Posted online October 23, 2007
Soybean sudden death syndrome (SDS): Multi-gene genealogies and pathogenicity of five Fusarium spp. associated with the disease and multiplex real-time PCR identification, detection and quantification of the causal agents. Z. K. ATALLAH (1), S. L. Giammaria (2), J. C. Rupe (2), and W. R. Stevenson (1). (1) Dept. Plant Pathology, University of Wisconsin, Madison, WI 53706; (2) Dept. Plant Pathology, University of Arkansas, Fayetteville, AR 72701.Five fungal species in the Fusarium solani complex are linked to SDS, namely: F. virguliforme, F. tucumaniae, F. brasiliense, F. cuneirostrum and F. phaseoli. Only F. virguliforme is described from soybean in North America. Disease etiology and virulence of some species on soybean are currently debated. In this study, we examined the phylogenetic relatedness amongst the 5 species using multi-gene genealogy (ITS and 7 protein-coding gene sequences) and compared their ability to cause SDS. All 5 species were resolved into individual monophyletic clades, with SNPs, consistent within a species, separating them. Isolates from each species were used to inoculate soybeans in a growth chamber. All species produced significant root rot, but only F. virguliforme and 2 F. brasiliense isolates caused SDS foliar symptoms. Foliar symptom severity correlated with top and root weight loss, but root rot severity did not correlate. SDS pathogens likely recently emerged from dry and mung beans repeatedly in North and South America. SNPs in the ITS sequences identifying F. virguliforme, F. tucumaniae and F. brasiliense were used to develop multiplex TaqMan probe and Plexor real-time assays to identify, detect and quantify the 3 pathogens.
A thirty-year evaluation of crabapples for durable apple scab resistance. J. Beckerman (1), J. Chatfield (2), and E. Draper (2). (1) Department of Botany and Plant Pathology, Purdue University; (2) The Ohio State University Extension Service.
Crabapples (Malus spp.) are a popular ornamental in the commercial and residential landscape. Over a thirty-year period at the Secrest Arboretum, 297 species, hybrids, and cultivated varieties of ornamental crabapple were evaluated for their resistance to apple scab, caused by the fungus Venturia inaequalis. Of these 297 cultivars, 31 were identified as resistant to apple scab for greater than a ten-year period. Of these 31, 14 possessed a stable resistance that was observed to break down by new virulent races of the pathogen. Notable amongst these changes of scab races is the identification of race 5, possessing virulence to the Vm gene, race 3 that infects ‘Geneva’ but not ‘Dolgo’, and the first identification and report of scab on a M. floribunda selection that was reported as resistant even prior to the first 25 years of the evaluation. The presence of scab on this cultivar in North America suggests the presence of race 7 in North America. Five named species or cultivars remained scab resistant for almost the entire thirty-year trial.
Evaluation of PCR-based methods for species-specific detection of Phytophthora sojae. J. C. BIENAPFL, J. A. Percich, and D. K. Malvick. Dept. of Plant Pathology, University of Minnesota, St. Paul, MN 55108.
Phytophthora rot, caused by Phytophthora sojae, is one of the most damaging soybean diseases in the U.S.A. However, other Phytophthora species can also infect soybean in the Midwestern U.S.A. Accurate diagnosis is important for management of Phytophthora rot. The goal of this study was to evaluate PCR methods for rapid and specific detection of P. sojae. Genomic DNA was extracted from P. sojae as well as P. cactorum, P. cinnamomi, P. nicotianae, and an unknown Phytophthora sp. isolated from soybean. PCR assays using two sets of primers reported by other researchers (PS1/PS2, Wang et al. 2006 and PSOJF1/PSOJR1, Hart 2004) that target the ITS region were evaluated to determine specificity to P. sojae. Results indicate that PS1/PS2 amplifies DNA from P. sojae as well as from the other Phytophthora spp. tested. Preliminary results using PSOJF1/PSOJR indicate these primers are specific for P. sojae. Results suggest the potential value of PCR-based assays using PSOJF1/PSOJR1 to detect P. sojae in plant tissue and soil, as well as to distinguish P. sojae from other Phytophthora spp.
Propiconazole movement into the roots of red oaks and Ceratocystis fagacearum survival following macroinfusion treatment. R. A. Blaedow (1) and J. Juzwik (2). (1) Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108; (2) Northern Research Station, USDA Forest Service, St. Paul, MN 55108.
