H. W. (Bud) Platt

Phylogenetics and taxonomy of the fungal vascular wilt pathogen Verticillium, with the descriptions of five new species.

Knowledge of pathogen biology and genetic diversity is a cornerstone of effective disease management, and accurate identification of the pathogen is a foundation of pathogen biology. Species names provide an ideal framework for storage and retrieval of relevant information, a system that is contingent on a clear understanding of species boundaries and consistent species identification. Verticillium, a genus of ascomycete fungi, contains important plant pathogens whose species boundaries have been ill defined. Using phylogenetic analyses, morphological investigations and comparisons to herbarium material and the literature, we established a taxonomic framework for Verticillium comprising ten species, five of which are new to science. We used a collection of 74 isolates representing much of the diversity of Verticillium, and phylogenetic analyses based on the ribosomal internal transcribed spacer region (ITS), partial sequences of the protein coding genes actin (ACT), elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD) and tryptophan synthase (TS). Combined analyses of the ACT, EF, GPD and TS datasets recognized two major groups within Verticillium, Clade Flavexudans and Clade Flavnonexudans, reflecting the respective production and absence of yellow hyphal pigments. Clade Flavexudans comprised V. albo-atrum and V. tricorpus as well as the new species V. zaregamsianum, V. isaacii and V. klebahnii, of which the latter two were morphologically indistinguishable from V. tricorpus but may differ in pathogenicity. Clade Flavnonexudans comprised V. nubilum, V. dahliae and V. longisporum, as well as the two new species V. alfalfae and V. nonalfalfae, which resembled the distantly related V. albo-atrum in morphology. Apart from the diploid hybrid V. longisporum, each of the ten species corresponded to a single clade in the phylogenetic tree comprising just one ex-type strain, thereby establishing a direct link to a name tied to a herbarium specimen. A morphology-based key is provided for identification to species or species groups.

Relationship between resistance to late blight in potato foliage and tubers of cultivars and breeding selections with different resistance levels

Due to changes in the pathogen population, late blight of potatoes caused byPhytophthora infestans has become a more difficult disease to manage and there is a need for new potato cultivars with greater and more stable disease resistance. Field studies are required to identify and characterize host resistance to late blight in both foliage and tubers but, as epidemics vary from year to year, combining multi-year results can be difficult. Residual maximum likelihood (REML) was successfully used to analyze data which demonstrated good correlations between foliar and tuber responses to late blight (US-1 genotype). In addition, studies on post-harvest inoculated tubers provided more reliable disease responses than field studies, and the disease resistance with this method correlated well with foliar disease responses. This tuber test would be a useful method of pre-screening selections for disease response prior to testing in the field.

Characterization of changes in populations of Phytophthora infestans in Canada using mating type and metalaxyl sensitivity markers

Isolates of Phytophthora infestans were obtained from individual fields of potato and tomato and from potato storages across Canada in 1994 (142 samples yielding 555 isolates), 1995 (185 samples yielding 914 isolates), and 1996 (300 samples yielding 1013 isolates). Characterization of these isolates according to mating type and sensitivity to metalaxyl revealed the changing nature of P. infestans populations in Canada. In 1994, isolates of the traditional Al, metalaxyl-sensitive (MS) phenotype were common, but by 1996, they were no longer recovered from any tissue samples. Isolates of the A2 mating type, which were predominantly insensitive to metalaxyl (MI), were prevalent in 1996, except in British Columbia (B.C.). In B.C., Al isolates highly insensitive to metalaxyl predominated. Isolates of the Al, MI phenotype were also found in a sample of tomatoes from Ontario in 1996. New populations composed of the A2 mating type were predominantly insensitive to metalaxyl, but they showed an intermediate respons...

