Determination of presumptive vegetative compatibility groups of Verticillium dahliae occurring on sunflower using molecular markers
Genetic diversity of a global population of Colletotrichum coccodes using amplified fragment length polymorphism markersK. ALANANBEH (1), L. Tsror (2), N. Gudmestad (1)(1) North Dakota State University, Fargo, ND, U.S.A.; (2) Plant Pathology Unit, Gilat Research Center, Mobile Post Negev 85280, ISRAELColletotrichum coccodes (Wallr.) Hughes is an imperfect fungus in which vegetative compatibility may serve as a means of genetic exchange. Vegetative compatibility grouping (VCG) has been useful for measuring genotypic diversity but has certain limitations that reduce its effectiveness and utility. In the case of C. coccodes, isolates from different continents anastomose reliably and it is a useful tool in studying global population except for cases where no anastomosis occurs between nit mutants, or in case isolates are not capable to produce nit mutants. Molecular markers have proven very effective in studying the genetic diversity of several plant pathogens. The main objective of this study was to study the genetic diversity of a global population of C. coccodes, including North American, European/Israeli, Australian, and South African isolates, using AFLP markers. A total of 788 C. coccodes isolates were studied using three primer pairs. NA-VCG5 was the most common VCG globally, followed by NA-VCG2. Among the four regions studied, there was a relatively low gene diversity (h = 0.22), relatively high population differentiation GST (0.30), and low but significant linkage disequilibrium (rBarD < 0.09) except for isolates from South Africa. Overall gene flow (Nm) was 1.16, meaning that one or more individuals are exchanged among the five regions each generation. Most of the variation among the four geographic regions originated from the within population differentiation (?PT) (52.50%) while variation among regions and among populations was 0.0% and 0.05% respectively. Based on geographic origin, the global C. coccodes population had five main groups NA-VCG1, NA-VCG2, NA-VCG3, NA-VCG4/5, and NA-VCG6/7. Conversely, C. coccodes differentiation based on ancestry using a Bayesian clustering approach showed six main subpopulations. Values of gene diversity, population differentiation, linkage disequilibrium, gene flow, variation among subpopulations, and variation within subpopulations were 0.22, 0.46, 0.04–0.09, 0.58, 43%, and 57% respectively. Based on those analyses, the population of C. coccodes exists as one large population with four main groups (NA-VCG1/3; NA-VCG2; NA-VCG4/5; and NA-VCG6/7). Our data suggest that global C. coccodes population is closely related, and probably of the same origin. It is likely that C. coccodes became established on each continent as the potato was moved around the globe. Due to the lack of intermixing of the populations after their introduction, the C. coccodes population on a continent became bottle-necked which may explain the loss of vegetative compatibility among regional populations. AFLP, a dominant marker, proved valuable in differentiating and studying the global population of C. coccodes, however, using co-dominant markers which can detect heterozygous alleles, such as simple sequence repeat markers (SSR) could provide additional information at the genetic level and could further differentiate the global C. coccodes population.
Understanding the epidemiology of Cercospora kikuchii on soybean using foliar fungicidesA. AMIRI (1), C. R. Grau (1), N. C. Koval (1), P. D. Esker (1)(1) University of Wisconsin, Madison, WI, U.S.A.Foliar fungicides may be effective for managing Cercospora leaf blight (CLB) caused by Cercospora kikuchii in soybean. To improve our understanding of the epidemiology of C. kikuchii and to test the hypothesis that early foliar fungicide applications can disrupt the infection process of the pathogen, multifactorial trials were conducted in 2008 and 2009 at the Arlington and West Madison Agricultural Research Stations. Azoxystrobin + propiconazole (Quilt) was applied to four soybean varieties (AG2422, DSR221, DSR234, K245) at one of three growth stages (V5,R1,R3). Disease assessments were made one week post-R3 application on a weekly basis and yield was determined at harvest. CLB severity was numerically different among varieties in 2008, and was significantly affected by the variety x fungicide interaction in 2009 (P = 0.0024). The varieties AG2422 and K245 had higher severity in both years. There were no significant differences in CLB incidence and severity for the application timings, and in 2009 the highest severity was observed when Quilt was applied at V5 stage with the exception of the DSR221 variety. In 2008, there was no evidence of an effect of fungicide or variety on grain yield, while in 2009, there was an effect of variety (P = 0.0015). Current results suggest that early applications of azoxystrobin + propiconazole may not disrupt C. kikuchii infections in soybean and that further research is needed to investigate the role of latent infections.
Management of anthracnose stem blight with foliar fungicidesN. BESTOR (1), D. Mueller (1), A. Robertson (1)(1) Iowa State UniversityAnthracnose stem blight, most often caused by Colletotrichum truncatum, has been documented to cause soybean yield loss and reduction in seed quality. There are several fungicides available in Iowa for use on soybean that includes anthracnose on their label. The effect of fungicides on anthracnose severity was evaluated at five locations across Iowa in 2008 and 2009. Two fungicides, Headline and Stratego Pro, were applied at one of two timings (growth stages R1 and R3). Percent anthracnose severity was estimated on 20 plants from each plot at growth stage R8. Soybeans were harvested using a plot combine and all yields were standardized bushels per acre and at 13% moisture. Fungicides reduced anthracnose severity in both 2008 and 2009. However, there was no correlation between anthracnose severity and yield. Furthermore, yield component data was collected from 80 to 100 plants in the untreated control and correlated to the level of anthracnose on each plant. Data collected included pods per plant, seeds per pod, and seed size. There was no correlation between anthracnose severity and yield component data.
