F oxysporum from ginseng with 21 isolates of F. oxysporum from a range of other hosts. Inoculum of F. Oxysporum originating from straw mulch or soil in ginseng gardens, together with seedborne inoculum, may be primary sources for infection of seedling roots.
75.Punja, ZK; Wan, A; Rahman, M; Goswami, RS; Barasubiye, T; Seifert, KA; Levesque, CA. (2008) Growth, population dynamics, and diversity of Fusarium equiseti in ginseng fields.European Journal of Plant Pathology 121: 173-184 Growth, population dynamics, and diversity of Fusarium equiseti in ginseng fields
epidemiology; floral infection; soilborne pathogen; straw mulch
Fusarium equiseti is prevalent in ginseng soil, straw mulch and in ginseng root tissues and is the cause of a root surface discolouration on ginseng grown in British Columbia. Population levels of the fungus in ginseng fields ranged from 3.8x10(3) cfu g(-1) soil to 1.4x10(4) cfu g(-1) soil and were highest at 0-5 cm soil depths compared to 10-15 cm. Soil population levels were negatively correlated with S content in soil and positively correlated with Zn levels. Barley or wheat straw added to soil significantly increased population levels under laboratory conditions. Mycelial growth in culture was highest at 26-30 degrees C and at pH 7.2-7.8. Samples of flowers and berries, and harvested seed, contained DNA of F. equiseti detected using a Fusarium-specific DNA array and the fungus was isolated from these tissues on agar medium. A high degree of genetic variation in the EF-1 alpha gene sequence was present among 52 isolates of F. equiseti which originated from ginseng fields. At least seven clades were identified. Inoculum dispersal from straw mulch used in ginseng gardens can result in seed contamination by the fungus. In addition, fungal growth near the soil surface under warm summer conditions can result in infection and crown discolouration of ginseng roots. DOI
74. Sumampong, G; Shamoun, SF; Punja, ZK. (2008) Occurrence of Phoma argillacea on Rubus spectabilis in British Columbia and an evaluation of its potential as a forest weed biological control agent.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 30: 74-84 Occurrence of Phoma argillacea on Rubus spectabilis in British Columbia and an evaluation of its potential as a forest weed biological control agent
Phoma argillacea; mycoherbicide; inundative biological control; Rubus spectabilis
A survey was conducted on Vancouver Island and in coastal mainland British Columbia of naturally occurring fungal pathogens of salmonberry (Rubus spectabilis), an endemic plant species considered to be undesirable in conifer regeneration sites. Fungi were recovered from necrotic lesions on foliage and stems and identified to genus level. An in vitro pathogenicity test was performed on 281 fungal isolates using detached salmonberry leaves. Nineteen isolates were found to be moderately to highly aggressive, causing >50% leaf area necrosis within 14 days. These included Botrytis cinerea (17 isolates), Phoma sp. (3 isolates), and Septoria rubi (1 isolate). The three isolates of Phoma, identified as P. argillacea, were selected for further testing on salmonberry plants under greenhouse conditions. Mycelial or conidial inoculum were applied to the foliage of plants, which were incubated for 48 It at 20-22 degrees C and 100% relative humidity (RH) and then grown at 20-22 degrees C and 55-65% RH, with a 16 h photoperiod, under greenhouse conditions. Extensive necrosis of leaves and stems developed within 14 days for all isolates using mycelia and conidia as inoculum. A minimum of 18 h of continuous leaf wetness and mycelial inoculum of 2.6 x 10(3) colony forming units/mL was required for infection to occur. In a small host range test, three economically important conifer species, namely Douglas-fir (Pseudotsuga menziesii), western hemlock (Tsuga heterophylla), and western red cedar (Thuja plicata), showed tolerance at the seedling stage with <5% disease severity. On red raspberry (Rubus idaeus), P argillacea caused some initial damage (leaf spots and chlorosis) but failed to cause cane blight. These results demonstrate the pathogenicity of P. argillacea on salmonberry and the conditions required for establishing disease.
73. Wally, O; Jayaraj, J; Punja, ZK. (2008) Comparative expression of beta-glucuronidase with five different promoters in transgenic carrot (Daucus carota L.) root and leaf tissues.Plant Cell Reports 27: 279-287 Comparative expression of beta-glucuronidase with five different promoters in transgenic carrot (Daucus carota L.) root and leaf tissues
carrot; beta-glucuronidase; promoter; organ specific expression
Tissue-specific patterns and levels of protein expression were characterized in transgenic carrot plants transformed with the beta-glucuronidase (GUS) gene driven by one of five promoters: Cauliflower mosaic virus 35S (35S) and double 35S (D35S), Arabidopsis ubiquitin (UBQ3), mannopine synthase (mas2) from Agrobacterium tumefaciens or the rooting loci promoter (rolD) from A. rhizogenes. Five independently transformed carrot lines of each promoter construct were assessed for GUS activity. In leaves, activity was highest in plants with the D35S, 35S and UBQ3 promoters, while staining was weak in plants with the mas2 promoter, and only slight visual staining was present in the leaf veins of plants containing rolD promoter . Strong staining was seen in the lateral roots, including root tips, hairs and the vascular tissues of plants expressing the 35S, D35S and UBQ3. Lateral roots of plants containing the rolD construct also showed staining in these tissues while the mas2 promoter exhibited heightened staining in the root tips. Relatively strong GUS staining was seen throughout the tap root with all the promoters tested.. When GUS expression was quantified, the UBQ3 promoter provided the highest activity in roots of mature plants, while plants with the D35S and 35S promoter constructs had higher activity in the leaves. Although plants containing the mas2 promoter had higher levels of activity compared to the rolD plants, these two promoters were significantly weaker than D35S, 35S and UBQ3. The potential for utilization of specific promoters to target expression of desired transgenes in carrot tissues is demonstrated. DOI
72. Jayaraj, J; Punja, ZK. (2007) Combined expression of chitinase and lipid transfer protein genes in transgenic carrot plants enhances resistance to foliar fungal pathogens.Plant Cell Reports 26: 1539-1546 Combined expression of chitinase and lipid transfer protein genes in transgenic carrot plants enhances resistance to foliar fungal pathogens
Daucus carota; pant transformation; disease resistance; leaf blights
Two pathogenesis-related (PR) protein genes consisting of a barley chitinase (chi-2) and a wheat lipid-transfer-protein (ltp) were introduced singly and in combination into carrot plants via Agrobacterium-mediated transformation using the phosphinothricin acetyl transferase (bar) gene as a selectable marker. Over 75% of regenerated plants were confirmed to be positive for the transgenes by PCR and RT-PCR and were resistant to the herbicide Liberty (0.2%, v/v). Northern analysis and immunoblotting confirmed the expression of the transgenes in about 70% of the plants, with variable expression levels among individual lines. Southern analysis revealed from one to three copies of each transgene. Transgenic plants were inoculated with two necrotrophic foliar fungal pathogens, Alternaria radicicola and Botrytis cinerea, and showed significantly higher resistance when both PR genes were expressed compared to single-gene transformants. The level of disease reduction in plants expressing both genes was 95% for Botrytis and 90% for Alternaria infection compared to 40-50% for single-gene transformants. The chi2 and ltp genes could be deployed in combination in other crop plants to significantly enhance resistance to necrotrophic fungal pathogens. DOI
71. Noshad, D; Riseman, A; Punja, Z. (2007) Evaluation of Daphne germplasm for resistance to Daphne sudden death syndrome caused by the soil-borne pathogen Thielaviopsis basicola.Hortscience 42: 1639-1643 Evaluation of Daphne germplasm for resistance to Daphne sudden death syndrome caused by the soil-borne pathogen Thielaviopsis basicola
Many daphne cultivars are susceptible to fungal root pathogens and require frequent fungicide applications during production. To identify taxon differences to disease susceptibility, we evaluated 32 Daphne species and cultivars for resistance to the soilborne pathogen, Thielaviopsis basicola (Berk. and Broome) Ferr., by both in vitro- and in vivo-based methods. Disease-free plant roots were inoculated with the pathogen through topical application of a spore suspension and observed weekly for disease development/progression. Significant variation for disease severity among the taxa evaluated was determined using a plant disease index. Plant reactions ranged from highly resistant, e.g., D. tangutica and D. retusa, to highly susceptible, e.g., D. cneorum. In addition, a high correlation was found between the in vitro and in vivo techniques for the seven selected species, indicating that they are comparable. However, the in vitro assay provided results in significantly less time than the in vivo assay.
70.Punja, ZK; Wan, A; Goswami, RS; Verma, W; Rahman, M; Barasubiye, T; Seifert, KA; Levesque, CA. (2007) Diversity of Fusarium species associated with discolored ginseng roots in British Columbia.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 29: 340-353 Diversity of Fusarium species associated with discolored ginseng roots in British Columbia
epidemiology; molecular detection; Panax quinquefolius; rusty root
Crown discoloration on roots of American ginseng (Panax quinquefolius) is a major problem that causes a reduction in quality and can be a limiting factor to ginseng production in some regions of Canada. The symptoms result from an accumulation of phenolic compounds within disorganized and disrupted cells, accompanied by the presence of fungal hyphae in affected cells. Twelve Fusarium species were recovered on isolation media during June-September of 2004 and 2005 from ginseng roots displaying root-surface discoloration in British Columbia. Many of the same species were also recovered from soil from ginseng fields and cereal-straw mulch used during ginseng production. Hybridization of labeled PCR amplicons from DNA samples from affected root tissue to a DNA array designed using oligonucleotides specific to 26 Fusarium species was used to identify the Fusarium species present in ginseng roots. Over 90% of symptomatic root samples contained F. equiseti and several other Fusarium species, including F.sporotrichioides and F. avenacemn, that were absent in nonsymptomatic tissues. An in vitro pathogenicity test was used to screen 180 Fusarium isolates originating front affected roots. Reddish brown lesions developed 10 days following inoculation with 58 isolates of F. equiseti and F. sporotrichioides and to a lesser extent with 7 isolates of F.avenacetan and F. culmorum. Greenhouse and field inoculations confirmed the pathogenicity of E equiseti and F.sporotrichioides isolates to ginseng roots. Pathogenicity tests conducted with 340 additional isolates representing 10 Fusarium species originating from wheat- and barley-straw mulch and soil from ginseng fields, as well as from cereal hosts worldwide, identified a proportion of isolates (52.5%) of F. equiseti, F. sporotrichioides, F. avenaceum, and F.culmorum that produced reddish brown lesioned areas. These cereal-infecting Fusarium species can induce a tissue response following infection that results in root-surface discoloration.
69. Rahman, M; Punja, ZK. (2007) Biological control of damping-off on American ginseng (Panax quinquefolius) by Clonostachys rosea f. catenulata (= Gliocladium catenulatum).Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 29: 203-207 Biological control of damping-off on American ginseng (Panax quinquefolius) by Clonostachys rosea f. catenulata (= Gliocladium catenulatum)
American ginseng; Panax quinquefolius; damping-off; soilborne fungi; Pythium; Fusarium; seed treatment; biological control; Clonostachys rosea f. catenulata
Seedling damping-off caused by several soilborne fungal pathogens is a recurring problem for commercial growers of American ginseng (Panax quinquefolius) throughout the major production areas of Canada. The pathogens isolated from diseased seedling roots during 2001 and 2002 included Pythium ultimum and other Pythium species, Phytophthora cactorum, Fusarium solani and other Fusarium species, Rhizoctonia solani, and Cylindrocarpon destructans. Four commercially formulated biocontrol agents - Trichoderma harzianum strain T-22 (RootShield (R) Drench (TM) WP (wettable powder)), Streptomyces griseoviridis strain K61 (Mycostop (R) WP), Trichoderma virens (synonym: Gliocladium virens) strain GL-21 (SoilGard (TM) 12G (granular)), and Clonostachys rosea f. catenulata (synonym: Gliocladium catenulatum) strain J1446 (PreStop (R) WP and PreStop Mix (TM) G (granular)) - were evaluated in commercial gardens of American ginseng at three locations during 2002 and 2003 for efficacy in reducing damping-off. Application of PreStop Mix G as a combination of seed treatment (applied in the fall season) and three soil drenches (applied in the spring season) significantly (P <= 0.05) increased seedling stand; the protection provided by this treatment was comparable to that of a seed treatment with Apron XL (R) LS (solution for seed treatment; active ingredient: metalaxyl-m). Captan (TM) 50 WP, as a seed treatment, showed the highest efficacy in reducing damping-off in this study. The other three biocontrol products did not protect ginseng seedlings against damping-off.
67. Rodriguez, G; Punja, ZK. (2007) Root infection of wasabi (Wasabia joponica) by Pythium species.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 29: 79-83 Root infection of wasabi (Wasabia joponica) by Pythium species
root loss; hydroponic culture; Pythium dissotocum; Pythium intermedium; wasabi; Wasabia japonica
Wasabi (Wasabia japonica) is cultivated under cool, humid conditions in many regions of the world for its rhizome, which is used as a culinary ingredient. In British Columbia, there is some wasabi production under semihydroponic conditions in polyethylene greenhouses. In January 2004, wasabi plants with browning and root loss, with no apparent foliar symptoms, were obtained. Pythium dissotocum and Pythium intermedium were consistently isolated from the affected roots. Wasabi plants produced from meristem-tip tissue culture were grown hydroponically ill Gamborg's B5 nutrient Solution at 18 degrees C for 6 months, inoculated with mycelial plugs of each Pythium sp., and incubated at 20 degrees C. After 2 months, the inoculated plants had significant root browning and loss, and the root dry mass of inoculated plants was reduced to 16% of that in the noninoculated controls. Both Pythium spp. were observed colonizing the roots and were recovered from inoculated plants. Pythium dissotocum and P. intermedium appear to be prevalent root pathogens of W. japonica without inducing apparent foliar symptoms. However, root loss can predispose the affected plants to other biotic and abiotic stresses, leading to plant mortality.
66. Verma, N; MacDonald, L; Punja, ZK. (2007) Environmental and host requirements for field infection of blueberry fruits by Colletotrichum acutatum in British Columbia.Plant Pathology 56: 107-113 Environmental and host requirements for field infection of blueberry fruits by Colletotrichum acutatum in British Columbia
anthracnose; disease prediction; epidemiology; ripe-rot; Vaccinium corymbosum; weather conditions
Higher recovery of Colletotrichum acutatum, the causal agent of anthracnose (ripe-rot), from blueberry tissues during the growing seasons of 2002 and 2003 was found at bloom and ripe berry than at other stages of plant development. The effects of leaf-wetness duration and ambient temperature on fruit infection frequency were determined during the growing seasons of 2001-03. Potted 2-year-old blueberry plants were exposed for 1-week periods to prevailing environmental conditions and natural inoculum in a commercial field, and grown to harvest, when fruit infection was assessed. Three peaks of infection were observed: early during bloom, mid-season during the mature green berry stage, and later in the season when berries had ripened. Weather data collected simultaneously indicated that a minimum of 10 h of leaf wetness at 11 degrees C was sufficient for fruit infection. These conditions preceded each peak of infection. To determine whether peaks of infection in the field were also caused by changes in host susceptibility or available inoculum, groups of potted blueberry plants were artificially inoculated at weekly intervals during the growing season of 2004, exposed to prevailing environmental conditions, and fruit infection assessed at harvest. Flowers and developing fruits were found to be susceptible throughout the season, indicating that specific peaks of infection were associated with environmental conditions and availability of inoculum. DOI
62. Feeney M, Punja ZK. (2006) Hemp (Cannabis sativa L.).Methods Mol Biol. 2006; 344:373-82. Hemp (Cannabis sativa L.).
Hemp (Cannabis sativa L.) suspension culture cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary plasmid pNOV3635. The plasmid contains a phosphomannose isomerase (PMI) selectable marker gene. Cells transformed with PMI are capable of metabolizing the selective agent, mannose, whereas cells not expressing the gene are incapable of using the carbon source and will stop growing. Callus masses proliferating on selection were screened for PMI expression using a chlorophenol red assay. Genomic DNA was extracted from putatively transformed callus lines and the presence of the PMI gene was confirmed using polymerase chain reaction and Southern hybridization. Using this method, an average transformation frequency of 31.23% +/- 0.14 was obtained for all transformation experiments, with a range of 15.1 to 55.3%.
