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.

Large genomic deletion linked to field-evolved resistance to Cry1F corn in fall armyworm (Spodoptera frugiperda) from Florida.

The fall armyworm (Spodoptera frugiperda) is a highly polyphagous lepidopteran pest of relevant food and fiber staple crops. In the Americas, transgenic corn and cotton producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have controlled and reduced the damage caused by S. frugiperda. However, cases of field-evolved S. frugiperda resistance to Bt corn producing the Cry1F insecticidal protein have been documented in North and South America. When characterized, field resistance to Cry1F is linked to insertions and mutations resulting in a modified or truncated ABC transporter subfamily C2 (SfABCC2) protein that serves as Cry1F receptor in susceptible S. frugiperda. In this work, we present detection of a large genomic deletion (~ 8 kb) affecting the SfABCC2 and an ABC transporter gene subfamily 3 -like gene (SfABCC3) as linked to resistance to Cry1F corn in a S. frugiperda strain from Florida (FL39). Monitoring for this genomic deletion using a discriminatory PCR reaction in field-collected S. frugiperda moths detected individuals carrying this allele in Florida, but not in surrounding states. This is the first report of a large genomic deletion being involved in resistance to a Bt insecticidal protein.

Genes controlling polyunsaturated fatty acid synthesis are developmentally regulated in broiler chicks.

1. The objective of this study was to characterise the regulation of the pathways that synthesise long-chain polyunsaturated fatty acids (PUFA) on developing adipose deposits in broiler embryos and chicks. Subcutaneous adipose depots were harvested from embryos and embryonic d E13, E15 and E17. Subcutaneous, abdominal and crop (neck) adipose, as well as liver, were collected at 7 and 14 d post-hatch. 2. Targeted RNA sequencing was used to quantify expression of 6 elongation of very long-chain fatty acid (ELOVL) genes, two isoforms of stearoyl-CoA desaturase (SCD and SCD5), and three fatty acid desaturases (FADS1, FADS2, and FADS6) in each depot and in the liver. Expression levels of marker genes for fatty acid oxidation and adipogenesis (peroxisome proliferator-activated receptor gamma (PPARG)) were quantified. Fatty acid composition of subcutaneous adipose was analysed using gas chromatograph-mass spectrometry (GC/MS). 3. Genes in the PUFA synthetic pathway were differentially expressed across developmental ages and between depots. These include elongase and desaturase genes, that have not previously been characterised in chicken. Correlation analyses identified subsets of co-regulated genes and fatty acids and highlighted relationships that may influence adipose metabolism and development. 4. It was concluded that PUFA synthesis is an active and dynamically regulated pathway in developing adipose deposits in the broiler chick. These data highlighted potential novel roles for specific elongase and desaturase genes in adipose deposition and metabolism.

Salisapiliaceae - a new family of oomycetes from marsh grass litter of southeastern North America.

Several filamentous oomycete species of the genus Halophytophthora have recently been described from marine environments, mostly from subtropical and tropical ecosystems. During a survey of oomycetes from leaf litter of Spartina alterniflora in salt marshes of southeastern Georgia, isolates of four taxa were recovered that bore similarity to some members of Halophytophthora but were highly divergent from isolates of Halophytophthora s.str. based on a combined sequence analysis of two nuclear loci. In phylogenetic analyses, these isolates were placed basal to a monophyletic group comprised of Pythium of the Pythiaceae and the Peronosporaceae. Sequence and morphology of these taxa diverged from the type species Halophytophthora vesicula, which was placed within the Peronosporaceae with maximum support. As a consequence a new family, the Salisapiliaceae, and a new genus, Salisapilia, are described to accommodate the newly discovered species, along with one species previously classified within Halophytophthora. Morphological features that separate these taxa from Halophytophthora are a smaller hyphal diameter, oospore production, lack of vesicle formation during sporulation, and a plug of hyaline material at the sporangial apex that is displaced during zoospore release. Our findings offer a first glance at the presumably much higher diversity of oomycetes in estuarine environments, of which ecological significance requires further exploration.

Molecular comparison of natural hybrids of Phytophthora nicotianae and P. cactorum infecting loquat trees in Peru and Taiwan.

