Anne E. Dorrance

Systemic Resistance Induced by Trichoderma spp.: Interactions Between the Host, the Pathogen, the Biocontrol Agent, and Soil Organic Matter Quality

ABSTRACT Several factors affect the ability of Trichoderma spp. to provide systemic disease control. This paper focuses on the role of the substrate in which plants are grown, resistance of the host to disease, and the ability of introduced Trichoderma inoculum to spread under commercial conditions. Several reports reveal that foliar disease control provided by Trichoderma spp. is more effective on plants grown in compost-amended media compared with in lower-in-microbial-carrying-capacity sphagnum peat media. In Rhododendron spp., host resistance affects control of Phytophthora dieback provided by Trichoderma spp. For example, T. hamatum 382 (T382) significantly (P = 0.05) suppressed the disease on susceptible cv. Roseum Elegans while plant vigor was increased. The disease was not suppressed, however, on highly susceptible cvs. Aglo and PJM Elite even though the vigor of these plants was increased. Using a strain-specific polymerase chain reaction assay under commercial conditions, it was demonstrated that introduced inoculum of T382 did not spread frequently from inoculated to control compost-amended media. Other Trichoderma isolates typically are abundant in control media within days after potting unless inoculated with a specific Trichoderma isolate. Thus, the low population of isolates that can induce systemic resistance in composting and potting mix environments may explain why most compost-amended substrates do not naturally suppress foliar diseases.

Evaluation of Fusarium graminearum Associated with Corn and Soybean Seed and Seedling Disease in Ohio

Fusarium graminearum is an important pathogen of cereal crops in Ohio causing primarily head blight in wheat and stalk and ear rot of corn. During the springs of 2004 and 2005, 112 isolates of F. graminearum were recovered from diseased corn and soybean seedlings from 30 locations in 13 Ohio counties. These isolates were evaluated in an in vitro pathogenicity assay on both corn and soybean seed, and 28 isolates were tested for sensitivity to the seed treatment fungicides azoxystrobin, trifloxystrobin, fludioxonil, and captan. All of the isolates were highly pathogenic on corn seed and moderately to highly pathogenic on soybean seed. Fludioxonil was the only fungicide that provided sufficient inhibition of mycelial growth; however, several fludioxonil-resistant mutants were identified during the sensitivity experiments. These results indicate that F. graminearum is an important pathogen of both corn and soybean seed and seedlings in Ohio, and that continued use of fludioxonil potentially may select for less sensitive isolates of F. graminearum.

Efficacy of Fungicides on Sclerotinia sclerotiorum and Their Potential for Control of Sclerotinia Stem Rot on Soybean

Sclerotinia stem rot of soybean, caused by Sclerotinia sclerotiorum, is a major disease in the north central region of the United States. One approach to managing Sclerotinia stem rot on soybean is the use of fungicides. S. sclerotiorum was assayed for sensitivity to benomyl, tebuconazole, thiophanate methyl, and vinclozolin in pure cultures on agar medium, inoculated soybean seedlings, detached inoculated leaves, and in experimental field plots. To evaluate the inhibitory effect of four fungicides on growth of S. sclerotiorum in vitro, potato dextrose agar (PDA) was amended with the fungicides at six concentrations. Based on measurements of fungal radial growth, vinclozolin was the most effective in inhibiting S. sclerotiorum mycelial growth at 1.0 μg a.i./ml of PDA. Ranges of reduction of radial growth of 91 isolates of S. sclerotiorum on PDA amended with thiophanate methyl and vinclozolin were 18 to 93% and 93 to 99%, respectively, when compared with the nonamended agar control. Benomyl, thiophanate methyl, and vinclozolin applied to greenhouse-grown seedlings prevented S. sclerotiorum from expressing symptoms or signs on leaf tissue. Detached leaves sprayed with thiophanate methyl and then inoculated with mycelial plugs of S. sclerotiorum did not express symptoms or signs. Of 13 different environments in Illinois, Indiana, Ohio, and Wisconsin from 1995 through 2000, six had low Sclerotinia stem rot incidence (<1%), three environments had low to moderate Sclerotinia stem rot incidence (5 to 25%), and four environments had high Sclerotinia stem rot incidence (>25%). When disease incidence was high, no consistent control of Sclerotinia stem rot was observed with benomyl or thiophanate methyl using different application systems. However, under low disease incidence, spray systems that were able to penetrate the canopy reduced the incidence of Sclerotinia stem rot an average of 50%.

