Phytophthora capsici: Recent Progress on Fundamental Biology and Disease Management 100 Years After Its Description.

Phytophthora capsici is a destructive oomycete pathogen of vegetable, ornamental, and tropical crops. First described by L.H. Leonian in 1922 as a pathogen of pepper in New Mexico, USA, P. capsici is now widespread in temperate and tropical countries alike. Phytophthora capsici is notorious for its capability to evade disease management strategies. High genetic diversity allows P. capsici populations to overcome fungicides and host resistance, the formation of oospores results in long-term persistence in soils, zoospore differentiation in the presence of water increases epidemic potential, and a broad host range maximizes economic losses and limits the effectiveness of crop rotation. The severity of disease caused by P. capsici and management challenges have led to numerous research efforts in the past 100 years. Here, we discuss recent findings regarding the biology, genetic diversity, disease management, fungicide resistance, host resistance, genomics, and effector biology of P. capsici.

Managing Onion Thrips can Limit Bacterial Stalk and Leaf Necrosis in Michigan Onion Fields.

Onion thrips (Thrips tabaci) is a major insect pest of onion and it has been identified as a likely vector of Pantoea agglomerans (bacterial stalk and leaf necrosis), a relatively new pathogen to Michigan's onion industry. Our objective was to develop an integrated insect and disease management program by examining the efficacy of bactericides and insecticides alone and in combination to limit bacterial stalk and leaf necrosis caused by P. agglomerans. We also examined the association of onion thrips and disease incidence in the field, because thrips are known to transmit this pathogen. In the pesticide trial, insecticides reduced both thrips abundance and bacterial stalk and leaf necrosis incidence whereas bactericides alone did not reduce disease severity. Positive correlations among thrips population density, numbers of thrips positive for P. agglomerans, and bacterial stalk and leaf necrosis incidence in onion fields were determined. This study suggests that onion thrips feeding can facilitate the development of bacterial stalk and leaf necrosis in Michigan's commercial onion fields, and results from the pesticide trials indicate that thrips feeding damage is positively correlated with disease incidence. Therefore, in order to reduce bacterial stalk and leaf necrosis incidence in onion, management efforts should include reducing onion thrips populations through the use of insecticides and other cultural practices.

Disease Resistance to Multiple Fungal and Oomycete Pathogens Evaluated Using a Recombinant Inbred Line Population in Pepper.

Incorporating disease resistance into cultivars is a primary focus of modern breeding programs. Resistance to pathogens is often introgressed from landrace or wild individuals with poor fruit quality into commercial-quality cultivars. Sites of multiple disease resistance (MDR) are regions or "hot spots" of the genome with closely linked genes for resistance to different pathogens that could enable rapid incorporation of resistance. An F2-derived F6 recombinant inbred line population from a cross between 'Criollo de Morelos 334' (CMM334) and 'Early Jalapeno' was evaluated in inoculated fruit studies for susceptibility to oomycete and fungal pathogens: Phytophthora capsici, P. nicotianae, Botrytis cinerea, Fusarium oxysporum, F. solani, Sclerotinia sclerotiorum, Alternaria spp., Rhizopus oryzae, R. stolonifer, and Colletotrichum acutatum. All isolates evaluated were virulent on pepper. Significant differences in disease susceptibility were identified among lines for each of the pathogens evaluated. P. capsici was the most virulent pathogen, while R. oryzae and one Sclerotinia isolate were the least virulent. Quantitative trait loci associated with resistance were identified for Alternaria spp. and S. sclerotiorum. Positive correlations in disease incidence were detected between Alternaria spp. and F. oxysporum, F. solani, and C. acutatum, as well as between C. acutatum and Botrytis spp., F. oxysporum, F. solani, and P. capsici. No sites of MDR were identified for pathogens tested; however, positive correlations in disease incidence were detected among pathogens suggesting there may be genetic linkage among resistance genes in CM334 and Early Jalapeno.

Regional and Temporal Population Structure of Pseudoperonospora cubensis in Michigan and Ontario.

