Genotyping-by-Sequencing to Predict Resistance to Lima Bean Downy Mildew in a Diversity Panel.

Lima bean is affected by severe downy mildew epidemics caused by the oomycete Phytophthora phaseoli. There are six documented races of P. phaseoli (A to F). Race F is currently predominant in the mid-Atlantic region, creating the need for resistant lima bean cultivars with desirable agronomic characteristics. In order to develop markers for detecting race F resistance, bulked segregant analysis (BSA) using genotyping-by-sequencing (GBS) was used on a biparental F2 population comprised of 216 lima bean progeny segregating for a dominant race F resistance phenotype. Data were analyzed using a custom bioinformatic analysis pipeline (redrep). Kompetitive allele-specific polymerase chain reaction assays were developed using 12 GBS markers associated with the race F resistance phenotype. Using these assays, the F2 population was used to map the race F resistance locus. Seven markers were in linkage and significantly associated with race F resistance that mapped between two markers located approximately 4.88 centimorgan (cM) apart. These assays were successfully used to genotype a newly acquired lima bean diversity panel consisting of 256 landraces, cultivars, and wild germplasm, and a haplotype consisting of two of the seven linked markers was demonstrated to accurately predict race F resistance. This confirmed the ability of our customized methods to accurately predict phenotypes in diverse lines of lima bean.

First Report of Boxwood Blight Caused by Calonectria pseudonaviculata in Delaware, Maryland, New Jersey, and New York.

Boxwood (Buxus spp.) are commercially important evergreen ornamental plants with an annual market value of over $103 million in the United States. The recent U.S. incursion of boxwood blight disease caused by the fungus Calonectria pseudonaviculata (syn. Cylindrocladium pseudonaviculatum, Cy. buxicola) threatens the health and productivity of boxwood in both landscape plantings and nurseries. The first confirmed U.S. reports of the disease were made from Connecticut and North Carolina in November 2011 (2,4), followed by diagnoses in 10 additional states during 2012 and 2013. By August 2013, symptoms consistent with boxwood blight had been observed from B. sempervirens in Delaware, Maryland, New Jersey, and southeastern New York. Affected plants showed rapid onset of disease symptoms: dark brown to black spots or diffuse dark areas on leaves, followed by defoliation. Narrow, elongate black cankers also formed on current season shoots. Symptomatic stems and leaves were placed in petri dishes with moistened filter paper at 22°C for 3 days under continuous light. Conidiophores were excised, then placed on potato dextrose agar amended with streptomycin and neomycin (0.3 g/l). Resultant colonies showed dark brown pigmentation at the colony center surrounded by tan to reddish brown rings with white mycelia at the advancing edge. Conidia (n = 30 per isolate) were hyaline, cylindrical, rounded at both ends, with a single septum (45 to 76 × 4 to 6 µm; avg. 63 × 5 µm). Conidiophores (n = 20 per isolate) comprised a stipe, a hyaline septate stipe extension (length 119 to 192 µm; avg. 150 µm) and a terminal ellipsoidal vesicle (diameter 4 to 10 µm; avg. 7 µm). Based on morphological characteristics, the causal agent was identified as C. pseudonaviculata (1,4). Voucher specimens were deposited in the U.S. National Fungus Collections (BPI 892698 to 701). To verify morphological diagnosis, genomic DNA was extracted from fungal biomass grown in liquid cultures of yeast extract peptone dextrose media. A portion of the ß-tubulin gene (TUB2) was PCR amplified and sequenced bi-directionally using primers Bta/Bt2b (3). BLASTn searches of NCBI GenBank databases using the TUB2 sequences (Accession Nos. KF785808 to 11) demonstrated 96 to 100% sequence identity with other C. pseudonaviculata isolates. To confirm pathogenicity, 5-month-old B. sempervirens and B. microphylla seedlings were spray-inoculated with a spore suspension of 1 × 104 conidia/ml. One isolate from each state was independently tested with four replicates each. Non-inoculated water-sprayed plants served as negative controls. Plants were maintained in growth chambers at 22°C under constant light. Blight symptoms developed 4 to 5 days post inoculation. C. pseudonaviculata was re-isolated from inoculated plants; no symptoms or signs were observed from control plants. To our knowledge, this is the first report of C. pseudonaviculata in the states of Delaware, Maryland, New Jersey, and New York. This report demonstrates that C. pseudonaviculata is now widespread across the United States eastern seaboard, and represents a substantial threat to boxwood plants in North American landscapes and nurseries. References: (1) P. Crous et al. Sydowia 54:23, 2002. (2) D. F. Farr and A. Y. Rossman. Fungal Databases, USDA-ARS. Retrieved from http://nt.ars-grin.gov/fungaldatabases , 30 August 2013. (3) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (4) K. L. Ivors et al. Plant Dis. 96:1070, 2012.

First Report of Downy Mildew of Lima Bean Caused by Phytophthora phaseoli in Virginia.

