Avirulence (AVR) Gene-Based Diagnosis Complements Existing Pathogen Surveillance Tools for Effective Deployment of Resistance (R) Genes Against Rice Blast Disease.

Avirulence (AVR) genes in Magnaporthe oryzae, the fungal pathogen that causes the devastating rice blast disease, have been documented to be major targets subject to mutations to avoid recognition by resistance (R) genes. In this study, an AVR-gene-based diagnosis tool for determining the virulence spectrum of a rice blast pathogen population was developed and validated. A set of 77 single-spore field isolates was subjected to pathotype analysis using differential lines, each containing a single R gene, and classified into 20 virulent pathotypes, except for 4 isolates that lost pathogenicity. In all, 10 differential lines showed low frequency (<24%) of resistance whereas 8 lines showed a high frequency (>95%), inferring the effectiveness of R genes present in the respective differential lines. In addition, the haplotypes of seven AVR genes were determined by polymerase chain reaction amplification and sequencing, if applicable. The calculated frequency of different AVR genes displayed significant variations in the population. AVRPiz-t and AVR-Pii were detected in 100 and 84.9% of the isolates, respectively. Five AVR genes such as AVR-Pik-D (20.5%) and AVR-Pik-E (1.4%), AVRPiz-t (2.7%), AVR-Pita (0%), AVR-Pia (0%), and AVR1-CO39 (0%) displayed low or even zero frequency. The frequency of AVR genes correlated almost perfectly with the resistance frequency of the cognate R genes in differential lines, except for International Rice Research Institute-bred blast-resistant lines IRBLzt-T, IRBLta-K1, and IRBLkp-K60. Both genetic analysis and molecular marker validation revealed an additional R gene, most likely Pi19 or its allele, in these three differential lines. This can explain the spuriously higher resistance frequency of each target R gene based on conventional pathotyping. This study demonstrates that AVR-gene-based diagnosis provides a precise, R-gene-specific, and differential line-free assessment method that can be used for determining the virulence spectrum of a rice blast pathogen population and for predicting the effectiveness of target R genes in rice varieties.

Assessment of the Virulence Spectrum and Its Association with Genetic Diversity in Magnaporthe oryzae Populations from Sub-Saharan Africa.

A collection of 122 isolates of Magnaporthe oryzae, from nine sub-Saharan African countries, was assessed for virulence diversity and genetic relatedness. The virulence spectrum was assessed by pathotype analysis with a panel of 43 rice genotypes consisting of differential lines carrying 24 blast resistance genes (R-genes), contemporary African rice cultivars, and susceptible checks. The virulence spectrum among isolates ranged from 5 to 80%. Five isolates were avirulent to the entire rice panel, while two isolates were virulent to ∼75% of the panel. Overall, cultivar 75-1-127, the Pi9 R-gene donor, was resistant to all isolates (100%), followed by four African rice cultivars (AR105, NERICA 15, 96%; NERICA 4, 91%; and F6-36, 90%). Genetic relatedness of isolates was assessed by single nucleotide polymorphisms derived from genotyping-by-sequencing and by vegetative compatibility tests. Phylogenetic analysis of SNPs of a subset of isolates (n = 78) revealed seven distinct clades that differed in virulence. Principal component analysis showed isolates from East Africa were genetically distinct from those from West Africa. Vegetative compatibility tests of a subset of isolates (n = 65) showed no common groups among countries. This study shows that blast disease could be controlled by pyramiding of Pi9 together with other promising R-genes into rice cultivars that are adapted to East and West African regions.

Multiplex real-time PCR assays for detection of four seedborne spinach pathogens.

AIMS: To develop multiplex TaqMan real-time PCR assays for detection of spinach seedborne pathogens that cause economically important diseases on spinach. METHODS AND RESULTS: Primers and probes were designed from conserved sequences of the internal transcribed spacer (for Peronospora farinosa f. sp. spinaciae and Stemphylium botryosum), the intergenic spacer (for Verticillium dahliae) and the elongation factor 1 alpha (for Cladosporium variabile) regions of DNA. The TaqMan assays were tested on DNA extracted from numerous isolates of the four target pathogens, as well as a wide range of nontarget, related fungi or oomycetes and numerous saprophytes commonly found on spinach seed. Multiplex real-time PCR assays were evaluated by detecting two or three target pathogens simultaneously. Singular and multiplex real-time PCR assays were also applied to DNA extracted from bulked seed and single spinach seed. CONCLUSIONS: The real-time PCR assays were species-specific and sensitive. Singular or multiplex real-time PCR assays could detect target pathogens from both bulked seed samples as well as single spinach seed. SIGNIFICANCE AND IMPACT OF THE STUDY: The freeze-blotter assay that is currently routinely used in the spinach seed industry to detect and quantify three fungal seedborne pathogens of spinach (C. variabile, S. botryosum and V. dahliae) is quite laborious and takes several weeks to process. The real-time PCR assays developed in this study are more sensitive and can be completed in a single day. As the assays can be applied easily for routine seed inspections, these tools could be very useful to the spinach seed industry.

First Report of Peronospora farinosa f. sp. spinaciae Causing Downy Mildew on Spinach in Egypt.