Natural Phytophthora hybrids (P. nicotianae x P. cactorum) infecting loquat in Peru and Taiwan were characterized with AFLP (amplified fragment length polymorphism) markers, the internal transcribed spacer (ITS) region and the phenol acid carboxylase gene (Pheca) and inheritance of the mitochondrial cytochrome oxidase I gene (coxI). AFLP profiles of two Taiwanese isolates recovered in 1995 were polymorphic in approximately 50% of the fragments whereas five Peruvian isolates, recovered 2002-2003 and 2007, showed no genotypic variation. Sequencing analysis of the cloned ITS region resulted in the identification of sequences with high homology to either P. nicotianae (99%) or P. cactorum (97%). Direct sequence analysis of the Pheca gene revealed 13 heterozygous sites suggesting the presence of both P. nicotianae and P. cactorum genes in P. hybrids isolates. Melting analyses of coxI suggested that all seven Phytophthora hybrids inherited the mitochondrial DNA from P. nicotianae. Our results suggest that Phytophthora hybrids from Peru might have originated from a single hybridization event and that the two isolates from Taiwan might have originated through different hybridization events. The Peruvian hybrids appear to have persisted at least 3 y at three locations. Possible factors influencing the population structure of Phytophthora hybrids infecting loquat are discussed.

Occurrence and characterization of a Phytophthora sp. pathogenic to asparagus (Asparagus officinalis) in Michigan.

A homothallic Phytophthora sp. was recovered from asparagus (Asparagus officinalis) spears, storage roots, crowns, and stems in northwest and central Michigan in 2004 and 2005. Isolates (n = 131) produced ovoid, nonpapillate, noncaducous sporangia 45 microm long x 26 microm wide and amphigynous oospores of 25 to 30 microm diameter. Mycelial growth was optimum at 25 degrees C with no growth at 5 and 30 degrees C. All isolates were sensitive to 100 ppm mefenoxam. Pathogenicity studies confirmed the ability of the isolates to infect asparagus as well as cucurbits. Amplified fragment length polymorphism analysis of 99 isolates revealed identical fingerprints, with 12 clearly resolved fragments present and no clearly resolved polymorphic fragments, suggesting a single clonal lineage. The internal transcribed spacer regions of representative isolates were homologous with a Phytophthora sp. isolated from diseased asparagus in France and a Phytophthora sp. from agave in Australia. Phylogenetic analysis supports the conclusion that the Phytophthora sp. isolated from asparagus in Michigan is a distinct species, and has been named Phytophthora asparagi.

Survival and spread of Phytophthora capsici in Coastal Peru.

Phytophthora capsici is a soilborne pathogen that causes significant losses to pepper production in Peru. Our objective was to investigate the mechanisms by which P. capsici is able to survive and spread. During 2005 to 2007, 227 isolates of P. capsici were collected from four species of pepper (Capsicum annum, C. baccatum, C. chinense, and C. pubescens) and tomato (Solanum lycopersicum) at 33 field sites in 13 provinces across coastal Peru. All 227 isolates were of the A2 mating type and amplified fragment length polymorphism (AFLP) analysis indicates that 221 of the isolates had the same genotype. Analyses of six polymorphic single nucleotide polymorphism (SNP) loci showed fixed heterozygosity suggesting a single clonal lineage is widely dispersed. Members of the same clonal lineage were recovered during 2005 to 2007 from geographically separate locations from each of the host types sampled. Our results indicate that clonal reproduction drives the population structure of P. capsici in Peru. The impact of continuous cropping and irrigation from common river sources on the population structure in Barranca Valley are discussed.

First Report of Mefenoxam Sensitivity and Pathogenicity of Phytophthora citricola Isolated from American Ginseng (Panax quinquefolium).