Cucurbit downy mildew (CDM), caused by the oomycete pathogen Pseudoperonospora cubensis, is a devastating disease that affects cucurbit species worldwide. This obligate, wind-dispersed pathogen does not overwinter in Michigan or other northern regions and new isolates can enter the state throughout the growing season. To evaluate the regional and temporal population structure of P. cubensis, sporangia from CDM lesions were collected from cucurbit foliage grown in Michigan and Ontario field locations in 2011. Population structure and genetic diversity were assessed in 257 isolates using nine simple sequence repeat markers. Genetic diversity was high for isolates from Michigan and Canada (0.6627 and 0.6131, respectively). Five genetic clusters were detected and changes in population structure varied by site and sampling date within a growing season. The Michigan and Canada populations were significantly differentiated, and a unique genetic cluster was detected in Michigan.

Resistance to Crown and Root Rot Caused by Phytophthora capsici in a Tomato Advanced Backcross of Solanum habrochaites and Solanum lycopersicum.

Phytophthora capsici causes devastating disease on many vegetable crops, including tomato and other solanaceous species. Solanum habrochaites accession LA407, a wild relative of cultivated tomato, has shown complete resistance to four P. capsici isolates from Michigan cucurbitaceous and solanaceous crops in a previous study. Greenhouse experiments were conducted to evaluate 62 lines of a tomato inbred backcross population between LA407 and the cultivated tomato 'Hunt 100' and 'Peto 95-43' for resistance to two highly virulent P. capsici isolates. Roots of 6-week-old seedlings were inoculated with each of two P. capsici isolates and maintained in the greenhouse. Plants were evaluated for wilting and plant death three times per week for 5 weeks. Significant differences were observed in disease response among the inbred tomato lines. Most lines evaluated were susceptible to P. capsici isolate 12889 but resistant to isolate OP97; 24 tomato lines were resistant to both isolates. Heritability of Phytophthora root rot resistance was high in this population. Polymorphic molecular markers located in genes related to resistance and defense responses were identified and added to a genetic map previously generated for the population. Resistant lines and polymorphic markers identified in this study are a valuable resource for development of tomato varieties resistant to P. capsici.

Evaluation of a Diverse, Worldwide Collection of Wild, Cultivated, and Landrace Pepper (Capsicum annuum) for Resistance to Phytophthora Fruit Rot, Genetic Diversity, and Population Structure.

Pepper is the third most important solanaceous crop in the United States and fourth most important worldwide. To identify sources of resistance for commercial breeding, 170 pepper genotypes from five continents and 45 countries were evaluated for Phytophthora fruit rot resistance using two isolates of Phytophthora capsici. Genetic diversity and population structure were assessed on a subset of 157 genotypes using 23 polymorphic simple sequence repeats. Partial resistance and isolate-specific interactions were identified in the population at both 3 and 5 days postinoculation (dpi). Plant introductions (PIs) 640833 and 566811 were the most resistant lines evaluated at 5 dpi to isolates 12889 and OP97, with mean lesion areas less than Criollo de Morelos. Genetic diversity was moderate (0.44) in the population. The program STRUCTURE inferred four genetic clusters with moderate to very great differentiation among clusters. Most lines evaluated were susceptible or moderately susceptible at 5 dpi, and no lines evaluated were completely resistant to Phytophthora fruit rot. Significant population structure was detected when pepper varieties were grouped by predefined categories of disease resistance, continent, and country of origin. Moderately resistant or resistant PIs to both isolates of P. capsici at 5 dpi were in genetic clusters one and two.

Susceptibility of Greenhouse Ornamentals to Phytophthora capsici and P. tropicalis.