Evaluating the prevalence of bacterial blight of dry beans in North Dakota and pathogens involvedY. CHANG (2), R. Lamppa (2), J. Prishmann (1), H. Bata (2), R. S. Goswami (2)(1) ND State Seed; (2) North Dakota State UniversityBacterial blight has been a prevailing concern in most dry edible bean growing regions including North Dakota. The three major bacterial blight pathogens of dry beans reported in this area and their respective diseases are - Xanthomonas axonopodis pv. phaseoli (Xap) - Common bacterial blight; Pseudomonas syringae pv. phaseolicola (Psp) - halo blight and Pseudomonas syringae pv. syringae (Pss)- Bacterial brown spot. These are primarily seed transmitted and highly regulated, making them a major threat to the dry bean seed industry; particularly in North Dakota which is the largest producer of dry edibles in the country. The objectives of this study were to ascertain the prevalence of each of these three pathogens in North Dakota and to evaluate possible race changes in Psp. A foliar disease survey conducted in 2008 and 2009 covered 39 and 58 dry bean fields respectively. Pathogens isolated were identified through biochemical tests and species specific PCR. Psp was isolated from 47% and 12% of the fields surveyed in 2008 and 2009 and Pss was detected from 50% and 26% of the fields in the two years respectively. Pathogenicity tests were conducted on Pss and Psp isolates from 2009. All isolates were found to be capable of causing disease on a susceptible variety of dry bean but a variation in aggressiveness among Psp isolates was observed. Preliminary race typing results indicate the presence of race 6 and 8 of Psp in this region.
A survey of fungicide resistance in the Venturia inaequalis populations of Indiana and MichiganK. S. CHAPMAN (2), G. Sundin (1), J. Beckerman (2)(1) Michigan State University, East Lansing, MI, U.S.A.; (2) Purdue University, West Lafayette, IN, U.S.A.Venturia inaequalis, the causal agent of apple scab, infects apple trees. Long-term and extensive fungicide use has led to multiple fungicide resistances developing with varying frequencies in different orchards. To assess fungicide resistance levels, isolates of V. inaequalis were collected from Indiana and Michigan orchards. Single-spore derived isolates were evaluated using mycelium growth assays with previously determined baseline concentrations of fungicides and corresponding thresholds for growth. Fungicides tested include: kresoxim-methyl, thiophanate-methyl, dodine and myclobutanil. We identified isolates which were classified as resistant or shifting towards resistant to each fungicide. Resistance to kresoxim-methyl and myclobutanil, the primary fungicides used for apple scab management, was present in both states. This is the first report of field resistance to kresoxim-methyl in the United States. A total of 19% of isolates from Indiana and 44% in Michigan were shifted to kresoxim-methyl. Resistance to myclobutanil occurred in over 55% of isolates. Isolates that tested resistant or shifted often tested this way for multiple chemicals. Of 199 isolates tested, 38% were identified as resistant or shifted to two fungicides and 12% were resistant or shifted to all four of the fungicides. Statistical analyses of spore counts and radial mycelial growth resulted in no statistical differences between sensitive isolates and isolates resistant to one or more fungicides, suggesting no fitness penalty is associated with resistance based on these two parameters. The presence of resistance to all major fungicides used for apple scab management leaves growers with fewer control options. However, preliminary data indicates that the “second generation” DMI fungicide difenoconazole, when tested at the highest field rate, inhibits the growth of isolates resistant or shifted to a “first generation” DMI, such as myclobutanil. Thus, “second generation” DMIs may be a viable short-term management option for growers with DMI-resistance present in their orchards.
Response of Phytophthora root rot differential lines to Northern stem canker in soybeanT. E. CHASE (1), U. R. Rosyara (1)(1) South Dakota State UniversityNorthern stem canker (NSC), caused by Diaporthe phaseolorum var. caulivora (DPC), has re-emerged as a soybean disease of concern among farmers and crop consultants in the North Central region. Effective practices for managing risk from NSC have not been developed or critically assessed to date. Published, as well as unpublished, studies suggest that traditional approaches such as crop rotation or tillage may have little or no effect in reducing risk. Therefore, availability of genetic resistance would be highly desirable for soybean producers wishing to reduce risk for losses from NSC. As part of a continuing series of studies on resistance to NSC, we screened the established set of Phytophthora root & stem rot differential lines for their reaction in greenhouse seedling inoculations. All fifteen lines were susceptible to infection by DPC, resulting in typical NSC symptoms, but there were quantitative differences in reaction. No differential resistance reactions were observed to NSC, but line L76-1988, carrying Rps2 was significantly less susceptible than other lines studied. Although resistance cannot be attributed to Rps2 based on this experiment, L76-1988 may nonetheless prove to be a useful source of quantitative or partial resistance to NSC.