Downy mildew, caused by Peronospora farinosa f. sp. spinaciae (= P. effusa) is an economically important disease in most areas where spinach is grown. This disease has become increasingly important in intensive production fields for pre-packaged salad mixes where plant densities typically are very high (2). However, little is known about race diversity of the downy mildew pathogen of spinach in smaller (<1 ha) production areas. Small (~0.1 ha) spinach production fields in Fayoum, Egypt, often intercropped with lettuce, were examined in February 2013. Downy mildew was observed in three spinach fields in the Fayoum area. Most of the cultivars being grown were traditional cultivars commonly grown from locally produced open pollinated seed. Disease incidence was relatively low with only about 10% of the plants showing symptoms of infection. Symptoms of downy mildew were observed on the cultivar Meky, and included chlorotic spots with blue-gray sporulation on the underside of the symptomatic leaves. Microscopic examination revealed sporangia, measuring 20.2 × 30.5 µm, and monopodial sporangiaphores of 180 to 330 µm length matching the description of P. farinosa f. sp. spinaciae (1). In addition, the pathogen was identified by examination of the nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) sequence, which had 100% identity to a 762-bp ITS sequence in GenBank of P. farinosa f. sp. spinaciae (Accession No. DQ643879.1). The Fayoum area of Egypt gets relatively low annual rainfall, typically <10 to 15 cm annually, often concentrated in the winter months of November to February, followed by very hot, dry summer months. Although downy mildew of spinach has been reported in Israel, adjacent to Egypt, the disease apparently is relatively rare in the arid Middle East (3). This is the first known report of downy mildew of spinach in Egypt. References: (1) Y. Choi et al. Mycol. Res. 111:318, 2007. (2) J. C. Correll et al. Eur. J. Plant Pathol. 2011. (3) T. Rayss. Palest. J. Bot. Jerusalem Ser. 1:313, 1938.

Confirming QTLs and Finding Additional Loci Responsible for Resistance to Rice Sheath Blight Disease.

Rice sheath blight disease, caused by Rhizoctonia solani AG1-1A, is one of the most destructive rice diseases worldwide. Utilization of host resistance is the most economical and environmentally sound strategy in managing sheath blight (ShB). Ten ShB quantitative trait loci (QTLs) were previously mapped in a Lemont × Jasmine 85 recombinant inbred line (LJRIL) population using greenhouse inoculation methods at an early vegetative stage. However, confirmation of ShB-resistant QTLs under field conditions is critical for their utilization in marker-assisted selection (MAS) for improving ShB resistance in new cultivars. In the present study, we evaluated ShB resistance using 216 LJRILs under field conditions in Arkansas, Texas, and Louisiana during 2008 and 2009. We confirmed the presence of the major ShB-QTL qShB9-2 based on the field data and also identified one new ShB-QTL between markers RM221 and RM112 on chromosome 2 across all three locations. Based on the field verification of ShB evaluations, the microchamber and mist-chamber assays were simple, effective, and reliable methods to identify major ShB-QTLs like qShB9-2 in the greenhouse at early vegetative stages. The markers RM215 and RM245 were found to be closely linked to qShB9-2 in greenhouse and field assays, indicating that they will be useful for improving ShB resistance in rice breeding programs using MAS.

First Report of Twig Canker on Willow Caused by Colletotrichum acutatum in California.

Following prolonged spring rains and cool summer weather in 2010, mature weeping willow trees (Salix babylonica L.) growing next to a manmade lake in Marin County, CA, showed symptoms of a previously undescribed disease. During summer, small branches developed dark brown to black, sunken cankers. Canker lengths ranged from 3 to 20 cm. Within the cankered areas, affected twigs, shoots, and leaves turned brown, collapsed, and died. The distal portions of infected branches also died, giving the trees a blighted appearance. Acervuli and pink sporulation were observed in the canker tissue. When placed on acidified potato dextrose agar (A-PDA), canker tissues consistently yielded one type of fungal organism. On A-PDA, isolates produced gray aerial mycelium, acervuli, and single-celled fusiform conidia. Two isolates were identified as Colletotrichum acutatum based on sequence analysis of the ITS region of the ribosomal DNA and the 1-kb intron of the glutamine synthase gene (1) and fungal morphology (2,3) (GenBank Accession Nos. JQ951597 and JQ951598). The willow isolates examined were identified as C. acutatum based on a 99% identity of the ITS sequence with accession FR716517 and a 98% identity of the 1-kb intron sequence with accession GQ387248 in GenBank. Interestingly, the isolates were confirmed to be homothallic producing perithecia from monoconidial cultures. To demonstrate Koch's postulates, inocula were prepared from 2-week-old colonies of each of four isolates grown on A-PDA. Using containerized weeping willow trees as test material, shallow slits were cut into the epidermis of small (1.5-cm diameter or less) branches; one colonized agar plug was placed within each cut area and the epidermis was resealed by wrapping the branch with Parafilm. Ten inoculations were made for each isolate and inoculated plants were maintained in a greenhouse. After 4 weeks, inoculated branches exhibited dark cankers and twig dieback. C. acutatum was reisolated from all symptomatic cankers and matched the characteristics of the original isolates. Control twigs, inoculated with sterile agar plugs, did not develop any blight symptoms. This experiment was repeated and the results were the same. To our knowledge, this is the first documentation of C. acutatum as a pathogen of weeping willow in California. The disease resulted in repeated defoliation of trees in the Santa Venetia area of Marin County. Badly infected trees declined as a result of repeated defoliation and twig loss. Discussions with parks personnel suggested that the disease may have been present at low levels in the area for some years, and that disease severity increased dramatically with weather that was atypically wet and cool (max. mean temps. 5.5°C cooler and 6 cm more total rainfall than the records of the previous two years) for the area during May and June 2010, when the disease was discovered. References: (1) J. C. Guerber et al. Mycologia 95:872, 2003. (2) P. S. Gunnell and W. D. Gubler. Mycologia 84:157, 1992. (3) B. J. Smith and L. L. Black. Plant Dis. 74:69, 1990.