In March of 2004, stratified ginseng seeds from commercial Wisconsin gardens were planted in sterilized silica sand in a research greenhouse at Michigan State University. Following emergence, seedlings exhibiting wilting, damping off, and black stem lesions were observed. In the laboratory, symptomatic seedlings were rinsed with distilled water. Tissue samples were excised and embedded in water agar amended with ampicillin (100 mg/liter) and incubated at 25°C. In addition to the isolation of Phytophthora cactorum, a known pathogen of ginseng, P. citricola, (five isolates) also was identified from single-zoospore cultures based on morphology (2). One-week-old, dilute V8 agar cultures were used to obtain single zoospores. Cultures were flooded with 20 ml of sterilized distilled water chilled to 10°C and incubated at 25°C for 25 min to induce zoospore release. Zoospore suspensions were spread onto water agar plates, and after 24 h at 25°C, single germinating zoospores were selected at random and transferred to benomyl, ampicillin, rifampicin, and pentachloronitrobenzene (BARP)-amended V8 agar plates. Sequence analysis of the internal transcribed spacer (ITS) region 1 and 2 of the rDNA was also used to distinguish P. citricola from P. cactorum. A representative sequence for the isolates of P. citricola (NCBI Accession No. FJ217388) matched (100% similarity) a P. citricola isolate deposited in GenBank (Accession No. DQ486661). To screen P. citricola for in vitro response to mefenoxam, agar plugs (7-mm diameter) from 1-week-old V8 agar cultures incubated at 25°C under fluorescent lighting were placed in the center of each of two V8 agar plates amended with 0 and 100 ppm of mefenoxam (Ridomil Gold EC, 48% a.i., suspended in sterile distilled water and added to V8 agar cooled to 49°C). The plates were incubated at 25°C for 3 days under fluorescent lighting. Isolates were assigned a mefenoxam sensitivity rating based on the percentage of radial mycelial growth on the amended V8 agar when compared with the unamended control. Each of the five isolates was scored as mefenoxam resistant with growth on 100-ppm plates >30% of the controls. Koch's postulates were conducted for the isolates of P. citricola recovered from ginseng seedlings to confirm pathogenicity. Previously, P. citricola was reported as nonpathogenic to ginseng (1). Three-week-old, healthy ginseng seedlings were planted into 89- × 64-mm pots filled with autoclaved medium-particle vermiculite and maintained in the greenhouse under 63% shade cloth with temperatures between 18 and 26°C. Pots were arranged in a completely randomized block design with eight seedlings per isolate as replicates and watered as needed. A 2-ml inoculum suspension (approximately 104 zoospores) was injected into the potting medium at the stem base of each seedling. All of the isolates were pathogenic to ginseng seedlings with 60% of inoculated seedlings per isolate exhibiting wilting, damping off, and blackened stems within 3 weeks after inoculation. P. citricola was reisolated from all inoculated plants. To our knowledge, this is the first report of P. citricola pathogenic on ginseng. References: (1) T. W. Darmono et al. Plant Dis. 75:610, 1991. (2) D. C. Erwin and O. K. Ribeiro. Page 96 in: Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN. 1996.

Characterization of Phytophthora capsici Causing Foliar and Pod Blight of Snap Bean in Michigan.

Green and yellow snap bean plants with water-soaked foliar lesions, stem necrosis, pod blight, and overall plant decline were observed in four commercial fields in three Michigan counties during 2003 to 2005. All fields were cropped to cucurbits that exhibited symptoms of Phytophthora capsici infection in recent years. In all, 680 isolates of P. capsici were obtained from bean stems, petioles, leaves, and pods; the pathogen was not recovered from roots. Koch's postulates were completed with representative isolates, confirming P. capsici as the causal organism. Select isolates also were pathogenic on cucumber fruit, causing symptoms consistent with P. capsici infection. The majority of the P. capsici isolates collected were sensitive to the fungicide mefenoxam and were of the A1 mating type. Under laboratory conditions, six P. capsici isolates from snap bean (2003) were pathogenic on 12 different commercial bean cultivars, including soybean. Infected bean plants exhibited water-soaked lesions, foliar necrosis, and wilting. We subjected 131 isolates collected from 2003 and 2004 to amplified fragment length polymorphism analysis to investigate diversity among isolates and geographical populations and to determine whether bean P. capsici isolates were similar to isolates from a cucurbit host. This is the first in-depth study of P. capsici on snap bean in Michigan. Although bean cultivars previously were considered a suitable rotation for crops susceptible to P. capsici, this is no longer a recommended practice.

First Report of Phytophthora nicotianae Causing Asparagus Spear and Root Rot in Peru.

