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.

Incidence and Severity of Cavity Spot of Carrot as Affected by Pigmentation, Temperature, and Rainfall.

Field trials to determine the effect of carrot pigmentation and weather parameters on cavity spot (CS) of carrot were conducted in the Holland/ Bradford Marsh region of Ontario between 2002 and 2009. In all, 23 colored carrot cultivars from the United States Department of Agriculture (USDA) Agricultural Research Service breeding program at the University of Wisconsin (n = 5) and commercial seed companies (n = 18) were seeded in organic soil (pH 6 to 7, 45 to 75% organic matter) in late May to early June and harvested in late October or early November. Carrot roots were assessed for CS severity midseason and postharvest. Evaluations postharvest indicated that the purple pigmented carrot from breeding line 'USDA 106-3' and cultivars 'Purple Rain' and 'Purple Haze' consistently had low CS severity. The orange-pigmented 'USDA 101-23', 'Cellobunch', 'YaYa', and 'Envy' had moderate CS; and the red-pigmented carrot breeding line 'USDA 104-3' and cultivars 'Atomic Red', 'Proline Red', 'Dragon', and an unnamed line from India had high CS. Differences in CS severity in carrot cultivars between evaluations at midseason and postharvest suggest that some carrot cultivars are more susceptible to Pythium spp. inoculum in soil (alloinfection) and others to secondary infection (autoinfection) that can be attributed to the Pythium sp. involved in CS. CS severity was positively correlated with total rainfall 2 and 3 months after seeding, and was negatively correlated with number of days with air temperature ≥30°C 3 and 4 months after seeding. Soil temperature and total rainfall were the best predictors of CS incidence and severity. These results could allow a forecast of disease incidence and severity at harvest.

First Report of Downy Mildew Caused by Peronospora belbahrii on Basil (Ocimum spp.) in Ontario.

Basil (Ocimum spp.) is one of the most commercially significant fresh culinary herb crops worldwide. In Ontario, basil is grown both in the field and in the greenhouse. In the summer of 2011, basil plants grown in a research field at the Simcoe Research Station in Norfolk County, Ontario, Canada (44°15'N, 77°35'W), were infected with downy mildew. Infected leaves exhibited interveinal chlorotic lesions on the upper surface and clear to black sporulation on the abaxial leaf surfaces. Leaf senescence and defoliation occurred at high disease severity, which reduced marketable yield. Basil downy mildew symptoms were severe on leaves of cultivars Genovese and Sweet Basil, with 40 to 100% disease incidence. Based on morphological characteristics, the basil downy mildew causal agent was identified as Peronospora belbahrii Thines (4). Infected leaves were collected and microscopic observations of the sporulating lesions were carried out and the structures measured. Sporangiophores (n = 20) were hyaline with relatively long, straight trunks and were monopodially branched, with a length of 150 to 360 µm (average 285 µm). Sporangiophores ended with two slightly curved branchlets, the longer one measuring 15 to 27 µm (average 19 µm) and the shorter one 5 to 15 µm (average 9 µm). Sporangia (n = 50) were round, or slightly ovoid, olive to brown in color, and measured 29 × 25 µm (25 to 35 × 20 to 30 µm). Genomic DNA was extracted from 10 isolates and the nuclear ribosomal internal transcribed spacer (ITS) region was amplified with ITS4 and ITS5 primers and sequenced. The sequences of the 10 isolates were nearly identical. A BLAST search of the NCBI database with the ITS sequences (GenBank Accession No. KC756923) revealed a 98 to 100% similarity to the sequences of P. belbahrii (HQ730979, FJ436024, and HQ702191) isolated from sweet basil in Florida (3), California (1), and Hungary (2), respectively. To confirm pathogenicity, 5-week-old 'Genovese' seedlings were sprayed with a suspension of 1 × 105 sporangia/ml. Plants were kept in a growth chamber maintained at 23/18°C, 60 to 85% relative humidity, and 12/12 h light/dark. Non-inoculated plants served as controls. Basil downy mildew symptoms developed after 8 days on the inoculated plants and the pathogen was identified in association with symptoms consistent with downy mildew. The non-inoculated controls remained healthy. In North America, the occurrence of basil downy mildew has been reported since 2007 (3) and the disease has spread into several U.S. states. To our knowledge, this is the first report of downy mildew on sweet basil in Canada. References: (1) C. L. Blomquist et al. Plant Dis. 93:968, 2009. (2) G. Nagy and A. Horvath. Plant Dis. 95:1034, 2011. (3) P. D. Roberts et al. Plant Dis. 98:199, 2009. (4) M. Thines et al. Mycol. Res. 113:532, 2009.