Use of foliar fungicides for control of gray leaf spot of corn across environmentsP. D. ESKER (4), C. A. Bradley (3), P. Paul (2), A. Robertson (1)(1) Iowa State University, Ames, IA, U.S.A.; (2) Ohio State University, Wooster, OH, U.S.A.; (3) University of Illinois, Urbana, IL, U.S.A.; (4) University of Wisconsin, Madison, WI, U.S.A.One of the most damaging foliar diseases of corn in the north-central region is gray leaf spot (GLS), caused by Cercospora zeae-maydis (Czm). The availability of data from coordinated trials on the need for foliar fungicides to control GLS is limited. Trials were established in IA, IL, OH, and WI to examine corn hybrid and fungicide application timing effects for control of GLS. Using a split plot arrangement, the whole plot factor was corn hybrid and the subplot factor was fungicide active ingredient (non-treated, pyraclostrobin, or tetraconzole) and timing. Four hybrids that differed in crop relative maturity and reaction to GLS were examined in each trial. Plants were inoculated using sterilized-sorghum infested with Czm from V9 to V12. Fungicide applications were made in 2008 at V12, VT, or R2, while in 2009, the timing was VT, R2, or R3. Trials were assessed for foliar disease incidence and severity on the ear leaf as well as grain yield and grain moisture. Preliminary analyses have focused on individual trials. Results to date have indicated large differences in yield across environments. Only in trials from IL was there evidence of an effect of either hybrid (P < 0.0001 and P = 0.0034 in 2008 and 2009, respectively) or fungicide (P = 0.0070 and P = 0.0019 in 2008 and 2009, respectively) on grain yield. Current results suggest that the risk of GLS differs across environments and impacts the likelihood that there is a yield response to fungicide.
Does previous crop history impact response to foliar fungicides in corn?P. D. ESKER (4), C. A. Bradley (3), P. Paul (2), A. Robertson (1)(1) Iowa State University, Ames, IA, U.S.A.; (2) Ohio State University, Wooster, OH, U.S.A.; (3) University of Illinois, Urbana, IL, U.S.A.; (4) University of Wisconsin, Madison, WI, U.S.A.Results from foliar fungicide trials across the north-central region have not conclusively shown that previous crop affects yield response in corn. To improve understanding of the effect of previous crop and foliar fungicide timing, trials were conducted in IA, IL, OH, and WI in 2008 and 2009. In 2008, a preliminary trial was established with a single replication of previous crop (corn or soybean) in each state. Corn hybrids were selected for local areas and management for weeds and insects followed local recommendations. There were four fungicide treatments (timings): non-treated, V12, VT, or R2. The fungicide was a pre-mix combination of propiconazole + trifloxystrobin. Trials were assessed for foliar disease incidence and severity on the ear leaf as well as grain yield and grain moisture. Based on a multi-environment analysis, there was no evidence of a difference in grain yield or grain moisture among treatments. In 2009, trials were modified slightly by location, including use of single previous crop, replicated previous crop, and multiple hybrids, and fungicide timing was also changed to VT, R2, or R3. Preliminary results for grain yield ranged from no effect of fungicide timing (IA), yield differences between previous crop soybean and corn (WI, P = 0.0572), and yield differences between hybrid (IL, P = 0.0443) and fungicide timing (IL, P = 0.0618). Current results suggest that previous crop may not be a major predictor for grain yield response to foliar fungicides.
Widespread latent and pathogenic infection of soybean by the Phomopsis disease complex in MinnesotaC. M. FLOYD (1), D. K. Malvick (1)(1) University of MinnesotaTop dieback and early-dying of soybean are common in August in Minnesota (MN). Occasional isolations suggested that the Phomopsis disease complex is often associated with these symptoms. This disease complex can cause significant damage to soybean. The goal of this study was to determine the distribution and potential importance of Phomopsis in MN soybean fields. For part A of this study, 70 symptomatic plants with top dieback, stem canker, and/or stem blight symptoms were collected from 50 fields in southern MN in 2006–2009. Fungi with morphological characteristics similar to Phomopsis and Diaporthe were isolated from stems. Genus was confirmed to be Diaporthe or Phomopsis using a standard PCR assay, and qPCR assays were used to identify species. ITS DNA sequences from a subset of the isolates all matched either P. longicolla or D. phaseolorum var. caulivora. P. longicolla was detected in 46% of the plant samples and D. phaseolorum var. caulivora was detected in 4% of the samples. For part B of this study, 150 asymptomatic plants were arbitrarily collected from 94 fields statewide in early August 2007, and the presence of these pathogens in stems was determined with PCR assays. P. longicolla was detected in 10% of the plants, and other unidentified Phomopsis or Diaporthe species were detected in 35%. For part C, four common soybean cultivars were inoculated individually with P. longicolla or D. phaseolorum var. caulivora isolates using the cut stem inoculation method. All cultivars inoculated with either pathogen developed sunken, dark brown cankers; and leaves wilted and became necrotic at infected nodes. The results confirm that Phomopsis and Diaporthe are frequently associated with stem blight and top-dieback symptoms in MN, and these fungi are widespread as a group of latent and potentially damaging pathogens in asymptomatic plants.
Evaluation of fungicides and fungicide timing on management of sunflower rust (Puccinia helianthi) at three locations in North Dakota in 2008 and 2009A. FRISKOP (4), B. Schatz (2), S. Halley (3), J. Schaefer (1), T. Gulya (5), F. Mathew (4), J. Jordahl (4), S. Meyer (4), K. Misek (3), P. Hendrickson (2), S. Markell (4)(1) Cenex Harvest States, Grandin, ND, U.S.A.; (2) NDSU-Carrington Research Extension Center, Carrington, ND, U.S.A.; (3) NDSU-Langdon Research Extension Center, Langdon, ND, U.S.A.; (4) North Dakota State University, Fargo, ND, U.S.A.; (5) USDA-ARS, Fargo, ND, U.S.A.Sunflower rust, caused by Puccinia helianthi (Sch.), incidence has increased in U.S. sunflower fields since 2002. With little genetic resistance, fungicides may be a practical management tool. However, the efficacy of many fungicides has not been assessed for sunflower rust management. Additionally, timing thresholds of newer fungicide chemistries (i.e. QoI) have not been investigated. The objective of this study was to evaluate fungicide efficacy and timing for management of sunflower rust. In separate but adjacent experiments, fungicide efficacy (fungicide trial) and timing trials (timing trial) were arranged in a randomized complete block design. Trials were conducted in 2008 and 2009 at the North Dakota State University Research Extension Centers in Carrington, ND and Langdon, ND, and at a Cenex Harvest States research plot in southeast ND. Four row plots were sown at each location with the confection hybrid ‘Jaguar’. To facilitate disease development, spreader rows were inoculated with P. helianthi race 336 and supplemental irrigation was used as needed. In the fungicide trials, six to eleven fungicides were applied at R5. In timing trials, pyraclostrobin and tebuconazole were applied at R3, R5, and R6, singularly or sequentially. To evaluate disease, pustule coverage on the top four fully-expanded leaves of ten randomly selected sunflower plants was visually estimated with the aid of disease assessment diagrams. Evaluations were conducted three to six times and area under disease progress curve (AUDPC) values were generated. Results from the fungicide trials showed all fungicides reduced AUDPC from that of the non-treated control. Results from the timing trials indicated the most efficacious time to make a fungicide application was at R5.2 when disease severity was at 1–3%. Our study suggests that fungicide applications are an effective management tool for rust. However, timing of application should be further investigated.