During 2006, spears, roots, and crowns of asparagus (Asparagus officinalis) exhibiting brown necrotic lesions with water soaking were collected from several sites across Peru (Ica, Lima, and Trujillo). Small infected tissue sections were washed thoroughly with tap and sterile distilled water and transferred to corn meal agar plates (CMA) amended with PARP (100 ppm of pimaricin, 100 ppm of ampicillin, 30 ppm of rifampicin, and 100 ppm of pentachloronitrobenzene) and incubated for five days at 25°C. Hyphal tips were subcultured from actively expanding mycelium. Sporangia produced on CMA were papillate and averaged 38 µm long × 29 µm wide. Chlamydospores were terminal or intercalary and averaged 35 µm in diameter. Isolates incubated in the dark for more than 3 weeks did not produce oospores in single culture. Mating with Phytophthora capsici tester isolates CBS 121656 = A1 and CBS 121657 = A2 indicate that all five isolates were A2. For pathogenicity tests, inoculum was generated by incubating 300 g of autoclaved wheat seeds with four agar plugs (7 mm) of expanding mycelium in polyethylene bags for 1 month at 25°C. Nine-week-old asparagus plants (UC151 F1) were transferred into pots containing autoclaved substrate (1 part sand, 1 part potting soil, and 1 part peat). Inoculum was added as 1 g of inoculum per kilogram of substrate. Plants were maintained in the greenhouse at 23°C and watered daily. Decline symptoms as well as root and spear rot were observed after 7 days and a Phytophthora sp. was reisolated from infected tissue. No symptoms were observed on asparagus plants inoculated with sterile inoculum. DNA was isolated from two representative isolates, and the nuclear ribosomal internal transcribed spacer (ITS) region was amplified with ITS4 and ITS6 primers and sequenced. ITS sequence was submitted for a BLAST search in the NCBI database, showing Phytophthora nicotianae strain UQ848 Accession No AF266776 as the closest match with 99% sequence similarity (1). The consensus ITS sequence was deposited in NCBI (Accession No. EU433396). These results, together with the morphological characteristics, indicate that the Phytophthora sp. isolated from asparagus in Peru is P. nicotianae (Breda de Haan) (2). To our knowledge, this is the first report of P. nicotianae infecting asparagus and represents a new threat for asparagus growers in Peru. Control methods such as moderate watering and metalaxyl application are being applied to reduce Phytophthora outbreaks. References: (1) D. E. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society. St Paul, MN, 1996.

First Report of Phytophthora cactorum Causing Root Rot of Processing Carrots (Daucus carota) in Michigan.

In the fall of 2005, processing carrot fields in Mason, Newaygo, and Oceana counties, Michigan, were surveyed for Phytophthora spp. Carrot roots were sampled from areas of fields that exhibited patches of chlorotic, blighted, or wilted foliage. Dark brown, firm, water-soaked lesions occurred near the middle and crown areas of diseased carrot roots. In the advanced stages of disease, carrot root tissue readily collapsed and a soft rot developed while petioles turned black. The internal portions of the diseased carrot roots were brown and rubbery. Roots with these symptoms are not suitable for processing. Carrot roots were washed with tap water and the tissue excised from the edge of developing lesions and plated aseptically onto BARP-amended (25 ppm of benomyl, 100 ppm of ampicillin, 30 ppm of rifampicin, and 100 ppm of pentachloronitrobenzene) regular V8 juice agar. Plates were incubated at 23 to 25°C for 7 days. Phytophthora sp. was isolated from carrot root samples from all surveyed areas. Ten representative single-sporangium isolates cultured on dilute V8 juice agar were examined for morphological characteristics. The homothallic Phytophthora sp. isolates produced papillate, obpyriform, caducous sporangia (35.0 to 45.2 × 26.2 to 33.2 µm) with 1 to 3 µm long pedicels, plerotic oospores (27.0 to 32.0 µm in diameter) with paragynous antheridia, and primarily terminally produced chlamydospores that were 30.0 to 40.0 µm in diameter. Radial growth on V8 juice agar was observed at temperatures between 10 and 30°C with optimum growth at 25°C and no growth at 5 and 35°C. Pathogenicity of the 10 isolates was tested by inoculating three of each wounded and nonwounded carrot roots with a 7-mm mycelial plug from the edge of actively growing 5-day-old cultures. Inoculated carrot roots were incubated for 7 days in a moist chamber at 23 to 25°C. Symptoms developed 3 to 7 days after inoculation, with non-wounded roots exhibiting firm, dark brown, water-soaked lesions and wounded roots exhibiting soft rot with dark brown margins. The Phytophthora sp. was always isolated from the inoculated roots. Controls remained healthy and no pathogen was isolated from these roots. On the basis of the morphological and physiological characteristics, the Phytophthora sp. isolated was identified as Phytophthora cactorum ((Lebert & Cohn) J. Schrot.) (2). Identity of these isolates was confirmed by sequencing of the internal transcriber spacers (ITS). Amplified fragment length polymorphism (AFLP) profiles for 37 isolates were >83% similar, which is expected for conspecific isolates. The ITS sequences from six representative isolates were identical and shared 100% homology to P. cactorum (GenBank Accession No. AF266772) isolated from Rubus idaeus (1). The consensus ITS sequence was deposited in NCBI (Accession No. EF052680). P. cactorum was reported in New York on field and stored carrot roots in 1952 (3), but to our knowledge, this is the first report in Michigan. Finding of P. cactorum on carrot roots represents a new and significant threat to the Michigan processing carrot industry, which ranks fourth in the United States. References: (1) D. E. L. Cooke et al. Fungal Gen. Biol. 30:17, 2000. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Disease Worldwide. The American Phytopathological Society. St. Paul, MN, 1996. (3) W. E. Rader. N Y State (Cornell) Agr. Exp. Stn. Bull. 889:5, 1952.

