Determination of Heterodera glycines Virulence Phenotypes Occurring in South Dakota.

The soybean cyst nematode (SCN), Heterodera glycines, is the most important yield-limiting pathogen of soybean in the United States. In South Dakota, SCN has been found in 29 counties, as of 2016, and continues to spread. Determining the virulence phenotypes (HG types) of the SCN populations can reveal the diversity of the SCN populations and the sources of resistance that would be most effective for SCN management. To determine the HG types prevalent in South Dakota, 250 soil samples were collected from at least three arbitrarily selected fields in each of the 28 counties with fields previously found to be infested with SCN. SCN was detected in 82 fields (33%), and combined egg and juvenile counts ranged from 200 to 65,200 per 100 cm3 of soil. Eggs and juveniles were extracted from each soil sample and were used to infest seven SCN HG type test indicator soybean lines and 'Williams 82' as the susceptible check. A female index (FI) was calculated based on the number of females found on each indicator line relative to those on the susceptible check. A FI equal to or greater than 10% in any line was assigned as that HG type. Out of 73 SCN populations for which HG type tests were done, 63% had FI ≥10% on PI 548316 (indicator line #7), 25% on PI 88788 (#2), 19% on PI 209332 (#5), 7% on PI 548402 (#1), 4% on PI 90736 (#3), and 4% on PI 89722 (#6). None of the SCN populations had FI ≥10% on PI 437654 (indicator line #4). The most prevalent HG types were 0, 2.5.7, and 7. These accounted for 81% of all the HG types determined for the samples tested. HG types with ≥10% reproduction on indicator lines PI 88788, PI 209332, and PI 548317 were most prevalent in the soil samples tested, suggesting that the use of these sources of resistance for developing SCN resistant cultivars should be avoided. For sustainable SCN management, use of resistant cultivars should be rotated with nonhost crops and cultivars with different sources of resistance.

First Report of Sudden Death Syndrome of Soybean Caused by Fusarium virguliforme in South Dakota.

During the 2012 soybean growing season, soybean (Glycine max (L.) Merr.) plants submitted to South Dakota State University Plant Diagnostic Clinic exhibited symptoms typical of sudden death syndrome (SDS) caused by Fusarium virguliforme (Aoki, O'Donnell, Homma, & Lattanzi). In the 2013 soybean growing season, a soybean survey targeting SDS-symptomatic plants was carried out in 20 eastern South Dakota counties between July and August when plants were at the beginning seed and beginning seed maturity growth stages. Soybean plants with SDS-like symptoms were found in eight counties at very low incidence (<3%). Approximately 15 plants per field that had symptoms resembling those of SDS were collected and fungal isolations were made. Leaf symptoms included some necrosis and slight interveinal chlorosis. The tap roots also had areas of necrosis and the vascular system was brown. Isolations were made from the symptomatic tap root sections. The tap root sections were surface sterilized using a 10% NaOCl for 1 min and then rinsed once for 1 min with sterile water before being placed on an acidified potato dextrose agar. Slow growing isolates of F. virguliforme with characteristic blue sporodochia were isolated from these symptomatic plant roots. The conidia were banana-shaped with 4 to 5 septae, a typical characteristic for F. virguliforme. Koch's postulates were performed using a modified layer test method (2). Briefly, the conidia from the isolate (PL1200158 from Yankton county, SD) was used to infest sterile sorghum seed. In the greenhouse, three holes were punched in the bottom of 32 oz. Styrofoam cups. The bottom 11 cm of the cup was then filled with vermiculite. A 2-cm layer of fully colonized sorghum seed was placed on top of the vermiculite. This was covered with a 2- to 5-cm layer of vermiculite. Fifteen soybean cv. Sloan seeds were placed on top of this vermiculite layer and covered with approximately 2 cm more vermiculite for each cup for a total of 12 cups. The temperature in the greenhouse was approximately 23°C with 14 h of light and 10 h of darkness for 21 days. Leaves began to show necrosis and the roots had brown, rotted lesions. Symptoms did not develop on non-inoculated controls. After 5 weeks under greenhouse conditions, the roots of infected plants were removed, surface sterilized, and F. virguliforme was re-isolated. SDS was further confirmed by PCR using primers designed from FvTox1 gene. FvTox1, a single-copy gene, has been found to be highly species specific and primers from this region delineate F. virguliforme from other Fusarium species (1). The PCR product size matched that of expected size. The PCR product was sequenced and a BLAST search matched (100%) only the sequences of F. virguliforme FvTox1 gene (GenBank Accession No. JF440964). The confirmation of SDS in eight counties in South Dakota indicates that SDS may be widespread and a concern for soybean production when conditions are conducive for SDS to develop. References: (1) G. C. Y. Mbofung, et al. Plant Dis. 95:1420, 2011. (2) A. F. Schmitthenner and R. G. Bhatt. Useful Methods for Studying Phytophthora in the Laboratory. Special Circular, Ohio Agricultural Research and Development Center, Wooster, OH, 1994.