In midwestern states, the systemic fungicide propiconazole (PPZL) is used to manage root graft transmission of the oak wilt fungus, Ceratocystis fagacearum in oak (Quercus) species within the red oak group. PPZL movement into oak roots and its ability to prevent infection via root grafts has not been demonstrated. Fungicide concentration and pathogen viability were determined in primary roots 2, 12, and 24 months after a PPZL application that either preceded or followed root inoculation with the fungus. The chemical was found to move basipetally into roots at least 1.0 m (maximum distance sampled) from the injection site and was detected on all sampling dates. PPZL concentration in root samples increased with proximity to the injection point and application date. C. fagacearum was successfully isolated from root samples up to 24 months after treatment; therefore PPZL likely suppresses disease development rather than eradicating the pathogen from roots or preventing root graft transmission.
The description and phylogenetic placement of two putative new species of Pythium. K. D. BRODERS and A. E. Dorrance. Dept. of Plant Pathology, The Ohio State University, Wooster, OH 44691.
During a survey of corn and soybean fields in Ohio for seed and seedling pathogens, several species of Pythium were recovered, as well as two distinct morphological groups. These two distinct morphological groups were evaluated for their pathogenicity on corn and soybean and sensitivity to fungicides used as seed treatments. The sequence of the ITS1, 5.8s, and ITS2 regions of the ribosomal DNA was used to determine the phylogenetic position of these two putative new species. Both morphological groups were pathogenic corn and soybean, and were insensitive to mefenoxam and azoxystrobin in vitro. These two mophological groups are being classified as new species based on the distinct morphological and sequence data, and are grouped with several other species of Pythium with proliferating sporangia, which appear to be more closely related to several Phytophthora species than to species of Pythium which possess globose or filamentous sporangia. Additionally, both of these species appear to be important pathogens of corn and soybean, and may be selected for their insensitivity to seed treatment fungicides.
Bean pod mottle virus spatial pattern and its relationship with bean leaf beetle winter mortality. E. BYAMUKAMA, A. Robertson, and F. W. Nutter, Jr. Department of Plant Pathology, Iowa State University, Ames, IA.
Bean pod mottle virus (BPMV) causes significant reductions in soybean yield and quality. In order to determine the spatial pattern of BPMV, a state-wide soybean survey was carried out during the 2005 and 2006 growing seasons. Plant samples from 8-20 soybean fields/county and 30 plants/field were tested for BPMV by ELISA. Both the field and county-level prevalence and incidence data were mapped and analyzed for spatial dependence. County-level BPMV prevalence was 41% and 91% in 2005 and 2006, respectively, whereas BPMV incidence ranged from 0 to 30% and 0 to 100%, respectively. There was significant spatial dependence for BPMV incidence (using Moran’s I), indicating clustering of counties with similar levels of BPMV incidence. There was a significant negative linear relationship (R(^2) = 37.4%) between BPMV county incidence and predicted bean leaf beetle mortality. These results indicate that multi-county-scale biotic and abiotic risk factors may be associated with BPMV risk and, therefore, management tactics should also be deployed (or not deployed) at a multi-county scale.
Spatial and temporal spread of bean pod mottle virus (BPMV) and relationship between time of BPMV infection and soybean yield. E. BYAMUKAMA, A. Robertson, and F. W. Nutter, Jr. Department of Plant Pathology, Iowa State University, Ames, IA.
Little quantitative information is available concerning the temporal and spatial spread of bean pod mottle virus. Moreover, the relationship between time of BPMV infection and yield is not well documented. To quantify temporal and spatial spread of BPMV, field plots were divided into 150, 30-cm quadrats and sampled every 10-11 days and then tested for BPMV by ELISA. At harvest, quadrats were grouped according to the date of first BPMV detection. The rate of BPMV infection in 2006 ranged from 0.09 to 0.17 logits/day, indicating that BPMV incidence was doubling every 4.1 to 8.3 days. Plots that had early onset of BPMV also had the highest BPMV incidence (R(^2) = 76.9%). Spatial pattern of BPMV within plots was highly aggregated. There was a strong relationship between time of BPMV infection (detection) and soybean yield, with yield loss ranging from 27.4% for early infection to 1.5% late season infection. For every 4.3 days that BPMV infection was delayed in 2006, soybean yield increased by a bushel. Thus tactics that delay time of infection can provide a substantial economic benefit to soybean producers.
A rapid method for identifying fungicide resistance. R. Deford (1), J. Beckerman (1), and K. Cox (2). (1) Department of Botany and Plant Pathology, Purdue University, 915 W. State St., West Lafayette, IN 47906, USA; (2) Department of Plant Pathology, New York State Agricultural Experiment Station, Geneva, NY 14456, USA.