Soil and rhizosphere populations of Fusarium and fluorescent Pseudomonas spp. associated with field-grown plants are affected by sorghum genotypeD. L. FUNNELL-HARRIS (1), J. F. Pedersen (1), S. E. Sattler (1)(1) USDA-ARS, Grain, Forage and Bioenergy Research Unit, University of Nebraska, LincolnSorghum [Sorghum bicolor (L.) Moench] is valued for bioenergy, feed and food. Potential of sorghum genotypes to support differing populations of root- and soil-associated fluorescent Pseudomonas spp. or Fusarium spp., in two soils, was assessed. Pseudomonad and Fusarium numbers were assessed from roots and soil of field-grown sorghum genotypes, RTx433 and Redlan. Possible biological control capabilities of Pseudomonas isolates, including hydrogen cyanide (HCN) and 2,4-diacetylphlorogluconol (phl) production, also were assessed. In dryland field conditions, RTx433 roots had greater numbers of pseudomonads than Redlan before anthesis but similar numbers after. There were no differences in numbers of pseudomonads from dryland soil or roots or soil of irrigated plants. Percentages of HCN-producing root isolates and phl soil isolates declined on irrigated Redlan plants, but percentages of HCN-producers increased in dryland conditions. Redlan roots had greater percentages of Fusarium isolates in the Gibberella fujikuroi species complex. Results indicated that sorghum genotype affected rhizosphere populations of fluorescent Pseudomonas spp. and Fusarium spp. across soil environments.
Management strategies for stem rust (Puccinia graminis f. sp. tritici) of wheat using fungicidesV. GUPTA (1), J. M. Stein (1)(1) South Dakota State University, Brookings, SDStem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is one of most devastating diseases of wheat throughout the world and is a growing threat to all wheat growing regions. The management of stem rust with fungicides is of particular interest due to the evolution of highly virulent races of Pgt in eastern Africa (e.g. Ug99). Herein we present results from studies evaluating fungicidal activity against Pgt. First, a laboratory experiment was conducted to evaluate inhibition of spore germination using seven different chemicals. EC50 values were calculated and it was found that strobilurins were best at inhibiting urediniospore germination followed by the newer triazoles. Second, application timing was evaluated in the greenhouse with fungicides applied 24 hr, 48 hr, 96 hr and 168 hr before or after inoculation. All of the pre-inoculation treatments inhibited disease development; however the triazoles were significantly better when applied after infection. Field experiments were conducted in 2008 and 2009 using commercial products. In 2008, all treatments had lower stem rust severity and higher yields in comparison to the untreated control. Tebuconazole and prothioconazole were most effective in controlling stem rust. In 2009, no significant differences were observed, however disease development was limited on the untreated plots due to non-conducive weather conditions.
Genetic structure of Mycosphaerella graminicola populations in the major wheat-growing regions of the United StatesS. GURUNG (2), S. Goodwin (4), M. Kabbage (3), W. Bockus (1), T. Adhikari (2)(1) Kansas State University, Manhattan, KS, U.S.A.; (2) North Dakota State University, Fargo, ND, U.S.A.; (3) Texas A&M University, College Station, TX, U.S.A.; (4) USDA-ARS, Purdue University, West Lafayette, IN, U.S.A.Septoria tritici blotch (STB), caused by Mycosphaerella graminicola, is one of the major diseases of wheat worldwide. However, there is little information available on the population genetic structure of this pathogen in the major wheat-growing regions of the United States. We analyzed the genetic structure of 334 isolates of M. graminicola from 11 populations, three from spring wheat in California and North Dakota and eight from winter wheat in Indiana and Kansas using 17 microsatellite markers and two mating type loci. Clone-corrected data revealed that most of the M. graminicola populations had high levels of gene diversity (H = 0.31 to 0.52) and genotype diversity (GD = 0.98 to 1). Both the gene (H) and genotype diversity (GD) was higher for both populations from Indiana and North Dakota than California and Kansas. Similarly, there was a high level of gene flow (Nm = 1.73 to 23.30) and very less genetic distance (D = 0.52 to 0.98) among populations. Equal frequencies of mating types (MAT1-1 and MAT1-2) were found in all populations except in the California population. No evidence of linkage disequilibrium (LD) was observed in all populations analyzed. Overall, these results suggest that there is frequent sexual recombination in the M. graminicola populations and the populations are likely a single large population of M. graminicola affecting wheat fields in the United States.
