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.

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.

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.

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.

Investigating the genetic structure of Phytophthora capsici populations.

Phytophthora capsici Leonian is a destructive soilborne pathogen that infects economically important solanaceous, cucurbitaceous, fabaceous, and other crops in the United States and worldwide. The objective of this study was to investigate the genetic structure of 255 P. capsici isolates assigned to predefined host, geographical, mefenoxam-sensitivity, and mating-type categories. Isolates from six continents, 21 countries, 19 U.S. states, and 26 host species were genotyped for four mitochondrial and six nuclear loci. Bayesian clustering revealed some population structure by host, geographic origin, and mefenoxam sensitivity, with some clusters occurring more or less frequently in particular categories. Bayesian clustering, split networks, and statistical parsimony genealogies also detected the presence of non-P. capsici individuals in our sample corresponding to P. tropicalis (n = 9) and isolates of a distinct cluster closely related to P. capsici and P. tropicalis (n = 10). Our findings of genetic structuring in P. capsici populations highlight the importance of including isolates from all detected clusters that represent the genetic variation in P. capsici for development of diagnostic tools, fungicides, and host resistance. The population structure detected will also impact the design and interpretation of association studies in P. capsici. This study provides an initial map of global population structure of P. capsici but continued genotyping of isolates will be necessary to expand our knowledge of genetic variation in this important plant pathogen.

Dynamics of Pseudoperonospora cubensis Sporangia in Commercial Cucurbit Fields in Michigan.

Major outbreaks of cucurbit downy mildew have been observed since 2005 in Michigan, the top state for pickling cucumber production in the United States. Airborne concentrations of Pseudoperonospora cubensis sporangia were monitored from 2006 to 2009 in a commercial cucurbit field in each of five Michigan counties. Temperature, relative humidity, leaf wetness, and rainfall were recorded on site in 2008 and 2009. Seasonal and daily trends for sporangial concentrations were examined using loess regression to determine when sporangia were present throughout the growing season. Airborne sporangia were detected from June through September, and concentrations increased during this period. Peak airborne sporangial concentrations occurred between 8:00 and 13:00 h. Airborne sporangial concentrations were positively correlated with temperature (r = 0.17 to 0.38, P < 0.0001) and negatively correlated with relative humidity (r = -0.16 to -0.45, P ≤ 0.0046) and leaf wetness (r = -0.14 to -0.39, P ≤ 0.0001) in 2008 and 2009 at all sites. In autoregressive moving average models, sporangial concentrations were negatively related to relative humidity (slope = -0.03 to -1.34, P < 0.0496) at three of five sites in 2008 and four of five sites in 2009. Hence, sporangia are likely to be airborne above the crop canopy during periods with high temperatures and low relative humidity and leaf wetness.

Temporal Genetic Structure of Phytophthora capsici Populations from a Creek Used for Irrigation in Michigan.

Irrigation water may harbor Phytophthora capsici, and irrigating susceptible vegetable crops with infested water can initiate epidemics. In this study, we evaluated the genetic structure of 106 P. capsici isolates collected from a creek used for irrigation (2002, 2003, and 2006) and from a field adjacent to the creek (2001) using six polymorphic nuclear loci. Bayesian clustering analysis detected four clusters in the sample, and some clusters occurred more or less frequently in certain years. In 2006, isolates belonging to cluster four predominated in the sampling. Mean pairwise FST values (0.008 to 0.065) indicated low differentiation between categories, but the most differentiation was observed when 2006 was compared to 2001 and 2002. Differences in isolate phenotypic traits were observed year-to-year. Isolates insensitive to mefenoxam were more common in 2006 and 2003 than in 2002 and 2001. The mating type ratio was approximately 1:1 in 2002 and 2003, but was skewed toward A1 in 2001 and toward A2 in 2006. Since irrigation water can remain infested or become reinfested annually with P. capsici for years after the adjacent fields are transitioned to nonsusceptible crops, growers are advised to avoid potentially infested irrigation water even after rotating to nonhost crops for several years.

Variation in Phenotypic Characteristics of Phytophthora capsici Isolates from a Worldwide Collection.

To determine variation within Phytophthora capsici, 124 P. capsici isolates from 12 countries were characterized for sporangial length and width, pedicle length, oospore diameter, sporangia and chlamydospore production, and growth at 32, 35, and 38°C. Sporangia were 23 to 35 µm wide and 38 to 60 µm long; differences in width and length were noted when isolates were grouped by genetic cluster and continent of origin. Length:breadth ratio (1.34 to 2.07) and pedicle length (20 to 260 µm long) varied widely among isolates; differences were apparent by continent and host family of origin. Oospore diameters varied among isolates (22 to 37 µm), but no differences were noted by isolate genetic cluster, host family of origin, continent of origin, mating type, or sensitivity to mefenoxam. Differences in sporangia production were observed among isolates grouped by continent, and isolates from nonvegetable hosts produced fewer sporangia than isolates from vegetable hosts. When cultures were incubated in a liquid medium, 35 P. capsici isolates formed chlamydospores. Most (122 of 124) of the isolates were able to grow at 35°C, but all of the isolates grew poorly at 38°C. The results of this study indicate substantial variation in morphological and physiological characteristics among P. capsici isolates.