59. Noshad, D; Punja, ZK; Riseman, A. (2006) First report of Thielaviopsis baskola on Daphne cneorum.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 28: 310-312 First report of Thielaviopsis baskola on Daphne cneorum
Daphne cneorum 'Ruby Glow'; Daphne sudden death syndrome; garland flower; mad daphne disease; root pathogen; Chalara elegans
A previously undescribed fungal disease, coined daphne sudden death syndrome (DSDS), was reported on Daphne cneorum from several nurseries and research centres in British Columbia, Canada. Diseased and healthy plants were obtained from several sources, and from these plants, the following fungi were isolated: Fusarium roseum, Fusarium oxysporum, Trichoderma sp., Aspergillus sp., and Thielaviopsis basicola. However, only Thielaviopsis basicola was recovered from every diseased plant while being absent from healthy plants. All isolated fungi were grown as pure cultures and individually inoculated, via topical application of a spore suspension, to the roots of either healthy, 2-year-old D. cneorum plants or healthy rooted plantlets grown in vitro. The Thielaviopsis basicola isolate was the only recovered fungus to induce symptoms that matched those previously observed from DSDS-infected plants. Therefore, we conclude that Thielaviopsis basicola is the causal agent of DSDS.
58. Park, Y; Chen, XB; Punja, ZK. (2006) Molecular and biological characterization of a mitovirus in Chalara elegans (Thielaviopsis basicola).Phytopathology 96: 468-479 Molecular and biological characterization of a mitovirus in Chalara elegans (Thielaviopsis basicola)
black root rot; fungal virus; mycovirus
A 2.8-kb double-stranded RNA (dsRNA) element in strain BK18 of Chalara elegans originally isolated from cotton soil in California was characterized by obtaining a full-length cDNA sequence (2,896 nucleotides in length) from a series of overlapping clones. Sequence analysis revealed the presence of one large open reading frame (ORF I) using the initochondrial genetic code, with 20 to 34% amino acid identity to the ORF I of other previously reported fungal mitochondrial RNA viruses. The ORF I encoded a putative protein of 705 amino acids and contained the conserved motif characteristic of RNA-dependent RNA polymerases. Purification of mitochondria from strain BK 18 confirmed the co-localization of this dsRNA. and northern blot hybridization with a strand-specific probe revealed the (+) single-stranded nature. This Chalara elegans mitovirus (CeMV) is designated as a new member of the genus Mitovirus of the family Narnaviridae. Using dsRNA-specific primers. the ORF I region (positions 427 to 2544) was obtained from an additional 2.8-kb dsRNA element in strain HA2 originating from carrot roots in the Netherlands. Both ORFs had 98% homology at the nucleotide and amino acid levels. CeMV was also found to be present in five additional strains of C. elegans from different geographic locations worldwide, and a 97 to 100% nucleotide sequence identity was observed within a 300-bp region of ORF I in these strains. To determine the biological effects of CeMV on C. elegans, attempts to cure strain BK 18 of the dsRNA were made. Sequential transfers of mycelium at 35 to 37 degrees C yielded a colony which lacked the 2.8-kb dsRNA when visualized on agarose gels and also in northern blot hybridization analysis. However. reverse transcription-polymerase chain reaction with specific primer sets revealed a band., indicating that dsRNA replication had been significantly repressed (latent). The wild type and latently infected strains were compared for colony morphology, growth rate, melanin production, various enzymatic assays (polyphenoloxidase, laccase. tyrosinase, and esterase), and virulence on carrot roots. Colony morphology on V8 agar was comparable between the two strains. while growth rate, melanin production, and virulence were enhanced in the latently infected strain. There were no detectable differences in enzymatic activity. Transmission electron microscopy of hyphae of the wild type and latently infected strains revealed differences in the number and size of the mitochondria, which were enhanced in the latently infected strain. Our results show that CeMV is a new member of the genus Mitovirus with some disruptive effects on its fungal host and is present in C. elegans strains from different locations worldwide.
57. Park, YJ; Chen, XB; Punja, ZK. (2006) Diversity, complexity and transmission of double-stranded RNA elements in Chalara elegans (synanam. Thielaviopsis basicola).Mycol Res 110: 697-704 Diversity, complexity and transmission of double-stranded RNA elements in Chalara elegans (synanam. Thielaviopsis basicola)
black root rot; mycovirus; dsRNA transmission
Double-stranded (ds) RNA banding patterns were determined in 21 wild-type strains of the soilborne plant pathogen Chalara elegans originating from different geographic regions worldwide. Five strains, each with a unique dsRNA pattern, were selected for cDNA cloning, northern blot analysis and dsRNA transmission experiments. Four strains contained multiple (up to 6) dsRNA elements (2.0 kbp to 12 kbp in size) and one strain contained a single 2.8 kbp fragment. These five strains were distinguished from one another by their unique RAPD-PCR patterns. Seven partial cDNA clones were derived from the predominant 2.8, 5.3, and 12 kbp dsRNA elements. Nucleotide sequence analysis and northern blot hybridizations revealed a high degree of genetic dissimilarity among the different molecular-size dsRNA elements, even those found within a single strain. Four clones from the 5.3 kbp dsRNA fragment showed a 23-43 % amino acid identity to either the coat protein or RNA-dependent RNA polymerase regions of viruses in the Totiviridae. One clone from the 2.8 kbp dsRNA fragment had a 55-57 % amino acid identity to the RdRp region of viruses in the Narnaviridae, Two clones from the 12 kbp dsRNA fragment showed no significant homology to any known virus group. Colonies derived from 100 single-conidia isolates of C. elegans strains with the 2.8, 5.3 and 12 kbp elements all contained the corresponding dsRNA element, indicating that dsRNA transmission through conidia was highly efficient, regardless of molecular size. However, transmission of dsRNA between the mycelium of strains of C. elegans could not be achieved in this study. Genetically unique strains carrying diverse dsRNA elements appear to have evolved within populations of C. elegans. Based on our findings, there are at least 3 groups of viruses present in C. elegans. (c) 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
56.Punja, ZK. (2006) Recent developments toward achieving fungal disease resistance in transgenic plants.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 28: S298-S308 Recent developments toward achieving fungal disease resistance in transgenic plants
antifungal proteins; antimicrobial peptides; biotechnology; disease-resistance genes; pathogenesis-related proteins; signaling pathways
Considerable progress has been made in the use of genetic engineering techniques to enhance fungal disease resistance in plants. As a result, a greater understanding of signaling pathways and molecules elicited by pathogen infection has been achieved. Furthermore, manipulation of signaling pathways involved in plant defense has been demonstrated. The expression of a number of disease-resistance (R) genes in transgenic plants has been illustrated. Strategies to detoxify microbial products involved in fungal pathogenicity have provided promising results. The continued use of naturally occurring and synthetic antimicrobial compounds has yielded interesting findings. The future potential of genetic engineering technology remains promising given the recent scientific advances that have been made in enhancing resistance to a broad range of pathogens in many plant species. Furthermore, the basic understanding of the interactions between fungal pathogens and their host plants has been greatly enhanced.
54. Rahman, M; Punja, ZK. (2006) Influence of iron on cylindrocarpon root rot development on ginseng.Phytopathology 96: 1179-1187 Influence of iron on cylindrocarpon root rot development on ginseng
Cylindrocarpon root rot, caused by Cylindrocarpon destructans, is an important disease on ginseng (Panax quinquefolius) in Canada. We studied the effects of iron (Fe) on disease severity and pathogen growth. When Hoagland's solution was amended with Fe at 56 and 112 mu g/ml compared with 0 mu g/ml, disease initiation and final severity on hydroponically maintained ginseng roots was significantly (P < 0.0001) enhanced. Under field conditions. wounding of roots with a fine needle followed by application of 0.05% FeNaEDTA to the rhizosphere of treated plants significantly enhanced Cylindrocarpon root rot in 2003 and 2004 compared with unwounded roots with Fe or wounded roots without Fe. Foliar applications of Fe (as FeNaEDTA) to ginseng plants three times during the 2002 and 2003 growing seasons significantly increased Fe levels in root tissues. These roots developed larger lesions following inoculation with C. destructons in vitro. When radioactive Fe (Fe-59) was applied to the foliage of ginseng plants, it was detected in the secondary phloem and in cortical and epidermal tissues within I week. Artificially wounded areas on the roots accumulated more Fe-59 than healthy areas. Diseased tissue also had threefold higher levels of phenolic compounds and Fe compared with adjoining healthy tissues. High-performance liquid chromatography analysis revealed enhanced levels of protocatechuic acid, chlorogenic acid, caffeic acid, ferulic acid, cinnamic acid, phloridizin, and quercetin. Phenolic compounds produced in diseased and wounded tissues sequestered Fe in vitro. The effects of Fe on mycelial growth, conidial germ tube length, and secondary branching of germ tubes of C destructans were examined in vitro. When grown on Chrome-azurol S medium, Fe also was sequestered by C. destructans through siderophore production, which was visualized as a clearing pigmented zone at the margin of colonies. Mycelial dry weight was significantly increased in glucose/yeast broth containing Fe at 56 or 112 mu g/ml. Conidial germ tube length and secondary branching of hyphae also were enhanced after 8 and 16 h by Fe. Colony growth of C. destructans was not enhanced by Fe, but significantly greater spore production was observed with Fe at 56 and 112 mu g/ml compared with no Fe in the medium. Although these levels of Fe had no effect on fungal pectinase enzyme activity, polyphenoloxidase (PPO) activity was significantly (P < 0.0001) enhanced. We conclude that Fe enhances Cylindrocarpon root rot through enhanced pathogen growth, sporulation, and PPO enzyme activity. Fe sequestered by phenolic cornpounds produced in wounded tissues can enhance Fe levels at the site of infection. The pathogen also has the ability to sequester Fe at these sites.
52. Verma, N; MacDonald, L; Punja, ZK. (2006) Inoculum prevalence, host infection and biological control of Colletotrichum acutatum: causal agent of blueberry anthracnose in British Columbia.Plant Pathol 55: 442-450 Inoculum prevalence, host infection and biological control of Colletotrichum acutatum: causal agent of blueberry anthracnose in British Columbia
biological control; diagnostics; epidemiology; Gliocladium catenulatum; Trichoderma harzianum; Vaccinium corymbosum
To identify the causal organism of anthracnose (ripe-rot), which reduces yield and postharvest quality of blueberries grown in British Columbia, Canada, 80 isolates were recovered from diseased fruits collected from commercial blueberry fields during 2002-04 and identified as Colletotrichum acutatum using colony morphology, growth rate and species-specific PCR primers. In vitro incubation of replicated sets of inoculated detached berries at various temperatures produced infection at temperatures of 7-30 degrees C, with an optimum at 20 degrees C. Colletotrichum acutatum could not survive on the soil surface as mummified berries but the pathogen was detected mostly within flower buds and less so in blueberry twigs and fruit trusses. Infection of developing flower buds in May-June of the preceding growing season gave the highest inoculum recovery in the following year. Two commercial fungal biocontrol agents, Prestop (Gliocladium catenulatum) and PlantShield (Trichoderma harzianum), each reduced anthracnose development in 2003 and 2004 by up to 45% when sprayed three times onto plants between flowering and fruit ripening.
51. Wally O, Jayaraman J, Punja ZK. (2006) Carrot (Daucus carota L.).Methods Mol Biol. 2006;344:3-12.Carrot (Daucus carota L.).
Plants are susceptible to infection by a broad range of fungal pathogens. Many horticulturally important crop species lack adequate genetic resistance to disease. Studies on potential mechanisms of disease resistance in plants have revealed the importance of a range of pathogenesis-related (PR) proteins with antifungal activity in reducing colonization of plant tissues by pathogens. We are evaluating a range of PR-proteins, through heterologous expression in transgenic carrot tissues, for their effects on fungal disease development. The protocols for carrot transformation with a thaumatin-like protein are described. In addition, the use of herbicide resistance as a selectable marker in carrot transformation is illustrated. In this protocol, petiole segments from carrot seedlings are exposed to Agrobacterium for 10-30 min and co-cultivated for 3 d, after which herbicide selection is imposed until embryogenic calli are produced after 8-12 wk. The transfer of the embryogenic calli to hormone-free medium yields transgenic plantlets. This genetic transformation protocol has supported the generation of transgenic carrot plants with defined T-DNA inserts at the rate of between 1 and 3 Southern positive independent events out of 100.
49. Wegener, LA; Martin, RR; Bernardy, MG; MacDonald, L; Punja, ZK. (2006) Epidemiology and identification of strains of Blueberry scorch virus on highbush blueberry in British Columbia, Canada.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 28: 250-262 Epidemiology and identification of strains of Blueberry scorch virus on highbush blueberry in British Columbia, Canada
Carlavirus; Vaccinium corymbosum; Vaccinium macrocarpon; epidemiology; coat protein; strain identification; virus detection
Blueberry scorch virus (BlScV), first discovered in British Columbia, Canada, in 2000, causes a potentially serious disease on highbush blueberry (Vaccinium corymbosum). The epidemiology of BlScV was studied over four consecutive years (2001-2004) in British Columbia. In 2001, leaf tissue sampled from 40 commercial fields tested positive for BIScV, when using a polyclonal antibody in a double antibody sandwich - enzyme-linked immunosorbent assay. In 2002, 2003, and 2004, BlScV was detected in an additional 19, 49, and 12 previously uninfected fields, respectively. Disease spread was studied in more detail by mapping BlScV distribution in three commercial blueberry fields. The percent increase in diseased plants ranged from 4.4% to 5.2% from 2001 to 2002, and from 4.2% to 9.6% from 2002 to 2004. Symptoms on blueberry cultivars in May included necrosis of blossoms and young leaves, leaf chlorosis, and shoot blight; line patterns on the leaves of some infected cultivars were observed in October. BlScV was also detected in two additional Vaccinium spp., namely cranberry (V macrocarpon) and black huckleberry (V. membranaceum). Reverse transcription - polymerase chain reaction with BlScV-specific primers was used to obtain partial sequences of the coat-protein gene for 12 BlScV isolates from blueberry in British Columbia. These sequences were compared with each other and with those of strains from United States (NJ-1, NJ-2, and WA-2) and two strains recently described in British Columbia (BC-1 and BC-2). The 12 British Columbia isolates shared 88%-100% homology with each other and were more closely related to BC-2 and NJ-2 than to BC-1, NJ-1, and WA-2. The high incidence of BlScV in highbush blueberry in British Columbia, the range of symptoms, the presence of BlScV in other Vaccinium spp., and the variability in sequences of the coat-protein gene among strains suggest that the virus is widespread and that different strains are now established in British Columbia.