Comparison of polymerase chain reaction based methods with plating on media to detect and identify verticillium wilt pathogens of potato

A polymerase chain reaction (PCR) based assay that utilizes specific primers was compared with assays involving plating on selective agar media for its ability to detect and identify verticillium wilt pathogens of potato following artificial inoculations under field conditions. The common Verticillium species were detected in stem tissues and soil using both methods. The PCR assay was faster and efficient, requiring only 2 days for positive species identification, whereas the media-plating method required more than 4 weeks. Verticillium albo-atrum strain 1 (VA1) was the most frequently recovered species in soil and stems that were assayed using PCR and plating methods. However, the weakly pathogenic V. albo-atrum strain 2 (VA2) could not be differentiated from the more aggressive VA1 strain using the plating on media method. Verticillium tricorpus was detected at very low levels in plots that had been inoculated with this species, whereas VA2 was not detected in assayed potato stems using either method. H...

Response of solanaceous cultivated plants and weed species to inoculation with A1 or A2 mating type strains of Phytophthora infestans

Hypersensitive response symptoms of late blight were induced when sporangia of Phytophthora infestans were inoculated on discs of excised leaf tissues of black nightshade, eggplant, green pepper, and tobacco. No symptoms due to disease or hypersensitive response were observed in stem piece tissues of these plant species, suggesting that these four are nonhosts for late blight. The Al (US-1), the A2 (US-8), and the more recent Al mating type (US-ll) strains infected both leaf and stem tissues of climbing and hairy nightshade, potato, and tomato but only leaf tissues of petunia. This is the first report of the new strains of P. infestans causing late blight symptoms on climbing nightshade and petunia in Canada. Based on infection levels, infection rates, and spore production, the US-8 and US-ll genotypes were similar and both were more aggressive than the US-1 genotype. Disease severity and sporulation were greater on potato inoculated with US-1 than for the other host plants and were the least on petunia. ...

Interspecific crossability and cytogenetic analysis of sexual progenies of Mexican wild diploid 1EBN speciesSolanum pinnatisectum andS. cardiophyllum

Mexican wild diploid species,Solanum pinnatisectum (S. pnt) (2n=2x=24, 1EBN: endosperm balance number) is a useful germplasm source of late blight and Colorado potato beetle (CPB) resistance in potato improvement. However, it is very difficult to cross this species with other 1EBNSolanum species. Sexual hybrids among three accessions ofS. pnt and two accessions ofS. cardiophyllum (S. cph) (2n=2x=24, 1EBN) were studied. There were large differences in the cross-compatibility among the genotypes and accessions from these two 2x-1EBN species. Interspecific incompatibility existed in the crosses betweenS. pnt andS. cph, in whichS. cph functioned only as the male parent. The crosses with accessions PI 275236 ofS. pnt produced 35 hybrids following the conventional propagation procedures. Seven hybrids were obtained with theS. pnt accession PI 275233 with the aid of embryo rescue, while hybridization involving accession PI 253214 resulted in no fruit. The cytogenetic analysis indicated that all of the hybrids derived from crosses of accession PI 275233 ofS. pnt withS. cph had 2n = 24 chromosomes and were pollen fertile. Crosses involving accession PI 275236 ofS. pnt produced both 2x (2n=24) and 3x (2n=36) hybrids. Meiotic analysis at metaphase I of pollen mother cells (PMCs) showed a fairly high rate of chromosome pairing that averaged between 10.10 and 11.10 bivalents per cell in most of the 2x hybrids, indicatingS. pnt andS. cph have the similar genome. A high frequency of trivalents and quadrivalents were observed in 3x hybrids derived fromS. pnt 2 ×S. cph 1, indicating homology exists among these chromosomes. These results suggest that specific gene(s) and not either differences between genomes or EBN controlled interspecific crossability and embryo development. The disease and insect tests on the 2x and 3x hybrids revealed that all have high levels of resistance to both late blight and CPB. Thus it could appear that selection of genotypes is a key for successful interspecific hybridization when using Mexican wild diploid species as a source of economic important traits.ResumenLa especie silvestre diploide mexicana,Solanum pinnatisectum (S. pnt) (2n=2x=24, 1EBN: número de balance del endospermo) es una fuente útil de germoplasma para resistencia al tizón tardío y al escarabajo (Colorado potato beetle: CPB) para el mejoramiento de la papa. Sin embargo, es muy difícil cruzar esta especie con otras especies deSolanum. Se estudiaron los híbridos sexuales entre tres accesiones deS. pnt y dos accesiones deS. cardiophyllum (S.cph) (2n=2x=24, 1 EBN). Hubo grandes diferencias en la compatibilidad de cruzamiento entre los genotipos y accesiones de estas dos especies 2x-1 EBN. Existió incompatibilidad en los cruzamientos entreS. pnt yS. cph, en los cualesS. cph funcionó solamente como progenitor masculino. Los cruzamientos con accesiones PI 275236 deS. pnt produjeron 35 híbridos después de los procedimientos convencionales de propagación. Se obtuvieron siete hibridos con la accesión PI 275233 deS. pnt con la ayuda de rescate de embrión. Mientras que la hibridación que involucró la accesión PI 253214 no produjo frutos. El análisis citogenético indicó que todos los híbridos derivados de cruzamientos de la accesión PI 275233 deS. pnt conS. cph tenían 2n=24 cromosomas y polen fértil. Los cruzamientos involucrando la accesión PI 275236 deS. pnt produjeron los híbridos 2x (2n=24) y 3x (2n=36). El análisis meiótico en la metafase I de las células madre del polen (PMC’s) mostraron una regular tasa alta de apareamiento de cromosomas, cuyo promedio estuvo entre 10.10 y 11.10 bivalentes por célula en la mayoría de los híbridos 2x, indicando queS. pnt yS. cph tienen genomas similares. Se observó una alta frecuencia de trivalentes y cuatrivalentes en hibridos 3x derivados deS. pnt 2xS. cph lx, indicando que existe homología entre estos cromosomas. Estos resultados sugieren que el o los genes específicos y no las diferencias entre genomas o la EBN controlaron el cruzamiento interespecífico y el desarrollo del embrión. Las pruebas para la enfermedad y el insecto en los híbridos 2x y 3x revelaron que todos ellos tienen niveles altos de resistencia a ambos, el tizón tardío y el CPB. Así, puede parecer que la selección de genotipos es la clave para una hibridación interespecífica exitosa cuando se utilizan especies mexicanas silvestres diploides como fuente de caracteristicas económicas importantes.