Archaeophytopathology of global soybean rustJ. S. HAUDENSHIELD (1), G. L. Hartman (1)(1) USDA-ARSPhakopsora pachyrhizi and P. meibomiae are two rust species that infect soybean (Glycine max). A number of other hosts support the uredinial growth of these Phakopsora, including Pachyrhizus erosus, Pueraria lobata, and Vigna unguiculata, but no aecial host is known. Traditionally, these two species are said to differ in geographic distribution, with P. pachyrhizi confined to Asia, Africa & Australia, and P. meibomiae confined to South & Central America. Several herbaria have accessions reported to contain one of the two species and include specimens from those locations, some nearly or over 100 years old. We sampled 38 of these archival specimens, and extracted & speciated the DNA of the fungus, if present, using quantitative PCR specific to either P. pachyrhizi, P. meibomiae, or to a third group inclusive of many rust species. Of the archival specimens, 11 were positive for P. pachyrhizi, including a 1912 specimen from Japan, but no P. pachyrhizi was found in specimens from before 1994 outside of Asia or Australia. Fifteen specimens were positive for P. meibomiae, including a 1928 specimen from Brazil and two 1923 specimens from the Philippines. Twelve specimens (including all African accessions) were found to be negative for both species, but six were positive in the more inclusive rust assay, and included specimens from Tanzania, Sao Tome, Nigeria, Sierra Leone, and China; all had been labeled as P. pachyrhizi and none were on G. max. These results demonstrate the feasibility of DNA genotyping in archaeophytopathological investigations and suggest that P. pachyrhizi may have been more recently introduced to Africa than previously believed.
Quick identification of Xanthomonas campestris pv. translucens, Bacterial leaf streak causing pathogen of small grainsY. R. KANDEL (1), L. E. Osborne (1), K. D. Glover (1), C. A. Tande (1)(1) South Dakota State University, Brookings, SD, U.S.A.In recent years, Bacterial leaf streak (BLS), caused by Xanthomonas campestris pv. translucens (Xct) has emerged as a potential threat to spring wheat production in the Northern Great Plains. Water-soaked, chlorotic or necrotic stripes on leaves are the characteristic symptom of the disease, however diagnosis based on field symptoms is not always accurate. Pathogen characterization and identification through conventional techniques is often time consuming, costly and inaccurate. Here, we examined application of the Biolog micro-plate system (Biolog, Inc., Hayward, Calif.) to identify the pathogen. Fifty bacterial strains were isolated from symptomatic wheat leaf tissues. Of the fifty strains, twenty five were discarded based on their color and morphology on KG medium. The remaining isolates were screened for pathogenicity on wheat in the greenhouse and tested with the Biolog system. Based on Biolog, three were identified as Xct, four were Pseudomonas syringae pv. syringae and rest were non-pathogens. The isolates identified as Xct produced bacterial leaf streak symptoms in pathogenicity tests while the other strains did not produce disease symptoms. Bacteria were re-isolated from symptomatic tissues to satisfy Koch’s postulates. Each of the three Xct isolates identified initially by the Biolog System were validated as Xct following inoculation, re-isolation and re-testing. Each Xct isolate caused water soaking in wheat seedlings within 4 days after inoculation. Those lesions were covered with bacterial ooze and bordered by a yellow margin. They were developed to yellow-brown longitudinal streaks within a week after inoculation. Results showed that the Biolog system was efficient in identifying the pathogen among numerous isolates. It can be combined with few conventional tests for preliminary identification and elimination of non-pathogens. Biolog system combining with limited conventional tests could be a less costly and more rapid technique in identifying plant pathogenic bacteria.
An Indiana survey of Phytophthora species in nurseries, greenhouses, and landscape plantingsA. J. LEONBERGER (1), C. Speers (1), G. Ruhl (1), T. Creswell (1), J. L. Beckerman (1)(1) Purdue University, West Lafayette, INFrom 2006 to 2008, samples with symptoms consistent with Phytophthora blight and crown rot were collected as part of the USDA-APHIS Phytophthora ramorum nursery survey and submitted by additional outside sources to the Purdue Plant and Pest Diagnostic Laboratory. From 22 sites, 93 Phytophthora isolates were obtained from 1657 host samples containing 15 plant genera. Comparison of the internal transcribed spacer (ITS) sequence of the ribosomal DNA identified 10 Phytophthora sp. A majority of the isolates were either P. citricola (39.8%) or P. citrophthora (28.0%). P. citricola isolates were collected at 12 sites from 5 host genera, and included Forsythia, Juglans, Pieris, Rhododendron, and Syringa. P. citrophthora was isolated from 11 sites on 7 host genera: Calycanthus, Forsythia, Ilex, Kalmia, Pieris, Rhododendron, and Syringa. The other identified Phytophthora sp. consisted of P. cactorum, P. cactorum x hedraiandra, P. cambivora, P. capsici, P. cryptogea, P. drechsleri, P. nicotianae, and P. syringae. Sixteen isolates showed signs of possible species hybridization. Four isolates were found to be hybrids of P. cactorum and P. hedraiandra as verified by cloning and sequencing the ITS regions. Three of the P. cactorum x hedraiandra isolates came from Rhododendron plants at the same site. The other hybrid isolate was recovered from Dicentra, which is not a known host of either of the parental species, P. cactorum or P. hedraiandra, and suggests an increase of host range due to species hybridization.