48. Park, YJ; James, D; Punja, ZK. (2005) Co-infection by two distinct totivirus-like double-stranded RNA elements in Chalara eleganse (Thielaviopsis basicola).Virus Research 109: 71-85 Co-infection by two distinct totivirus-like double-stranded RNA elements in Chalara eleganse (Thielaviopsis basicola)
Chalara elegans; dsRNA sequence; Totiviridae; fungal viruses
A full-length cDNA clone was developed from a 5.3 kb double-stranded (ds) RNA element present in strain CKP of the plant pathogenic fungus Chalara elegans. The complete nucleotide sequence was 5310bp in length and sequence analysis revealed that it contained three large putative open reading frames (ORFs). ORFI was initiated at nucleotide position 329 and encoded a putative coat protein, which shared some homology (35-45% amino acid identity) to other dsRNAs in the family Totiviridae. Both ORF2 and ORF3 were initiated at nucleotide positions 2619 and 4071, respectively, and encoded a putative RNA-dependent RNA polymerase (RdRp). Sequence comparison using deduced amino acid sequences of both ORF2 and ORF3 revealed that all RdRp conserved motifs shared highest homology (41% identity) to that of SsRNA1 of Totiviridae. This dsRNA in C. elegans was designated Chalara elegans RNA Virus 1 (CeRV1). During the development of the full-length cDNA clone of CeRV1, several partial cDNA clones from an additional dsRNA fragment in strain CKP were obtained, which when aligned with each other, produced one linear fragment which was 2336 bp long. Northern blot and sequence analysis of this second clone showed it differed in sequence composition from CeRV1. This dsRNA in C. elegans was designated Chalara elegans RNA Virus 2 (CeRV2). Sequence analysis of CeRV2 showed it contained all conserved motifs and shared some homology (45% amino acid identity) to RdRp regions of Totiviridae. The nucleotide and amino acid sequences of the conserved motifs of the RdRp regions between CeRV1 and CeRV2 showed an identity of 56% and 50%, respectively. These findings suggest that co-infection of two distinct totivirus-like dsRNAs (CeRV1 and CeRV2) in C. elegans, a first report in this fungus. Transmission electron microscopy of strain CKP of C. elegans revealed the presence of putative virus-like particles in the cytoplasm, which were similar both in shape and size to viruses in the Totiviridae. (C) 2004 Elsevier B.V. All rights reserved.
47.Punja, ZK. (2005) Transgenic carrots expressing a thaumatin-like protein display enhanced resistance to several fungal pathogens.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 27: 291-296 Transgenic carrots expressing a thaumatin-like protein display enhanced resistance to several fungal pathogens
disease resistance; pathogenesis-related protein; Daucus carota; alternaria leaf blight; grey mould; white mould
Two transgenic carrot lines (T-33 and T-47) were developed through Agrobacterium-mediated transformation. Both lines constitutively expressed a rice thaumatin-like protein and the phosphinothricin acetyltransferase protein for herbicide resistance, used as a selectable marker. Both lines were propagated in tissue culture and transferred to soil and maintained under growth-chamber conditions. Detached leaves of the transgenic lines and nontransformed 'Nanco' were inoculated in vitro with eight different fungal pathogens, and the extent of leaf necrosis was assessed. The two transgenic lines both had significantly lower disease (P 5 <= 0.05) when compared with the nontransformed 'Nanco' for select pathogens. Line T-33 had significantly lower leaf necrosis when inoculated with Alternaria dauci, Alternaria petroselini, Alternaria radicini, Botrytis cinerea, Rhizoctonia solani, and Sclerotinia sclerotiorum. Line T-47 had significantly lower disease when inoculated with A. dauci, A. radicini, B. cinerea, Sclerotium rolfsii, and Sclerotinia selerotiorum. The detached leaf method was a rigorous approach for assessing the transgenic carrot lines for resistance to fungal infection. The enhanced resistance observed in this study to fungal pathogens as a consequence of expression of a thaumatin-like protein in carrot tissues is the first to demonstrate efficacy against six different pathogens.
46. Rahman, M; Punja, ZK. (2005) Factors influencing development of root rot on ginseng caused by Cylindrocarpon destructans.Phytopathology 95: 1381-1390 Factors influencing development of root rot on ginseng caused by Cylindrocarpon destructans
disappearing root rot; epidemiology; Panax quinquefolius; pathogenicity
The fungus Cylindrocarpon destructans (Zins) Scholten is the cause of root rot (disappearing root rot) in many ginseng production areas in Canada. A total of 80 isolates of C. destructans were recovered from diseased roots in a survey of ginseng gardens in British Columbia from 2002-2004. Among these isolates, 49% were classified as highly virulent (causing lesions on unwounded mature roots) and 51% were weakly virulent (causing lesions only on previously wounded roots). Pectinase and polyphenoloxidase enzymes were produced in vitro by C. destructans isolates when they were grown on pectin and phenol as a substrate, respectively. However, highly virulent isolates produced significantly (P < 0.001) higher enzyme levels compared with weakly virulent isolates. Histopathological studies of ginseng roots inoculated with a highly virulent isolate revealed direct hyphal penetration through the epidermis, followed by intracellular hyphal growth in the cortex. Subsequent cell disintegration and accumulation of phenolic compounds was observed. Radial growth of highly and weakly virulent isolates on potato dextrose agar was highest at 18 and 21 degrees C, respectively and there was no growth at 35 degrees C. Mycelial mass production as significantly (P <= 0.01) lower at pH 7.0 compared with pH 5.0. To study the effects of pH (5.0 and 7.0) and wounding on disease development, ginseng roots were grown hydroponically in Hoagland's solution. Lesions were significantly larger (P < 0.001) at pH 5.0 compared with pH 7.0 and wounding enhanced disease by a highly virulent isolate at both pHs. In artificially infested soil, 2-year-old ginseng roots were most susceptible to Cylindrocarpon root rot among all root ages tested (I to 4 years) when evaluated using a combined scale of disease incidence and severity. Root rot severity was significantly (P < 0.002) enhanced by increasing the inoculum density from 3.45 x 10(2) CFU/g of soil to 1.86 x 10(3) CFU/g of soil. Disease severity was higher at 20 degrees C compared with 15 and 25 degrees C and at -0.02 MPa soil moisture compared with -0.005 and -0.001 MPa. A significant interaction between soil moisture and temperature was observed for root rot severity.
45. Rahman, M; Punja, ZK. (2005) Biochemistry of ginseng root tissues affected by rusty root symptoms.Plant Physiol Biochem 43: 1103-1114 Biochemistry of ginseng root tissues affected by rusty root symptoms
chitosan; defense response; iron-sequestration; Panax quinquefolius; phenolics; phenylalanine ammonia-lyase; peroxidase; polyphenoloxidase; wounding
Ginseng rusty root, a disorder of unknown cause (s), in which reddish-brown to orange-brown areas develop on the surface of field-grown roots, was studied at the cellular and biochemical levels. Using light microscopy, the affected areas were shown to comprise of the epidermis and underlying 6-8 cell layers of the cortical tissues. Rusty root areas ranged fro small clusters of 3-4 cells to larger expanding areas of > 80 cells. These cells appeared golden-brown and stained a bluish-green with Toluidine Blue indicating the presence of phenolic compounds. Energy-dispersive X-ray spectroscopy and atomic emission spectrometry of affected epidermal cells revealed a significant accumulation of Fe, Al, Si, Mg and other cations when compared to adjoining healthy cells. The concentrations of the six most common ginsenosides found in ginseng roots (Rg(1), Re, Rb-1, Rc, Rb-2, and Rd) were reduced by 40-50% in rusty root-affected epidermal and cortical tissues when compared to adjacent healthy tissues. Total phenolic compounds were increased by up to threefold in affected tissues and HPLC analysis revealed significantly higher levels of quercetin, cinnamic acid, vanillic acid, p-coumaric acid, benzoic acid, chlorogenic acid and catechin. In vitro phenolic-metal binding assays confirmed that phenolic compounds were able to sequester positively-charged metal ions, in particular Fe, to form a phenolic-metal ion complex. In ginseng callus cultures, accumulation of phenolic compounds was increased threefold within 12 It of treatment with chitosan (M), and to a lesser extent by wounding. Specific defense enzymes, namely phenylalanine ammonia-lyase (PAL, E.C. 4. 3. 1. 5.), polyphenoloxidase (PPO, E.C. 1. 10. 3. 1.) and peroxidase (POD, E.C. 1. 11. 1. 7.), were also significantly enhanced in treated callus tissues and in rusty root tissues. On field-grown ginseng roots, application of chitosan induced symptoms similar to rusty root, whereas wounding and ethylene treatments did not. Based on these results, rusty root symptoms on ginseng are proposed to result from an induction of host defense responses, especially phenolic production, in epidermal and underlying cortical cells. This induction is likely due to attempted invasion by as-yet uncharacterized chitin-containing soil fungi, which were observed in many of the affected cells. Subsequent oxidation of phenolic compounds and sequestration of metal ions, in particular Fe, appear to be largely responsible for the symptoms observed. (c) 2005 Elsevier SAS. All fights reserved.
44.Punja, ZK. (2004) Virulence of Chalara elegans on bean leaves, and host-tissue responses to infection.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 26: 52-62 Virulence of Chalara elegans on bean leaves, and host-tissue responses to infection
black root rot; Thielaviopsis basicola; Chalara elegans; virulence; Phaseolus vulgaris
Chalara elegans (syn. Thielaviopsis basicola) is a soilborne plant pathogen with a wide host range, which initially establishes a hemibiotrophic relationship, following infection of its host. To assess the differences in virulence among isolates and to study colonization of host tissues, detached bean leaves were used in an inoculation assay. Two-week-old 'Kentucky Wonder' leaves were sprayed with 0.8-1.0 mL of inoculum (10(5) phialospores/mL) and incubated under moist conditions for 5 days at 20-23 degreesC. Necrotic lesions that developed were enumerated and measured for 27 isolates of C. elegans from a broad range of hosts and geographic locations. Lesion numbers were affected by culture age, with 2-week-old cultures providing the most lesions compared with older cultures. There were pronounced differences in virulence among isolates, which were correlated with differences in colony morphology. The isolates were grouped into high, moderate, and low virulence classes based on the number of lesions produced. Highly virulent isolates were darkly pigmented, while moderately and weakly virulent isolates had reduced colony pigmentation. Bean cultivars influenced the number of lesions that developed, and 'Kentucky Wonder' was the most susceptible compared with 'Royal Burgundy' and 'Kentucky Blue'. In infected leaf tissues, the pathogen was initially localized to epidermal cells, which became necrotic and accumulated phenolic compounds. Callose deposits, cell-wall thickening, and accumulation of polyphenol oxidase were also observed in these epidermal cells after 36-48 h. Spread of hyphae to underlying mesophyll cells occurred after 48-72 h. Chlamydospores and phialospores developed on lesions within 5 days after inoculation. Most lesions were limited in size to less than 1 mm and resembled local lesions resulting from a virus inoculation. The ability of C. elegans to infect and induce defense responses in bean leaves has not been previously reported. This host-pathogen system provides an interesting model for further studies on the cellular responses and potentially induced resistance in beans, resulting from a soilborne pathogen.
43.Punja, ZK; Feeney, M; Schluter, C; Tautorus, T. (2004) Multiplication and germination of somatic embryos of American ginseng derived from suspension cultures and biochemical and molecular analyses of plantlets.In Vitro Cellular & Developmental Biology-Plant 40: 329-338 Multiplication and germination of somatic embryos of American ginseng derived from suspension cultures and biochemical and molecular analyses of plantlets
cell culture; embryogenesis; ginsenosides; Panax quinquefolius; RAPD banding patterns
Seedlings from 11 seed sources (lines) of American ginseng from different geographic regions were evaluated on Murashige and Skoog medium (MS) containing 10 muM (alpha-naphthaieneacetic acid (NAA) and 9 [muM 2,4-dichlorophenoxyacetic acid (2,4-D) for callus development and somatic embryo formation. Leaf and stem explants call used it a frequency of 18.2-100%, while somatic embryos were produced from these calluses at a frequency of 2587.5% after 5 mo. Suspension cultures of nine lines were established by transferring embryogenic callus to MS liquid medium with NAA and 2,4-D at 2.5 and 2.25 muM, respectively, and maintained by subcultures even, 8 wk. Globular somatic embryos from these cultures were germinated on half-strength MS containing 1% activated charcoal, and roots > 5 turn in length developed within 3 wk. A 7-d exposure to 3 muM gibberellic acid and 5 muM 6-benzylaminopurine significantly enhanced shoot development and promoted further root development. The chromosome number, profiles of the common triterpenoid saponins (ginsenosides), and random amplified polymorphic DNA (RAPD) banding patterns in plantlets derived from suspension culture were compared to those of zygotic seedlings. The chromosome number in root tip cells and suspension cultured cells was 48. Patterns of the six major ginsenosides, determined by thin-layer chromatography, in leaves of tissue culture-derived plantlets were identical to those in seedlings. RAPD patterns among plantlets originating from the same tissue-cultured line were mostly identical; however, altered patterns were observed in some lines that had been maintained in suspension culture for almost 4yr. The results from this study indicate that propagation of desired ginseng genotypes in suspension culture can be achieved, and that biochemical and molecular markers can be used for authentication of resulting plantlets.
42. Rose, S; Punja, ZK. (2004) Greenhouse cucumber cultivars differ in susceptibility to fusarium root and stem rot.Horttechnology 14: 240-242 Greenhouse cucumber cultivars differ in susceptibility to fusarium root and stem rot
Fusarium oxysporum f.sp radicis-cucumerinum; disease resistance; screening of cultivars
Eighteen cucumber (Cucumis sativus L.) cultivars (long English type) were screened for their susceptibility to fusarium, root and stem rot caused by Fusarium oxysporum Schlechtend.: Fr. f.sp. radicis-cucumerinum D.J. Vakalounakis using seedlings at the third true-leaf stage. Roots were trimmed and dipped into a spore suspension (10(5) spores/mL) of the pathogen and the plants were re-potted. A disease severity index (DSI) was used to assess disease responses 4 or 8 weeks later based on plant mortality and the height of surviving plants compared to the noninoculated controls. 'Sienna', 'Amazing' and 'Dominica' were most susceptible to infection and the resulting DSI values were significantly (P less than or equal to 0.05) higher compared to noninoculated control plants. The cultivars 'Korinda', 'Euphoria' and 'Aviance' displayed significantly lower DSI values which were not significantly different from noninoculated control plants. The remaining 12 cultivars displayed DSI values which were intermediate between the above two classes of responses. The results from this study indicate there is the potential to identify and develop cultivars and breeding lines of greenhouse cucumbers with enhanced resistance to fusarium root and stem rot.
41. Feeney, M; Punja, ZK. (2003) Tissue culture and Agrobacterium-mediated transformation of hemp (Cannabis sativa L.).In Vitro Cellular & Developmental Biology-Plant 39: 578-585 Tissue culture and Agrobacterium-mediated transformation of hemp (Cannabis sativa L.)
callus; suspension culture; Agrobacterium tumefaciens; mannose selection; phosphomannose isomerase; regeneration; transgenic hemp
Hemp (Cannabis sativa L.) is cultivated in many parts of the world for its fiber, oil, and seed. The development of new hemp cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the propagation of hemp in tissue culture and to establish a protocol for Agrobacterium-mediated transformation for foreign gene introduction. Stem and leaf segments from seedlings of four hemp varieties were placed on Murashige and Skoog medium with Gamborg B5 vitamins (MB) supplemented with 5 muM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 muM kinetin, 3% sucrose, and 8 g l(-1) agar. Large masses of callus were produced within 4 wk for all cultivars. Suspension cultures were established in MB medium containing 2.5 muM 2,4-D. To promote embryogenesis or organogenesis, explants, callus, and suspension cultures derived from a range of explant sources and seedling ages were exposed to variations in the culture medium and changes to the culture environment. None of the treatments tested were successful in promoting plantlet regeneration. Suspension cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary vector pNOV3635 with a gene encoding phosphomannose isomerase (PMI). Transformed callus was selected on medium containing 1-2% mannose. A chlorophenol red assay was used to confirm that the PMI gene was expressed. Polymerase chain reaction and Southern hybridization detected the presence of the PMI gene. Copy number in different lines ranged from one to four.
