Seasonal Progression and Agronomic Impact of Tobacco streak virus on Soybean in Wisconsin.

Pod necrosis and dieback of terminal portions of stems, commonly called bud blight, are observed in soybean fields and associated with Tobacco streak virus (TSV), an understudied virus of soybean. Furthermore, many TSV-infected plants are asymptomatic. The objectives of this study were to characterize the distribution and seasonal progress of TSV-infected plants in both natural and controlled epidemics, and the agronomic impact of TSV on soybean in plots with controlled introduction of inoculum. Incidence of TSV-positive samples ranged from 17 to 56% in a general survey. In the presence of natural sources of inoculum, the incidence of TSV-infected plants ranged from 10 to 95% depending on cultivar and location, and peaked at growth stage R2, but detection dropped dramatically at R5. During 2001, significant yield loss and incidence of mottled seed were associated with TSV, but results were confounded by a high incidence of SMV. In 2002, SMV was controlled by cultivar selection, and a 25% reduction in grain yield was attributable to TSV. The incidence of mottled seed and green stem syndrome were low in the presence of TSV. Reductions in plant density and final plant height contributed to reduced yields. However, no significant differences were found in seed number per plant and 100-seed weight. Data indicate that plant mortality was the main mechanism by which TSV caused yield loss in induced epidemics.

Resistance to Brown Stem Rot in Soybean Germ Plasm with Resistance to the Soybean Cyst Nematode.

The soybean cyst nematode (SCN) and Phialophora gregata f. sp. sojae, the causal agent of brown stem rot (BSR), are two pathogens of soybean commonly found in the same field throughout the north-central United States. Field experiments designed to study the role of SCN-resistant germ plasm in soybean production have led to data suggesting that some sources of SCN resistance also may provide resistance to BSR. Soybean germ plasm with resistance to SCN was evaluated in greenhouse and field environments for resistance to BSR development based on the percentage of host tissue symptomatic of BSR. Comparison of SCN-resistant cultivars and plant introductions (PI) to standard BSR-resistant and -susceptible checks were conducted in two greenhouse experiments using a root-dip inoculation with a single isolate of P. gregata. For both greenhouse experiments, PI 209332 was the only source of SCN resistance with resistance to BSR similar to standard BSR-resistant checks. Nine other sources of SCN resistance, including PI 88788 and Peking, expressed BSR symptom severity similar to BSR-susceptible checks. Cultivars derived from most SCN-resistant sources, including PI 209332, also were susceptible to BSR development, while four of the five cultivars derived from PI 88788 were highly resistant to BSR development. SCN-resistant cultivars derived from PI 88788, Peking, and PI 209332 were planted along with standard BSR-resistant and -susceptible checks at two field locations naturally infested with P. gregata and SCN or P. gregata alone. As in greenhouse experiments, four of the five cultivars derived from PI 88788 expressed resistance to BSR development equal to or better than standard BSR-resistant checks at both locations. In contrast, cultivars derived from PI 209332 and Peking expressed varying levels of disease development depending on field environment. Yields observed for PI 88788-derived cultivars were higher than BSR-resistant checks regardless of the presence of SCN. Data from both greenhouse and field experiments suggest that cvs. Williams and Williams 82 may contain a gene or genes for BSR resistance that require one or more modifier genes, possibly located in the genome of PI 88788, for complete expression.

Occurrence of Soybean Stem Canker (Diaporthe phaseolorum var. meridionalis) in Wisconsin.

