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.

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.

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.