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.

First Report of Colletotrichum coccodes Causing Leaf and Neck Anthracnose on Onions (Allium cepa) in Michigan and the United States.

In July of 2010, dry, oval lesions, each with a salmon-colored center and bleached overall appearance, were observed on the leaves and neck of onions plants growing in production fields of Newaygo, Ottawa, Kent, and Ionia counties, Michigan. Acervuli and setae that are characteristic of Colletotrichum spp. were observed with a dissecting microscope, and elliptical conidia (8 to 23 × 3 to 12 µm) with attenuated ends were observed with a compound microscope. Symptomatic tissues were excised and cultured onto potato dextrose agar amended with 30 and 100 ppm of rifampicin and ampicillin, respectively. The cultures produced pale salmon-colored sporulation after growing for 5 days at 22 ± 2°C and black microsclerotia after 2 weeks. Six isolates were confirmed as C. coccodes based on sequence analysis of the internal transcribed (ITS) region of the ribosomal DNA and a 1-kb intron of the glutamine synthase gene (GS) (2). Sequences were submitted to GenBank (Accession Nos. JQ682644 and JQ682645 for ITS and GS, respectively). Pathogenicity tests were conducted on two- to three-leaved 'Stanley' and 'Cortland' onion seedlings. Prior to inoculation, seedlings were enclosed in clear plastic bags overnight to provide high relative humidity. The bags were removed, and seedlings were sprayed inoculated with a C. coccodes conidial suspension (5 × 105 conidia/ml and 25 ml/plant) in sterile double-distilled water. Control seedlings were sprayed with sterile double-distilled water. Tween (0.01%) was added to the conidial suspension and the water. Plants were enclosed in bags for 72 h postinoculation and incubated in growth chambers at 28°C day/23°C night with a 12-h photoperiod. Sunken, oval lesions were observed on the foliage of the onion seedlings inoculated with C. coccodes 4 days postinoculation. Lesions coalesced and foliage collapsed 7 days postinoculation. Control plants remained asymptomatic. When five leaf samples per replication were detached and incubated in a moist chamber for 3 days at 21 ± 2°C, abundant acervuli and setae were observed on the symptomatic tissue but not on control tissue. C. coccodes was consistently recovered from the onion seedling lesions. Six different Colletotrichum spp. have been reported to cause diseases on onions worldwide (1,4). C. circinans, which causes smudge, is an occasional onion pathogen in Michigan, while C. gloeosporioides has only been reported to be infecting onions in Georgia (3). To our knowledge, this is the first report of C. coccodes infecting and causing disease in onions plants. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , August 6, 2010. (2) J. C. Guerber et al. Mycologia 95:872. 2003. (3) C. Nischwitz et al. Plant Dis. 92:974. 2008. (4) H. F. Schwartz, and K. S. Mohan. Compendium of Onion and Garlic Diseases and Pests, 2nd ed. The American Phytopathological Society, St. Paul, MN. 1995.

Characterization of White Rust of Perennial Pepperweed Caused by Albugo candida in California.