Effects of Temperature, Humidity, and Wounding on Development of Phytophthora Rot of Cucumber Fruit.

The effects of temperature (10, 15, 20, 25, 30, and 35°C) and relative humidity (~35, 60, 70, 80, and 100%) on development of Phytophthora fruit rot, caused by Phytophthora capsici, of pickling cucumber (Cucumis sativus) were investigated in controlled growth chamber studies. The effect of wounding on disease development was characterized for small (2.0 to 2.5 cm diameter × 8 to 9 cm long), medium (3.0 to 4.0 cm diameter × 12.0 to 13.0 cm long), and large (>4.5 cm diameter × >14 cm long) pickling cucumbers. Lesions developed on cucumbers incubated at all temperatures tested except 10 and 35°C. Disease severity was greatest on cucumbers incubated at 25°C at 4 days postinoculation (dpi). Lesions formed on cucumbers incubated at all relative humidities tested. The diameter of water-soaking and pathogen growth increased as the incubation relative humidity increased. Wounding lessened age-related resistance in pickling cucumbers. The diameter of water-soaking was similar for all wounded cucumbers at 4 dpi regardless of fruit size. Sporangial production was greater on small and medium fruits than on large fruits. These results indicate that P. capsici is capable of infecting cucumbers over a wide range of temperature and relative humidity conditions. Wounding increases the susceptibility of pickling cucumbers to P. capsici.

Influence of Environment on Airborne Spore Concentrations and Severity of Asparagus Purple Spot.

Environmental conditions, airborne concentrations of Pleospora herbarum ascospores and conidia, and purple spot disease severity on spears and fern were monitored for 3 years in two no-till asparagus fields in Michigan. Purple spot lesion development on spears was correlated with low temperature and vapor pressure deficit and high rainfall. Low vapor pressure deficit and high rainfall were also correlated with airborne ascospore concentrations at both sites. Lesion development on the fern was favored by longer periods of leaf wetness and low vapor pressure deficit and rainfall. Daily airborne conidia concentrations were positively correlated with average temperature and the number of hours of leaf wetness per day and negatively correlated with vapor pressure deficit. Airborne conidia concentrations displayed a diurnal periodicity with greater concentrations between 700 and 1300 h. The results of this study indicate that a model to predict purple spot on asparagus spears should include temperature, vapor pressure deficit, and rainfall; a model for disease on the fern should include temperature, leaf wetness, vapor pressure deficit, and rainfall.

Effects of Temperature, Concentration, Age, and Algaecides on Phytophthora capsici Zoospore Infectivity.

Controlled laboratory studies were undertaken to determine the effects of water temperature (2, 9, 12, 19, 22, and 32°C), inoculum concentration (1 × 102, 1 × 103, 5 × 103, 1 × 104, 2 × 104, and 4 × 104 zoospores/ml), and zoospore suspension age (0, 1, 3, and 5 days old) on infection of pickling cucumbers (Cucumis sativus) by Phytophthora capsici. Zoospore motility and mortality in response to commercial algaecides were also investigated. Cucumbers became infected at all temperatures tested, except 2°C, and the highest infection incidence was observed for cucumbers incubated in suspensions held at ≥19°C. Fewer fruit (<40% at ≥19°C, 0% at ≤12°C) became infected when water contained 1 × 102 zoospores/ml. Almost 100% of fruit were infected when water contained ≥5 × 103 zoospores/ml at temperatures ≥12°C. While the incidence of fruit infection declined with the zoospore suspension age, infection still occurred when 5-day-old suspensions were used. Commercial algaecides inhibited zoospore motility and caused significant zoospore mortality in laboratory assays, and show promise for treatment of infested irrigation water. Avoidance of infested irrigation water throughout the growing season is warranted until effective and economically acceptable water treatments are developed for field use.

Dispersal and movement mechanisms of Phytophthora capsici sporangia.

Understanding the mechanisms of Phytophthora capsici sporangial dissemination is paramount to understanding epidemic initiation and development. Direct laboratory observations showed P. capsici sporangial dispersal occurred in water with capillary force, but did not occur in response to wind or a reduction in relative humidity. Atmospheric sporangial concentrations were monitored under field conditions using a volumetric spore sampler in a commercial cucurbit field and in an experimental setting where copious sporangia were continuously available in close proximity to the spore trap. Dispersal was infrequent (0.7% of total hours monitored) during sampling in a commercial field; 14 sporangia were detected during a 7.5-week sampling period. In the experimental field situation, dispersal occurred in 4.6% of the hours sampled and 438 sporangia were impacted onto tapes during a 7-week sampling period. Airborne sporangial concentrations were positively associated with rainfall at both sites, but not vapor pressure deficit. Furthermore, in the experimental field situation, wind speed was not significant in regression analysis. Wind speed was not measured in the commercial field. Hence, both direct laboratory observations and volumetric spore sampling indicate that dispersal of sporangia via wind currents is infrequent, and sporangia are unlikely to be naturally dispersed among fields by wind alone.