40.Punja, ZK; Utkhede, RS. (2003) Using fungi and yeasts to manage vegetable crop diseases.Trends in Biotechnology 21: 400-407 Using fungi and yeasts to manage vegetable crop diseases
Vegetable crops are grown worldwide as a source of nutrients and fiber in the human diet. Fungal plant pathogens can cause devastation in these crops under appropriate environmental conditions. Vegetable producers confronted with the challenges of managing fungal pathogens have the opportunity to use fungi and yeasts as biological control agents. Several commercially available products have shown significant disease reduction through various mechanisms to reduce pathogen development and disease. Production of hydrolytic enzymes and antibiotics, competition for plant nutrients and niche colonization, induction of plant host defense mechanisms, and interference with pathogenicity factors in the pathogen are the most important mechanisms. Biotechnological techniques are becoming increasingly valuable to elucidate the mechanisms of action of fungi and yeasts and provide genetic characterization and molecular markers to monitor the spread of these agents.
39.Punja, ZK; Yip, R. (2003) Biological control of damping-off and root rot caused by Pythium aphanidermatum on greenhouse cucumbers.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 25: 411-417 Biological control of damping-off and root rot caused by Pythium aphanidermatum on greenhouse cucumbers
biocontrol; Cucumis sativus; Gliocladium catenulatum; root rot; damping-off; Pythium aphanidermatum; greenhouse cucumber
Damping-off and root rot of greenhouse cucumbers grown in rock wool, caused by Pythium aphanidermatum, is a recurring problem for growers throughout the major production areas of Canada. Four commercially formulated biocontrol agents, Streptomyces griseoviridis strain K61 (Mycostop(R)), Trichoderma harzianum strain T-22 (Rootshield(TM) Drench), Trichoderma virens strain GL-21 (SoilGard(TM) 12G), and Gliocladium catenulatum strain J1446 (PreStop(TM) WP, Prestop mix) were evaluated in growth-chamber and greenhouse trials conducted over 2 years for efficacy against this disease. The agents were applied twice, once at seeding time and again 11 days later, as a drench (or incorporated into the sawdust medium for SoilGard). This was followed by inoculation with a P. aphanidermatum mycelial suspension 12 days after seeding. In growth-chamber trials conducted at 28-30 degreesC and 90% relative humidity, seedling mortality in the treatment receiving P. aphanidermatum alone was over 80%. Only G. catenulatum (Prestop WP) significantly reduced disease incidence and enhanced plant height and fresh mass under these conditions. In greenhouse experiments conducted in the fall seasons of 2001 and 2002, mortality in the treatments receiving P. aphanidermatum alone was about 60%. The most effective biocontrol agent was G. catenulatum (Prestop WP, Prestop mix), followed by S. griseoviridis (Mycostop), which both significantly reduced plant mortality and increased plant height. These results indicate that G. catenulatum, when applied as a preventative treatment, has the potential to significantly reduce root rot and damping-off caused by P. aphanidermatum on greenhouse cucumbers, under conditions of high disease pressure.
38. Rose, S; Parker, M; Punja, ZK. (2003) Efficacy of biological and chemical treatments for control of fusarium root and stem rot on greenhouse cucumber.Plant Disease 87: 1462-1470 Efficacy of biological and chemical treatments for control of fusarium root and stem rot on greenhouse cucumber
Potential disease control methods were evaluated against root and stem rot of cucumber (Cucumis sativus) caused by Fusarium oxysporum f. sp. radicis-cucumerinum. Crab/shrimp shell chitin; three composted media; the biological control agents Pseudomonas chlororaphis strain 63-28, Trichoderma harzianum (RootShield Drench), Streptomyces griseoviridis (Mycostop), Gliocladium catenulatum (Prestop WP, Prestop Mix), and Trichoderma (Gliocladium) virens (SoilGard); and the fungicides thiram or benomyl were added at seeding time followed by inoculation with the pathogen. The addition of chitin (4%, vol/vol) to a peat-based medium significantly (P less than or equal to 0.05) enhanced seedling growth, increased soil pH, and reduced F oxysporum f. sp. radicis-cucumerinum populations, but the severity of disease was increased. The addition of composted media (greenhouse compost, windrow composted dairy solids, and vermi-composted dairy solids) to the seeding cavity in a rock wool block medium, followed 48 h later by inoculation with F oxysporum f. sp. radicis-cucumerinum, reduced seedling mortality when measured after 37 days. Greenhouse compost was significantly (P:! 0.05) more suppressive than the other two composts, and the suppression was partially eliminated by sterilization of the compost. The biological control agent G. catenulatum (formulated as Prestop WP and Prestop Mix) significantly reduced seedling mortality when it was applied at seeding 24 h prior to inoculation with the pathogen in the rock wool block medium. None of the other biological control agents reduced disease incidence when compared with control plants under these experimental conditions. Pseudomonas chlororaphis and the fungicide thiram both significantly reduced plant mortality at 17 and 24degreesC when pathogen-infested seed was treated, or when bacteria-treated and fungicide-treated seed were planted into pathogen-infested peat medium at 24degreesC. Under semicommercial propagation conditions, treatments consisting of Prestop WP, RootShield Drench Mycostop, and windrow composted dairy solids reduced the severity of disease caused by oxysporum f. sp. radicis-cucumerinum in two out of three trials. The efficacy of the biological control agents was affected by seasonal differences in growing conditions, which affected the incidence and severity of the disease. The results from this study indicate that several different approaches can be used at seeding to control Fusarium root and stem rot on greenhouse cucumber.
37. Chen, WP; Punja, ZK. (2002) Agrobacterium-mediated transformation of American ginseng with a rice chitinase gene.Plant Cell Reports 20: 1039-1045 Agrobacterium-mediated transformation of American ginseng with a rice chitinase gene
Panax quinquefolius; genetic transformation; pathogenesis-related protein; selectable marker; fungal resistance
Transformation of American ginseng (Panax quinquefolius L.) with Agrobacterium strain LBA4404 containing a rice chitinase gene under control of the maize ubiquitinI promoter and the phosphinothricin acetyltransferase (bar) and hygromycin phosphotransferase (hpt) genes as selectable markers is described. Epicotyl explants from 2- to 3-week-old ginseng seedlings were pre-cultured for 5-7 days on MS medium supplemented with 10 muM alpha-naphthaleneacetic acid and 9 muM 2,4-dichlorophenoxyacetic acid (ND medium) prior to Agrobacterium infection. The explants were either immersed in a bacterial suspension for 20 min or received a 10-mul or 15-mul droplet of bacteria. A co-culture period of 3 or 4 days was provided on ND medium with or without acetosyringone and ascorbic acid. Selection for transformed calli was conducted on ND medium containing 20 mg/l phosphinothricin or 100 mg/l hygromycin over a 10-month period. A maximum callusing frequency of 27.7% was achieved on selection medium when explants were infected by the droplet method and co-cultured on ND medium without acetosyringone and ascorbic acid. Almost 90% of the 32 lines that survived selection were shown to be transformed. Immersion of explants reduced the callusing frequency to 9.3%. The presence of the transgenes was detected by Southern hybridization and polymerase chain reaction (PCR) analysis. The expression of the chitinase gene was demonstrated by reverse transcription PCR and Western analysis. One hundred and two ginseng plantlets were recovered from 11 confirmed transgenic lines. The transgene integration in plantlets of two lines was demonstrated by Southern analysis. This is the first report of Agrobacterium-mediated transformation of this important medicinal plant.
36. Chen, WP; Punja, ZK. (2002) Transgenic herbicide- and disease-tolerant carrot (Daucus carota L.) plants obtained through Agrobacterium-mediated transformation.Plant Cell Reports 20: 929-935 Transgenic herbicide- and disease-tolerant carrot (Daucus carota L.) plants obtained through Agrobacterium-mediated transformation
fungal resistance; bar; hpt; tissue culture; pathogenesis-related protein
Transgenic carrot (Daucus carota L.) plants expressing a rice thaumatin-like protein (tlp), phosphinothricin acetyltransferase (bar) and the hygromycin phosphotransferase (hpt) genes were obtained by Agrobacterium-mediated transformation. Petiole and hypocotyl segments of three carrot cultivars were used as the explant sources. Following infection, selection was achieved on Murashige and Skoog medium with 1 mg/l phosphinothricin or 25 mg/l hygromycin B, which was increased after 2 weeks to 10 mg/l phosphinothricin and 100 mg/l hygromycin B. The presence of the tlp and bar transgenes was confirmed by polymerase chain reaction and Southern blot analyses, and the expression of the thaumatin-like protein was demonstrated by Western blot analysis. Among 45 primary transformants, 13 were selected for assessment of herbicide and/or disease tolerance. The transgenic plants showed varying levels of tolerance to the herbicide phosphinothricin, depending on the transformation events in different lines. Four transgenic lines also showed significantly enhanced tolerance to the foliar and root pathogen Botrytis cinerea or Sclerotinia sclerotiorum when inoculated under controlled environment conditions. Two lines had significantly enhanced tolerance to the herbicide phosphinothricin as well as to both pathogens. These results demonstrate the feasibility of introducing two potentially useful agronomic traits into carrot through genetic engineering.
35. Deeks, SJ; Shamoun, SF; Punja, ZK. (2002) Histopathology of callus and germinating seeds of Arceuthobium tsugense subsp tsugense infected by Cylindrocarpon cylindroides and Colletotrichum gloeosporioides.International Journal of Plant Sciences 163: 765-773 Histopathology of callus and germinating seeds of Arceuthobium tsugense subsp tsugense infected by Cylindrocarpon cylindroides and Colletotrichum gloeosporioides
Arceuthobium; biocontrol; Colletotrichum; Cylindrocarpon; dual culture; histopathology; in vitro
Two fungi that parasitize western hemlock dwarf mistletoe (Arceuthobium tsugense subsp. tsugense), Cylindrocarpon cylindroides and Colletotrichum gloeosporioides, were evaluated for their virulence on germinating seeds and callus grown in vitro. Mistletoe seeds were germinating on Harvey's tissue culture medium in one-half of a petri plate, while the other half contained water agar on which fungal growth was initiated from a mycelial plug. Callus tissue was initiated on Harvey's medium or modified White's medium and challenged with fungi on Harvey's medium (C. cylindroides) or modified White's medium (C. gloeosporioides) because fungal growth rates were found to be moderate on these media. At 0.5, 1, 2, 3, and 7 d postcontact with fungi, mistletoe tissues were investigated by light microscopy. In seeds, both endosperm and radicle were colonized, and cushion development, cell wall degradation, and intercellular and intracellular colonization were evident with both fungi. Both fungi inhibited callus growth, degraded cell wall, and colonized inter- and intracellular spaces. Cells infected with C. cylindroides were disorganized and plasmolyzed. The in vitro screening method developed in this study was useful to elucidated host-pathogen interactions and showed that C. cylindroides was more virulent in colonizing dwarf mistletoe tissues than C. gloeosporioides.
34. Schluter, C; Punja, ZK. (2002) Genetic diversity among natural and cultivated field populations and seed lots of american ginseng (Panax quinquefolius L.) in Canada.International Journal of Plant Sciences 163: 427-439 Genetic diversity among natural and cultivated field populations and seed lots of american ginseng (Panax quinquefolius L.) in Canada
ginseng; molecular markers; genetic polymorphisms; random amplified polymorphic DNA
Genetic diversity within Canadian-grown North American ginseng (Panax quinquefolius L.) was evaluated using random amplified polymorphic DNA (RAPD) markers. Fifteen primers that produced 35 highly repeatable polymorphic markers were used to screen over 600 plant samples from various Canadian ginseng farms and seed lots. Ten samples from a Wisconsin seed lot and 58 samples from three natural ginseng populations in Quebec were also included for comparison. Genetic distance values, estimated as the complement to the simple matching coefficient, within cultivated populations ranged from 0.21 for a population in Nova Scotia to 0.34 for a British Columbia population, with an overall mean of 0.3. Distance values within three natural populations were either similar (0.33) or lower (0.12, 0.19) when compared with cultivated populations, indicating that populations under cultivation have not undergone a reduction in overall genetic diversity. However, one RAPD marker was polymorphic only in natural populations. Monotonic multidimensional scaling and x 2 analyses indicated that natural populations were genetically distinct from cultivated ones. Individual plants originating as seeds from the same mother plant had much lower genetic diversity (mean of 0.18) compared with individual field-grown plants chosen at random from the same farm. Segregation of some RAPD markers was observed among the progeny, indicating that parental plants have some degree of heterozygosity and that a level of outcrossing may be present. Estimates of the component for genetic diversity between populations (G'(ST)) were 18.0% and 28.0% for cultivated and natural populations, respectively; much of the variation was detected within and not between populations. These results imply that North American ginseng is a heterogeneous mix of genetic material and that the observed genetic diversity in cultivated populations in Canada results largely from the mixing of different seed lots. In addition, heterozygosity within the parent plants and cross-pollination appear also to contribute to genetic variation in this species.
33. Urquhart, EJ; Punja, ZK. (2002) Hydrolytic enzymes and antifungal compounds produced by Tilletiopsis species, phyllosphere yeasts that are antagonists of powdery mildew fungi.Canadian Journal of Microbiology 48: 219-229 Hydrolytic enzymes and antifungal compounds produced by Tilletiopsis species, phyllosphere yeasts that are antagonists of powdery mildew fungi
biological control; antibiotics; chitinases; glucanascs; powdery mildew
Isolates of five species of the yeast-like fungus Tilletiopsis Derx (Tilletiopsis albescens Gokhale, Tilletiopsis fulvescens Gokhale, Tilletiopsis minor Nyland, Tilletiopsis pallescens Gokhale, and Tilletiopsis washingtonensis Nyland) were screened for exo- and endo-beta-1,3-glucanase and chitinase production in a liquid broth used to produce inoculum for biological control studies. There were significant differences among the species, and highest overall enzyme activity was present in T. albescens and T. pallescens and lowest in T. washingtonensis. A time-course study of beta-1,3-glucanase and chitinase production in T. pallescens ATCC 96155 in broth culture with 2.5% glucose as the carbon source showed that enzyme activity gradually increased over a 3- to 21-day period. Maximum enzyme activity was found between pH 4.0 and 5.0. SDS-PAGE of beta-1,3-glucanase isozymes revealed a range of molecular masses from 18 to 29 kDa. Five isozymes were present in both T. albescens and T. pallescens and two in T. washingtonensis. Antifungal compounds were also detected in ethyl acetate extracts of culture filtrates of T. pallescens ATCC 96155 after 6 days of incubation, while no activity was detected at 14 days. One active fraction was selected following fractionation and preparative chromatography and was bioassayed against Podosphaera (sect. Sphaerotheca) xanthii (Castagne) U. Braun & N. Shishkoff and a number of other fungi. A concentration of 130 mug/mL inhibited germ tube development in P. xanthii, and mildew spores appeared plasmolyzed. Other fungi were inhibited at higher concentrations. Collapse of hyphae and conidiophores was also observed on mildewed leaves treated with the active fraction. Proton NMR analysis indicated that the inhibitory compound was a fatty acid ester. In 3- to 6-day-old cultures of T. pallescens ATCC 96155 demonstrating biological control activity, antifungal compound production may have a primary role in restricting growth of mildew fungi and other competitors when applied to leaves.