In California, perennial pepperweed (Lepidium latifolium) is an introduced Brassicaceae plant that is invasive, highly competitive, and listed as a noxious weed that grows in areas such as marshes, meadows, roadsides, and irrigation ditches. From 2008 through 2010, perennial pepperweed growing near farms in Monterey and Santa Clara counties was infected with white rust. Symptoms were light green-to-chlorotic spots on adaxial leaf surfaces and corresponding white, blister-like sori growing underneath the raised leaf epidermis on the abaxial surface. Sporangia were collected from lesions and used for DNA extraction. The internal transcribed spacer (ITS) region was amplified with primers ITS1/ITS4 and sequenced. The sequence matched with Albugo candida by BLAST against GenBank. On the basis of morphological and molecular data, the pathogen was confirmed to be A. candida. Pathogenicity was tested by scraping sporangia from infected leaves and spraying a suspension (1 × 105 sporangia/ml) onto pepperweed seedlings grown in pots. Plants were placed in an incubator at 100% relative humidity and 12°C for 48 h to induce zoospore release. Plants were subsequently maintained in a greenhouse. After 15 to 17 days, inoculated plants developed white rust symptoms and signs. Control plants sprayed with water did not become diseased. The experiment was completed two times with the same results. To determine the race of A. candida from perennial pepperweed, 4- to 5-week-old plants and 1- to 2-week-old seedlings of differential hosts (1-4) were inoculated in a similar fashion. The differential hosts were the following: Raphanus sativus (race 1), Brassica juncea cv. Burgonde (race 2A), B. juncea cv. Cutlass (race 2V), Armoracia rusticana (race 3), Capsella bursa-pastoris (race 4), Sisymbrium officinale (race 5), Rorippa islandica (race 6), B. rapa (B. campestris) cv. Torch (race 7A), B. rapa cvs. Reward, Cutlass, and AC Parkland (race 7V), B. nigra (race 8), B. oleracea (race 9), Sinapis alba (race 10), B. carinata (race 11), and perennial pepperweed as a control. White rust developed on pepperweed 10 to 14 days later but was not found on any of the differential hosts, indicating that this pathogen is not one of the currently described 11 races. The following commercial crop species were inoculated using the same method: arugula (Eruca sativa), Japanese mustard (B. campestris subsp. nipposinica), red mustard (B. juncea subsp. rugosa), tah tsai (B. campestris subsp. narinosa), cauliflower (B. oleracea subsp. botrytis), Chinese cabbage (B. campestris subsp. pekinensis), bok choy (B. rapa Chinensis group), broccoli raab (B. rapa subsp. rapa), and perennial pepperweed as a control. Only the perennial pepperweed developed white rust. To our knowledge, this is the first characterization of A. candida infecting perennial pepperweed in California. The disease has been documented on this plant in Colorado and also in Bulgaria, Portugal, and Spain. The host range information is important to growers because it indicates that the race currently infecting perennial pepperweed will not infect commercial crucifers. References: (1) P. A. Delwich and P. H. Williams. Cruciferae Newsl. 2:39, 1977. (2) C. B. Hill et al. Cruciferae Newsl. 13:112, 1988. (3) S. R. Rimmer et al. Can. J. Plant Pathol. 22:229, 2000. (4) P. R. Verma et al. Can. J. Bot. 53:1016, 1975.

Mapping quantitative trait Loci responsible for resistance to sheath blight in rice.

Rice sheath blight (ShB), caused by the soilborne pathogen Rhizoctonia solani, annually causes severe losses in yield and quality in many rice production areas worldwide. Jasmine 85 is an indica cultivar that has proven to have a high level of resistance to this pathogen. The objective of this study was to determine the ability of controlled environment inoculation assays to detect ShB resistance quantitative trait loci (QTLs) in a cross derived from the susceptible cv. Lemont and the resistant cv. Jasmine 85. The disease reactions of 250 F(5) recombinant inbred lines (RILs) were measured on the seedlings inoculated using microchamber and mist-chamber assays under greenhouse conditions. In total, 10 ShB-QTLs were identified on chromosomes 1, 2, 3, 5, 6, and 9 using these two methods. The microchamber method identified four of five new ShB-QTLs, one on each of chromosomes 1, 3, 5, and 6. Both microchamber and mist-chamber methods identified two ShB-QTLs, qShB1 and qShB9-2. Four of the ShB-QTLs or ShB-QTL regions identified on chromosomes 2, 3, and 9 were previously reported in the literature. The major ShB-QTL qShB9-2, which cosegregated with simple sequence repeat (SSR) marker RM245 on chromosome 9, contributed to 24.3 and 27.2% of total phenotypic variation in ShB using microchamber and mistchamber assays, respectively. qShB9-2, a plant-stage-independent QTL, was also verified in nine haplotypes of 10 resistant Lemont/Jasmine 85 RILs using haplotype analysis. These results suggest that multiple ShB-QTLs are involved in ShB resistance and that microchamber and mist-chamber methods are effective for detecting plant-stage-independent QTLs. Furthermore, two SSR markers, RM215 and RM245, are robust markers and can be used in marker-assisted breeding programs to improve ShB resistance.

Anthracnose of Sunflower Sprouts Caused by Colletotrichum acutatum in California.