The susceptibility of fabaceous (Lupinus and Lathyrus spp.) and solanaceous (Calibrachoa, Browallia, Nicotiana, Nierembergia, and Petunia spp.) ornamental plants compared with straightneck squash (Cucurbita pepo) inoculated with Phytophthora capsici and P. tropicalis was investigated in greenhouse studies. Four P. capsici isolates and one P. tropicalis isolate were evaluated. Flowering tobacco (Nicotiana × sanderae), sweet pea (Lathyrus latifolius), lupine (Lupinus polyphyllus), squash, and million bells (Calibrachoa × hybrida) were susceptible to P. capsici and P. tropicalis. Bush violet (Browallia speciosa) and cup flower (Nierembergia scoparia) were not susceptible to either pathogen. Petunia (Petunia × hybrida) was susceptible to P. capsici but not P. tropicalis. Area under the plant growth curve (AUPGC) was also affected in some susceptible plants. AUPGC was significantly different in inoculated plants compared with the untreated controls of Nicotiana and Calibrachoa. In addition, six Calibrachoa cultivars were evaluated for susceptibility to P. capsici and P. tropicalis in a separate experiment. Although all Calibrachoa cultivars were susceptible to P. capsici and P. tropicalis, 'Celebration Purple Star' displayed limited disease following inoculation with either pathogen. Calibrachoa 'Cabaret' and 'Can-Can' inoculated with P. capsici or P. tropicalis displayed significant differences in AUPGC compared with the uninoculated controls. 'Callie', 'Million Bells Cherry Pink', and 'Superbells' had significantly reduced AUPGC compared with the controls when inoculated with P. tropicalis but not P. capsici. These results expand the host range of P. capsici to include Calibrachoa spp., L. polyphyllus, and Lathyrus latifolius, and P. tropicalis to include L. latifolius, Nicotiana spp., and straightneck squash.

Field Response of Cucurbit Hosts to Pseudoperonospora cubensis in Michigan.

Downy mildew, caused by Pseudoperonospora cubensis, is a severe foliar disease of many cucurbit crops worldwide. Forty-one cucurbit cultigens (commercial cultivars and plant introductions) from five genera (Cucumis, Citrullus, Cucurbita, Lagenaria, and Luffa) were assessed for susceptibility to P. cubensis in a research field exposed to natural inoculum in Michigan. Eight cultigens from a differential set for pathotype determination were included within the 41 cultigens to detect changes in dominant P. cubensis pathotypes present. No pathotype differences were found between 2010 and 2011. Cucumis melo cultigen MR-1 was less susceptible to Michigan P. cubensis populations than other C. melo cultigens. No symptoms or signs of infection were detected on cultigens of Cucurbita moschata and C. pepo. Disease onset was later in 2011 than 2010; greater than 90% disease severity in pickling cucumber 'Vlaspik' was observed in both years. This study confirmed that Cucumis is the most susceptible cucurbit genus, while Citrullus and Cucurbita cultigens were the least susceptible genera to populations of P. cubensis in Michigan. Area under the disease progress curve values indicated that disease progress was limited on all Citrullus cultigens compared with Cucumis cultigens, and pathogen sporulation was not detected under field conditions. Future studies should evaluate the ability of a reduced fungicide program to control downy mildew on less susceptible Cucumis melo 'Edisto 47', 'Primo', 'Athena', 'Strike', 'Ananas', 'Banana', and 'Tam-Dew'. Many of the melon cultivars evaluated were selected on the basis of reported resistance to downy mildew, yet they showed significant disease symptoms. It is important to evaluate candidate cultigens for resistance to local P. cubensis populations.

Relationships Between Airborne Pseudoperonospora cubensis Sporangia, Environmental Conditions, and Cucumber Downy Mildew Severity.

Airborne Pseudoperonospora cubensis sporangia were collected 0.5 m above the ground from May to late September or early October 2010 and 2011 in unsprayed cucumber (Cucumis sativus) fields in Benton Harbor and Frankenmuth, MI. Cucumber downy mildew incidence and severity were evaluated weekly within each field from June until September or October. The first airborne sporangium was detected before the crop was planted for each site-year. The greatest numbers of airborne sporangia were detected when moderate to high disease severity (≥5% symptomatic leaf area) was detected within the field. Fewer airborne sporangia were present with low disease severity (<5% symptomatic leaf area), and even fewer were detected prior to planting the cucumber crop. The number of airborne sporangia detected, time post planting, planting number (first versus second versus third planting), temperature, and leaf wetness were positively associated and solar radiation was negatively associated with disease occurrence for at least one site-year. Michigan growers currently use an aggressive, calendar-based fungicide program to manage cucumber downy mildew. Because airborne sporangium concentrations were one of the most important factors identified in this study, the current fungicide recommendation of decreasing the spray interval following disease detection in an area is warranted.