Fungicide resistance is a problem that has had a tremendous impact on agriculture worldwide. Effective tests must not only be accurate, but must get the results to the producer quickly. Traditional poison agar assays to determine fungicide resistance often require several days to weeks to achieve results. We have developed an approach to detect resistance that has the capability of producing reliable results in as little as a day. Alamar blue dye, which turns pink in the presence of respiration, was used to detect respiration in 96-well tissue culture plates. Each well contained 100 µl potato dextrose broth, 10 µl alamar blue, 10(^5) spore solution of Monlinia fruticola, and either 0, 0.0003, 0.003, or 0.03 µg fenbuconzole. Ten isolates were used that had previously been classified in regard to fenbuconazole sensitivity, of which the evaluator was unaware during the time of the tests. Results were determined visually by recording colorimetric changes regardless of fungicide concentration. Our results using Alamar blue dye corroborated the results determined by traditional poison agar assays, but in 24 hours compared to 3 days. We estimate reagent and time costs to be significantly less than traditional assays. This technology could drastically improve the efficiency of our response to producers with local fungicide resistance concerns.
Transducin beta-like gene 1 (TBL1) in Magnaporthe grisea is essential for infectious growth. S.-L. Ding and J.-R. Xu. Department of Botany and Plant Pathology, Purdue University.
The TBL1 gene was identified by random insertional mutagenesis in Fusarium graminearum to be required for wheat head infection. It is well conserved among Pyrenomycetes and may represent a novel fungal pathogenicity factor. To further characterize its role in plant infection, we generated a tbl1 deletion mutant in Magnaporthe grisea. The tbl1 mutant was significantly reduced in conidiation and failed to cause typical blast lesions on rice seedlings. At 7 days post inoculation, the tbl1 mutant had no defect in germination and appressorium formation. However, appressoria formed by the tbl1 mutant were reduced in the efficiency of appressorial penetration. For those appressoria that were successful in penetration, the primary infectious hyphae had limited and failed to differentiate into secondary infectious hyphae. In penetration assays with rice leaf sheath epidermal cells, extensive host defense responses and local plant cell death were often observed in cell penetrated by the tbl1 mutant. These data indicate that TBL1 is required for the differentiation of secondary infectious hyphae and plant colonization in M. grisea. TBL1 may be involved in overcoming plant defense responses pre- and post-penetration.
Yield loss associated with Septoria brown spot. A. E. Dorrance (1), D. Mills (1), J. Davlin (2), B. Eisley (2), and C. CRUZ (1). (1) Dept. of Plant Pathology, The Ohio State University, Wooster, OH 44691; (2) Western Research Station, The Ohio State University, S. Charleston, OH 45368.
Septoria brown spot has been thought of as a minor disease in soybeans, affecting primarily the lower canopy. The objective of these studies was to determine if fungicide applications may manage Septoria brown spot and improve yield. Four cultivars, Pioneer 93M92, Dekalb DKB38-52, Midwest GR 3931, and Asgrow AG3905 were planted in two locations during 2006 at the Western and Northwest Research Stations. In one study, tebuconazole and pyraclostrobin were evaluated at R3 and R5; while in a second study, chlorothalonil was applied at almost weekly intervals. At Northwest, tebuconazole plus pyraclostrobin applied alone or in combination significantly reduced brown spot when applied at R3 but not at R5 compared to one nontreated control. Corresponding yields averaged 3 and 0.5 bu/A greater in fungicide treatment than nontreated at Western and Northwest, respectively. Chlorothalonil applied at 6 to 10 consecutive times significantly reduced brown spot but the corresponding yield increases averaged 2.2 bu/acre.
Armillaria diversity in two Michigan Montmorency tart cherry orchards. M. L. Ellis (2), T. J. Proffer (1), G. W. Sundin (2), J. L. Jacobs (2), and R. Hammerschmidt (2). (1) Dept. Biological Sciences, Kent State Univ., Salem, OH 44460; (2) Dept. Plant Pathology, Michigan State Univ., E. Lansing, MI 48824.
Armillaria spp. cause shoestring root rot, an important disease of Montmorency tart cherry trees in Michigan. In 2006, two tart cherry orchards in the northwest region of Michigan were intensively surveyed to examine the strain diversity of Armillaria. The collected isolates were analyzed using somatic incompatibility tests and molecular techniques. Six clones of A. ostoyae were detected from 22 isolates from the first orchard, and 22 clones representing at least three different species were detected from 81 isolates from the second orchard. Isolates were tentatively identified as A. ostoyae, A. mellea, A. gallica and/or A. calvescens using established protocols for PCR and RFLP of the IGS-1 region. This study has revealed the presence of previously unreported species of Armillaria in the major cherry producing region of Leelanau County. These findings expand available information on population diversity of Armillaria and will provide growers with valuable information on whether a site is suitable for orchards or vineyards.
Early growth of a nonhost pathogen, Mycosphaerella graminicola, on barley. E. HELLIWELL (1) and S. B. Goodwin (2). (1) Dept. Botany & Plant Pathology and (2) USDA-ARS, 915 W. State St., Purdue Univ., W. Lafayette, IN 47907.