Characterization of Pythium spp. Associated with Corn and Soybean Seed and Seedling Disease in Ohio

Cool, moist conditions in combination with minimum tillage, earlier planting, and recent shifts in commercial fungicide seed-treatment active ingredients have led to an increase in corn (Zea mays) and soybean (Glycine max) seedling establishment problems. This situation resulted in an investigation of Pythium spp. associated with seed and seedling diseases. Samples of diseased corn and soybean seedlings were collected from 42 production fields in Ohio. All isolates of Pythium recovered were identified to species using morphological and molecular techniques and evaluated in an in vitro pathogenicity assay on both corn and soybean seed, and a subset of the isolates was tested for sensitivity to fungicides currently used as seed treatments. Eleven species and two distinct morphological groups of Pythium were identified, of which six species were moderately to highly pathogenic on corn seed and nine species were highly pathogenic on soybean seed. There was significant variation (P < 0.05) in sensitivity to mefenoxam, azoxystrobin, trifloxystrobin, and captan both across and within species. Multiple species of Pythium had the capacity to reduce germination of both corn and soybean seed. Results indicated that mefenoxam, azoxystrobin, trifloxystrobin, or captan, when used individually, may not inhibit all pathogenic species of Pythium found in Ohio soils.

Effect of Partial Resistance on Phytophthora Stem Rot Incidence and Yield of Soybean in Ohio

Phytophthora root and stem rot of soybean commonly causes losses in both stand and yield in Ohio. Environmental conditions which favor the pathogen typically occur in many areas of the state during late spring and summer. This study examined the performance of 12 soybean cultivars with partial resistance, with or without Rps genes, to different populations of Phytophthora sojae and various levels of disease pressure. The soybean cultivars were evaluated in seven field environments with and without metalaxyl over 4 years. There was a highly significant genotype-environment interaction which was due in part to variable disease pressure. The incidence of Phytophthora stem rot in subplots ranged from 0 to 10 plants in the most susceptible cultivar, Sloan, while significantly less stem rot developed in cultivars with high levels of partial resistance or partial resistance combined with an Rps gene in three of the seven environments. Metalaxyl applied in-furrow had a significant effect on early and final plant populations as well as yield (P < 0.001) in two of the seven environments, and for yield (P = 0.05) in one environment. This indicates that at these two environments, 2001 Lakeview and VanBuren, early season Phytophthora disease was controlled with the in-furrow fungicide treatment. When diverse populations of P. sojae were present, yields from soybean cultivars with high levels of partial resistance were significantly higher than those with low levels of partial resistance. Soybean cultivars with specific resistance genes Rps1k, Rps1k + Rps6, or Rps1k +Rps3a had higher yields than plants with only partial resistance in environments where race determination indicated that the populations of P. sojae present were not capable of causing disease on plants with the Rps1k gene. However, in an environment with very low disease pressure, yields of soybean cultivars with partial resistance were not significantly different from those with single Rps genes or Rps gene combinations. These results demonstrate that genetic traits associated with high levels of partial resistance do not have a negative effect on yield. Soybean cultivars that had the most consistent ranking across environments were those with moderate levels of partial resistance in combination with either Rps1k or Rps3a.

Assessment of Greenhouse and Laboratory Screening Methods for Evaluating Potato Foliage for Resistance to Late Blight

Greenhouse and laboratory screening methods for assessing potato foliage for resistance to late blight were compared using 15 cultivars and advanced breeding selections with known field response to late blight. Screening methods included greenhouse inoculation of plants in several age classes, and laboratory assays of detached leaflets, leaf disks, and stem cuttings. Greenhouse inoculation of plants 7 to 11 weeks after planting, near the time of flowering, corresponded best to results obtained in field evaluations, but there were significant differences in disease severity between separate greenhouse tests. This is consistent with variation in late blight severity on a year-to-year basis when cultivars are compared in the field. The greenhouse inoculation method allowed for testing of several components of partial resistance, such as infection efficiency and lesion growth rate, which may exist for each cultivar. Laboratory assays proved less reliable than greenhouse assays for overall ratings of partial resistance, but could be useful for measuring specific components of resistance. Screening evaluations for late blight resistance should include standard cultivars with known reaction to Phytophthora infestans to reference the disease potential within the screening evaluation.