In California, sunflower (Helianthus annuus) is commercially grown and marketed as edible, germinated sprouts. Sunflower seeds are soaked overnight in water, drained, and seeded into a compost-based medium in flats. The flats are incubated for 3 days in low-light conditions until roots develop. Flats are then moved under filtered shade, with air circulation provided by fans, for 3 to 5 days. The germinated seedlings are finally grown in full sunlight for another 3 to 7 days and are then ready for market. Such sprouts can be consumed raw or cooked. In 2009, in Placer County, CA, a previously undescribed disease occurred on commercial organic sunflower sprouts. Symptoms were dark brown, irregularly shaped lesions on seedling cotyledons and stems. Fungal fruiting structures were not observed in diseased tissues. Isolations were carried out on symptomatic seedlings that were surface sanitized in 1.2% NaOCl and then rinsed in sterile distilled water (SDW). When placed on acidified potato dextrose agar (A-PDA), tissues consistently yielded one type of fungal organism. On A-PDA, isolates produced dark gray, aerial mycelium, acervuli, and single-celled fusiform conidia. Eight isolates were examined for sequence variation in the internal transcribed spacer (ITS) region and the intron in the glutamine synthetase (GS) gene (2). On the basis of spore morphology and a complete match (100%) of the ITS sequence and the GS intron sequence, all eight isolates were identified as Colletotrichum acutatum subgroup C1 represented by isolate ATCC MYA-662 (2). Six isolates were each tested for pathogenicity on sunflower by spraying a spore suspension (1 × 106 conidia/ml) onto sets (six-pack trays; one plant per cell) of plants at either cotyledon or first true-leaf stage. Each plant set was sprayed with approximately 5 to 8 ml of inoculum. Plants were incubated for 48 h in a humidity chamber and then maintained in a greenhouse. Control sunflower plants were sprayed with distilled water and otherwise treated similarly. After 14 days, inoculated cotyledons and true leaves developed lesions similar to those observed on commercial samples; C. acutatum was reisolated from these lesions. Control plants developed no symptoms. The experiment was repeated and the results were the same. To test for possible seedborne C. acutatum, sunflower seeds of the same commercial seed lot used to plant the affected crop were surface sanitized (1.2 % NaOCl for 60 s), rinsed in SDW, placed in incubation boxes (10.2 × 10.2 cm, clear acrylic, Hoffman Manufacturing, Inc., Jefferson, OR), and then processed according to a previously described freeze blotter assay (1). After testing 10 replications of 100 seeds each, no C. acutatum was detected. To our knowledge, this is the first report of anthracnose of sunflower sprouts in California and the first report of a sunflower disease caused by C. acutatum. This sprout production environment, consisting of high humidity, prolonged periods of high moisture, and low light, likely favored the disease. For the affected commercial sprouts crops, disease reached as high as 25% incidence and continues to be an intermittent problem. References: (1) L. J. du Toit et al. Plant Dis. 89:4, 2005. (2) J. C. Guerber et al. Mycologia 95:872, 2003.

First Report of Fruit Rot of Pumpkin Caused by Fusarium solani f. sp. cucurbitae in Arkansas.

During 2008, fruit rot of pumpkin (Cucurbita pepo L.) occurred on several cultivars in commercial fields in northeast and northwest Arkansas. Disease incidence ranged from 50 to 75% of the fruit, which were unmarketable. Symptoms included large (>10 cm), brown, corky lesions where the fruit was in contact with the soil. Initially, the lesions were water soaked. A cross section of the symptomatic fruit rind revealed a dry, brown, spongy rot with a light brown halo. Lesions finally became soft and wet, causing infected fruit to collapse. Masses of white mycelia surrounded advanced lesions. No rot symptoms were observed on the vines. Fusarium spp. were isolated from symptomatic fruit. Macroconidia obtained from field-infected fruit and pure potato dextrose agar (PDA) cultures of the predominant Fusarium sp. were morphologically similar. The straight, cylindrical, and robust macroconidia contained between five and seven septa. The apical cell was rounded and blunt and the basal cell was rounded. All three morphological types were tested for pathogenicity on mature fruit of cv. Sorcerer. Fruit were surface disinfected in 70% ethanol. Wounds were made (4 mm deep) in the fruit surface with a cork borer. Three wounds per isolate per fruit were inoculated with a PDA plug colonized with mycelium from a 3-day-old culture. Three replicated wounds were inoculated per isolate and four replicate fruit were used. After inoculation, the wounds were covered with saran wrap. The fruit were incubated at approximately 24°C and evaluated after 7 days. An uncolonized PDA plug was used as a negative control. After 7 days, only the predominant Fusarium sp. produced typical lesions, which were brown, water soaked, and approximately 3 cm in diameter. Fusarium spp. were recovered from the inoculated lesions. The colonies on PDA and macroconidia of the pathogenic Fusarium sp. were morphologically similar to the isolate inoculated and the ones recovered from field lesions. DNA was extracted from the same three isolates used in the pathogenicity test. A portion of the translation elongation factor 1α (TEF) gene was sequenced to verify the identity of the pathogenic isolates. On the basis of a comparison of the Fusarium-ID database at Pennsylvania State University (3), the pathogenic isolates had a 100% match with Fusarium solani f. sp. cucurbitae race 1, teleomorph Nectria haematococca mating population I, isolate NRRL 22098. F. solani f. sp cucurbitae was previously identified as the causal agent of crown and foot rot and a fruit rot of cucurbits and responsible for outbreaks on pumpkin fruit in Connecticut, Missouri, New York, and Ohio from 2001 to 2003 and again in Ohio in 2005 (2). In 2008, a higher average total of monthly precipitation was recorded late in the growing season in Arkansas, (13.7 cm in August and 23.7 cm in September). Although F. equiseti has previously been reported as a fruit rot pathogen of pumpkin in Arkansas (1), to our knowledge, this is the first report of F. solani f. sp cucurbitae as causal agent of pumpkin fruit rot in the state. Reference: (1) J. C. Correll et al. Plant Dis. 75:751, 1991. (2) W. H. Elmer et al. Plant Dis. 91:1142, 2007. (3) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004.

Characterization of a resistance locus (Pfs-1) to the spinach downy mildew pathogen (Peronospora farinosa f. sp. spinaciae) and development of a molecular marker linked to Pfs-1.