Mycosphaerella graminicola causes Septoria tritici blotch of wheat, but not barley. Septoria passerinii causes speckled leaf blotch of barley and is closely related to M. graminicola, but does not infect wheat. Our goal was to analyze the early growth of M. graminicola during nonhost interactions with barley compared to R-gene and susceptible interactions of barley inoculated with S. passerinii, and R-gene and susceptible interactions of wheat inoculated with M. graminicola. Trypan blue staining showed that M. graminicola germinates on barley leaves and enters via the stomata similarly to wheat, but fungal growth stagnates shortly after penetration. Staining with 3,3-diaminobenzidine showed an accumulation of H(2)O(2) around stomatal cells and, later, epidermal cells, indicating a possible hypersensitive response. Quantitative real-time PCR showed differences in fungal biomass among the interactions. These data show that M. graminicola penetrates cells and triggers production of reactive oxygen species, providing further evidence for an active defense response of barley to this wheat pathogen.
Characterization of Michigan isolates of Armillaria using endo-polygalacturonase and phenoloxidizing enzymes. S. I. HOLLOSY and R. Hammerschmidt. Dept. Plant Pathology, Michigan State University, East Lansing, MI 48824.
Armillaria is a complex genus of basidiomycetes that is composed of both plant pathogenic and saprobic species. How Armillaria species differ in their ability to produce degradative enzymes needed for parasitic and/or saprophytic growth has not been defined by previous research. This study involved biochemical characterization of Michigan isolates of Armillaria gathered from orchards and forested areas. Armillaria spp. were grown on various carbon sources and culture filtrates were analyzed for endopectinolytic and phenoloxidizing enzymes. Polygalacturonase (PG) activity was greatest in A. mellea filtrates. A. ostoyae and A. gallica exhibited moderate PG activity. Thus far, laccase has only been detected in A. gallica culture filtrates. Pathogenic species (A. mellea and A. ostoyae) have shown a tendency to produce greater activities of pectinolytic enzymes and less laccase activity than saprobic Armillaria species. These results indicate that there may be a difference in the degradative enzymes produced by pathogenic and saprophytic Armillaria species. This information may be helpful in determining those characteristics that are essential for an isolate of Armillaria to be pathogenic.
Brown stem rot development and stem populations of Phialophora gregata f. sp. sojae are influenced by soybean monocultures. T. J. HUGHES, N. C. Koval, and C. R. Grau. Dept. of Plant Pathology, University of Wisconsin-Madison, 53706.
Ten soybean genotype-induced monocultures were established in a location never previously seeded to soybean at Arlington, WI. The objective was to study the effects of continuous cropping of brown stem rot (BSR) resistant and susceptible soybean cultivars on populations of Phialophora gregata f. sp. sojae (Pgs) genotypes A and B and the disease BSR. Presence of Pgs genotypes A and B within stems was determined by isolation of the pathogen followed by PCR to identify genotype. BSR foliar and stem symptom severity were generally consistent within each BSR-resistant and susceptible cultivar irrespective of the monoculture. Genotype B was predominate in BSR-resistant (4.3:1) and BSR-susceptible (3.5:1) soybean cultivars grown on a BSR-resistant monoculture. Similar results were obtained for BSR-resistant (4.5:1) cultivars grown on a BSR-susceptible monoculture. Pgs genotypes A and B were observed in similar frequency for BSR-susceptible cultivars grown on a BSR-susceptible monoculture. The data supports the preference of Pgs genotype B for cultivars resistant to BSR.
Effects of latent infection by Phialophora gregata on physiology and growth of soybean. A. E. IMPULLITTI and D. K. Malvick. University of Minnesota, St. Paul, MN 55108.
Latent infection of soybean by the brown stem rot pathogen, P. gregata (PG), begins by the V3 growth stage and continues until symptoms appear during reproductive growth stages. To investigate if PG is a parasite or survives as a neutral symbiont during latent infection, we assessed leaf area (LA) and stomatal resistance of one resistant (R) and one susceptible (S) cultivar infected with PG. Plants were inoculated with type A of PG, known to cause leaf and stem symptoms, and type B, which causes stem symptoms only. Two weeks after inoculation (WAI), LA of R and S cultivars infected with type A was reduced 20% or 40% compared to controls, respectively, whereas LA was not affected by type B. Four WAI (end of V growth stages), the LA of the R cultivar infected with type A was similar to controls, however, the LA of the S cultivar was reduced 60%. Type B reduced the LA of the S cultivar 35%, but did not reduce the LA of the R cultivar four WAI. Stomatal resistance, as measured with a porometer, did not differ regardless of treatment. Preliminary results suggest latent infection reduces photosynthetic area but has negligible effects on stomatal resistance (water stress).
AREA, nitrogen regulatory gene, influences fumonisin B1 production of Fusarium verticillioides. H. Kim and C. P. Woloshuk. Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907.