32. Deeks, SJ; Shamoun, SF; Punja, ZK. (2001) In vitro germination and development of western hemlock dwarf mistletoe.Plant Cell Tissue and Organ Culture 66: 97-105 In vitro germination and development of western hemlock dwarf mistletoe
Arceuthobium tsugense subsp tsugense; callus; holdfast; parasite; radicle; Viscaceae
A procedure for in vitro culture of the parasitic flowering plant western hemlock dwarf mistletoe, Arceuthobium tsugense (Rosend.) G.N. Jones subsp. tsugense, is described. A factorial experiment evaluated the effects of media (Harvey's medium (HM) and modified White's medium (WM)), temperatures (15 degreesC and 20 degreesC), presence or absence of light, and plant growth regulators (the auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and the cytokinin 6-benzylaminopurine (BAP) at varying concentrations (0.001 mg l(-1) to 1 mg l(-1))). Seed explants germinated in less than one week in culture and produced radicles. Optimal conditions for radicle elongation were WM at 20 degreesC in the presence of light and without plant growth regulators. Some of the radicles split at the tip to yield callus while others swelled to become spherical holdfasts. Holdfasts were also produced at the tips of radicles, and callus arose from split holdfasts. Factors that promoted holdfast production were Harvey's medium, light, and 2,4-D at 1 mg l(-1). Callus development from split radicles and split holdfasts was optimal on WM with 0.5 mg l(-1) 2,4-D and 1 mg l(-1) BAP at 20 degreesC in the dark.
31.Punja, ZK. (2001) Genetic engineering of plants to enhance resistance to fungal pathogens - a review of progress and future prospects.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 23: 216-235 Genetic engineering of plants to enhance resistance to fungal pathogens - a review of progress and future prospects
antifungal proteins; antimicrobial peptides; biotechnology; elicitors; hypersensitive response; pathogenesis-related proteins; phytoalexins; resistance genes; transgenic plants
Recent applications of techniques in plant molecular biology and biotechnology to the study of host-pathogen interactions have resulted in the identification and cloning of numerous genes involved in the defense responses of plants following pathogen infection. These include: genes that express proteins, peptides, or antimicrobial compounds that are directly toxic to pathogens or that reduce their growth in situ; gene products that directly inhibit pathogen virulence products or enhance plant structural defense genes, that directly or indirectly activate general plant defense responses; and resistance genes involved in the hypersensitive response and in the interactions with avirulence factors. The introduction and expression of these genes, as well as of antimicrobial genes from nonplant sources, in a range of transgenic plant species have shown that the development of fungal pathogens can be significantly reduced. The extent of disease reduction varies with the strategy employed as well as with the characteristics of the fungal pathogen, and disease control has never been complete. Manipulation of salicylic acid, ethylene, and cytokinin levels in transgenic plants have provided some interesting results with regard to enhanced disease tolerance or susceptibility. The complex interactions among the expressed gene product, plant species, and fungal pathogen indicate that the response of transgenic plants cannot be readily predicted. Combinations of defense gene products have shown considerably more promise in reducing disease than single-transgene introductions. The use of tissue-specific or pathogen-inducible promoters, and the engineered expression of resistance genes, synthetic antimicrobial peptides, and elicitor molecules that induce defense responses have the potential to provide commercially useful broad-spectrum disease resistance in the not-too-distant future. The issues and challenges that will need to be addressed prior to the widespread utilization of these transgenic plants are highlighted.
30.Punja, ZK; Parker, M; Elmhirst, J. (2001) Fusarium wilt of field-grown muskmelon in British Columbia.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 23: 403-410 Fusarium wilt of field-grown muskmelon in British Columbia
Fusarium orysporum f. sp melonis race 1; Cucumis melo var. cantalupensis
A wilt disease of field-grown muskmelon (Cucumis melo L. var. cantalupensis) was observed during the 1999 and 2000 growing seasons on three farms in the semi-arid interior region near the Okanagan Valley of British Columbia. Initial symptoms were yellowing and wilting of leaves, which sometimes occurred unilaterally; in some cases, this was followed by plant death prior to flowering. The crown area of diseased plants had dark brown lesions that progressed into lateral branches for distances of 45-75 cm. Some lesions also extended into the developing fruit, which frequently became infected and rotted. Stem lesions were accompanied by dark brown exudate droplets and necrosis extended into the cortical and vascular regions. Plants with fruit collapsed when temperatures exceeded 25-30degreesC in mid-July and early August. White mycelium developed on the infected crown area and on fruits collected from mature plants in the field, after incubation under high humidity conditions, and macroconidia and microconidia of Fusarium oxysporum Schlechtend.:Fr. were produced. A number of muskmelon cultivars were affected, and the most susceptible were Galia and Castella, while Athena appeared unaffected in the field. Pathogenicity tests demonstrated that muskmelon was the only susceptible host and no disease symptoms developed on cucumber (Cucumis sativus L.), watermelon (Citrullus vulgaris Eckl. & Zeyh), or squash (Cucurbita pepo L.). Based on symptomology and host range, the pathogen was identified as F. oxysporum Schlechtend. f. sp. melonis (Leach & Currence) W.C. Snyder & H.N. Hans. Inoculation of a set of differential muskmelon cultivars revealed that the pathogen was F. oxysporum f. sp, melonis race 1. The pathogen was recovered from diseased transplants in a propagation greenhouse and from seed recovered from naturally infected fruits on mature plants in the field. This is the first report of fusarium wilt of muskmelon in British Columbia, and the first report of race 1 of F. oxysporum f. sp. melonis in Canada.
29. Kannangara, T; Utkhede, RS; Paul, JW; Punja, ZK. (2000) Effects of mesophilic and thermophilic composts on suppression of Fusarium root and stem rot of greenhouse cucumber.Canadian Journal of Microbiology 46: 1021-1028 Effects of mesophilic and thermophilic composts on suppression of Fusarium root and stem rot of greenhouse cucumber
Fusarium oxysporum f. sp radicis-cucumerinum; biological control
Three composts were tested for their ability to suppress root and stem rot caused by the soil borne fungal pathogen Fusarium oxysporum f. sp. radicis-cucumerinum (FORC) on cucumber. Two of the composts were prepared from separated dairy solids either by windrow (WDS) or vermicomposting (VMC) while the third, obtained from International Bio-Recovery (IBR), was prepared from vegetable refuse using aerobic digestion. Three sets of potting mixes were prepared by mixing the composts with sawdust at varying ratios, and seeded with cucumber cv. Corona. After 14 days of growth in the greenhouse, inoculum of FORC (20 mL of 5 x 10(6) micro-conidia per mL) was applied to each pot at three different times (14, 21, and 35 days). In unamended inoculated pots, the pathogen caused stunted growth and reduced flowers. Amendment of WDS in the potting mix suppressed these symptoms, while VMC and IBR had no effect. All three composts reduced the FORC colony forming units (cfu) at the end of the experiment (10 weeks). There was a large increase of fluorescent bacteria near the vicinity of roots particularly in WDS amended potting mixes. When water extracts of the composts were plated onto acidified potato dextrose agar (APDA), only IBR contained a potent thermostable inhibitor to FORC. This inhibitor was removed by activated charcoal but was not partitioned into petroleum ether at acid, basic, or neutral pH. Inhibition of FORC by IBR was not due to electrical conductivity or trace elements in the compost. Contrasting effectiveness of the WDS and VMC made from the same waste suggests that composting method can influence the disease suppression properties of the finished compost.
28.Punja, ZK; Parker, M. (2000) Development of fusarium root and stem rot, a new disease on greenhouse cucumber in British Columbia, caused by Fusarium oxysporum f. sp radicis-cucumerinum.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 22: 349-363 Development of fusarium root and stem rot, a new disease on greenhouse cucumber in British Columbia, caused by Fusarium oxysporum f. sp radicis-cucumerinum
Cucumis sativus; Fusarium oxysporum; crown rot
A root and stem rot of greenhouse cucumber was first observed in the Eraser Valley of British Columbia in 1994 and has since increased in frequency and severity. Affected plants wilted at the fruit-bearing stage, especially at temperatures over 27 degreesC, and mycelial growth and orange spore masses developed on the crown and stem. The pathogen was isolated from roots and crowns, and from cortical and vascular tissues up to 75 cm from the crown. Reactions of 25 cucumber cultivars ranged from highly susceptible to moderately resistant; the widely-grown long English cultivars Flamingo, Mustang, and Serami were all highly susceptible. The pathogen was identified as Fusarium oxysporum Schlechtend.:Fr. forma specialis radicis-cucumerinum D.J. Vakalounakis (F.o.r.c.), which has not previously been reported in Canada but occurs in Greece and the Netherlands. Muskmelon (Cucumis melo L.), squash (Cucurbita pepo L.), watermelon (Citrullus vulgaris Schrad.), and gourd (Luffa aegyptiaca Mill.) developed root and stem symptoms similar to those on cucumber when inoculated using a root dip method. Random amplified polymorphic DNA analysis using eight primers revealed that the pathogen was distinct from Fusarium oxysporum f. sp. cucumerinum J.H. Owen (F.o.c., cause of fusarium wilt). Pectolytic enzyme production in vitro was greater in F.o.r.c. than F.o.c. The optimal temperature range for growth of F.o.r.c. on potato dextrose agar was 24-27 degreesC. Disease developed at 17 and 24 degreesC but not at 32 degreesC. The pathogen was recovered on Komada's medium at a frequency of 10(5) colony-forming units (cfu)/cm(3) from the growing substrate and run-off water, but at a low frequency from the air. The initial sources of inoculum may be contaminated seed or growing medium, and infection of seedlings can occur within the first 4 weeks of growth. Artificial seed inoculation caused damping-off on cucumber and muskmelon seedlings. Wounding of roots and low temperatures during seedling development and fruit load on mature plants enhance disease severity. Fusarium root and stem rot has the potential to spread to other regions of Canada.
27. Schluter, C; Punja, ZK. (2000) Floral biology and seed production in cultivated North American ginseng (Panax quinequefolius).Journal of the American Society for Horticultural Science 125: 567-575 Floral biology and seed production in cultivated North American ginseng (Panax quinequefolius)
flower development; pollination; seed set; berries
Morphological characteristics of flowers, duration of flowering, degree of self-pollination, and extent of berry and seed production in North American ginseng (Panax quinquefolius L.) were studied under controlled environmental conditions as well as under field conditions. A comparison was also made between plants of 3 and 4 years of age at two field locations. The duration of flowering was 3 weeks and was similar in plants of both age groups grown in the two environments; however, 4-year-old plants produced an average of 40% more flowers (100 per plant in total) compared to 3-year-old plants, Flowers were comprised of five greenish-colored petals, five stamens, and an inferior ovary consisting of predominantly two fused carpels and stigmatic lobes, Anthers dehisced in staggered succession within individual flowers, and flowering began with the outermost edge of the umbel and proceeded inwards, At any given time during the 4-neek flowering period, 10% of the flowers in an umbel had recently opened and mere producing pollen, Stigma receptivity was associated with separation of the stigmatic lobes; this occurred at some time after pollen release. Growth of pollen tubes through the style in naturally pollinated flowers was most evident when the stigmatic lobes had separated. The proportion of flowers that developed into mature berries (pollination success rate) was in the range of 41% to 68% for both 3-year-old and 4-year-old plants. However, when the inflorescence was bagged during the flowering period, berry formation was increased by 13% to 21% in 4-year-old plants, depending on location. 4 majority of the berries (92% to 99%) contained one or two seeds in an almost equal frequency, with the remaining berries containing three seeds. In l-year-old plants, the frequency of two-seeded berries was increased by 13% by bagging the inflorescence, These observations indicate that P. quinquefolius is highly self-fertile and that several physiological and environmental factors can affect seed production.
26. Deeks, SJ; Shamoun, SF; Punja, ZK. (1999) Tissue culture of parasitic flowering plants: Methods and applications in agriculture and forestry.In Vitro Cellular & Developmental Biology-Plant 35: 369-381 Tissue culture of parasitic flowering plants: Methods and applications in agriculture and forestry
broomrape; dodder; figwort; laurel; mistletoe; sandalwood
Parasitic flowering plants, from 23 genera in 7 families (Convolvulaceae, Lauraceae, Loranthaceae, Orobanchaceae, Santalacene. Scrophulariaceae and Viscaceae) have been cultured in vitro. These plants include both hemiparasites and holoparasites that parasitize stems and roots of angiosperms and gymnosperms. This review highlights relevant information on each genus with regard to its biology, distribution, host range, and tissue culture procedures. Tissue culture has been used to study aspects of the development, metabolism, reproduction, physiology and nutritional requirements of these plants under controlled conditions. Studies of host-parasite relationships, including potential roles of signal/receptors that influence host development and physiology, and factors influencing seed germination and haustorium formation; have been conducted. The effects of chemicals and herbicides on the physiology and biochemistry of parasite embryo and seedling development have been studied, as well as the influence of inhibitors or stimulants on seed germination. Tissue culture has provided a method for propagation and genetic improvement of plants with commercial value.
25.Punja, ZK; Sun, LJ. (1999) Morphological and molecular characterization of Chalara elegans (Thielaviopsis basicola), cause of black root rot on diverse plant species.Canadian Journal of Botany-Revue Canadienne de Botanique 77: 1801-1812 Morphological and molecular characterization of Chalara elegans (Thielaviopsis basicola), cause of black root rot on diverse plant species
black root rot; RAPD analysis; Thielaviopsis basicola
The extent of variation in colony morphology and chlamydospore size, septation, and pigmentation was studied in 50 isolates of Chalara elegans Nag Raj et Kendrick (syn. Thielaviopsis basicola (Berk. et Br.) Ferr.) originating from 12 different geographic areas and substrates. In addition, the extent of genetic variation among these isolates was determined using random amplified polymorphic DNA (RAPD) analysis. Five general morphological groups could be distinguished among the isolates, two of which were aberrant phenotypes (albino and mycelial) that were derived upon continuous subculture of some wild-type isolates in the laboratory. The isolates with the most variation in phenotype originated from British Columbia and California. Six primers (10-mers) were used to generate 90 bands in RAPD-PCR, of which 75 were polymorphic. A high degree of diversity was apparent within C. elegans, and some banding patterns generated by specific primers were unique to certain isolates, thereby generating fingerprints. Distinct groups (clusters) were obtained following UPGMA analysis and, generally, these were composed of isolates from similar geographic regions or hosts. However, isolates from some areas, for example, British Columbia, were also found to belong to different clusters. There was generally a good relationship between groups assigned on the basis of morphology and those derived from cluster analysis, that is, isolates within a cluster tended to have similar morphology. In a few isolates, the aberrant phenotypes (albino and mycelial) could be distinguished using RAPDs from the wild type by the absence of 1 or 2 bands, indicating that changes in the nucleotide sequence had occurred, possibly through mutation. The average similarity index among all 50 isolates of C. elegans was 87%. An outgroup species (Chalara thielaviodes) had a similarity value of 40%.