Changes in mating types, resistance to metalaxyl, and Gpi-allozyme genotypes of Phytophthora infestans in Canadian provinces from 1996 to 1998.

Variations within Phytophthora infestans populations in Canada, in terms of mating type, metalaxyl resistance, and glucose-6-phosphate isomerase allozyme genotypes, are presented for the period 1996-1998. During 1996, only the A2 mating type was found in most Canadian provinces. However, both Al and A2 mating types were found in Ontario and British Columbia (BC). In 1997, samples of late blight-infected plants were received from five provinces only. The A2 mating type was isolated from samples from Prince Edward Island, New Brunswick, and Quebec (QC), while both Al and A2 mating type isolates were found in BC and Manitoba. In 1998, late blight was confirmed in all provinces, and all samples were of the A2 mating type, except those from BC and QC, which had both mating types. Changes in levels of metalaxyl-sensitive (MS), metalaxyl-intermediate (MI), and metalaxyl-resistant (MR) strains of P. infestans in Canada were also observed. Within the A2 mating type, the frequency of MS isolates increased in most provinces, except in BC, where this rate is decreasing. For the Al mating type, there is an increase in MR individuals that have been displacing the MI strains from 1996 to 1998. A high variability was recorded either among Canadian provinces or from year to year inside each province in terms of mating types, Gpi-allozyme genotypes, and especially metalaxyl response. In general, during 1996-1998, samples from four Canadian provinces have included both Al and A2 mating types. In addition, beside US-8 and US-11, isolates with characteristics of US-7. US-10, and US-14 were found during 1998, with different levels of resistance to metalaxyl.

Changes in metalaxyl resistance among glucose phosphate isomerase genotypes of Phytophthora infestans in Canada during 1997 and 1998.