Fumonisin B1 (FB1) is nitrogen-containing, a secondary metabolite produced by Fusarium verticillioides. Previous studies have indicated that FB1 biosynthesis is repressed in cultures containing ammonium phosphate (AP) as a nitrogen source. The fungus also fails to produce FB1 when grown on blister stage kernels. The growth rate of a disrupted-AREA mutant, AREA11, on defined media containing either AP or casein hydrolysate was similar to the wild type, but was substantially lower on media containing sodium nitrate or bovine serum albumin. On mature maize kernels, the mutant did not produce FB1, most likely because of poor growth. When AP was added to kernels, the mutant grew as well as the wild-type strain, but neither strain accumulated FB1. This result indicates that the AREA gene is required for growth on mature kernels and possibly for FUM gene expression. The AREA11 strain also grew well on blister kernels, indicating that the elevated levels of free amino acids present in early stages of kernel development are sufficient to support growth of the mutant. We will present results from additional experiments that demonstrate how different nitrogen and carbon sources affect FB1 biosynthesis in a strain that over-expresses AREA.
A transducin beta-like gene tbl1 is necessary for pathogenicity in Fusarium graminearum. C. Koten. Dept. of Pathology, Purdue University, West Lafayette, IN 47907.
Fusarium head blight (FHB) or scab caused by Fusarium graminearum is an important disease of wheat and barley. In addition to yield losses, infected grains are reduced in quality and contaminated with mycotoxins. In our previous study, we identified several mutants with reduced virulence by random insertional mutagenesis. In one of these mutants, the transforming vector was inserted in a predicted gene named tbl1 (for transducin beta-like gene 1). Tbl1 is homologous to the mouse tblr1, which encodes a putative nuclear receptor corepressor. The yeast homologue is sif2, a factor in the SET3 complex which is involved in sporulation. In humans, tbl1 functions as a transcriptional co repressor and is vital during neuronal differentiation. The TBL1 protein contains three WD40 repeats and an N-terminal LisH domain, which is involved in protein-protein interactions. We generated the tbl1 deletion mutant by the ligation PCR gene replacement approach. The delta tbl1 mutant was non-pathogenic and significantly reduced in conidiation. It was defective in colonizing flowering wheat heads and more sensitive to a plant defensin MsDef1. Conidium germination was delayed in the tbl1 mutant. Interestingly, tbl1 is the only LisH domain-containing gene in the F. graminearum genome. To determine its function, we generated a tbl1 (^delta LisH) allele and transformed it into the delta tbl1 mutant. Phenotype analysis of the resulting transformants expressing tbl1 (^delta LisH) suggests that the LisH domain is essential for the tbl1 function and plant infection. To examine the differences between wild type and mutant, we generated a tbl1- GUS construct and examined GUS expression on wheat heads and isolated kernels. Our results indicate that tbl1 plays a critical role in conidium germination, response to plant defense compounds, and colonization of wheat tissues.
Assessing effects of temperatures on occurrence of Asian soybean rust with biogeography information of rust diseases in North America. X. Li and X. B. Yang. Department of Plant Pathology, Iowa State University, Ames, IA 50011.
Optimal infection temperatures of rust diseases and the average/maximum July mean temperatures along the northern boundary of geographic distribution range of each rust disease in North America were examined. Linear correlation between optimum infection temperatures and July mean temperatures along these northern boundaries of disease range were modeled. With optimal infection temperature of soybean rust (Phakopsora pachyrhizi, SBR), SBR potential distribution range was calculated using the regression models based on rusts with similar disease cycle to SBR. This range was defined by either of two contours: where average or maximum July mean temperature was 21°C or 24°C respectively for the period of 1968 to 1996. This range includes most of the U.S. soybean production areas. Temperature favorability for SBR infection was assessed using daily data from 1961 to 2000. The southeastern U.S. has the highest temperature favorability. The southern Midwest in west of Mississippi River is less favorable. The North Central region is moderate. Texas and the northern states have low favorability.
Ecological grouping of soybean fungal diseases in the United States: Implications to the risk of Asian soybean rust. X. LI and X. B. Yang. Department of Plant Pathology, Iowa State University, Ames, IA 50011.
Optimal temperature of disease development, host ranges, moisture requirement for infection, secondary infection, means of dispersal, and capacities of pathogen survival, were used to describe 34 soybean fungal diseases in the U.S. quantitatively. Gower’s similarity coefficient suggested similar epidemiology of soybean rust (Phakopsora pachyrhizi) in North America to that of downy mildew. Principal coordinate analysis projected these diseases in a 2-dimension space with significant patterns identified for variables of means of inoculum dispersal and free moisture requirement for pathogen infection. Cluster analysis identified three major disease clusters. The first mainly consisted of leaf diseases. The second consisted of leaf/pod diseases, including soybean rust, brown spot, frogeye leaf spot, Phyllosticta leaf spot, purple seed stain, etc. Seedborne root diseases formed the third cluster. Diseases in the second cluster which historically only caused light to moderate yield losses in the North Central imply that soybean rust would probably cause light yield losses as well in this region.