Characterization of Phytophthora capsici from Michigan Surface Irrigation Water.

ABSTRACT Phytophthora capsici infects cucurbitaceous and solanaceous crops worldwide. In free water, P. capsici sporangia release zoospores that may be disseminated by moving surface water. Surface irrigation sources (river system, ponds, and ditches) in three Michigan counties with a history of P. capsici-susceptible crop production were monitored for the pathogen during four growing seasons (2002 to 2005). Pear and cucumber baits were suspended in water at monitoring sites for 3- to 7-day intervals and water temperature was recorded. Baits were washed and lesions were excised and cultured on water agar amended with rifampicin and ampicillin. P. capsici was detected at monitoring sites in multiple years, even when non-host crops were planted nearby. Recovered isolates (N = 270) were screened for sensitivity to the fungicide mefenoxam and characterized for mating type (MT). P. capsici isolates resistant to mefenoxam were common in water sources from southwest and southeast Michigan. Most monitoring sites yielded isolates of a 1:1 ratio of A1:A2 MTs. Amplified fragment length polymorphism analysis of select isolates from 2002 to 2004 indicated a lack of similarity groups persisting over time and in specific geographical locations. Data suggest that P. capsici did not overwinter in any of the surface water sources monitored. Water temperatures were correlated to positive P. capsici detection from all monitoring sites. The frequent detection of P. capsici in surface water used for irrigation in the primary vegetable growing regions in Michigan suggests that this is an important means of pathogen dissemination.

A Detached Cucumber Fruit Method to Screen for Resistance to Phytophthora capsici and Effect of Fruit Age on Susceptibility to Infection.

Identification and utilization of resistance to Phytophthora capsici could provide the basis for a viable management strategy against cucumber fruit rot, a persistent threat in cucumber (Cucumis sativus) production. Our objectives were to develop a method for testing detached, nonwounded, cucumber fruit for resistance to P. capsici, and to screen cucumber cultivars and plant introductions (collectively referred to as cultigens) for resistance. Four P. capsici isolates (differing in their sensitivity to the fungicide mefenoxam and compatibility type) were compared for their fruit infection capability in 1999 and 2000. No significant differences were found among isolates, and a single isolate was used for all subsequent screens. From 1999 to 2004, 480 cucumber cultigens were grown according to standard practices at Michigan State University research farms in four fields with no history of P. capsici. Commercially mature fruit were harvested, inoculated with P. capsici, and rated for lesion diameter, pathogen sporulation diameter, and density of pathogen sporulation. Although no fruit exhibited complete resistance to P. capsici, some cultigens exhibited limited pathogen sporulation. In the process of screening, it was observed that younger, smaller fruit were comparatively more susceptible than older, larger fruit. Replicated trials with hand-pollinated fruit showed that the transition from susceptible to more resistant appeared to coincide with the transition from the period of rapid fruit elongation to the period of increased fruit diameter. This is the first report using a nonwounded fruit screen to analyze cucumber resistance to P. capsici.

Detection of a Phytophthora sp. Causing Asparagus Spear and Root Rot in Michigan.