24. Ramsfield, TD; Shamoun, SF; Punja, ZK; Hintz, WE. (1999) Variation in the mitochondrial DNA of the potential biological control agent Chondrostereum purpureum.Canadian Journal of Botany-Revue Canadienne de Botanique 77: 1490-1498 Variation in the mitochondrial DNA of the potential biological control agent Chondrostereum purpureum
Chondrostereum purpureum; mitochondrial DNA; genetic variation
The mitochondrial DNA (mtDNA) of Chondrostereum purpureum (Pers.:Fr.) Pouzar was extracted and purified, and the size ranged from 51.8 to 66.4 kb. One isolate each from British Columbia, Alberta, Finland, the Netherlands, and New Zealand were found to have identical BamHI mtDNA restriction patterns, resulting in a mitochondrial genome of 63.8 kb. An additional isolate from British Columbia and one from Switzerland had different banding patterns, however, resulting in mitochondrial genomes of 66.4 kb and 51.8 kb, respectively. A sequence-characterized amplified region (SCAR) assay, based on a polymerase chain reaction, was developed to rapidly screen a larger population of 84 isolates from North America, Europe, and New Zealand. Two SCARs, one encoding the NADH 4 gene (3 kb) and the second encoding the ATPase VI and cytochrome b genes (5.1 kb), were digested with 24 restriction enzymes. There were no polymorphisms in the NADH 4 containing SCAR, while a single polymorphism was detected by NsiI in the ATPase VI - cytochrome b containing SCAR. Two mitochondrial haplotypes that were distributed throughout the sample population were thus identified. The coancestry coefficient (theta) for all subpopulations of the sample population was calculated to be 0.0353. The level of gene diversity in the mtDNA of C. purpureum suggested that the chance introduction of novel mitochondrial genes following biological control applications of the fungus is relatively low.
23. Descalzo, RC; Punja, ZK; Levesque, CA; Rahe, JE. (1998) Glyphosate treatment of bean seedlings causes short-term increases in Pythium populations and damping off potential in soils.Applied Soil Ecology 8: 25-33 Glyphosate treatment of bean seedlings causes short-term increases in Pythium populations and damping off potential in soils
Pythium-herbicide interaction; Pythium root rot; predisposition; root pathogens; herbicide; damping off
The effect of bean root residues on populations of known isolates and unidentified Pythium species in soils was assessed, with special regard to herbicide treatment of bean seedlings. The general Pythium population in a muck soil was significantly increased 6 days after foliar treatment of bean seedlings with glyphosate, and by soil amendment with heat-killed bean roots, but not by amendment with roots of healthy bean seedlings. The enhanced populations returned to near initial levels 4 days Later. Isolated populations of Pythium ultimum (a glyphosate-synergistic isolate) and Pythium coloratum (a non-synergistic isolate) in a sandy loam soil were enhanced approximately 10-fold over control by roots of healthy bean seedlings and by roots of seedlings killed with either glyphosate or paraquat. Distinct peaks in the population responses of P. ultimum and P. coloratum occurred at 9 and 18 days after treatment, respectively. Strong positive correlations were observed between the population estimates obtained by dilution plating and damping off of sunflower for both P. ultimum and P. coloratum. These results suggests that herbicide treatment of plants can cause temporary increases in both Pythium, populations and damping off potential of soils. (C) 1998 Elsevier Science B.V. All rights reserved.
22. Goodwin, SB; Smart, CD; Sandrock, RW; Deahl, KL; Punja, ZK; Fry, WE. (1998) Genetic change within populations of Phytophthora infestans in the United States and Canada during 1994 to 1996: Role of migration and recombination.Phytopathology 88: 939-949 Genetic change within populations of Phytophthora infestans in the United States and Canada during 1994 to 1996: Role of migration and recombination
DNA fingerprinting; fungicide resistance; late blight; migration; population genetics
Dramatic changes occurred within populations of Phytophthora infestans in the United States and Canada from 1994 through 1996. Occurrence of the US-8 genotype, detected rarely during 1992 and 1993, increased rapidly and predominated in most regions during 1994 through 1996. US-7, which infected both potato and tomato and made up almost 50% of the sample during 1993, was detected only rarely among 330 isolates from the United States analyzed during 1994. It was not detected at all in more limited samples from 1996. Thus, ability to infect both potato and tomato apparently did not increase the fitness of this genotype relative to US-8, as predicted previously. US-I, the previously dominant genotype throughout the United States and Canada, made up 8% or less of the samples analyzed during 1994 through 1996. A few additional genotypes were detected, which could indicate the beginnings of sexual reproduction of FI infestans within the United States and Canada. However, clonal reproduction still predominated in all locations sampled; opportunities for sexual reproduction probably were limited, because the Al and A2 mating types usually were separated geographically. The high sensitivity of the US-I genotype to the fungicide metalaxyl also could have reduced opportunities for contact between the mating types in fields where this compound was applied. The previous correlation between metalaxyl sensitivity and genotype was confirmed and extended to a new genotype, US-17: all US-1 isolates tested were sensitive; all isolates of the US-7, US-8, and US-17 genotypes tested to date have been resistant. Isolates of P. capsici and P. erythroseptica, two other species often found on tomato and potato, could be easily distinguished from each other and from P. infestans using a simple allozyme assay for the enzyme glucose-6-phosphate isomerase. This technique could be useful for rapid identification of species, in addition to genotype of P. infestans. It generally was not possible to predict which genotypes would be present in a location from 1 year to the next. Long-distance movement of US-8 in seed tubers was documented, and this was probably the primary means for the rapid spread of this genotype from 1993 through 1996.
21. Oleskevich, C; Shamoun, SF; Vesonder, RF; Punja, ZK. (1998) Evaluation of Fusarium avenaceum and other fungi for potential as biological control agents of invasive Rubus species in British Columbia.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 20: 12-18 Evaluation of Fusarium avenaceum and other fungi for potential as biological control agents of invasive Rubus species in British Columbia
wild red raspberry; thimbleberry; salmonberry; mycoherbicide
Fungi were isolated from naturally infected Rubus strigosus, R. parviflorus, and R, spectabilis plants in an attempt to identify biological control agents for these invasive species in reforestation sites. Three endemic fungi, Fusarium avenaceum, Colletotrichum dematium, and a Phomopsis sp., were selected for further study after they were found to induce >50% leaf area necrosis when inoculated onto detached Rubus leaves using in vitro pathogenicity tests. However, when inoculum was applied to intact Rubus plants under shadehouse conditions, significant foliar necrosis was not observed. Inoculum production methods, amendment of inocula with adjuvants, and application of low doses of glyphosate were investigated for their effects on pathogenicity. Foliar infection was increased significantly when inoculum of F. avenaceum was sown on a rice-grain substrate and applied in combination with an organosilicone surfactant (0.4% Silwet L-77(R)) to R, strigosus and R, parviflorus plants. Extraction and analysis of infested rice filtrates for metabolite production showed that a single toxin, moniliformin, was present at levels of 3300 ppm. Pathogenicity of the other two fungi was not enhanced under any conditions assayed. The potential for further development of F. avenaceum as a biological control agent of weedy Rubus species is discussed.
20.Punja, ZK; Forster, H; Cunningham, I; Coffey, MD. (1998) Genotypes of the late blight pathogen (Phytophthora infestans) in British Columbia and other regions of Canada during 1993-1997.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 20: 274-282 Genotypes of the late blight pathogen (Phytophthora infestans) in British Columbia and other regions of Canada during 1993-1997
metalaxyl resistance; late blight; sexual recombination
Isolates of Phytophthora infestans were collected from British Columbia at various times during 1993-1997 and from eight other provinces in Canada during 1993-1995 and characterized for mating type (either Al or A2), growth response to metalaxyl (either sensitive-MS or insensitive-MI), and random amplified polymorphic DNA (RAPD) patterns. Among the isolates tested, 312 were from diseased potato cultivars, while 14 originated from tomato or hairy nightshade plants with blight symptoms. A composite of RAPD patterns generated by nine primers following PCR amplification was used to identify novel genotypes based on presence of polymorphic bands. The occurrence in Canada of genotypes that corresponded to US 1, US 6, US 7, and US 8 was confirmed during this study. The RAPD primers also identified 15 novel genotypes from British Columbia (BC) and 4 from New Brunswick, regions in which both Al and A2 mating types of P. infestans have previously been found and oospore production reported. In 1993, genotypes US 6 and US 7 were found in British Columbia, while US 1 was found in five other provinces (Alberta, Manitoba, New Brunswick, Prince Edward Island, and Quebec). In 1994, genotype US 8 was found in New Brunswick and US 1 in Alberta, Prince Edward Island, Quebec, and Saskatchewan. In 1995, US 8 was identified from all provinces except Alberta and British Columbia, while genotype US 1 was found in Alberta, Manitoba, and Prince Edward Island. Among the novel genotypes from British Columbia and New Brunswick, many were localized to specific geographic areas and most were ephemeral and not recovered in subsequent years. Only two genotypes from British Columbia - BC 1 (A2, MI) and BC 11 (Al, MI) - were recovered over several years and from different locations, indicating they had overwintered and likely spread through movement of infected plant materials or inoculum. Genotypes US 7 and BC 11 were recovered from tomato, and BC 11, BC 13, BC 14, and BC 15 from hairy nightshade, illustrating the importance of other solanaceous hosts as potential sources of inoculum. The results from this study illustrate the complex and changing genotypic structure of P. infestans, which may, in part, be responsible for the increased prevalence of late blight on potato and tomato in Canada. A complex genotype structure, such as that observed in British Columbia, may be characteristic of other regions in which both mating types are in close proximity. The diversity observed is likely the outcome of sexual recombination after oospore formation and germination under natural conditions.
19. Tirajoh, A; Kyung, TS; Punja, ZK. (1998) Somatic embryogenesis and plantlet regeneration in American ginseng (Panax quinquefolium L.).In Vitro Cellular & Developmental Biology-Plant 34: 203-211 Somatic embryogenesis and plantlet regeneration in American ginseng (Panax quinquefolium L.)
medicinal plant; propagation; tissue culture
Somatic embryogenesis in American ginseng (Panax quinquefolium L.) was investigated from three explant sources (root, leaf and epicotyl) with Murashige and Skoog (MS) medium containing different growth regulators. Mature roots and leaves obtained from 3- to 5-yr-old field-grown plants, and seedling leaves and epicotyls from plantlets grown in vitro, were evaluated. From root and epicotyl explants, callus development Tvas optimal with 3,6-dichloro-o-anisic acid (dicamba) (9.0 mu M) and kinetin (KN) (5.0 mu M) as the growth regulators. When these calluses were transferred after 3 mo. to dicamba alone (9.0 mu M), somatic embryo formation was observed at an average frequency of 15.6% in root explants;after an additional 3 mo., and 2% in epicotyl explants after an additional 6 mo. No plantlets were recovered because the embryos germinated to form shoots with no roots. From leaf explants, callus growth was optimal,vith alpha-naphthaleneacetic acid (NAA) at 10.0 mu M and 2,4-dichlorophenoxyacetic acid (-2,4-D) at 9.0 mu M. Somatic embryos developed on this medium, with the highest frequency (40%) obtained after 3 mo. from seedling-leaf explants. Calluses on mature leaves formed somatic embryos after 7 mo. with NAA/2,4-D at an average frequency of 30%. Transfer of these somatic embryos to 6-benzyladenine/gibberellic acid (4.4/2.9 mu M) promoted shoot development but no roots were observed. Up to 100% of germination was observed within 6 wk on half-strength MS salts containing activated charcoal (1%) and on NAA/2,4-D (5.0/4.5 mu M) with charcoal (1%). On the latter medium, somatic embryos enlarged and frequently gave rise to new somatic embryos after a brief callusing phase. The embryos germinated through a two-stage process. involving the elongation of the root followed by the formation of a shoot. The highest recovery of ginseng plantlets from germinated embryos was 61.0%. Following transfer to potting medium and maintenance under conditions of high humidity and low light intensity, the plantlets elongated and developed new leaves, A high percentage (50%) of these plants have been acclimatized to soil.
18. Cruickshank, MG; Morrison, DJ; Punja, ZK. (1997) Incidence of Armillaria species in precommercial thinning stumps and spread of Armillaria ostoyae to adjacent Douglas-fir trees.Canadian Journal of Forest Research-Revue Canadienne de Recherche Forestiere 27: 481-490 Incidence of Armillaria species in precommercial thinning stumps and spread of Armillaria ostoyae to adjacent Douglas-fir trees
The frequency of Armillaria species in precommercial thinning stumps and the interaction at root contacts between Douglas-fir (Psendotsuga menziesii (Mirb.) France) crop trees and stumps colonized by Armillaria ostoyae (Romagn.) Herink were investigated at sites in four biogeoclimatic zones along a transect from the coast through the southern interior of British Columbia. The frequency of stumps colonized by A. ostoyae and Armillaria sinapina Berube & Dessureault varied among lower, mid, and upper slope transects. On coastal sites, A. sinapina dominated fresh hygrotopes and A. ostoyae dominated slightly dry hygrotopes, and the frequency of both fungi was low on moist hygrotopes. On interior sites, A. ostoyae was found over all hygrotopes, but with lower frequency on the driest sites. The distribution of the two Armillaria species on sites is apparently determined by anoxia associated with periodic soil saturation, by drying of the soil, and by host response limiting spread of pathogenic species, At root contacts between colonized stump roots and crop tree roots, transfer and infection by A. ostoyae occurred more frequently in moist biogeoclimatic zones than dry ones. Lesion size on crop tree roots was related to inoculum volume at some sites and to stump root diameter at others. The percentage of lesions on roots at which crop trees farmed callus was associated with tree bole volume. The results indicate that there will be crop tree mortality following precommercial thinning, especially where inoculum levels are high in the Interior Cedar-Hemlock and Interior Douglas-fir biogeoclimatic zones.
17. Liu, L; Punja, ZK; Rahe, JE. (1997) Altered root exudation and suppression of induced lignification as mechanisms of predisposition by glyphosate of bean roots (Phaseolus vulgaris L.) to colonization by Pythium spp.Physiological and Molecular Plant Pathology 51: 111-127 Altered root exudation and suppression of induced lignification as mechanisms of predisposition by glyphosate of bean roots (Phaseolus vulgaris L.) to colonization by Pythium spp.
Several possible mechanisms for the glyphosate-induced predisposition of bean roots (Phaseolus vulgaris L.) to colonization by Pythium spp. were investigated. Glyphosate at 0.1 and 1.0 mu g ml(-1) from the surfactant-containing formulation Roundup(R) and the non surfactant-containing formulation Accord(R) did not affect mycelial growth of Pythium ultimum and Pythium splvaticum on water agar and cornmeal agar. One microgram per millilitre of glyphosate from both formulations significantly stimulated germination of sporangia of P. ultimum. Germination and growth of germ tubes of P. ultimum were significantly greater in root exudates from bean plants whose primary leaves had been treated with glyphosate than in exudates from non-treated plants. The lignin content of roots was increased significantly when P. ultimum or P. sylvaticum was added to the hydroponic system in which the roots were growing. When glyphosate was applied 2 days prior to Pythium, deposition of lignin in response to Pythium in the bean roots was significantly reduced. These results suggest that predisposition by glyphosate of bean roots to colonization by Pythium spp. may involve changes in root exudates that enhance germination and growth of pathogen propagules, and suppression of a pathogen-induced lignification response by plant roots. (C) 1997 Academic Press Limited.