Inoculation timing, mist duration and isolate effects on Fusarium head blight and deoxynivalenol in two hard red spring wheat cultivarsM. McMullen (1), S. M. MEYER (1), J. Jordahl (1)(1) North Dakota State University, Fargo, ND, U.S.A.The effects of inoculation timing, mist duration, and the Fusarium graminearum isolate’s trichothecene genotype on Fusarium head blight (FHB) severity and deoxynivalenol (DON) content in grain were evaluated in a greenhouse on two spring wheat cultivars (Glenn, rated MR to FHB, and Trooper, rated MS to FHB). Growth stages at inoculation timing were early flowering (Feekes 10.51), kernel watery ripe (Feekes 10.54) or kernel medium to late milk (Feekes 11.1). Spores from North Dakota isolates of a 3ADON or a 15ADON trichothecene genotype were atomized onto wheat heads. Following inoculation, plants were placed in a mist chamber for two, five or 10 days, and then were moved to a greenhouse bench. Each treatment had 10 plants per replicate, four replicates per trial, and each trial was repeated four times. Plants were rated 14 to 21 days post inoculation for disease FHB index [(incidence × head severity)/100]. At maturity, all inoculated heads were harvested and grain was ground and analyzed for DON content using gas chromatography and electron capture. Results indicated that the highest DON level occurred with the 3ADON inoculation of Trooper wheat, when analyzed across all mist and growth stage treatments. The 10 day mist period resulted in the highest FHB index and DON production, regardless of isolate, growth stage or cultivar used. The Feekes 10.51 and 10.54 inoculations with the 3ADON isolate did not differ significantly in FHB index or DON production, but the Feekes 10.54 inoculations with the 15ADON isolate resulted in higher FHB and DON than Feekes 10.51 inoculations. The late inoculations, at Feekes 11.1 growth stage, did result in FHB infection, but had the lowest FHB and DON among growth stage inoculations, over all mist durations, isolates and cultivar treatments.
Corn ear molds and mycotoxins in North Dakota, 2009M. MCMULLEN (1), K. Kinzer (1), M. Mostrom (1), J. Ransom (1)(1) North Dakota State University, Fargo, ND, U.S.A.In 2009, North Dakota grew 2 million acres of corn. Temperatures in 2009 averaged below normal across the state during the growing season, with accumulated corn growing degree units ranging from 0 to 180 units below normal statewide. By the second week of October, only 23% of corn fields had mature corn, and 0% was harvested. A killing frost in the second week of October and subsequent cold temperatures and rains contributed to high moisture corn and development of corn ear molds. NDSU coordinated a corn mold survey in response to grower concerns about corn ear molds. County and area extension specialists submitted samples of 6–12 ears per field from fields in 24 counties representing major corn growing areas. A total of 94 corn samples from the NDSU extension survey effort and private submissions were evaluated for molds and mycotoxins. The predominant fungus identified via microscopy was Cladosporium species, but some samples also had visible hyaline or pink hyphae, with some of these confirmed to be species of Fusarium. The 94 samples were screened by the NDSU Veterinary Diagnostic Laboratory for 17 trichothecenes (mycotoxins produced by Fusarium species), using gas chromatography/mass spectrometry techniques. Aflatoxin was not assayed because this mycotoxin is associated with stress conditions brought on by hot, dry conditions, a situation which we did not have in North Dakota in 2009. Mycotoxins were not detected in 73% of samples. Of the remaining 27% with detectable mycotoxins, deoxynivalenol (vomitoxin) was predominant (60% of positive samples). However, only 4 of the 15 samples positive for deoxynivalenol had levels greater than 1 ppm. Additional mycotoxins detected included zearalenone (in 3 samples); T-2 toxins (in 10 samples); and nivalenol (in 3 samples).
Genetic and morphologic diversity of Streptomyces species that cause potato common scab in MichiganQ. MENG (1), A. O. Adesemoye (1), W. W. Kirk (1), J. Hao (1)(1) Michigan State UniversityPotato common scab is a worldwide disease caused by several Streptomyces spp. In Michigan (MI), Streptomyces scabies is the predominant pathogenic species. A survey was conducted to analyze the distribution and diversity of Streptomyces species in MI. Potato tubers with typical commons scab symptoms were collected from various locations. One hundred and fifty isolates of Streptomyces were isolated from tuber lesions and purified by transferring single colonies on Streptomyces selective media. Morphology of the isolates was examined on different International Streptomyces Project media. To date, 49 isolates have been identified as Streptomycetes and processed as follows. Pathogenicity of the isolates was determined in the greenhouse using potato plants grown from seed tubers. Soils were inoculated with individual isolates and common scab assessed on daughter tubers using a categorical scale (0–5 where 0 = no scab symptoms and 5 = pitted scab on >50% of the tuber surface). Genomic DNA was extracted from the isolates and polymerase chain reaction (PCR) was conducted using primers of marker genes such as txtAB, TomA, and nec1 for pathogenic Streptomyces spp. Gene of 16S rRNA was amplified, sequenced and analyzed for each isolate using the BLAST algorithm against the NCBI Genbank. Some of the sequences have been submitted to the GenBank. The capability of isolates to produce thaxtomin was determined from oat brain medium culture and confirmed with high performance liquid chromatography. Four isolates were S. stelliscabiei, six were S. scabies, and the remaining 39 were classified as Streptomyces species. This is the first report of S. stelliscabiei in MI. All 49 isolates were pathogenic and txtAB, nec1, and tomA were present in all isolates except 3 of the isolates (DS21, KRUF21, and LEN02) that lacked either nec1 or tomA. Overall, the Streptomyces spp. isolates examined were phenotypically and genetically heterogeneous, indicating diversity.