Evaluation of Pepper Fruit for Resistance to Phytophthora capsici in a Recombinant Inbred Line Population, and the Correlation with Fruit Shape.

Phytophthora capsici causes fruit, root, and foliar blight on pepper (Capsicum annuum) in field production. Breeding for disease-resistant commercial pepper cultivars is essential to long-term management of P. capsici. In this study, the severity of Phytophthora fruit rot was evaluated in an F6 recombinant inbred line population between CM334, a landrace from Mexico, and the commercial 'Early Jalapeño'. The two parents and 67 progeny lines were evaluated for fruit rot resistance at 3 and 5 days post inoculation (dpi) using three P. capsici isolates. Fruit shape was also evaluated for each line, and the correlation between shape and disease symptoms was investigated. Significant differences were detected among lines in lesion area measured 3 and 5 dpi, and in phenotypic traits (fruit length, width, and shape index). Of the fruit phenotypic traits measured, only fruit shape index had a significant, albeit weak (r = 0.2892, P = 0.02), correlation with lesion area when inoculated, and with only one of the three isolates of P. capsici evaluated. These results suggest that breeding for fruit rot resistance in pepper will have minimal linkage with fruit shape in the CM334 background.

QTL mapping of fruit rot resistance to the plant pathogen Phytophthora capsici in a recombinant inbred line Capsicum annuum population.

Phytophthora capsici is an important pepper (Capsicum annuum) pathogen causing fruit and root rot, and foliar blight in field and greenhouse production. Previously, an F6 recombinant inbred line population was evaluated for fruit rot susceptibility. Continuous variation among lines and partial and isolate-specific resistance were found. In this study, Phytophthora fruit rot resistance was mapped in the same F6 population between Criollo del Morelos 334 (CM334), a landrace from Mexico, and 'Early Jalapeno' using a high-density genetic map. Isolate-specific resistance was mapped independently in 63 of the lines evaluated and the two parents. Heritability of the resistance for each isolate at 3 and 5 days postinoculation (dpi) was high (h(2) = 0.63 to 0.68 and 0.74 to 0.83, respectively). Significant additive and epistatic quantitative trait loci (QTL) were identified for resistance to isolates OP97 and 13709 (3 and 5 dpi) and 12889 (3 dpi only). Mapping of fruit traits showed potential linkage with few disease resistance QTL. The partial fruit rot resistance from CM334 suggests that this may not be an ideal source for fruit rot resistance in pepper.

Age-Related Resistance to Phytophthora Fruit Rot in 'Dickenson Field' Processing Pumpkin and 'Golden Delicious' Winter Squash Fruit.

Phytophthora fruit rot, caused by Phytophthora capsici, is a major constraint to cucurbit production for the processing industry in Michigan. Age-related resistance to Phytophthora fruit rot has been identified in pepper and some cucurbit fruit. In this study, 'Dickenson Field' processing pumpkin (Cucurbita moschata) and 'Golden Delicious' winter squash (C. maxima) were evaluated for age-related resistance to Phytophthora fruit rot. Hand-pollinated fruit were harvested 3, 7, 10, 14, 21, 28, 42, or 56 days post pollination (dpp), and inoculated with P. capsici isolate 12889. Susceptibility to Phytophthora fruit rot decreased with fruit age in Dickenson Field processing pumpkin, whereas Golden Delicious winter squash remained susceptible to fruit rot even as fruit reached full physiological maturity. Less than 15% of Dickenson Field fruit 21 dpp or older became diseased. Conversely, about 80% of Golden Delicious fruit 21 dpp or older became diseased. Lesion diameter and pathogen growth density ratings differed significantly (P < 0.0001) among fruit ages for both cultivars, and were negatively correlated (ρ = -0.37 to -0.87) with fruit age. Lesion diameter and pathogen growth were generally greater on younger fruit than older fruit. Lesion diameter was greatest on 7- and 10-dpp-old fruit of Dickenson Field and Golden Delicious, respectively. Pathogen growth density ratings were greatest on 3-dpp-old fruit of both cultivars. Several morphological and physiological changes were observed as fruit matured. Soluble solids content and exocarp firmness of both cultivars increased with fruit age. Lesion diameter and pathogen growth density ratings were negatively correlated (ρ = -0.29 to -0.73) with soluble solids content and exocarp firmness.