Assessment of Laboratory Methods for Evaluating Potato Tubers for Resistance to Late Blight

Two laboratory methods (whole tuber and tuber slice) were evaluated and compared with field assessment of potato tubers for resistance to late blight caused by Phytophthora infestans. Altogether, the resistance responses of 20 cultivars and advanced selections were compared by the three methods. All three assays separated materials into resistant and susceptible groups. The majority of cultivars and advanced selections did not react in the same way within the field and tuber-slice assays but did react similarly within the whole-tuber assay. Some cultivars were susceptible in the field at harvest but were resistant in the whole-tuber assay. The differences may be due to the extent of lenticel and eye development during tuber formation. In contrast, other cultivars resistant in the field at harvest were susceptible in the whole-tuber assay. In this case, placement in the hill may affect whether or not the tuber is exposed to inoculum or whether aging of tubers during storage affects susceptibility to tuber blight. Our study shows that high levels of tuber resistance are available in certain commercial cultivars and advanced potato selections. For laboratory assessments to be reliable, however, both pre- and poststorage evaluations may be necessary.

Robust RNAi-Based Resistance to Mixed Infection of Three Viruses in Soybean Plants Expressing Separate Short Hairpins from a Single Transgene

Transgenic plants expressing double-stranded RNA (dsRNA) of virus origin have been previously shown to confer resistance to virus infections through the highly conserved RNA-targeting process termed RNA silencing or RNA interference (RNAi). In this study we applied this strategy to soybean plants and achieved robust resistance to multiple viruses with a single dsRNA-expressing transgene. Unlike previous reports that relied on the expression of one long inverted repeat (IR) combining sequences of several viruses, our improved strategy utilized a transgene designed to express several shorter IRs. Each of these short IRs contains highly conserved sequences of one virus, forming dsRNA of less than 150 bp. These short dsRNA stems were interspersed with single-stranded sequences to prevent homologous recombination during the transgene assembly process. Three such short IRs with sequences of unrelated soybean-infecting viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus) were assembled into a single transgene under control of the 35S promoter and terminator of Cauliflower mosaic virus. Three independent transgenic lines were obtained and all of them exhibited strong systemic resistance to the simultaneous infection of the three viruses. These results demonstrate the effectiveness of this very straight forward strategy for engineering RNAi-based virus resistance in a major crop plant. More importantly, our strategy of construct assembly makes it easy to incorporate additional short IRs in the transgene, thus expanding the spectrum of virus resistance. Finally, this strategy could be easily adapted to control virus problems of other crop plants.

Infection of Soybean Seed by Fusarium graminearum and Effect of Seed Treatments on Disease Under Controlled Conditions

Fusarium graminearum causes seed decay and damping-off of soybean. This study evaluated the effect of inoculum density of F. graminearum, temperature, and fungicide seed treatments on disease development. To determine the optimum conditions for disease development, individual soybean seed was inoculated with 100 μl of a suspension of 2.5 × 102, 2.5 × 103, 2.5 × 104, or 2.5 × 105 macroconidia/ml in a rolled-towel assay at temperatures of 18, 22, and 25°C. Inoculum concentrations of 2.5 × 104 macroconidia/ml or higher were necessary for optimum disease development at all temperatures. The efficacy of captan, fludioxonil, mefenoxam + fludioxonil, azoxystrobin, trifloxystrobin, and pyraclostrobin as seed treatments was then evaluated with the same assay at 2.5 × 104 and 2.5 × 105 macroconidia/ml. Seed treated with captan at 61.9 g a.i. or fludioxonil at 2.5 or 5.0 g a.i. per 100 kg developed smaller lesions than other seed treatments and the nontreated control. Based on these results, there are limited choices in fungicide seed treatments for managing this seedling disease, and it is possible that shifts in seed treatment products may have played a role in the recent emergence of this soybean pathogen.

Analysis of genes underlying soybean quantitative trait loci conferring partial resistance to Phytophthora sojae.

Few quantitative trait loci (QTL) have been mapped for the expression of partial resistance to Phytophthora sojae in soybean and very little is known about the molecular mechanisms that contribute to this trait. Therefore, the objectives of this study were to identify additional QTL conferring resistance to P. sojae and to identify candidate genes that may contribute to this form of defense. QTL on chromosomes 12, 13, 14, 17, and 19, each explaining 4 to 7% of the phenotypic variation, were identified using 186 RILs from a cross of the partially resistant cultivar ‘Conrad’ and susceptible cultivar ‘Sloan’ through composite interval mapping. Microarray analysis identified genes with significant differences in transcript abundances between Conrad and Sloan, both constitutively and following inoculation. Of these genes, 55 mapped to the five QTL regions. Ten genes encoded proteins with unknown functions, while the others encode proteins related to defense or physiological traits. Seventeen genes within the genomic region that encompass the QTL were selected and their transcript abundance was confirmed by quantitative reverse transcription polymerase chain reaction (qRT‐PCR). These results suggest a complex QTL‐mediated resistance network. This study will contribute to soybean resistance breeding by providing additional QTL for marker‐assisted selection as well as a list of candidate genes which may be manipulated to confer resistance.