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.

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.

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.

First Report of Black Rot of Carrots Caused by Alternaria radicina in Michigan.

In April 2005, an Alternaria sp. was isolated from carrot (Daucus carota) roots harvested in the fall of 2004 and held at 1 to 3°C in a storage facility in Newaygo County, MI. The pathogen was readily isolated on water agar from root tissue exhibiting grayish black, sunken lesions. Morphological characteristics were noted 5 to 7 days after single-conidium cultures were established on potato dextrose agar (3). Sixteen Alternaria sp. isolates were recovered. Cultures were dark olive brown, and conidia were pigmented, ellipsoidal, and produced singly or in chains of two. Conidia were 35 to 45 µm long and 15 to18 µm in diameter, usually with three to eight transverse and one to four longitudinal septa. Pathogenicity of isolates was tested on carrot roots in the laboratory and carrot seedlings (cv. Goliath) in the greenhouse. In the laboratory, four surface-sterilized, whole carrot roots were sprayed until runoff with 2 × 106 conidia/ml of each isolate and incubated at 23 to 25°C in a moist chamber for 10 days. Controls were sprayed with sterile distilled water. Ten to fifteen days after inoculation, inoculated carrots exhibited grayish black, sunken lesions, and an Alternaria sp. was reisolated from the margin of the lesions. Controls remained healthy. In the greenhouse, seven pots containing one 2-week-old carrot seedling were watered to saturation and plants were sprayed until runoff with 2 × 106 conidia/ml for each isolate. Control plants were sprayed with sterile distilled water. After inoculation, plants were enclosed in clear plastic bags, placed under 63% woven shade cloth and watered regularly. Black lesions were observed on the foliage 7 days after inoculation, and wilt and death of plants were observed 15 to 30 days after inoculation. Alternaria sp. was reisolated from the foliage of symptomatic plants. Control plants remained healthy. DNA was extracted from all isolates, and the nuclear ribosomal internal transcribed spacer (ITS) region amplified with primers ITS4 and ITS5 and sequenced. A portion of the ITS sequence has been deposited in the NCBI database (GenBank Accession No. DQ394073). A BLAST search of the NCBI database with the ITS sequences revealed A. radicina, Accession No AY154704, as the closest match with 100% sequence similarity. In September 2005, an Alternaria sp. was isolated from black lesions on carrot roots, crowns, and foliage that were collected from fields in Newaygo and Oceana counties, MI. The recovered isolates were morphologically similar to A. radicina isolates obtained from stored carrots in April 2005. First isolated and identified on stored carrots in New York (3), A. radicina is also present in other carrot-producing areas of the United States (1) and was isolated not only from stored carrots but also from carrots in the field (2) and carrot seeds (4). To our knowledge, this is the first report of A. radicina on stored and field carrots in Michigan, which signifies a serious risk to a carrot industry that ranks among the top five in the United States. References: (1) D. F. Farr et al. Fungi on Plants and Plant Produce in the United States.The American Phytopathological Society, St. Paul, MN, 1989. (2) R. G. Grogan and W. C. Snyder. Phytopathology 42:215, 1952. (3) F. C. Meier and E. D. Eddy. Phytopathology 12:157, 1922. (4) B. M. Pryor and R. L. Gilbertson. Plant Dis. 85:18, 2001.

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.