Using Soil-Applied Fungicides to Manage Phytophthora Crown and Root Rot on Summer Squash.

Phytophthora crown and root rot (Phytophthora capsici) of summer squash is especially difficult to manage because all commercial cultivars are highly susceptible to P. capsici. Producers have traditionally relied on foliar fungicide applications to control Phytophthora crown and root rot despite their limited efficacy. Soil fungicide applications, including via subsurface drip chemigation, have recently gained interest as a method of improving control of P. capsici infections. In this study, soil drenches and foliar applications of 11 fungicides were compared for control of Phytophthora crown and root rot of summer squash in replicated field and greenhouse trials. Fungicides were applied at 7-day intervals. Incidence (%) of plant death was assessed from 7 to 42 days post inoculation (dpi) in field trials. Crown rot severity was rated on a scale of 1 (no wilting) to 5 (plant death) from 5 to 21 dpi in greenhouse trials. Results of field and greenhouse trials were similar. Plant death of 'Cougar' following inoculation with P. capsici isolate 12889 occurred at all growth stages from first true-leaf to full maturity in field trials. Plant death 42 dpi differed significantly (P ≤ 0.0001) among fungicides and application methods. The fungicide-application method interaction also was significant. Some fungicides were ineffective regardless of application method. In general, soil drenches were more effective than foliar applications at limiting plant death but no treatment completely controlled disease symptoms. Mean plant death 42 dpi was 41% for soil drenches and 92% for foliar sprays. Drenches of fluopicolide, mandipropamid, or dimethomorph limited plant death to ≤10% and prevented yield loss associated with root and crown rot. Foliar applications generally did not reduce plant death compared with the untreated, inoculated control, and were unable to prevent yield loss in field trials. In greenhouse trials, crown rot severity differed significantly (P ≤ 0.0001) among fungicides, application methods, and cultivars when plants were inoculated with P. capsici isolate 12889 or SP98. Crown rot was less severe and disease progress was slower following soil drenches than foliar applications. Some fungicide treatments were more effective on 'Leopard,' which was less susceptible to P. capsici than 'Cougar.' Soil application methods, including soil drench and drip chemigation, should be evaluated when fungicides are registered for soilborne disease control, because these methods provide better control of Phytophthora crown and root rot than foliar application.

Evaluation of Eggplant Rootstocks and Pepper Varieties for Potential Resistance to Isolates of Phytophthora capsici from Michigan and New York.

Phytophthora capsici is a soilborne pathogen of major economic importance in pepper, and of less importance in tomato and eggplant production. As soil fumigation becomes more expensive and limited, and fungicide insensitivity of P. capsici isolates becomes more prevalent, grafting is quickly becoming an industry-favored method to control soilborne diseases. Greenhouse experiments were performed to evaluate an eggplant cultivar (Classic), two eggplant lines (EG195, EG203), a pepper line (CM334), and three pepper cultivars (Paladin, Camelot, and Red Knight) for root rot resistance to 14 P. capsici isolates. The isolates showed various degrees of virulence between pepper and eggplant in both experiments. Both eggplant and one pepper lines showed moderate resistance to the most virulent isolates tested in experiment one. The partially resistant pepper cultivar, Paladin, was significantly more susceptible than CM334 and the eggplant lines, but was still resistant to most isolates. In the second experiment, the eggplant cultivar Classic and the susceptible pepper cultivar Red Knight were both susceptible to most isolates tested, while EG203 and EG195 were resistant to most isolates. The two eggplant breeding lines, EG195 and EG203, showed moderate resistance to all isolates tested in both experiments. This is the first reported evaluation of eggplant resistance to P. capsici. Further research is warranted to test eggplant lines EG195 and EG203 for resistance to a wide range of soilborne pests and to evaluate their usefulness as P. capsici-resistant rootstocks for peppers, tomatoes, and eggplants.