16. Ng, KK; MacDonald, L; Punja, ZK. (1997) Biological control of rose powdery mildew with the antagonist yeast Tilletiopsis pallescens.Hortscience 32: 262-266 Biological control of rose powdery mildew with the antagonist yeast Tilletiopsis pallescens
Sphaerotheca pannosa var rosae; greenhouse diseases; Rosa species; yeast
The efficacy of Tilletiopsis pallescens Gokhale, a naturally occurring ballistospare-forming yeast isolated from mildew-infected leaves, was evaluated as a biological control agent against rose powdery mildew [Sphaerotheca pannosa (Wallr.:Fr.) Lev. var. rosae Woronichin]. Two trials were conducted on potted rose (Rosa sp.) plants (1-year-old cv. Cardinal Pink) under commercial greenhouse-growing conditions during the summer (June to September) when mildew was most severe. Mildew-infected plants were subjected to one of four treatments: a T. pallescens spore suspension applied three times (3-4 d apart), distilled mater (applied three times), one application of T. pallescens spore suspension or one application of culture filtrate without spores. Two weeks after treatment began, mildew development was evaluated by enumerating conidial density on sampled leaflets. Sporulation was significantly reduced (by 97%-98%) on plants treated with three applications of T. pallescens spore suspension, compared to a 47%-57% reduction on plants treated with three applications of distilled water. There was no significant difference in conidial density between plants treated with one application of T. pallescens spore suspension and plants treated with one application of its culture filtrate, with a 78%-94% reduction in conidia, which was significantly higher than for the water treatment. The mode(s) of action of T. pallescens appears to be eradicant and associated with enzymes or metabolites produced in the culture filtrate. The results from this study demonstrate the potential for biological control of rose powdery mildew under commercial growing conditions in British Columbia.
15.Punja, ZK. (1997) Fungal pathogens of American ginseng (Panax quinquefolium) in British Columbia.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 19: 301-306 Fungal pathogens of American ginseng (Panax quinquefolium) in British Columbia
Major fungal pathogens of American ginseng (Panax quinquefolium) in British Columbia were identified. Isolations were made from roots (235 samples) and leaves (25 samples) collected from ginseng-growing gardens in the Okanagan-Kootenay and Thompson-Cariboo regions during 1992-1996. The fungi recovered from damped-off 1- to 2-year old seedlings and from decayed mature roots were Pythium ultimum (35.6% of total isolates), species of Fusarium (30.5% frequency) (primarily F. solani, F. oxysporum, F. avenaceum, and F. equiseti), Rhizoctonia solani AG 4 (19.6% frequency), and Cylindrocarpon destructans (9.8% frequency). The fungi recovered from diseased leaf and petiole tissues were Phytophthora cactorum, Alternaria panax, A. alternata, and Botrytis cinerea. When tested in vitro, P. ultimum and F. solani were the most pathogenic to seedling roots, followed by R. solani and P. cactorum. On detached leaves, the most pathogenic fungi were P. cactorum, B. cinerea, and A. alternata, followed by A. panax and R. solani. The results of this study show the incidence, distribution, species identity, and pathogenicity of some of the commonly encountered fungi causing diseases of ginseng in British Columbia.
14.Punja, ZK. (1997) Comparative efficacy of bacteria, fungi, and yeasts as biological control agents for diseases of vegetable crops.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 19: 315-323 Comparative efficacy of bacteria, fungi, and yeasts as biological control agents for diseases of vegetable crops
The potential applications for use of bacteria, fungi, and yeasts as biological control agents of fungal diseases on vegetable crops are reviewed. The most extensively studied bacterial organisms, including Pseudomonas spp., Bacillus subtilis, and Enterobacter cloacae, have been reported to reduce many seedling diseases and root rots on several vegetable crop species. The bacteria are easy to grow and can be applied as a seed treatment or as a soil drench, and mechanisms by which they reduce diseases have been studied. The bacteria appear to protect plants against a wide range of pathogens and the potential for commercial utilization is promising. Fungal biocontrol agents, including the extensively studied Gliocladium and Trichoderma species, in addition to other weakly virulent or saprophytic fungi, have been reported to reduce infection or reproduction of many pathogens. Most of the applications of these fungi are for soilborne diseases, with a few reports of reduction of foliar fungal pathogens. The fungi are also relatively easy to grow and formulate for large-scale application and several are now available commercially. The yeast organisms have shown the greatest potential for reducing foliar diseases, especially those diseases caused by mildew fungi. Their use for postharvest disease control has not been investigated extensively and is worthy of research. All three groups of biocontrol agents require development of appropriate formulations to enhance application and survival, and they require additional study to elucidate the various modes of action against the fungal pathogens. The biocompatibility of these agents with fungicides is expected to enhance their efficacy, and there are a few examples available to demonstrate this. There are several areas in which additional research is needed, and these include the application of molecular techniques to characterize and/or modify strains, evaluation of ecological competence of the agents, and methods to enhance survival.
13. Urquhart, EJ; Punja, ZK. (1997) Epiphytic growth and survival of Tilletiopsis pallescens, a potential biological control agent of Sphaerotheca fuliginea, on cucumber leaves.Canadian Journal of Botany-Revue Canadienne de Botanique 75: 892-901 Epiphytic growth and survival of Tilletiopsis pallescens, a potential biological control agent of Sphaerotheca fuliginea, on cucumber leaves
antagonism; biological control; powdery mildew; yeast
The influence of low (70%) and high (90%) relative humidity on epiphytic growth, development, and survival of Tilletiopsis pallescens, a ballistospore-forming yeast-like fungus, on cucumber leaves was investigated. In addition, survival of the fungus in the presence or absence of powdery mildew (Sphaerotheca fuliginea) colonies was determined. Growth and development were visualized by scanning electron microscopy of the leaf surface, and survival was quantified as colony-forming units recovered on a semiselective medium. Development of T. pallescens from blastospores that were applied to healthy leaves at 70% relative humidity was limited to small colonies that grew adjacent to leaf veins 7 days after application. At 90% relative humidity, extensive hyphal networks had developed within 3 days of blastospore germination, and ballistospores were produced within 7 days. Growth and sporulation of T. pallescens were most extensive at the base and on the surface of leaf trichomes. In the presence of S. fuliginea, T. pallescens mycelium developed adjacent to hyphae and conidiophores of the pathogen within 3 days at both 70 and 90% relative humidity. However, at 90% relative humidity, growth of T. pallescens was more extensive and ballistospores were produced within 5 days, and there was visible collapse of mildew hyphae. There was no evidence of penetration of the leaf or mildew hyphae by T. pallescens. Survival of T. pallescens was significantly (P = 0.05) increased at 1 and 5 days postapplication at 70% relative humidity when blastospores were amended with 1% (v/v) canola oil - lecithin. Survival at 90% relative humidity was also significantly increased with canola oil - lecithin and by the presence of S. fuliginea. The addition of liquid paraffin - lecithin or liquid paraffin - Tween had no effect on survival when compared to the control. The results from this study indicate that growth and survival of T. pallescens are enhanced by high relative humidity and by the presence of powdery mildew, and canola oil - lecithin amendment improved survival on the leaf surface at reduced ambient humidity.
12. Urquhart, EJ; Sun, LJ; Punja, ZK. (1997) Identification of species of Tilletiopsis using random amplified polymorphic DNA analysis.Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie 19: 380-389 Identification of species of Tilletiopsis using random amplified polymorphic DNA analysis
biological control; DNA polymorphisms; yeast
Tilletiopsis is a genus of dimorphic yeast-like fungi containing six defined species, several of which have shown marked potential as biological control agents of a number of powdery mildew diseases. Identification of isolates of Tilletiopsis is currently based on colony and spore morphology, patterns of carbon and nitrogen utilization, and differences in guanine c cytosine ratios. Since these identification criteria can be time-consuming the use of RAPD analysis of PCR-generated DNA using random primers was evaluated as a method for species identification. Isolates of three Tilletiopsis species from British Columbia - T. minor, T. pallescens and T. washingtonensis - as well as type cultures of these and two additional species, T. albescens and T. fulvescens, were included in this study. The isolates originated from diverse plant hosts and geographical areas. Two isolates of Pseudozyma flocculosa, a biological control agent of rose powdery mildew, were included as an outgroup. The morphological characteristics of the colonies of these isolates are described. Following amplification of DNA using four RAPD primers, the data were subjected to UPGMA cluster analysis. Each of the known Tilletiopsis species could be clearly separated from one another, forming five distinct groups. T. albescens and T. pallescens were grouped closely together, while T. minor and T. washingtonensis formed separate clusters. T.fulvescens grouped closest to T. washingtonensis. The greatest intraspecific variation was observed among isolates of T. washingtonensis, followed by T. minor. Three isolates of Tilletiopsis sp. that were morphologically different from the known species, and which formed a separate cluster, may represent an undescribed species. Isolates of P. flocculosa were readily distinguished from the Tilletiopsis species (similarity coefficient of 0.6) and also formed a distinct group. An isolate tentatively identified as a Tilletiopsis sp. was found to be a Pseudozyma sp. by RAPD analysis. The RAPD-PCR technique resolved the identity of several anomalous isolates of Tilletiopsis that were difficult to identify using current morphological criteria. In addition, unique DNA fingerprints were generated for some isolates that may be useful for monitoring the distribution and spread of specific isolates. The RAPD results indicated that a high degree of genetic variation exists within the genus Tilletiopsis.
11. Chen, G; Zhang, Y; Li, J; Dunphy, GB; Punja, ZK; Webster, JM. (1996) Chitinase activity of Xenorhabdus and Photorhabdus species, bacterial associates of entomopathogenic nematodes.Journal of Invertebrate Pathology 68: 101-108 Chitinase activity of Xenorhabdus and Photorhabdus species, bacterial associates of entomopathogenic nematodes
endochitinase; exochitinase; Xenorhabdus nematophilus; X-bovienii; Photorhabdus luminescens
Xenorhabdus nematophilus (three strains),Xenorhabdus bovienii (one strain), and Photorhabdus luminescens (one strain) showed both exo- and endochitinase activity using p-nitrophenyl-N-acetyl-beta-D-glucosaminide and p-nitrophenyl-beta-D-N,N',N ''-triacetylchitotriose, respectively, as substrates. One to three bands were detected on PAGE gel with glycol chitin after electrophoresis. Variation in exo- and endochitinase activity among different species and strains was detected with the strongest activity in X. nematophilus and the weakest in P. luminescens. The primary form of X. bovienii had significantly greater chitinase activity than the secondary form, whereas their growth rate and total protein released into culture medium were similar The partially purified chitinase of X. bovienii showed significant antimycotic activity against conidial germination and germ tube elongation of Botrytis cinerea. (C) 1996 Academic Press, Inc.
10. Chycoski, CI; Punja, ZK. (1996) Characteristics of populations of Phytophthora infestans from potato in British Columbia and other regions of Canada during 1993 to 1995.Plant Disease 80: 579-589 Characteristics of populations of Phytophthora infestans from potato in British Columbia and other regions of Canada during 1993 to 1995
epidemiology; fungicide resistance; late blight; sexual recombination; Solanum tuberosum
The incidence and distribution of A1 and A2 mating types of Phytophthora infestans in Canada and of metalaxyl-sensitive (MS) and -insensitive (MI) strains were monitored during 1993, 1994, and 1995. Diseased leaves from about 1,600 plants were collected from 36 cultivars in 168 potato fields from five provinces at various times (June to September) during the growing season. The most extensive sampling (88% of total) was conducted in British Columbia (B.C.). About 500 isolates of P. infestans were characterized from over 1,000 collected. In 1993, the A1 mating type was found in all provinces sampled, while western Canada (B.C.) was the only region in which the A2 mating type was detected. In 1994, the A2 mating type was also found in eastern Canada (New Brunswick [N.B.]). In B.C., 23 fields sampled in 1993 had both A1 and A2 mating types, while in N.B. in 1994, both mating types originated from the same plant in seven different fields. When lesions from two of these fields were examined by microscope, sporangia of P. infestans and a single oospore were seen in leaf tissues, demonstrating the potential for oospore production in naturally infected leaves in western and eastern Canada if both mating types are present. Metalaxyl sensitivity tests measuring relative growth of isolates with metalaxyl at 0 and 50 mu g/ml revealed that all isolates collected in 1993 from Alberta, Manitoba, N.B., Nova Scotia, Ontario, Prince Edward Island, and Quebec were MS. In B.C., isolates showed a range of variation in growth with and without metalaxyl, and 76% of the isolates were MI. There was no correlation between recovery of these MI strains and use or nonuse of metalaxyl during the same growing season. Both MS and MI strains were present together in six fields. A low frequency of MS strains of both mating types was always recovered in B.C. When the frequency of occurrence of MS and MI strains throughout the growing season was examined, a high proportion of isolates collected early (June to July) from B.C. and N.B. in 1993 and 1994 was found to be MI. At the end of the season (September), MI isolates also occurred at a higher frequency in both locations and years. When the frequency of mating types was examined, the A2 type occurred at a higher proportion throughout the season in B.C. during 1993 acid 1994. However, in 1995, the A2 mating type was rarely recovered and the A1 type predominated. In N.B. in 1994, the A1 mating type was recovered at a higher frequency than the A2 type in July, but most collections later were of mixed mating type. When the isolates were grouped into A1, A2, MS, and MI categories and growth rates were compared, isolates from populations in B.C. and N.B. in 1994 of A2 mating type grew significantly faster than A1; isolates that were MI from populations in B.C. in 1993 grew significantly faster than MS isolates. However, no differences could be detected in the extent of leaf colonization or sporulation between isolates representing A1, A2, MS, or MI. A preliminary survival study showed that P. infestans could overwinter under B.C. conditions on artificially inoculated tubers. These results illustrate the dynamic nature of populations of P. infestans within and between growing seasons in western and eastern Canada, and demonstrate higher variation in the population in B.C.
9. Descalzo, RC; Punja, ZK; Levesque, CA; Rahe, JE. (1996) Assessment of host specificity among different species of glyphosate synergistic Pythium.Mycological Research 100: 1445-1453 Assessment of host specificity among different species of glyphosate synergistic Pythium
A total of 39 Pythium isolates representing 14 species of Pythium was assessed for host specificity as glyphosate synergists. This was done using three groups of Pythium isolates from roots of glyphosate-treated bean (PBI), wheat (PWI), and isolates from various glyphosate-untreated hosts. PBI consisted of 15 isolates and included P. ultimum, P. sylvaticum, P. coloratum, P. irregulare and P. group 'HS'. PWI consisted of 14 isolates representing the first four of these species. Pythium from glyphosate-untreated hosts included single isolates representing P. aphanidermatum, P. spinosum, P. paroecandrum, P. hypogynum, P. splendens, P, sulcatum, P. vanterpooli, P. acanthicum, P. arrhenomanes and P. coloratum. The glyphosate synergistic potential of the Pythium isolates was determined by treating a-week-old seedlings growing in soil infested with individual isolates of Pythium with different doses of glyphosate. LD(50) values associated with each isolate were estimated by logistic regression analysis of plant mortalities recorded 4 wk after treatment with glyphosate, and compared with LD(50) values for plants grown in the absence of Pythium. Host specificity was assessed by comparing the glyphosate synergistic potential of PBI on bean and PWI on wheat seedlings, with the potential of these same isolates of PBI on wheat and PWI on bean seedlings. Glyphosate synergistic potential of PBI was also estimated on sunflower and pepper, to test whether PBI were capable of glyphosate synergistic interaction (GSI) on other unrelated dicot yledonous species. Pythium isolates from glyphosate-untreated hosts were tested on bean to determine if Pythium species not represented in the PBI and PWI groups were capable of GSI. The glyphosate synergistic potentials of the PBI and PWI on wheat seedlings were low and inconsistent compared to those observed on dicot plants. AU PWI and 12 of the 15 PBI were glyphosate synergists on beans, and all the PBI were glyphosate synergistic on sunflower and pepper seedlings. All Pythium isolates from glyphosate-untreated sources tested were also glyphosate synergists on bean seedlings. These various tests of glyphosate synergistic potential of Pythium isolates from diverse sources on various plant species revealed no evidence of host specificity among the isolates and species tested.