Ascospore germination and infection efficiency of Sclerotinia sclerotiorum in relation to temperatureA. NEPAL (1), L. del Rio Mendoza (1)(1) North Dakota State UniversityThe effect of constant temperature on S. sclerotiorum ascospore germination and its infection efficiency were assessed on canola plants in controlled environment. The germination study had three replications and five temperatures (10–35°C, at 5°C intervals). For every temperature, 20 µl of a spore suspension (105 spores ml–1) of three North Dakota isolates were deposited on 15 cover slips each. The slips were placed in five Petri dishes (three isolates per dish) lined at their bottom with moist filter paper. One dish per replication was retrieved after 2, 6, 12, 18, and 24 hrs of incubation to estimate germination and germ tube length. The infection efficiency study had three replications and was conducted on 30-days old canola plants using the same temperatures, isolates, and ascospore concentrations. A 20 µl ascospore suspension from a mix of the three isolates was inoculated on 60 canola petals. Inoculated petals were placed on leaves of three plants per rep and incubated for at least 10 days. Plants were misted periodically to promote infection. Infection efficiency was assessed by calculating the percentage of petals that produced a leaf lesion and measuring lesion size every two days after inoculation (DAI). Optimum conditions for ascospore germination and growth occurred between 20 and 25°C. After 24 hrs of incubation at these conditions, germination was 77% and germ tubes were 31 μ long. Further, 50% of ascospores germinated within two hrs and had germ tubes 7 μ in length. First disease symptoms were observed within four and nine DAI on plants incubated at 20°C and 10°C, respectively. The fungus destroyed the entire leaves within nine and twenty DAI when incubated at 20°C and 10°C, respectively. No symptoms developed at >30°C.
Uniform fungicide timing and efficacy trials for management of common bean rust in North DakotaL. OLSON (3), S. Halley (2), B. Schatz (1), S. Meyer (4), R. Goswami (4), F. Mathew (4), R. Lamppa (4), P. Hendrickson (1), S. Markell (4)(1) Carrington Research Extension Center, Carrington, ND, U.S.A.; (2) Langdon Research Extension Center, Langdon, ND, U.S.A.; (3) North Dakota State University Area Office, Grand Forks, ND, U.S.A.; (4) North Dakota State University Plant Pathology Department, Fargo, ND, U.S.A.The most recent common bean rust (Uromyces appendiculatus Pers.:Pers.) epidemics in North Dakota’s dry edible bean (Phaseolus vulgaris L.,) crop caused in excess of $10 million in losses in 1994 and 1996. Rust has been managed with the resistance gene Ur-3, which was effective to all then-known races of rust in North Dakota, and widely deployed in commonly used cultivars. In 2008, a new race of rust conferring virulence to Ur-3 was detected for the first time in North Dakota, rendering the North Dakota crop susceptible to rust. Fungicides are a potential management tool for rust, however, many fungicides have not been evaluated for efficacy or to identify the most effective application timing on bean rust. The objective of this research was to evaluate the efficacy and timing of fungicides for rust management. Two separate experiments were planted to the pinto bean cultivar GTS-900, which lacks the Ur-3 gene, in RCBD designs at three North Dakota locations in 2009. To facilitate disease development, plots were inoculated with urediniospores of an U. appendiculatus isolate collected in 1996 known to be avirulent to Ur-3 and virulent on GTS-900. Disease was evaluated by visually estimating pustule coverage from a sample of ten leaves per plot. Plots were rated 4 to 6 times and Area Under Disease Progress Curve (AUDPC) was calculated. Results indicate that 1) most fungicides reduced AUPDC values compared to the check; 2) timing of these fungicides is a critical factor in reducing disease levels and increasing yield, and 3) the greatest reduction in AUDPC occurred with sequential applications.
Effect of multiple Ceratcystis smalleyi infections on stem water conductance in maturing bitternut hickoryJ. PARK (2), J. Cavender-Bares (2), J. Juzwik (1)(1) US Forest Service, St. Paul, MN, U.S.A.; (2) University of Minnesota, St. Paul, MN, U.S.A.Hundreds of cankers caused by Ceratocystis smalleyi are associated with declining, bark beetle-attacked bitternut hickory in the northeastern and north central U.S.A. Sapwood infections associated with the cankers are hypothesized to disrupt xylem water transport and contribute to crown wilt and tree death. In a field study, stems of healthy bitternut hickory (12.6 to 23.4 cm diameter at 1.4 m) were inoculated at 50 points (between 1.8 and 3.7 m stem height) with isolates of C. smalleyi or with sterile water in July 2008. In September 2009, sap flow in the study trees was monitored for 18 days using heat dissipation sensors. Although no crown wilt symptoms were observed, cankers were commonly found around inoculation points on the fungus-inoculated trees. All trees were felled, and measurements were made of inner bark necrosis, sapwood discoloration, and several xylem vessel features. Infected trees exhibited reductions in sap flux and daily water loss compared to controls (water inoculated and non-inoculated trees). Daily water loss for representative days was inversely correlated with the relative proportion of cankered bark area and numbers of tyloses in the outer two annual rings of the study trees. Measurements of other vessel features did not differ between inoculated and control trees. Preliminary results suggest multiple stem infections of C. smalleyi impair water transport in otherwise healthy bitternut hickory. A replicate field experiment is underway.