Fungal Planet description sheets: 107-127.

Novel species of microfungi described in the present study include the following from Australia: Phytophthora amnicola from still water, Gnomoniopsis smithogilvyi from Castanea sp., Pseudoplagiostoma corymbiae from Corymbia sp., Diaporthe eucalyptorum from Eucalyptus sp., Sporisorium andrewmitchellii from Enneapogon aff. lindleyanus, Myrmecridium banksiae from Banksia, and Pilidiella wangiensis from Eucalyptus sp. Several species are also described from South Africa, namely: Gondwanamyces wingfieldii from Protea caffra, Montagnula aloes from Aloe sp., Diaporthe canthii from Canthium inerne, Phyllosticta ericarum from Erica gracilis, Coleophoma proteae from Protea caffra, Toxicocladosporium strelitziae from Strelitzia reginae, and Devriesia agapanthi from Agapanthus africanus. Other species include Phytophthora asparagi from Asparagus officinalis (USA), and Diaporthe passiflorae from Passiflora edulis (South America). Furthermore, novel genera of coelomycetes include Chrysocrypta corymbiae from Corymbia sp. (Australia), Trinosporium guianense, isolated as a contaminant (French Guiana), and Xenosonderhenia syzygii, from Syzygium cordatum (South Africa). Pseudopenidiella piceae from Picea abies (Czech Republic), and Phaeocercospora colophospermi from Colophospermum mopane (South Africa) represent novel genera of hyphomycetes. Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa.

First Report of Root and Crown Rot of Wasabi (Wasabia japonica Matsum.) Caused by Phytophthora cryptogea in Michigan.

In September 2011, a Phytophthora sp. was isolated from wasabi (Wasabia japonica Matsum.) grown commercially in hydroponic culture in a large production facility in southwest Michigan. Approximately 20% of the plants were affected, resulting in serious losses for the grower. Plants exhibited severe wilting and root and crown rot, with soft water-soaked lesions on the crown and dark lesions on the roots. Small pieces of root tissue with dark lesions were excised and plated onto potato dextrose agar and unclarified V8 agar plates amended with 25 ppm of benomyl, 100 ppm of ampicillin, 30 ppm of rifampicin, and 100 ppm of pentachloronitrobenzene. Isolates of a Phytophthora sp. were recovered from root tissue. Isolates produced sporangia abundantly in culture. Sporangia averaged 48 µm long × 34 µm wide and were ellipsoid to ovoid, occasionally obpyriform, and were nonpapillate and noncaducous. Distinct hyphal swellings were noted and chlamydospores were observed rarely in culture. The isolate used for inoculations did not produce oospores alone in culture but was able to produce oospores when paired with an A1 culture of P. capsici and incubated in the dark. Oospores were not observed when the isolate was paired with an A2 culture of P. capsici. No growth was observed at 35°C, and the isolate was identified as Phytophthora cryptogea based on morphological and physiological traits. Pathogen identity was further confirmed using PCR primers specific to P. cryptogea (1). In addition, a BLAST search was conducted using the nucleotide database collection in GenBank comparing our isolate against Phytophthora spp., with 99% sequence similarity to P. cryptogea in two sequenced genes, beta tubulin and cytochrome c oxidase 1 (2). Sequences for the isolate were deposited in the GenBank database under accession numbers JX041520 and JX041521. To fulfill Koch's postulates, six small, potted wasabi seedlings were inoculated by placing 3 g of 1-month-old infested millet (100 g of millet, 72 ml of distilled water, 0.08 g of asparagine, and 10 7-mm diameter V8 agar plugs with actively growing P. cryptogea) onto the soil surface of each pot under coconut coir mulch. Plants were watered heavily after soil infestation and as needed thereafter. Three control plants were inoculated with sterile millet seed. The experiment was repeated once. Wilting was observed within 5 and 7 days, respectively, in the first and second experiment. All six inoculated plants were severely wilted within 25 and 56 days, respectively, except for a single plant in the second experiment that never wilted. Root and crown rot was observed on wilted plants and dark lesions could be observed on root tissue. P. cryptogea was recovered from five of the six plants inoculated in each experiment. None of the control plants in either experiment displayed symptoms of wilting, and the pathogen was not recovered from these plants when pieces of root tissue were excised and plated onto amended V8 agar. To our knowledge, this is the first report of P. cryptogea causing crown and root rot of wasabi. References: (1) D. Minerdi et al. Eur. J. Plant Pathol. 122:227, 2008. (2) L. M. Quesada-Ocampo et al. Phytopathology 101:1061, 2011.