Downy mildew is a destructive disease of spinach worldwide. There have been 10 races described since 1824, six of which have been identified in the past 10 years. Race identification is based on qualitative disease reactions on a set of diverse host differentials which include open-pollinated cultivars, contemporary hybrid cultivars, and older hybrid cultivars that are no longer produced. The development of a set of near-isogenic open-pollinated spinach lines (NILs), having different resistance loci in a susceptible and otherwise common genetic background, would facilitate identification of races of the downy mildew pathogen, provide a tool to better understand the genetics of resistance, and expedite the development of molecular markers linked to these disease resistance loci. To achieve this objective, the spinach cv. Viroflay, susceptible to race 6 of Peronospora farinosa f. sp. spinaciae, was used as the recurrent susceptible parent in crosses with the hybrid spinach cv. Lion, resistant to race 6. Resistant F(1) progeny were subsequently backcrossed to Viroflay four times with selection for race 6 resistance each time. Analysis of the segregation data showed that resistance was controlled by a single dominant gene, and the resistance locus was designated Pfs-1. By bulk segregant analysis, an amplified fragment length polymorphism (AFLP) marker (E-ACT/M-CTG) linked to Pfs-1 was identified and used to develop a co-dominant Sequence characterized amplified region (SCAR) marker. This SCAR marker, designated Dm-1, was closely linked ( approximately 1.7 cM) to the Pfs-1 locus and could discriminate among spinach genotypes that were homozygous resistant (Pfs-1Pfs-1), heterozygous resistant (Pfs-1pfs-1), or homozygous susceptible (pfs-1pfs-1) to race 6 within the original mapping population. Evaluation of a wide range of commercial spinach lines outside of the mapping population indicated that Dm-1 could effectively identify Pfs-1 resistant genotypes; the Dm-1 marker correctly predicted the disease resistance phenotype in 120 out of 123 lines tested. In addition, the NIL containing the Pfs-1 locus (Pfs-1Pfs-1) was resistant to multiple races of the downy mildew pathogen indicating Pfs-1 locus may contain a cluster of resistance genes.

Severe and Widespread Clubroot Epidemics in Nepal.

Cultivation of brassica vegetables has the highest potential for generating income among more traditional rice and maize farmers in Nepal. Among brassica vegetables, the most important are cauliflower (Brassica oleracea var. botrytis L.) and cabbage (B. oleracea var. capitata L.). Although clubroot disease, caused by Plasmodiophora brassicae Woronin, has been observed in Nepal since 1993, severe and widespread epidemics have been observed since 2004 in the Bhaktapur, Kathmandu, Lalitpur, and Palung Valley production areas. Typical disease symptoms (1) are widespread, and disease severity has been particularly severe in the Kathmandu Valley and Palung/Daman area of the Makwanpur District. Many cauliflower fields in these areas have had as much as 100% yield loss between 2004 and 2006 with an estimated 40% overall loss from clubroot. Estimates from interviews with growers in the Palung production area during an intensive farmers' interaction program indicated that cauliflower production was reduced from 5 to 6 metric tons per household (1,500 m2) prior to 2004 to <300 kg per household in 2004 and beyond. The economic loss in this area alone was estimated at $1.4 million in 2004 and 2005. Examination of transplant nurseries indicated that frequently >80% of the seedlings have symptoms of clubroot at the time of transplanting. Soil samples from throughout the production areas indicated that the sandy loam soils were predominately acidic (pH range of 4.2 to 7.2 with >90% below 6.0). Several management practices are being employed to reduce disease severity, including the use of clubroot resistant cultivars, raising the soil pH to >7.0 by using dolomitic lime, testing of the fungicide flusulfamide (Nebijin) and biopesticide Sanjeevani (Trichoderma viride), and biofumigation and solarization of the nursery beds in an effort to reduce disease pressure on transplant material. References: (1) G. R. Dixon. Compendium of Brassica Diseases. S. R. Rimmer et al., eds. American Phytopathological Society, St. Paul, 2007.

Comparison of Colletotrichum orbiculare and Several Allied Colletotrichum spp. for mtDNA RFLPs, Intron RFLP and Sequence Variation, Vegetative Compatibility, and Host Specificity.

ABSTRACT Based on spore morphology, appressorium development, sequence similarities of the rDNA, and similarities in amplified restriction fragment length polymorphism (AFLP), it has been proposed that Colletotrichum orbiculare, C. trifolii, C. lindemuthianum, and C. malvarum represent a single phylogenetic species, C. orbiculare. In the current study, the phylogenetic relationship among isolates in the C. orbiculare species complex was reassessed. In all, 72 isolates of C. orbiculare from cultivated cucurbit or weed hosts, C. trifolii from alfalfa, C. lindemuthianum from green bean, and C. malvarum from prickly sida (Sida spinosa) were examined for mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs), RFLPs and sequence variation of a 900-bp intron of the glutamine synthetase gene and a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase gene, and vegetative compatibility. In addition, host specificity was examined in foliar inoculations on cucurbit, bean, and alfalfa hosts. Inoculations also were conducted on cucumber fruit. Genetically distinct isolates, based on vegetative compatibility, within the species complex (C. orbiculare, C. trifolii, and C. malvarum) had an identical mtDNA haplotype (haplotype A) when examined with each of three different restriction enzymes. Isolates of C. lindemuthianum had a very similar mtDNA haplotype to haplotype A, with a single polymorphism detected with the enzyme HaeIII. The four species represent a phylogenetically closely related group based on a statistical analysis of the 900- and 200-bp intron sequences. However, distinct RFLPs in the 900-bp intron were consistently associated with each species and could be used to qualitatively and quantitatively distinguish each species. Furthermore, each of the species showed distinct host specificity, with isolates of C. orbiculare (from cucurbits), C. lindemuthianum, and C. trifolii being pathogenic only on cucurbits, green bean, and alfalfa, respectively. Consequently, distinct and fixed nucleotide, or genotypic (intron sequences and RFLPs) and phenotypic (host specificity) characteristics can be used to distinguish C. orbiculare, C. lindemuthianum, and C. trifolii from one another; therefore, they should be recognized as distinct species. This species delineation is consistent with the most current species concepts in fungi. More isolates and further characterization is needed to determine whether C. orbiculare from cocklebur and C. malvarum represent distinct species. RFLPs of the 900-bp intron may represent a relatively inexpensive, reliable, and useful diagnostic tool for general species differentiation in the genus Colletotrichum.