In the spring of 2004, a Phytophthora sp. was isolated from asparagus (Asparagus officinalis) spears, roots, and dormant crowns from several fields in Oceana and Ingham counties in Michigan. Symptomatic spears were often curved, had water-soaked lesions slightly above or below the soil line or were shriveled at the site of infection or both. Infected storage roots had water-soaked lesions but were not soft at the lesion site. Infected crowns had fewer roots than healthy crowns. In the laboratory, plant tissues were rinsed in tap water and blotted dry. Sections from the edge of lesions were placed aseptically onto BARP (25 ppm of benomyl, 100 ppm of ampicillin, 30 ppm of rifampicin, and 100 ppm of pentachloroni-trobenzene) amended unclarified V8 juice agar and incubated at 25°C for up to 7 days. Phytophthora sp. isolates recovered from the infected material produced ovoid, nonpapillate, noncaducous sporangia and amphigy-nous oospores on isolation media. Single-sporangium cultures made for each isolate were stored long term in sterile 2-ml microcentrifuge tubes containing two 7-mm mycelial plugs, two sterile hemp seeds, and 1 ml of sterile distilled water. Sporangia produced on dilute V8 juice agar averaged 45 µm long × 26 µm wide and oospores were 25 to 30 µm in diameter. Chlamydospores were not observed. Five detached 'Jersey Knight' spears were inoculated with a 7-mm mycelial plug from the edge of actively growing 5-day-old cultures and incubated at 23 to 25°C for 5 to 7 days in a moist chamber. After 3 days, water-soaked lesions and shriveling and curving of the spears were visible on all inoculated spears. The pathogen was always reisolated from the lesion edge. No symptoms were observed when spears were inoculated with sterile V8 juice agar plugs. DNA was extracted from representative isolates, and the nuclear ribosomal internal transcribed spacer (ITS) region was amplified with ITS6 and ITS4 primers and sequenced. A BLAST search of the NCBI database with the ITS sequence revealed Phytophthora sp. UQ2141, Accession No. AF266795, as the closest match with 99% sequence similarity. These results, coupled with the morphological characteristics of the isolates, indicate that the Phytophthora sp. isolated from asparagus in Michigan is among the constituents of Phytophthora spp. included in the P. megasperma clade 6 (2), whose taxa are currently being reevaluated. Although a Phytophthora sp. has been described previously on asparagus (1,3), this is the first report, to our knowledge, of a Phytophthora sp. on asparagus in Michigan. The occurrence of excessive rainfall in the spring of 2004 is likely responsible for widespread disease and considerable yield losses in production fields. References: (1) P. A. Ark and J. T. Barrett. Phytopathology 28:754, 1938. (2) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (3) V. Vujanovic et al. Plant Dis. 87:447, 2003.

Susceptibility of Mefenoxam-Treated Cucurbits to Isolates of Phytophthora capsici Sensitive and Insensitive to Mefenoxam.

The susceptibility of mefenoxam-treated cucurbit plants to infection by isolates of Phytophthora capsici that were sensitive (S), intermediately sensitive (IS), and fully insensitive (I) to mefenoxam in vitro was investigated to determine if in vitro screening will accurately predict the response of mefenoxam-treated cucurbit plants to infection by P. capsici. Isolates were recovered from a cross between IS parents or from naturally infected cucumbers, squash, pumpkins, and peppers in Michigan, New York, and North Carolina. Pumpkin or yellow squash seedlings were treated with water or mefenoxam at either 19.17 µg/ml or 57.51 µg/ml 24 h prior to inoculation. Four days after inoculation, all of the isolates of P. capsici exhibited similarly high virulence on the water-treated seedlings. The diameter of the lesions produced by S, IS, and I isolates on mefenoxam-treated seedlings ranged from no visible lesions for S isolates to lesions indistinguishable from those produced on water-treated plants for I isolates. These findings are consistent with the expectations based on in vitro screening and suggest that in vitro screening of mefenoxam sensitivity using a single high rate of mefenoxam (100 ppm) provides useful information for predicting the response of natural populations of P. capsici to mefenoxam.

Effect of Crop Rotation on the Survival of Phytophthora capsici in Michigan.