First Report of Colletotrichum acutatum sensu lato Causing Leaf Curling and Petiole Anthracnose on Celery (Apium graveolens) in Michigan.

In September 2010, celery plants with leaf cupping and petiole twisting were observed in commercial production fields located in Barry, Kent, Newago, and Van Buren Counties in Michigan. Long, elliptical lesions were observed on petioles but signs (mycelia, conidia, or acervuli) were not readily observed. Celery petioles were incubated in humid chambers (acrylic boxes with wet paper towels). After 24 h, conidia corresponding to the genus Colletotrichum were observed. Isolations were performed by excising pieces of celery tissue from the lesion margin and placing them on potato dextrose agar (PDA) amended with 30 ppm of rifampicin and 100 ppm of ampicillin. Plates were incubated at 21 ± 2°C under fluorescent light for 5 days. Fungal colony morphology was gray with salmon-colored masses of spores when viewed from above, and carmine when viewed from below. Isolates were single-spored and placed on 30% glycerol in -20°C, and cryoconservation media (20% glycerol, 0.04% yeast extract, 0.1% malt extract, 0.04% glucose, 0.02% K2HPO4) at -80°C. Conidia were 8.5 to 12.0 × 2.8 to 4.0 µm and straight fusiform in shape. Three isolates were confirmed as C. acutatum sensu lato based on sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA and the 1-kb intron of the glutamine synthase gene (3), both with 100% similarity with Glomerella acutata sequences. Sequences were submitted to GenBank (Accession Nos. JQ951599 and JQ951600 for ITS and GS, respectively). Additionally, C. acutatum specific primer CaIntg was used in combination with the primer ITS4 on 54 isolates from symptomatic celery plants, obtaining the expected 490-pb fragment (1). Koch's postulates were completed by inoculating 4-week-old celery seedlings of cultivars Sabroso, Green Bay, and Dutchess using three plants per cultivar. Prior to inoculation, seedlings were incubated for 16 h in high relative humidity (≥95%) by enclosing the plants in humid chambers. Seven-day-old C. acutatum s. l. colonies were used to prepare the inoculum. Seedlings were spray-inoculated with a C. acutatum s. l. conidial suspension of 1 × 106 conidia/ml in double-distilled water plus Tween 0.01%. Two control seedlings per cultivar were sprayed with sterile, double-distilled water plus 0.01% Tween. Plants were enclosed in bags for 96 h post inoculation and incubated in a greenhouse at 27°C by day/20°C by night with a 16-h photoperiod. Leaf curling was observed on all inoculated plants of the three cultivars 4 days after inoculation (DAI). Petiole lesions were observed 14 to 21 DAI. Conidia were observed in lesions after incubation in high humidity at 21 ± 2°C for 24 to 72 h. Symptomatic tissue was excised and cultured onto PDA and resulted in C. acutatum colonies. Control plants remained symptomless. C. acutatum (4) and C. orbiculare (2) were reported to cause celery leaf curl in Australia in 1966 (2,4). To our knowledge, this is the first report of C. acutatum s. l. infecting celery in Michigan. References: (1) A. E. Brown et al. Phytopathology 86:523, 1996. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab., USDA-ARS. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , 10 September 2010. (3) J. C. Guerber et al. Mycologia 95:872, 2003. (4) D. G. Wright and J. B. Heaton. Austral. Plant Pathol. 20:155, 1991.