Soybean (Glycine max) developed symptoms characteristic of stem canker during the 2000 to 2003 growing seasons in Wisconsin. Symptoms were widespread in 2003 and were associated with yield losses of ≈1% statewide and as much as 25% in individual fields. Affected plants expressed dieback of foliage beginning at growth stage R3 and progressed until the R6 growth stage. Dark brown lesions were frequently observed at a single node on the lower portion of stems of plants expressing foliage dieback. Fungi were isolated from symptomatic plants collected from seven growers' fields in Rock, Sauk, Veron, and Walworth counties and the Arlington and Marshfield Agricultural Research Stations. Stems with lesions were cut into ≈5-mm pieces, surface disinfested with a 0.5% NaOCl solution for 3 min, rinsed three times in sterile distilled water, and placed on water agar (WA) or potato dextrose agar (PDA) at pH 4.5. Hy-phal tips from colonies of interest were excised and placed on acidified PDA at 25°C under continuous light for 25 to 30 days. In addition to Diaporthe phaseolorum var. caulivora (the cause of northern stem canker), four isolates of D. phaseolorum var. meridionalis (the cause of southern stem canker) were isolated. Colonies of D. phaseolorum var. meridionalis isolates were white, lanose, and became tan with age as previously described for D. phaseolorum var. meridionalis (1). Pycnidia with alpha conidia (no beta conidia) and perithecia with 3.1 to 3.4 × 9.5 to 9.8 µm ascospores formed on oat flakes on acidified WA after 30 days. Stromata were brown to black and irregularly shaped. Four isolates of D. phaseolorum var. meridionalis were tested for pathogenicity in a controlled environment using a cut stem inoculation method (2). Stems of 3-week-old seedlings of cv. Sturdy were cut at the midpoint between the second and third node, and a PDA mycelial plug (4 mm in diameter) was placed on the surface of the cut stems. This method was used to inoculate 15 plants in three replicates for each isolate tested. Inoculated plants were placed in a mist chamber in the dark at 25°C for 4 days and later moved to a greenhouse with a 16-h photoperiod at 24 ± 3°C for 3 days. All plants challenged by this method exhibited stem lesions that were 2 to 3 cm long and of similar color to lesions observed in field-grown plants. For each isolate tested, D. phaseolorum var. meridionalis was reisolated from three randomly selected symptomatic plants. Negative controls with a PDA plug did not produce lesions. To our knowledge, this is the first report of D. phaseolorum var. meridionalis on soybean in Wisconsin. The significance of this report relates to the potential spread of D. phaseolorum var. meridionalis beyond its known southern range in the United States. References: (1) F. A. Fernandez et al. Stem Canker. Pages 33-35 in: Compendium of Soybean Diseases, 4th ed. G. L. Hartman et al., eds. The American Phytopathological Society, St. Paul, MN, 1999. (2) S. Li et al. Plant Dis. 85:1031, 2001.

Impact of Herbicides on Heterodera glycines Susceptible and Resistant Soybean Cultivars.

Several abiotic and biotic stresses can affect soybean in a growing season. Heterodera glycines, soybean cyst nematode, reduces yield of soybean more than any other pathogen in the United States. Field and greenhouse studies were conducted to determine whether preemergence and postemergence herbicides modified the reproduction of H. glycines, and to determine the effects of possible interactive stresses caused by herbicides and H. glycines on soybean growth and yield. Heterodera glycines reproduction factor (Rf) generally was less on resistant than susceptible cultivars, resulting in a yield advantage for resistant cultivars. The yield advantage of resistant cultivars was due to more pods per plant on resistant than susceptible cultivars. Pendimethalin reduced H. glycines Rf on the susceptible cultivars in 1998 at Champaign, Illinois, and in greenhouse studies reduced dry root weight of H. glycines-resistant and susceptible cultivars, therefore reducing Rf on the susceptible cultivars. The interactive stresses from acifluorfen or imazethapyr and H. glycines reduced the dry shoot weight of the resistant cultivar Jack in a greenhouse study. Herbicides did not affect resistant cultivars' ability to suppress H. glycines Rf; therefore, growers planting resistant cultivars should make herbicide decisions based on weeds present and cultivar tolerance to the herbicide.

First Report of Frogeye Leaf Spot (Cercospora sojina) in Wisconsin.