First Report of Ascochyta Blight of Field Pea Caused by Ascochyta pisi in South Dakota.

Tan lesions approximately 1.7 × 0.8 cm with distinct dark brown margins and small pycnidia were observed on leaves of field peas (Pisum sativum L. 'Agassiz') growing in Campbell County, South Dakota (45°45.62'N, 100°9.13'W) in July 2008. Small pieces of symptomatic leaves were surface sterilized (10% NaOCl for 1 min, 70% EtOH for 1 min, and sterile distilled H2O for 2 min) and placed on potato dextrose agar (PDA) for 7 days under fluorescent lights with a 12-h photoperiod to induce sporulation. A pure culture was established by streaking a conidial suspension on PDA and isolating a single germinated spore 3 days later. The culture was grown on clarified V8 media for 10 days. Conidia were 10 to 16 × 3 to 4.5 µm and uniseptate with a slightly constricted septum, similar to those of Ascochyta pisi Lib. The exuding spore mass from pycnidia growing on the medium was carrot red. No chlamydospores or pseudothecia were observed (1,2). To confirm the identity of A. pisi, DNA was extracted from the lyophilized mycelium of the 10-day-old culture with the DNeasy Plant Mini Kit (Qiagen, Valencia, CA). Internal transcribed spacer (ITS) regions I and II were amplified with PCR primers ITS 5 and ITS 4 (3). PCR amplicons were cleaned and directly sequenced in both directions using the primers. A BLASTN search against the NCBI nonredundant nucleotide database was performed using the consensus sequence generated by alignment of the forward and reverse sequences for this region. The consensus sequence (GenBank Accession No. GU722316) most closely matched A. pisi var. pisi strain (GenBank Accession No. EU167557). These observations confirm the identity of the fungus as A. pisi. A suspension of 1 × 106 conidia/ml of the isolate was spray inoculated to runoff on 10 replicate plants of 2-week-old, susceptible green field pea 'Sterling'. Plants were incubated in a dew chamber for 48 h at 18°C and moved to the greenhouse bench where they were maintained at 20 to 25°C with a 12-h photoperiod for 1 week. Tan lesions with dark margins appeared 7 days after inoculation and disease was assessed after 10 days (4). No symptoms were observed on water-treated control plants. A. pisi was reisolated from lesions and confirmed by DNA sequencing of the ITS region, fulfilling Koch's postulates. Currently, states bordering South Dakota (North Dakota and Montana) lead the United States in field pea production. Although acreage is limited in South Dakota, the identification of A. pisi in this region is serious. The disease is yield limiting and foliar fungicides are used for disease management (1). To our knowledge, this is the first report of Ascochyta blight on P. sativum caused by A. pisi occurring in South Dakota and the MonDak production region (the Dakotas and Montana). References: (1) T. W. Bretag et al. Aust. J. Agric. Res. 57:88, 2006. (2) A. S. Lawyer. Page 11 in: The Compendium of Pea Diseases. D. J. Hagedorn, ed. The American Phytopathological Society, St Paul, MN, 1984. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990. (4) J. M. Wroth. Can. J. Bot. 76:1955, 1998.