Delaware, the eastern shore of Maryland, and southern New Jersey have been the center of lima bean (Phaseolus lunatus L.) production in the eastern United States for nearly 50 years (1). Downy mildew has been the most important disease of lima bean in the humid eastern United States over that period. The causal agent of downy mildew, the oomycete pathogen Phytophthora phaseoli Thaxt., was first identified on lima bean in Connecticut in 1887 by Thaxter. Signs and symptoms of lima bean downy mildew include infection, necrosis and abscission of flowers, and shepherd's crooking of racemes, shoot tips, and petioles (1). Sporangia develop on shoot tips, petioles, pins (small pods), and pods in the field and on hypocotyls in-vitro. Since 2005, approximately 50% of the baby lima beans processed in the United States have been grown in Delaware and the eastern shore of Maryland. In 2008, commercial lima bean production began on the eastern shore of Virginia in Accomack County but no downy mildew was reported in that season. In 2009, approximately 1,825 ha in Accomack and Northampton counties were planted to baby lima bean. Weather conditions in 2009, including above average rainfall, were conducive for the development of downy mildew on the Delmarva Peninsula. Downy mildew was widespread in growers' fields in August and September in butter bean in southern New Jersey and baby lima bean in Sussex County, DE. In August 2009, a home gardener in Rappahannock, VA sent samples of infected lima bean pods from baby, Fordhook, and pole lima bean plants to the Virginia Tech Plant Disease Clinic in Blacksburg. On the basis of morphometric analysis, samples were determined microscopically to be infected by a Phytophthora sp. with rather uniform sporangia averaging 39 × 22 µm and short pedicels, diagnostic for P. phaseoli (1). On October 27, 2009, field scouts in Accomack County, VA identified two lima bean fields planted to cv. C-Elite-Select exhibiting moderate symptoms of downy mildew. Samples were brought to the Plant Diagnostic Clinic at the University of Delaware under USDA-APHIS permit and determined to be P. phaseoli based on morphometric analysis. Samples were inoculated onto a lima bean cultivar differential to determine pathogenicity to complete Koch's postulates and to determine their physiological race. Samples were inoculated onto lima bean cvs. 184-185 and C-Elite-Select, which are susceptible to race F and resistant to race E, Eastland and 8-78, which are susceptible to race E and resistant to race F, and Concentrated Fordhook, susceptible to all known races (1). Three pots containing five emerging seedlings each were inoculated with sporangia (approximately 103 per ml) prepared by soaking infected pods in 500 ml of sterile distilled water for 1 min with gentle agitation. Plants were placed in a Percival dew chamber with intermittent misting and set at 19. Infection and disease development were assessed daily and signs developed 7 days postinoculation in cvs. 184-85, C-Elite-Select, and Concentrated Fordhook, but not in Eastland and 8-78. Cultivar differential tests indicated that the isolates were P. phaseoli race F. Hypocotyls of infected plants were scraped, and isolations made on lima bean pod agar confirmed the presence of P. phaseoli. To our knowledge, this is the first time that downy mildew of lima bean has been reported in Virginia. Reference: (1) T.A. Evans et al. Plant Dis. 91:128, 2007.

First Report of the Telial Stage of Japanese Apple Rust on Juniperus chinensis in North America and the Aecial Stage on Malus domestica.

Following a report in April 2009 of the presence of Gymnosporangium yamadae Miyabae ex G. Yamada on crabapple (Malus toringo Siebold) in Wilmington, DE (2), University of Delaware, State of Delaware, and USDA/APHIS PPQ personnel collaborated to confirm and document the pathogen. G. yamadae is the causal agent of Japanese apple rust. The fungus is known from Asia with an aecial state on economically important Malus species and telial state on Juniperus chinensis. During the April 2009 site visit, ornamental J. chinensis were observed near the original crabapples. On May 7, 2009, telial galls were collected from the ornamental J. chinensis at the Wilmington site. The telia were confirmed to be G. yamadae by morphometric analysis and molecular data. The rDNA large subunit (LSU) sequence derived from the collected telial galls (GenBank Accession No. GU058012) was identical to the eight G. yamadae LSU sequences (GenBank Accession Nos. FJ848760-FJ848765, FJ559373, and FJ559375) reported from Korea by Yun et al. (3). Teliospores were 45 to 54 µm long with pedicels that were wide (7.0 to 8.4 µm) along the full length. The G. yamadae telial gall collected from Wilmington, DE was deposited into the U.S. National Fungus Collection (BPI 879273). Leaves of M. domestica on the University of Delaware farm in Newark were confirmed to have Japanese apple rust on Aug 4, 2009. Identification was made on the morphological presence of unique roestelioid aecia with long cornulated peridia that lacerate along the sides. The aecia differ from those of G. juniperi-virginianae, the causal agent of cedar apple rust, which has aecial peridia that fimbriate to the base and are strongly recurved (1). Following release of a USDA Pest Alert, subsequent samples submitted to USDA/APHIS PPQ indicated widespread incidence of the G. yamadae aecial state in the northeast, including Maryland, Maine, New Hampshire, New Jersey, New York, Pennsylvania, and Rhode Island. Japanese apple rust likely went undetected for several years because of similar symptomatology to cedar apple rust. To our knowledge, this is the first report of the telial stage of G. yamadae in North America and the first report of this pathogen on Malus domestica in the United States. Knowledge of the geographic distribution of G. yamadae is of significance because of the actionable regulatory status of the pathogen and its potential impact on ornamental and fruit growers of Malus spp. in the United States. References: (1) F. D. Kern. A Revised Taxonomic Account of Gymnosporangium. Pennsylvania State University Press, University Park, PA, 1973. (2) H. Y. Yun et al. Plant Dis. 93:430, 2009. (3) H. Y. Yun et al. Mycologia 101:790, 2009.