Frogeye leaf spot, caused by Cercospora sojina, is an economically important foliar disease of soybean (Glycine max (L.) Merr.) in areas where growing conditions are warm and humid. During a survey conducted in 2000 and 2001 in soybean fields in Wisconsin, reddish brown, circular to angular spots varying in diameter from 1 to 5 mm were observed on soybean leaves in four fields in Dane and Iowa counties, and in five and six fields in Lafayette and Green counties, respectively. Soybean plants were in growth stages between R3 and R5 during sampling. Disease incidence ranged from 30 to 100% with 5 to 10% of leaf area covered with leaf spot in 2000. In 2001, trace levels of the disease were detected in Dane County, but no symptomatic plants were present in the other counties. Symptomatic leaves were collected from all locations in 2000 and Dane county in 2001. Ten leaves were randomly picked from all samples for each year, placed in a 100 × 15 mm petri dish dampened with Whatman No.1 filter paper, and incubated overnight at 24°C. Fungal sporulation developed after 24 h. Fifteen spores were removed from the 10 leaves, placed on acidified potato dextrose agar (APDA), and incubated in the dark at 24°C. Cultures with dark pigmentation and associated conidia and conidiophores were observed after 3 weeks. The conidiophore, spore type, and leaf symptoms correspond to the description of C. sojina (1). Conidiophores were light-to-dark brown, one to four septate, and fasciculate. The conidiophores were also geniculate and measured 52 to 120 x 4 to 6 µm. Conidia were 0 to 10 septate, hyaline, elongate to fusiform, and measured 40 to 60 x 6 to 8 µm. Cultures were maintained on APDA, and spores for inoculations were produced on this medium. Spores from the 2000 cultures were harvested, bulked together, and used for pathogenicity tests. Pathogenicity tests were conducted in a growth chamber using a known susceptible soybean cultivar, Blackhawk. Ten-cm-diameter pots each containing 4 plants was used. Twenty plants were inoculated and 20 served as noninoculated controls. Ten-day-old plants were inoculated with a spore suspension of 3 × 105 spores/ml by spraying inoculum over the entire leaf surfaces with a spray atomizer. Control plants were sprayed similarly with sterile distilled water. Plants were incubated in an enclosed, transparent fiberglass box with a humidifier that provided 95 to 100% humidity. Lighting in the growth chamber was adjusted to 18-h light and 6-h dark during the inoculation period. Plants were removed from the box after 48 h and placed in a growth chamber with a 12-h photoperiod. The light output in the growth chamber was 300 µmol·m-2·s-1 and the temperature was maintained at 24 ± 3°C. The experiment was repeated once. Typical field symptoms appeared on each of the inoculated plant 8 days after inoculation, while the controls expressed no leaf symptoms. C. sojina was reisolated from all symptomatic plants. To our knowledge, this is the first report of C. sojina from soybean in Wisconsin. Reference: (1) D. V. Phillips. Frogeye leaf spot. Page 20 in: Compendium of Soybean Diseases. 4th ed. G. L. Hartman, J. B. Sinclair, and J. C. Rupe, eds. American Phytopathological Society, St. Paul, MN, 1999.

First Report of Aster Yellows Phytoplasma in Alfalfa.

Samples of alfalfa (Medicago sativa L.) leaves and stems showing symptoms of inter-veinal chlorosis and purpling, commonly associated with insect feeding, were collected from 8 sites in central and southern Wisconsin in autumn of 1998. Samples were frozen within 24 h of collection. Approximately 0.3 g of plant tissue from each sample was used for total DNA extraction according to the protocol of Zhang et al. (4), with minor modifications in grinding procedures and reagent volumes to optimize results. Nested polymerase chain reaction (PCR) was carried out by amplification of 16S rDNA with the universal primer pairs R16mF2/R16mR1 followed by R16F2n/R16R2 as described by Gunder-sen and Lee (1). Undiluted total sample DNA was used for the first amplification; PCR products were diluted (1:30) in sterile water prior to final amplification. Alfalfa DNA and sterile water were used as negative controls; DNA from phytoplasma causing X-disease in peach (CX) served as a positive control. Fragments of 16S rDNA from putative phytoplasmas amplified by PCR with the primer pair R16F2n/R16R2 were characterized by restriction endonuclease digestion (3). The resulting restriction fragment length polymorphism (RFLP) patterns were compared with patterns for known phytoplasmas described by Lee et al. (3). Products of nested PCR were also purified and sequenced with primers R16F2n/R16R2 and an automated DNA sequencer (ABI 377XL; C. Nicolet, Biotechnology Center, University of Wisconsin-Madison). Of 51 samples of alfalfa assessed, one sample from Evansville, WI, yielded a nested PCR product of the appropriate size (1.2 kb), indicating the presence of phytoplasma. Digestion of this product with various restriction enzymes produced RFLP patterns that were identical to those for phytoplasmas in the aster yellows phytoplasma subgroup 16SrI-A (3). Alignment of the DNA sequence of the nested PCR product from the positive sample with sequences found in the GenBank sequence data base (National Center for Biotechnology Information, Bethesda, MD) with the BLAST sequence similarity function confirmed this result. Although other phytoplasma strains (particularly those causing witches'-broom) have been reported to infect alfalfa (2), this is the first report of the presence of the aster yellows phytoplasma in the alfalfa crop. Vectors involved in transmission and the potential agronomic impacts of aster yellows phytoplasma in alfalfa are topics of current investigation. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) A.-H. Khadhair et al. Microbiol. Res. 152:269, 1997. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (4) Y.-P. Zhang et al. J. Virol. Methods 71:45, 1998.