Effects of monoculture on SCN population densities and SDS severity in field microplots. H. L. MEHL, L. J. Xing, and A. Westphal. Dept. Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907.
Fusarium solani f. sp. glycines (Fsg) causes sudden death syndrome (SDS) of soybean. The interaction of soybean cyst nematode (SCN) and Fsg was studied in field microplots. In 2005, field soil was placed in microplots and either non-treated or methyl bromide-fumigated; in factorial design, plots were infested with Fsg and/or SCN. Plots were planted to soybean cv. Williams 82 in spring 2005 and 2006. Foliar symptoms of SDS in 2006 were greatest in plots infested with both Fsg and SCN. There was a significant interaction of the fumigation, watering, SCN, and Fsg factors. In plots without the SCN-infestation treatment, SDS foliar symptoms were greater in non-fumigated plots; in SCN-infested plots, SDS foliar symptoms were greater in fumigated plots. Fumigated plots that were irrigated had greater SDS foliar symptoms than plots that were not watered; the opposite was observed in non-fumigated plots. At harvest, SCN egg numbers averaged across SCN infestation treatments were higher in fumigated soil compared to non-treated soil. The low numbers of eggs in the non-fumigated SCN-infested plots in 2006 suggest that the soil had become suppressive to SCN. The trial demonstrates modulation of the SDS and SCN disease complex by abiotic and biotic factors.
The MADS-box transcription factor Rlm1 is required for invasive growth in Magnaporthe grisea. R. Mehrabi and J.-R. Xu. Dept. Botany and Plant Pathology, Purdue University, West Lafayette, IN 47906.
Magnaporthe grisea is an ascomycetous fungus that causes devastating blast disease in graminaceous hosts such as rice, wheat, barley, and millet. We previously characterized the M. grisea MPS1 MAP kinase gene (the homolog of yeast SLT2) and found that appressoria formed by the mps1 mutant failed to penetrate the plant cells. We are currently investigating transcription factors putatively regulated by Mps1 MAP kinase. RLMl is one of the transcription factors found in the genome sequence of M. grisea and contains a MADS box type DNA binding domain and a MAP kinase docking site that possibly is the docking site for the cell wall integrity MAP kianse Mps1. Unlike the mps1 mutant, rlm1 deletion mutant shows normal conidiation and colony morphology. In the infection assays rlm1 mutant failed to cause typical blast lesion on the rice and barley seedlings. Interestingly rlm1 mutant is able to form appressoria and penetrate plant cells but failed to develop infectious hyphae. However, rlm1 mutant is able to penetrate and develop infectious hyphae when the plant cell responses are prevented by heat inactivation. Our data may indicate that RLM1 is involved in invasive growth possibly through overcoming plant defense responses.
Detached leaf assay a rapid screening technique to study foliar diseases of soybean. J. Mo (1), S. S. Navi (2), X. Li (2), T. Guo (1), and X. B. Yang (2). (1) Plant Protection Institute, Guangxi Academy of Agricultural Sciences (GXAAS), Nanning, 530 007, P. R. China; (2) Department of Plant Pathology, Iowa State University (ISU), Ames, IA 50011.
A detached leaf screening technique was developed to evaluate soybean for resistance to powdery mildew (Microsphaera diffusa) at ISU and rust (Phakopsora pachyrhizi) at GXAAS. In powdery mildew, first fully opened trifoliate leaves were collected excising at the base of petiole from three varieties, placed two leaves per 14-cm dia × 2-cm ht sterile Petridish containing a layer of wet wahtman#1 filter paper and sponge (10-cm L × 5-cm W × 1-cm H). Leaves placed in Petri dishes for detached leaf assay (DLA) and the seedlings grown in plastic cups for whole plant assay (WPA) were spray inoculated separately with 1 × 10(^3) and 1 × 10(^4), and 1 × 10(^5) conidia ml(^–1) and distilled water spray maintained as control. Inoculated leaves and seedlings were incubated at 24°C in fluorescent light (radiation of 5.48 J/second × m(^2) area) with 16h photoperiod for two weeks and were evaluated for disease reaction. Of the three varieties tested, Pioneer 93M42 showed higher severities compared with Latham 2485 and Pioneer 92M40RR and of the three concentrations, 1 × 10(^5) showed higher severities compared with other two. The DLA and WPA were tested for soybean rust pathogen using 10 cultivars and six isolates collected from three locations in China. Of the ten genotypes tested Ye-Qing × Bu-Lei-Mei showed S-S reaction across all the isolates and reactions of other genotypes against the isolates tested are presented in this paper. Also, we discuss results of the techniques tested against P. pachyrhizi, and M. diffusa. Main advantage of the DLA method is to facilitate assaying soybean response to multiple fungal pathogens in a short period.