A total of 930 isolates ofPhytophthora infestans collected during 1997 and 1998 from commercial potato and tomato fields, home gardens, and potato storages in Canada was analyzed for glucose phosphate-isomerase (Gpi) genotype, mating type, and resistance to metalaxyl. Both mating types and diversity in resistance to metalaxyl were detected within and amongGpi-genotypes. Pathogen populations were dominated by the 100:111:122Gpi-genotype (characteristic of US-8) in all the provinces where late blight was detected, except in 1997 in British Columbia (BC) where the 100:100:111Gpi-genotype (characteristic of US-11) predominated.Gpi-genotypes 100:111, 111:122, and 100:122 (characteristic of US-7, US-10 and US-14/US-17, respectively) were less common. Nearly all of the isolates with the 100:100:111Gpi-genotype were the A1 mating type, except for a few collected in BC in 1997. A majority of isolates with the 100:111:122Gpi-genotype were A2 except for a few found in Québec in 1998. Based on colony growth on media amended with 1,10, or 100 μg/ml metalaxyl, the 100:100:111Gpi-genotype isolates had higher levels of resistance to the fungicide in 1998 compared to 1997. In 1998, level of metalaxyl resistance was higher within the 100:100:111Gpi-genotype whereas it was lower within the 100:111: 122Gpi-genotype, when compared to 1997. BC was the only Canadian province where an increase in metalaxyl resistance within the 100:111:122Gpi-genotype was recorded between 1997 and 1998. Isolates from leaves had higher metalaxyl resistance levels than stem or tuber isolates, regardless of genotype.

VIABILITY OF OOSPORES OF PHYTOPHTHORA INFESTANS UNDER FIELD CONDITIONS IN NORTHEASTERN NORTH AMERICA

Studies were conducted in potato fields on Prince Edward Island (PE) and New Brunswick (NB) to determine the overwintering viability, germination, and infectivity of oospores of Phytophthora infestans. Oospore viability was determined using plasmolysis (4 M NaCl) and tetrazolium bromide (MTT) tests. After seven months exposure to winter conditions in potato fields, the viability of oospores buried on PE and NB ranged from 2.5 to 13% and from 1 to 28%, respectively, with the plasmolysis test and from 13 to 44% and from 2.5 to 40%, respectively, with the MTT test. In other studies on PE, oospore viability ranged from 5 to 15% 12 months after burial. In laboratory studies, oospores exposed to temperatures of -20 and -50°C for up to 90 days had viabilities greater than 50%, whereas exposure to 0, 4, or 15°C resulted in average viabilities from 23 to 36%. The lowest viability, 22%, was recorded at the highest temperature tested, 36°C. Oospore germination was low, ranging from 6 to 19%. However, this level was ...

Genetic Composition of Phytophthora infestans in Canada Reveals Migration and Increased Diversity

A dramatic increase in the incidence of late blight and changes within populations of Phytophthora infestans have been observed in various regions of Canada. In this study, the occurrence of several new genotypes of the pathogen was documented with associated phenotypes that dominated pathogen populations. Genotype US-23, previously detected only among isolates from the United States, dominated in the western Canadian provinces of British Columbia, Alberta (AB), Saskatchewan, and Manitoba (MB). Although isolates of US-23 infect both potato and tomato, these isolates were the only genotype recovered from commercial garden centers in Canada. Isolates of genotype US-8, previously dominant throughout Canada, represented the only genotype detected from the eastern Canadian provinces of New Brunswick and Prince Edward Island. Isolates of other genotypes detected in Canada included US-11 in AB, US-24 in MB, and US-22 in Ontario (ON). An additional genotype was detected in ON which appears to be a derivative of US-22 that may have arisen through sexual reproduction. However, evidence of clonal reproduction dominated among the isolates collected, and opportunities for sexual reproduction were probably limited because of a surprising geographic separation of the A1 and A2 mating types in Canada. Sensitivity of the US-22, US-23, and US-24 isolates to the fungicide metalaxyl, movement of potato seed and transplants, and weather conditions may have contributed to reduced opportunities for contact between the mating types in fields in Canada. All P. infestans isolates were readily distinguished from other related oomycetes with RG57 restriction fragment length polymorphism analysis. Long-distance movement in seed tubers and garden center transplants may have contributed to the rapid spread of the P. infestans genotypes across Canada. Tracking pathogen movement and population composition should improve the ability to predict the genotypes expected each year in different regions of Canada.