First Report of Race 8 of Downy Mildew, Caused by Peronospora farinosa f. sp. spinaciae, of Spinach in the United States.

Downy mildew, caused by Peronospora farinosa f. sp. spinaciae, is the most economically important disease of spinach (Spinacia oleracea) in the United States and the European Union. In the United States, 23,000 ha of spinach, with a crop value of approximately $170 million, were grown during 2005 (1; http://www.nass.usda.gov/index.asp ). Additionally, per capita, fresh-market spinach consumption has increased 214% in the past decade (1; http://www.nass.usda.gov/index.asp ). Increased demand for fresh-market spinach has led to changes in spinach production practices such as higher planting densities and year-round production. There are currently 10 described races (races 1 to 10) of P. farinosa f. sp. spinaciae. Race 8 was recovered from the Netherlands in 2004 (B. M. Irish, J. Correll, S. T. Koike, and T. Morelock. Plant Dis. [In press]), but has not been previously identified in the United States. In February 2007, several commercial fresh-market spinach fields in central Arizona were severely affected with downy mildew. Symptoms consisted of bright yellow leaf lesions ranging in size from 1 to 3 cm in diameter that supported dense purple sporulation of the pathogen on the corresponding abaxial leaf surface. Affected fields were primarily planted with spinach cv. Parrot, which is reported to be resistant to races 1 to 7 and 9. As much as 32 ha were affected and disease incidence reached as high as 25 to 30%. An isolate (PAR1) of the pathogen was obtained and used to inoculate a standard set of 10 differential spinach cultivars for race identification as previously described (B. M. Irish, J. Correll, S. T. Koike, and T. Morelock. Plant Dis. [In press]). Briefly, a spore suspension (1 × 105 sporangia per ml) was misted onto test plants; plants were then incubated in a dew chamber (20°C, 100% relative humidity) for 24 h and maintained in a greenhouse. Inoculation tests were conducted at least twice at each of two different locations (Arkansas and California), with each test including two replications of 15 plants per differential cultivar. The selective development of downy mildew on specific differentials indicated that the isolate was race 8 (B. M. Irish, J. Correll, S. T. Koike, and T. Morelock. Plant Dis. [In press]). To our knowledge, this is the first report of race 8 in the United States. Since there are a number of commercial spinach cultivars available with resistance to race 8, the economic impact of this race in the United States is expected to be low if resistant cultivars are grown (B. M. Irish, J. Correll, S. T. Koike, and T. Morelock. Plant Dis. [In press]). Reference: (1) R. N. Acharya and I. Molina. NFAPP Newsl. Second Quarter, 2005.

First Occurrence of Anthracnose Caused by Colletotrichum gloeosporioides on Pitahaya.

Pitahaya, Hylocereus undatus Britt. & Rose, is a columnar, climbing cactus that produces a commercially important fruit. In December 2004, a new disease was found on the crop in Miami-Dade County, FL. Reddish brown lesions with conspicuous chlorotic haloes developed concentrically on the edges of vine ribs. Lesion centers became white and coalesced to rot much of the vine column, and in severe cases, only the vascular column in the vine center was not diseased. Salmon-colored spores and waxy, subepidermal acervuli, typically with setae and simple, short, erect conidiophores, were observed in lesion centers. Tissue from lesion margins was surface disinfested and plated on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI). Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. was isolated from all samples. Colonies produced abundant conidia that were hyaline, one celled, straight, cylindrical, and averaged 14.7 × 5.0 µm with ranges of 12.5 to 17.5 × 3.8 to 7.5 µm (1). Cultural and morphological characteristics of isolates matched those for C. gloeosporioides except for appressoria and hyphopodia (1,2); pitahaya isolates had a spherical rather than lobed hyphopodia reported for C. gloeosporioides and averaged 10.9 (8.5 to 12.7) × 9.1 (7.1 to 10.3) µm. Internal transcribed spacer sequences for the pitahaya isolates were nearly identical (98% homology) to those for C. gloeosporioides isolates occurring on Euphatorium thymifolia in Thailand (GenBank Accession No. AY266393). Koch's postulates were examined in greenhouse trials at the Tropical Research and Education Center, Homestead, FL. Treatments consisted of a noninoculated control, four C. gloeosporioides isolates, and an Alternaria sp. All isolates came from symptomatic pitahaya tissue collected in Miami-Dade County. Fungi were grown on PDA for 7 days at 27°C. A sterile dissecting needle was used to gently pinprick the epidermis of the stem and 2-mm-diameter plugs of C. gloeosporioides, an Alternaria sp., or clean PDA were placed over wounds. Plants were placed in a plastic tent in a greenhouse where the temperature was held at 25°C, and free moisture was maintained on plant surfaces with a household humidifier for 48 h following inoculation. Two isolates of C. gloeosporioides were shown, in repeated greenhouse experiments, to cause reddish brown lesions with conspicuous chlorotic haloes that coalesced to rot much of the vine column, and Koch's postulates were completed with the reisolation of isolates that were used to inoculate plants. The age of vine segments had no significant effect on lesion development. To our knowledge, this is the first report of C. gloeosporioides as a pathogen of pitahaya. References: (1) J. A. Bailey and M. J. Jeger. Colletotrichum: Biology, Pathology and Control. CAB International, Wallingford, UK, 1992. (2) M. Du et al. Mycologia 97:641, 2005.