8. Descalzo, RC; Punja, ZK; Levesque, CA; Rahe, JE. (1996) Identification and role of Pythium species as glyphosate synergists on bean (Phaseolus vulgaris) grown in different soils.Mycological Research 100: 971-978 Identification and role of Pythium species as glyphosate synergists on bean (Phaseolus vulgaris) grown in different soils
Five Pythium species, P. ultimum, P. sylvaticum, P. irregulare, P. coloratum, and Pythium 'HS' group, were identified using morphological characteristics out of 65 isolates obtained from roots of glyphosate-treated bean seedlings grown in five different soils. Various genotypes within the Pythium species were determined from RFLP patterns of total DNA. There were six RFLP types represented in P. sylvaticum, three in P. ultimum, and two each for P. irregulare, P. coloratum and P. 'HS' group. The potential of a representative isolate from each RFLP group to enhance the herbicidal action of glyphosate was quantified by estimating glyphosate LD,, values on bean seedlings growing in sterilized soils amended with each isolate separately. The LD,, values were computed by logistic regression using plant mortality data gathered 4 wk after treatment of 2-wk-old seedlings with different doses of glyphosate. Twelve of the 15 isolates of Pythium tested were glyphosate synergists on beans. The efficacy of the different isolates as glyphosate synergists varied both between species and among different RFLP types within the same species. The pathogenicity of the representative isolates to beans without glyphosate treatment was also determined. All Pythium species tested were pathogenic to varying degrees on germinating bean seeds and on 2-wk-old bean seedlings. The results indicate that several Pythium species can function as glyphosate synergists and that five different soils all yielded glyphosate synergistic Pythium isolates.
7. Gilbert, MO; Zhang, YY; Punja, ZK. (1996) Introduction and expression of chitinase encoding genes in carrot following Agrobacterium-mediated transformation.In Vitro Cellular & Developmental Biology-Plant 32: 171-178 Introduction and expression of chitinase encoding genes in carrot following Agrobacterium-mediated transformation
Daucus carota; genetic transformation; hydrolytic enzyme; tissue culture
Transgenic carrot (Daucus carota L.) plants were obtained following transformation with disarmed Agrobacterium tumefaciens strains MOG 101 (octopine type) and EHA 105 (leucinopine type). The strains harbored a binary plasmid that contained either an acidic chitinase gene from petunia (pMOG196), or a basic chitinase gene from tobacco (pMOG198) or bean (pGA492-CHN), transcriptionally fused to the constitutive 35S promoter from cauliflower mosaic virus. Each construct also contained the neomycin phosphotransferase gene (npt II) conferring kanamycin resistance. The influence of the Agrobacterium strains, plasmids, carrot cultivars, ages of explant, and cocultivation times were evaluated. The frequency of transformation (i.e., development of somatic embryos on Murashige and Skoog medium with 4.5 mu M 2,4-D and 100 mg/liter of kanamycin) was highest (12.1%) with 2-5-wk-old epicotyl segments of the cultivar Nanco cocultivated for 2-3 d with the supervirulent A. tumefaciens strain EHA 105. Transformation efficiency was not influenced by explant age or binary plasmid, but was significantly influenced by cocultivation period, Agrobacterium strain, and carrot cultivar. Transformed embryogenic calluses from five independent transformation events (out of about 15) were multiplied in suspension cultures (liquid MS medium with 0.5 mu M 2,4-D and 50 mg/liter of kanamycin). Within 4-6 wk following plating of cell suspensions onto MS medium without growth regulators or kanamycin, plantlets developed. Excised shoots were rooted on MS medium with kanamycin (50 mg/liter) before transferring to soil. Transformation was confirmed in the five independent lines by polymerase chain reaction (PCR)-amplification of the npt II coding region and Southern hybridization analysis using an 800 bp Digoxigenin-UTP labeled probe specific for the npt II gene. One to four hybridizing bands were seen in the transgenic plants, indicating the integration of one to four T-DNA copies in the carrot genome. Transgenic plants of cultivars Golden State, Danvers Half Long, and Nanco, which contained either the acidic or basic chitinase genes were obtained. Expression of the introduced basic chitinase genes was confirmed by protein dot-blot analysis and immunostaining with anti-bean and anti-tobacco antibodies.
6. McCullagh, M; Utkhede, R; Menzies, JG; Punja, ZK; Paulitz, TC. (1996) Evaluation of plant growth-promoting rhizobacteria for biological control of Pythium root rot of cucumbers grown in rockwool and effects on yield.European Journal of Plant Pathology 102: 747-755 Evaluation of plant growth-promoting rhizobacteria for biological control of Pythium root rot of cucumbers grown in rockwool and effects on yield
Pseudomonas fluorescens; Pseudomonas corrugata; Serratia plymuthica; Pythium aphanidermatum; PGPR; biological control
Three strains of Pseudomonas fluorescens (63-49, 63-28, and 15), one strain of Pseudomonas corrugata (13) and one strain of Serratia plymuthica (R1GC4) were tested on rockwool-grown cucumbers for their ability to reduce Pythium root-rot caused by Pythium aphanidermatum. These strains were previously selected for biocontrol ability from collections of >4000 bacteria. Strains 63-49 and 63-28 were tested on cucumber plants grown in rockwool in two replicated Pythium-inoculated trials conducted in British Columbia (B.C). Another inoculated, replicated trial was conducted in Quebec with all five strains. Cucumber yields (fruit number and weight) were measured over a ten-week harvest period. Strain 63-49 caused an early promotion of plant growth and increased cucumber yields at early harvests. No measurable effect of Pythium inoculation on disease development was observed in the Quebec trial, due to unfavourable cool weather. However, 63-49 significantly increased the total number of cucumbers (12%) and cucumber weight (18%), compared to the non-treated control. Strains 13, 15 and R1GC4 slightly increased the cumulative cucumber yields, but strain 63-28 had no effect. In the B.C. trial, inoculation with P. aphanidermatum reduced the number and weight of cucumbers by 27%. Treatments of Pythium-inoculated cucumbers with 63-49 significantly increased fruit number and weight by 18%, compared to the Pythium-inoculated control. Strain 63-28 increased the cumulative number of cucumbers over time, compared to the Pythium-inoculated control, but the increase was less than with 63-49. The use of Pseudomonas spp. in rockwool-grown cucumbers can increase yields, both in the presence and absence of Pythium root rot, and with variable seasonal conditions and disease pressures.
5. Oleskevich, C; Shamoun, SF; Punja, ZK. (1996) The biology of Canadian weeds .105. Rubus strigosus Michx, Rubus parviflorus Nutt, and Rubus spectabilis Pursh.Canadian Journal of Plant Science 76: 187-201 The biology of Canadian weeds .105. Rubus strigosus Michx, Rubus parviflorus Nutt, and Rubus spectabilis Pursh
Rubus strigosus; Rubus idaeus; Rubus parviflorus; Rubus spectabilis; wild red raspberry; thimbleberry; salmonberry; forest weed biology; competition; distribution
Wild raspberry (Rubus strigosus Michx.), thimbleberry (Rubus parviflorus Nutt.), and salmonberry (Rubus spectabilis Pursh) are native perennial deciduous shrubs that rapidly invade disturbed areas. Through prolific vegetative growth, these shrubs form dense, multilayered, and monospecific stands and form extensive clonal colonies. They create habitat and supply food sources for a variety of forest fauna and are important in nutrient cycling and reducing soil erosion, These Rubus shrubs may effectively outcompete economically valuable regenerating conifers. A review of chemical, manual, and biological control methods is presented. Reproductive biology, growth and development, and population dynamics are discussed in detail.
4.Punja, ZK; Damiani, A. (1996) Comparative growth, morphology, and physiology of three Sclerotium species.Mycologia 88: 694-706 Comparative growth, morphology, and physiology of three Sclerotium species
basidiomycetes; histochemistry; oxalic acid; Sclerotium coffeicola; Sclerotium delphinii; Sclerotium rolfsii
Isolates of S. coffeicola, S. delphinii and S. rolfsii from diverse geographical areas were compared for differences in morphology (colony characteristics and sclerotial formation), growth response to different temperatures and media (radial growth, dry weight and sclerotial production), and ability to produce oxalic acid and pectinase enzymes. The best medium for colony growth of S. coffeicola and S. rolfsii was V8 agar and for S. delphinii it was PDA. Greatest sclerotial production in all three species was achieved on PDA at incubation temperatures of 20, 35, and 20 C, respectively. Under these conditions, the total sclerotia produced in was 7, 1043, and 47 per petri dish and the average sclerotial diam was 5.1, 0.6, and 3.5 mm for S. coffeicola, S. rolfsii and S. delphinii, respectively. The species all formed clamp connections on leading hyphae, and the number of nuclei in hyphal cells ranged fro 1 to 6, with an average of 2 per cell for each species. The basidial stage was induced only in S. rolfsii (Athelia rolfsii) on PDA containing charcoal and was not observed in the other two species. The sclerotia of all species were comprised of three layers: an outer rind, a middle cortex and an inner medulla. The cortical layer was 15-20 cell layers thick in S. coffeicola compared to 4-10 layers in the other two species. Histochemical staining of sclerotia showed that the concentration of carbohydrates and metachromatic granules was greatest in the medulla, followed by the cortex, and was mostly in the cytoplasm. Proteins were also distributed in both the cortex and medulla of S. rolfsii and S. delphinii, but were more concentrated in the medulla of S. coffeicola, and occurred in both the walls and cytoplasm. Phenolic compounds were observed in the rind cells of all sclerotia, with S. coffeicola containing the least amount, and were localized to the walls. All three species produced oxalic acid, pectinase and polyphenoloxidase in culture, with highest production in S. rolfsii, followed by S. delphinii and S. coffeicola. The results from this study show a close similarity among the three Sclerotium species for the parameters measured. The species can be differentiated by colony characteristics, such as morphology and size of sclerotia, by differences in histochemical staining and composition of sclerotia, and by growth responses to temperature.
3.Punja, ZK; Raharjo, SHT. (1996) Response of transgenic cucumber and carrot plants expressing different chitinase enzymes to inoculation with fungal pathogens.Plant Disease 80: 999-1005 Response of transgenic cucumber and carrot plants expressing different chitinase enzymes to inoculation with fungal pathogens
antifungal; hydrolytic enzymes
Three lines of cucumber cv. Endeavor, each transformed with a chitinase gene originating from petunia (acidic), tobacco (basic), or bean (basic) using Agrobacterium tumefaciens, were compared with nontransgenic plants for response to inoculation with Alternaria cucumerina, Botrytis cinerea, Colletotrichum lagenarium, and Rhizoctonia solani. In both growth chamber studies using whole plants and in vitro inoculations conducted with detached leaves, no differences in disease development (rate and final levels) were detected between transgenic and nontransgenic plants. Carrot cvs. Nanco and Golden State transformed with two chitinase genes (from petunia and tobacco) were also evaluated for response to inoculation with the pathogens Alternaria radicini, B. cinerea, R. solani, Sclerotium rolfsii, and Thielaviopsis basicola. A detached petiole inoculation method was used to compare nontransgenic and transgenic plants. The rate and final extent of lesion development after 7 days were significantly (P = 0.01) lower in the transgenic plants expressing the tobacco (basic) chitinase gene upon inoculation with B. cinerea, R. solani, and S. rolfsii, but not in plants expressing the petunia (acidic) chitinase gene. There were no detectable differences with A. radicini or T. basicola in either group of transgenic plants. These results demonstrate the in planta efficacy of a basic chitinase protein in providing enhanced tolerance of carrot to three fungal pathogens; however, the efficacy of chitinase gene transformation as a strategy for enhancing disease tolerance in plants can be influenced by the plant species used, the type of chitinase protein expressed, and the characteristics of the fungal pathogen.
2. Raharjo, SHT; Hernandez, MO; Zhang, YY; Punja, ZK. (1996) Transformation of pickling cucumber with chitinase-encoding genes using Agrobacterium tumefaciens.Plant Cell Reports 15: 591-596 Transformation of pickling cucumber with chitinase-encoding genes using Agrobacterium tumefaciens
Transformation of cucumber cv. Endeavor was attempted using three Agrobacterium tumefaciens strains (a supervirulent leucinopine type, an octopine type and a nopaline type), each harbouring one of three binary vectors which contained an acidic chitinase gene from petunia, and basic chitinase genes from tobacco and bean, respectively, driven by the CaMV 35S promoter. Petiole explants were inoculated with a bacterial suspension (10(8) cells . ml(-1)), cocultivated for 48-96 h and placed on Murashige and Skoog (MS) medium with 5.0 mu M each of 2,4-D and BA, 50 mg . l(-1) kanamycin and 500 mg . l(-1) carbenicillin. The frequency of embryogenic callus formation ranged from 0 to 12%, depending on strains/vectors used and length of cocultivation, with the highest being obtained using the leucinopine strain with petunia acidic chitinase gene. The kanamycin-resistant embryogenic calli were used to initiate suspension cultures (in liquid MS medium with 1.0/1.0 mu M 2,4-D/BA, 50 mg . l(-1) kanamycin) for multiplication of embryogenic cell aggregates. Upon plating of cell aggregates onto solid MS medium with 1.0/1.0 mu M NAA/BA and 50 mg . l(-1) kanamycin, calli continued to grow and later differentiated into plantlets. Transformation by the leucinopine strain and all three vectors was confirmed by PCR amplification of the NPT II gene in transgenic calli and plants, in addition to Southern analysis. Expression of the acidic chitinase gene (from petunia) and both basic chitinase genes (from tobacco and bean) in different transgenic cucumber lines was confirmed by Western analyses.
1. Zhang, YY; Haunerland, NH; Punja, ZK. (1996) Chitinase profiles in mature carrot (Daucus carota) roots and purification and characterization of a novel isoform.Physiologia Plantarum 96: 130-138 Chitinase profiles in mature carrot (Daucus carota) roots and purification and characterization of a novel isoform
antifungal; carrot; chitinase; Daucus carota; isoforms; pathogenesis-related protein
The profile of chitinases (EC 220.127.116.11) in mature carrot (Daucus carota L. cv. Eagle) roots was studied. Multiple chitinase bands (8-10) were observed in native and sodium dodecylsulfate-denaturing polyacrylamide gels. The molecular masses of these chitinases were estimated to be from 20 000 to 40 000. One major chitinase was purified and found to be an acidic protein with pI at 4.3 and a molecular mass of 39 500. The optimum pH for enzymatic activity was around 5 and the optimum temperature was 25 degrees C. The enzyme was stable at pHs below 8 and temperatures below 60 degrees C. The protein did not have a chitin-binding domain, but showed some similarity to the amino acid composition of tobacco class I chitinase. The N-terminal amino acid sequence did not resemble any of the described classes of chitinases. This chitinase did not possess lysozyme activity and showed antifungal activity when tested against Trichoderma sp.