The role of plant pathogens in suppressing growth and development of Abutilon theophrasti (velvetleaf). J. A. Okalebo, J. L. Lindquist, G. Yuen, R. Drijber, and E. Blankenship, Univ. of Nebraska – Lincoln.
Weed-suppressive soils may develop over time. Populations of pathogens specific to a weed host may increase naturally with the growth and spread of the weed. Velvetleaf death and growth suppression observed in a field (A) was investigated for the role that plant pathogens may play in this phenomenon. Soils were collected from five fields (including A) and tested in the greenhouse for biological suppressiveness. In the first experiment, surface-sterilized velvetleaf seeds were sown in pots containing the 5 soils and after 8 weeks of growth, velvetleaf mortality was greatest (84%) and dry biomass was smallest (0.3 g pot(^–1)) in soilA compared to the other soils. In a second experiment, velvetleaf growth in soilA subjected to heat-treatment (autoclaving) was compared to that of untreated soilA in its natural state. After 5 weeks, plants in heat-treated soilA yielded an average dry plant biomass of 0.14 g and leaf area of 45.2 cm(^2) compared to 0.03 g plant(^–1) and 5.8 cm(^2) plant(^–1) in the untreated soil. The high mortality and reduced growth observed may be due to pathogenic soilborne species such as Rhizoctonia solani, Pythium and Fusarium species. Further research is needed to identify soilborne microbes and the pathogenic effects on velvetleaf. Corn and soybean production in Nebraska, may potentially benefit from the development of a biological control agent/s for velvetleaf, a problem weed.
Characterization and screening of a bacterial artificial chromosome (BAC) library of soybean Pl399073. W. PIPATPONGPINYO and A. E. Dorrance. Dept. of Plant Pathology, The Ohio State University, Wooster, OH 44691.
A BAC library of soybean Pl399073 was constructed with 3.4% and 1.6% contamination from chloroplast and mitochondrial DNA, respectively; and represents 18X genome equivalents. This library demonstrated either a fast or slow colony growth-rate phenotype. Our objective was to characterize the library inserts in fast- and slow- growing library members. The libraries were arrayed into a total of 156,672 wells, of which approximately 25% were from slow-growing colonies. The average insert size of the slow library was 167 Kb. The average insert size of the fast library was 161 Kb. Microsatellite markers, which map to Rps8, were used to screen members of the ‘fast’ and ‘slow’ libraries. The slow library has the highest percentage of positive clones compared to the fast library. These results indicate that the slow-growing library does have a higher representation of this particular gene-rich region of the soybean genome.
Plant stress amelioration by phlD positive pseudomonads. R. E. RAUDALES, E. Stone, and B. B. McSpadden Gardener. Department of Plant Pathology, The Ohio State University-OARDC, Wooster, OH 44691.
During a field trial of bacterial seed treatments, plants inoculated with phlD positive pseudomonads were observed to be healthier than untreated plants in a section of the field characterized by low soil pH. To understand the effect of artificial sources of inoculum of these bacteria on corn plants grown under acid conditions; soils were collected from untreated subplots that had the tallest and the shortest plants for use in a greenhouse assays. Corn seeds were inoculated with the strain Wood1R of phlD positive pseudomonads independently and in association with a commercial source of mycorrhiza. Unexpectedly, the mycorrhiza increased the white lesions that are caused under acid conditions. However, when the mycorrhiza was co-inoculated with Wood1R the number of lesions was reduced. In addition, high levels of manganese (Mn) were detected in the plant tissue. These levels were significantly diminished in the plants that had been inoculated with Wood1R. Strong and positive correlations were observed between the Mn and the white lesions; suggesting that the mechanism by of plant protection was related to reduction of Mn uptake. This study provides evidence for the ability of inoculated populations of phlD positive pseudomonads to ameliorate the stress in corn caused by low soil pH.
Effect of tillage on population levels of Heterodera glycines in a crop sequence of corn and a nematode-susceptible or -resistant cultivar of soybean. A. SEYB (1), L. J. Xing (1), T. J. Vyn (2), and A. Westphal (1). Depts of (1) Botany and Plant Pathology, and (2) Agronomy, Purdue University, West Lafayette, IN 47907.