An Evaluation of Cucurbits for Susceptibility to Cucurbitaceous and Solanaceous Phytophthora capsici isolates.

Cucumber (Cucumis sativus) and squash (Cucurbita spp.) production in Michigan is limited by the oomycete pathogen Phytophthora capsici. Cucumber, summer squash, and winter squash fruit were evaluated for susceptibility to five isolates of P. capsici. Detached fruit were inoculated with a 5-mm-diameter culture plug of mycelia and sporangia and were incubated in a laboratory or greenhouse. Lesion and pathogen growth diameters were measured and pathogen growth density was visually assessed. All P. capsici isolates incited rot, with significant differences found among fruit type and pathogen isolate. Straightneck squash (Cucurbita pepo), slicing cucumber, and butternut squash (C. moschata) exhibited more severe symptoms than the other fruit tested. Summer and winter squash seedlings were evaluated in greenhouse experiments, in which P. capsici-infested millet seed (approximately 1 g) were placed on the surface of soilless potting media. Disease severity was visually assessed every 2 days for 14 days post inoculation. Crop type, pathogen isolate, or the crop type-pathogen isolate interaction term were significant for symptom appearance and area under the disease progress curve values. Differences in susceptibility of butternut squash and zucchini cultivars were observed following inoculation with solanaceous isolate 13351. Results from this study can refine management programs for Phytophthora rot.

The Genetic Structure of Pseudoperonospora cubensis Populations.

Pseudoperonospora cubensis is a destructive foliar pathogen of economically important cucurbitaceous crops in the United States and worldwide. In this study, we investigated the genetic structure of 465 P. cubensis isolates from three continents, 13 countries, 19 states of the United States, and five host species using five nuclear and two mitochondrial loci. Bayesian clustering resolved six genetic clusters and suggested some population structure by geographic origin and host, because some clusters occurred more or less frequently in particular categories. All of the genetic clusters were present in the sampling from North America and Europe. Differences in cluster occurrence were observed by country and state. Isolates from cucumber had different cluster composition and lower genetic diversity than isolates from other cucurbits. Because genetic structuring was detected, isolates that represent the genetic variation in P. cubensis should be used when developing diagnostic tools, fungicides, and resistant host varieties. Although this study provides an initial map of global population structure of P. cubensis, future genotyping of isolates could reveal population structure within specific geographic regions, across a wider range of hosts, or during different time points during the growing season.

Genetic Structure of Clavibacter michiganensis subsp. michiganensis Populations in Michigan Commercial Tomato Fields.

Clavibacter michiganensis subsp. michiganensis, causal agent of bacterial canker of tomato, is distinguished into four fingerprint types (A, B, C, and D) using BOX-polymerase chain reaction (PCR). To characterize the variation within the C. michiganensis subsp. michiganensis population in Michigan, 718 strains of C. michiganensis subsp. michiganensis were isolated from infected foliage and fruit collected from 14 and 9 Michigan commercial tomato fields in 1997 and 1998, respectively. The frequency of PCR types detected with BOX-PCR in all strains, and Bayesian cluster analysis, pairwise differentiation index comparisons, and genetic diversity estimates of 96 strains genotyped for six virulence-related genes revealed that C. michiganensis subsp. michiganensis populations in Michigan tomato fields are geographically structured. A multilocus haplotype cladogram was also consistent with geographic stratification in C. michiganensis subsp. Michiganensis populations. Some regions had strains predominantly of only one PCR type or belonging mostly to one genetic cluster, while other regions presented more diversity of occurrence of PCR types and genetic clusters. Results derived from this study provide information about the genetic structure of C. michiganensis subsp. michiganensis populations in Michigan and genetic diversity of C. michiganensis subsp. michiganensis inocula, which is key in developing disease management strategies.