Lima Bean Downy Mildew Epiphytotics Caused by New Physiological Races of Phytophthora phaseoli.

Before 1995, race D of Phytophthora phaseoli, the causal agent of downy mildew on lima bean (Phaseolus lunatus), was the prevalent physiological race in the mid-Atlantic region of the United States. Since 1995, however, new physiological races of P. phaseoli have been responsible for downy mildew outbreaks in previously resistant cultivars in this region. Cultivar differential testing of 180 isolates of P. phaseoli collected between 1994 and 2005 from Delaware and the eastern shore of Maryland has confirmed the presence of two new physiological races. The detection of race E in 1995 and race F only 5 years later in 2000, plus the lack of resistant cultivars to manage the epiphytotics in lima bean, have led to millions of dollars of crop losses. Intra- and interspecific genetic variation of Phytophthora spp. and isolates were assessed using amplified fragment length polymorphism DNA fingerprinting. Primer groups EcoRI+AG and MseI+C distinguished P. phaseoli and P. capsici from P. infestans but did not distinguish among different races of P. phaseoli.

First Report of Mefenoxam-Resistant Isolates of Phytophthora capsici from Lima Bean Pods in the Mid-Atlantic Region.

Phytophthora capsici Leonian, the causal agent of lima bean pod rot, was first identified as a pathogen of lima bean in 2002 (1) and poses a new threat to lima bean (Phaseolus lunatus L.) production in the Mid-Atlantic Region. The phenylamide fungicide mefenoxam (Ridomil Gold; Syngenta Crop Protection) is widely used in the region for controlling foliar and soilborne diseases caused by Oomycetes. Isolates of P. capsici were collected from lima bean pods from production fields in Delaware, Maryland, and New Jersey from 1998 to 2004. These isolates originated from survey samples of lima bean fields for another pathogen, P. phaseoli, in 1999 and 2000 and diagnostic samples were submitted to the Plant Disease Clinic. Isolates were from lima bean, except for one from pepper (basal stem). Identification was made on the basis of morphometric characteristics. No known sensitive or insensitive isolates were included in the evaluation. Single zoospore cultures were evaluated for mefenoxam sensitivity on V8 agar plates amended with 100 ppm of mefenoxam, a previously tested concentration (2). Seven-millimeter-diameter agar plugs of each isolate were cut from the edge of actively expanding cultures of P. capsici with a cork borer and transferred to three V8 agar plates amended with mefenoxam and three unamended V8 plates. The plates were arranged in a completely randomized design and incubated at 25°C in the dark for 3 days. After incubation, colony growth was measured in millimeters and averaged for the three replicate plates of each isolate and percent growth relative to the unamended control was calculated. Mefenoxam sensitivity was assigned according to methods of Lamour et al. (2). The experiment was repeated once, and also run with a treatment of 200 ppm of mefenoxam. Of sixteen isolates screened, nine were rated as sensitive, four were intermediately resistant, and three were resistant. There was no difference between the 100 and 200 ppm results, except for a slight increase in sensitivity for one isolate. A subsequent experiment tested five isolates at concentrations of 1, 10, 100, and 1,000 ppm. Results were consistent with previous tests, with resistant isolates exhibiting some growth at the highest concentration of mefenoxam. One resistant isolate was from a field in Delaware previously cropped to slicing cucumbers with a history of mefenoxam applications. The second was from Caroline County, MD, which is heavily cropped to pickling cucumbers and likely to have been exposed to mefanoxam applications for the control of fruit rot; the origin of the third insensitive isolate from lima bean is unknown. Mefanoxam usage on lima bean is usually limited to one foliar application of mefenoxam+copper hydroxide to control downy mildew in the fall crop in wet seasons. This study indicates that mefenoxam resistance is present in populations of P. capsici in lima bean fields in the Mid-Atlantic Region, presumably as a result of mefenoxam applications to other vegetable crops, principally cucurbits, which are planted in rotation with lima beans or from nearby cucurbit fields. Implementing strategies to minimize fungicide resistance in other vegetables is important to slow resistance development associated with this emerging pathogen on lima beans. Lima bean pod rot continues to be seen sporadically each year in fields with a history of P. capsici and abundant rainfall or excessive irrigation. References: (1) C. R. Davidson et al. Plant Dis. 86:1049, 2002. (2) K. H. Lamour et al. Phytopathology 90:396, 2000.