Phytophthora capsici is a limiting factor for some vegetable producers in Michigan despite crop rotation and fungicide applications. Our objective was to assess the effect of crop rotation on the survival of P. capsici at a naturally infested site in Michigan planted to cucumbers in 1998, corn in 1999 and 2000, and tomatoes in 2001. Isolates were characterized for compatibility type (CT), mefenoxam sensitivity, and amplified fragment length polymorphism (AFLP) marker profiles, and compared with isolates recovered from other locations in Michigan during the same time period. The A1:A2 CT ratio was 1:1, and approximately 60% of the isolates recovered in both years were intermediately sensitive or insensitive to mefenoxam. The majority of the isolates (89%) had unique AFLP fingerprints, and no members of the same clonal lineage were recovered among years. Isolates from this location were more similar to each other than to isolates from other locations in Michigan, and it was not possible to distinguish isolates based on the year of isolation. Genetic similarity analyses indicate that isolates from this location are part of a genetically distinct outcrossing population. These data indicate that P. capsici persisted as oospores for the 2 years between cucumbers and tomatoes, and that crop rotation and mefenoxam are not likely to provide economic control.

Etiology of Phytophthora drechsleri and P. nicotianae (=P. parasitica) Diseases Affecting Floriculture Crops.

Phytophthora nicotianae and P. drechsleri isolates (n = 413) recovered from eight floricultural hosts at 11 different production sites were described according to compatibility type, resistance to mefenoxam, and amplified fragment length polymorphism (AFLP) profiles. Sample sizes ranged from 2 to 120. In all cases, isolates recovered from a single facility had the same compatibility type and resistance to mefenoxam. AFLP analysis indicated that six clonal lineages of P. nicotianae and two clonal lineages of P. drechsleri were responsible for the 11 epidemics and that isolates recovered from the same facility were identical. A single clone of P. nicotianae was recovered from snapdragons at two field production sites in the southeastern United States receiving seedlings from the same source. This clone persisted at one site from 2000 to 2001. Another clone was recovered from verbena at three separate greenhouse facilities where one facility was supplying verbena to the other two. These results suggest that asexual reproduction of these pathogens plays an important role in epidemics and spread may occur between distant facilities via movement of plants.

The Dynamics of Mefenoxam Insensitivity in a Recombining Population of Phytophthora capsici Characterized with Amplified Fragment Length Polymorphism Markers.

ABSTRACT Recent findings from Michigan suggest that recombination may play a role in the survival and evolution of sensitivity to the fungicide mefenoxam in populations of Phytophthora capsici on cucurbit hosts. In 1998, 63 mefenoxam insensitive isolates were recovered from a squash field in which mefenoxam had been applied. Additional isolates were recovered from untreated squash fields planted at this location in 1999 (200 isolates) and the spring of 2000 (34 isolates). Isolates from 1998 and 1999 were characterized using fluorescent amplified fragment length polymorphism (AFLP) markers and all isolates were screened for compatibility type and mefenoxam sensitivity. In 1998 and 1999, 92 and 71% of the isolates, respectively, had unique multilocus AFLP genotypes with no identical isolates recovered between years. Seventy-two identical AFLP markers were clearly resolved in both the 1998 and 1999 sample sets, and fixation indices for the 37 polymorphic AFLP loci indicate little differentiation between years. There was no decrease in the frequency of resistant isolates during the 2 years without mefenoxam selection. We conclude that oospores play a key role in overwintering and that the frequency of mefenoxam insensitivity may not decrease in an agriculturally significant time period (2 years) once mefenoxam selection pressure is removed.

Investigating the Spatiotemporal Genetic Structure of Phytophthora capsici in Michigan.

ABSTRACT Phytophthora capsici isolates were recovered from pepper and cucurbit hosts at seven locations in Michigan from 1998 to 2000. Isolates were characterized for compatibility type (CT), mefenoxam sensitivity (MS), and amplified fragment length polymorphism (AFLP) marker profiles. In total, 94 AFLP bands were resolved. Individual populations were highly variable. Within populations, 39 to 49% of the AFLP bands were polymorphic and estimated heterozygosities ranged from 0.16 to 0.19. Of the 646 isolates fingerprinted, 70% (454) had unique AFLP profiles. No clones were recovered between years or locations. Pairwise F statistics (Phi(ST)) between populations from different locations ranged from 0.18 to 0.40. A tree based on unweighted pair-group method with arithmetic average cluster analysis indicates discrete clusters based on location. Isolates from the same location showed no clustering based on the year of sampling. Analysis of molecular variance partitioned variability among (40%) and within populations (60%). The overall estimated Phi(ST) was 0.34 (SD = 0.03). A1/A2 CT ratios were approximately 1:1, and MS frequencies were similar between years for the two locations sampled over time. These data suggest that P. capsici persists in discrete outcrossing populations and that gene flow among locations in Michigan is infrequent.