Three New Races of the Spinach Downy Mildew Pathogen Identified by a Modified Set of Spinach Differentials.

Spinach downy mildew, caused by Peronospora farinosa f. sp. spinaciae, is the most economically important disease of spinach worldwide. During the past few years, spinach cultivars resistant to the seven previously described races of P. farinosa f. sp. spinaciae were observed to be severely affected by downy mildew in both the United States and the European Union. Four new isolates of P. farinosa f. sp. spinaciae were collected from California and The Netherlands and characterized based on disease reactions on two modified sets of spinach differentials. The results led to the description of three new races of the downy mildew pathogen, designated races 8, 9, and 10. Four differential cultivars with resistance to races 1 to 7 were used to distinguish the three new races. Dolphin was susceptible to races 8 and 10 but resistant to race 9; Lion was susceptible to race 10 but resistant to races 8 and 9; Lazio was resistant to races 1 to 7 as well as races 8, 9, and 10; and Tarpy was susceptible to all three new races. The three new races also were used to evaluate the disease reactions on 43 contemporary commercial spinach cultivars in greenhouse trials. A survey of 58 isolates of P. farinosa f. sp. spinaciae collected in California and Arizona between 2004 and 2006 revealed that race 10 predominated in the areas sampled.

Rapid Determination of Rice Cultivar Responses to the Sheath Blight Pathogen Rhizoctonia solani Using a Micro-Chamber Screening Method.

An accurate greenhouse screening method has not been developed previously to identify host response to sheath blight disease caused by Rhizoctonia solani Kühn that causes significant economic losses in rice yield worldwide. The unavailability of a robust screening system in the greenhouse has made it difficult to quantify disease reactions to R. solani, and has hampered studies on the genetics of resistance and plant breeding efforts to improve resistance. In an effort to develop a standardized laboratory micro-chamber screening method to quantify resistance to R. solani in rice, five rice cultivars, representing a wide range of observed disease reactions under field conditions, were examined in a blind inoculation test at three locations (Arkansas, Texas, and Colombia). Rice seedlings were inoculated at the three- to four-leaf stage with potato dextrose agar plugs containing mycelium and then covered with a 2- or 3-liter transparent plastic bottle for maintaining high humidity after inoculation. Two cultivars, Jasmine 85 and Lemont, that consistently have shown the highest and lowest levels of resistance, respectively, in previous field and greenhouse studies, were used as standards. Concurrent field experiments in Arkansas and Texas also were performed to compare the greenhouse disease ratings with those observed under field conditions. Overall, the relative disease ratings of the seven test cultivars were consistent between test locations and with field evaluations. Thus, the micro-chamber screening method can be used as an effective approach to accurately quantify resistance to the sheath blight pathogen under controlled greenhouse conditions and should help expedite the selection process to improve resistance to this important pathogen.

First Report of Powdery Mildew Caused by Leveillula taurica on Tomato and Pepper in Bolivia.

Chlorotic and necrotic lesions typical of powdery mildew caused by L. taurica were observed in several tomato (Lycopersicon esculentum) and pepper (Capsicum annuum) fields in Santa Cruz State, Bolivia near the town of Mairana during September 2004. The tomato cultivars affected were Santa Clara, Superman, and Cool 45. Symptoms included bright yellow chlorotic lesions or brown necrotic lesions on different age leaves. Examination of samples collected from several fields revealed sporulation of L. taurica on abaxial leaf surfaces. The fungus had branched conidiophores, a tapered or pyriform apical conidium, with other conidia being more cylindrical (1,2). Conidial size was approximately 60 × 18 µm. Only the Oidiopsis stage was observed. Disease severity was high and caused a significant amount of leaf necrosis and partial defoliation on tomato. Only sporadic lesions were observed on pepper cv. YoloWonder and no significant foliar damage was observed. The growing region receives approximately 75 mm of rainfall annually with most of the rainfall occurring between October and April. Thus, powdery mildew was observed near the end of the normal 5-month dry season. It is likely that the disease has been in the region for some time based on observations from field personnel. Although reported from several other South American countries, to our knowledge, this is the first report of this disease in Bolivia. References: (1) H. J. Boesewinkel. Bot. Rev. 46:167, 1980; (2) J. C. Correll et al. Plant Dis. 71:248, 1987.