Heterodera glycines is one of the most damaging parasites of soybean. Sustainable management of this nematode is challenging and alternative agronomic practices are urgently needed. In long-term tillage plots at the Agricultural Center for Research and Education, Purdue University, the effects of tillage on the levels of H. glycines in a corn-soybean rotation under a susceptible or a resistant cultivar of soybean were investigated. The tillage treatments were: (A) moldboard plow and secondary tillage, (B) chisel plow and secondary tillage, (C) ridge tillage, and (D) no-tillage. Within the tillage treatments, subplots were planted to the susceptible cultivar, Williams 82 or the resistant cultivar, Pioneer 93M80. Soil samples to a depth of 30 cm were taken in spring and fall of 2005. In all tillage treatments, numbers of cysts were lower in soil planted to the resistant cultivar. Similar effects of the tillage treatments were found with both cultivars. In spring, the numbers of cysts and eggs were lower under no-tillage and ridge tillage than under chisel plow and moldboard plow. In fall, lower numbers of cysts and eggs were found in the no-tillage treatment than under chisel plow and mold board plow. Continuous low tillage intensity can reduce population densities of H. glycines and thus may improve sustainable production of soybean.
Multiplex real-time PCR assay to detect and quantify three genera of mycotoxigenic fungi. Y. Suanthie and C. P. Woloshuk. Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907.
Mycotoxins are fungal secondary metabolites that cause severe health-related problems when consumed by humans and animals. The most important mycotoxins that contaminate agricultural commodities are produced by species of Aspergillus, Fusarium, and Penicillium. Aspergillus species produce aflatoxins, ochratoxins, and sterigmatocystin; Fusarium species produce fumonisins, trichothecenes, and zearalenone; and Penicillium species produce ochratoxins, patulins, and citrinins. As part of a comprehensive approach for monitoring the presence of mycotoxigenic fungi, we are developing protocols based on Real-Time PCR techniques that will detect, identify, and quantify mycotoxigenic fungi. In this study, our objectives are to develop genus-specific Taqman® probes for Aspergillus, Fusarium, and Penicillium and use them in a multiplex assay. As the first step in our monitoring system, this assay will guide subsequent decisions for toxin-specific PCR and mycotoxin analyses.
Resistance to azoxystrobin and pyraclostrobin present in Ascochyta rabiei in North Dakota. K. A. WISE, C. A. Bradley, J. S. Pasche, and N. C. Gudmestad. Dept. Plant Pathology, North Dakota State University, Fargo, ND 58105.
Ascochyta blight (Ascochyta rabiei), is the most important disease of chickpea in the United States. Producers in North Dakota have relied on applications of QoI fungicides to manage this disease since 2002. Isolates of A. rabiei were collected from chickpea fields in North Dakota in 2005 and 2006 to monitor shifts in sensitivity to azoxystrobin and pyraclostrobin. An in vitro spore germination assay was used to determine EC(50) values for isolate sensitivities to azoxystrobin and pyraclostrobin. EC(50) values for collected isolates were compared to mean EC(50) values of baseline isolates. Results indicate that resistance to QoI fungicides was present in North Dakota in 2005 and 2006. Isolates collected in 2006 are up to 1000x less sensitive to azoxystrobin and pyraclostrobin than baseline isolates. A strong correlation was observed between isolate EC(50) values for azoxystrobin and pyraclostrobin, indicating that cross-sensitivity to these fungicides exists in the North Dakota A. rabiei population.
Expression of a dominant active MgRas2 bypassed surface recognition and attachment signals for appressorium formation in Magnaporthe grisea. X. Zhao, C. Xue, Y. Kim, and J.-R. Xu. Department of Botany and Plant Pathology, Purdue University.
Ras proteins are low molecular weight GTP-binding proteins that function as a molecular switch in signal transduction. We have identified two Ras homologues in Magnaporthe grisea. The mgras1 deletion mutant had no detectable phenotypes but the efforts to generate a mgras2 mutant failed, indicating that MgRAS2 may be an essential gene. Expression of a dominant active MgRas2(^G23V) allele in the wild-type strain had no effect on vegetative growth but enhanced conidiation. Transformants expressing MgRas2(^G23V)(DA66) efficiently formed appressoria on both hydrophobic and hydrophilic surfaces. Interestingly, DA66 formed melanized appressorium- like structures on the tip of aerial hyphae. We then transformed the MgRas2(^G23V) construct into other appressorium-forming fungi. In the resulting transformants, appressorium-like structures were observed at hyphal tips. When an appressorium-specific reporter was transformed into DA66, GFP signals were observed in these appressorium-like structures. DA66 had an elevated intracellular cAMP level and enhanced phosphorylation of the Pmk1 MAP kinase. However, expressing MgRas2(^G23V) had no effect in the mutants that blocked the Pmk1 pathway. These data indicated that MgRas2 may function upstream from both the cAMP/PKA and Pmk1 MAP kinase pathways. The G23V mutation in MgRas2 enables M. grisea to bypass surface recognition and attachment signals for appressorium formation.