Relative aggressiveness of Contans WG® and native Coniothyrium minitans isolates from Wisconsin soybean fieldsA. J. PELTIER (1), Y. Chen (1), P. D. Esker (1)(1) University of Wisconsin, Madison, WI, U.S.A.Sclerotinia stem rot (SSR) of soybean, caused by Sclerotinia sclerotiorum (Ss), can cause large yield loss in soybean, particularly in Wisconsin. Crop rotation is not used to control of SSR, as sclerotia of Ss are long lived in the soil. Coniothyrium minitans (Cm) parasitizes Ss sclerotia and a commercial formulation of Cm, Contans WG, has been approved for use on soybean. It is not known how well the Contans WG isolate can compete with native Cm populations. Experiments were designed to compare native and Contans WG isolate aggressiveness. Cm was isolated from soil from the Marshfield (Isolates 1 and 2) and Arlington (Isolates 3 and 4) Agricultural Research Stations in WI. Treatments in Trial 1 included Isolates 2 and 4, Contans WG and a negative control, while Trial 2 included Isolates 1 and 3, Contans WG and a negative control; each treatment was replicated four times. For each trial, sclerotia were individually inoculated with 10 µL of a 2 × 106/mL spore suspension or water and incubated in a petri dish in moist, sterile soil. After 7 and 14 days, 10 sclerotia were removed, surface sterilized, halved, and plated onto amended PDA. After 14 days, sclerotia were rated for Cm colonization. Spores were counted for three colonized sclerotia from each treatment/replicate combination. In Trial 1, Isolate 4 colonized more sclerotia (P = 0.01) and produced more spores (P = 0.04) than the Contans WG isolate. In Trial 2, although Isolate 3 colonized more sclerotia than Contans WG (P = 0.01), Contans WG produced more spores on colonized sclerotia than Isolate 3 (P < 0.0001). Implications for the biocontrol potential of Contans WG in WI are discussed.
Method and timing of azoxystrobin application to control Rhizoctonia root rot of sugar beetS. POORAN DESOUZA (1), M. Bolton (2), M. Khan (1)(1) NDSU, Fargo, ND, U.S.A.; (2) USDA, Fargo, ND, U.S.A.Rhizoctonia solani (Kühn) is the causal agent of Rhizoctonia root rot of sugar beet (Beta vulgaris L.) in North Dakota (ND) and Minnesota (MN). Studies were conducted to (i) determine the best method of applying azoxystrobin for controlling R. solani and (ii) determine the effect of azoxystrobin on disease control when applied pre- and post inoculation. Sugar beet plants were inoculated at the four-leaf stage and fungicides were applied subsequently as a hypocotyl drench or banded. Hypocotyl drench gave similar control as the banded application. To determine the best time to apply azoxystrobin, hypocotyls were drenched at the four-leaf stage at 0, 3, 10, 14, and 21 days post-inoculation and 0, 7, 14, and 28 days pre-inoculation. Azoxystrobin was not effective at controlling Rhizoctonia root rot when applied after inoculation. Azoxystrobin when applied prior to or on the day of inoculation provided effective Rhizoctonia root rot control.
Depth at which Rhizoctonia solani causes infection of sugar beetS. POORAN DESOUZA (1), M. Bolton (2), M. Khan (1)(1) NDSU, Fargo, ND, U.S.A.; (2) USDA, Fargo, ND, U.S.A.Rhizoctonia solani (Kühn) is the causal agent of Rhizoctonia root rot of sugar beet (Beta vulgaris L.). Typically, Rhizoctonia root rot symptoms appear to be initiated on the plant at the soil line. Recently, sugar beet plants were observed with Rhizoctonia root rot infections close to the root tips and R. solani AG 2-2 IIIB was identified as the causal agent. There were concerns that AG 2-2 IIIB may be causing infections lower on the roots which may make fungicide control difficult. Our objective was to determine the depth which is most favorable for infection. Plants were inoculated at the four-leaf stage using two grains of barley inoculated with R. solani AG 2-2 IIIB. Inoculum was buried at 2.54, 7.62, and 12.70 cm depth. Plants were rated for root rot disease severity using the 0–7 rating scale fourteen days after inoculation. Results indicated that infections by R. solani AG 2-2 IIIB occurred at all depths. However, R. solani appears to favor the area on the roots just below the soil line irrespective of the depth of placement.
Characterizing Phytophthora infestans US#22 for fungicide resistance, and pathogenicity and virulence on tomato cultivarsA. SEIDL (1), R. Clark (1), A. J. Gevens (1)(1) University of Wisconsin, Madison, WI, U.S.A.Late blight of tomato and potato, caused by the oomycetous pathogen Phytophthora infestans (Mont.) deBary, is one of the most devastating plant diseases worldwide. In 2009, a new type of P. infestan was identified in multiple U.S. states, primarily on tomato, and has been designated as US#22. Fourteen isolates of P. infestans were collected from tomato and potato throughout Wisconsin during the 2009 growing season. Each isolate was characterized for resistance to the fungicides mefenoxam and metalaxyl. All isolates exhibited sensitivity to 100 ppm mefenoxam and 100 ppm of the enantiomer metalaxyl in amended rye agar. Fifteen commercially available tomato cultivars with supposed resistance to late blight were screened for foliar resistance with a sporangial suspension of a single zoospore derived isolate of P. infestans collected from tomato in southern Wisconsin. Detached leaves of ‘Matt’s Wild Cherry’ and ‘Wapsipinicon’ were fully resistant at 9 days post inoculation (dpi). Thirteen of the tested cultivars were susceptible and exhibited pathogen growth and water soaked lesions as early as 5 dpi. Several cultivars exhibited a hypersensitive response (HR) at the site of inoculation, with ‘Pruden’s Purple’ exhibiting the most dramatic and consistent response. Our findings indicate the potential utility of metalaxyl or mefenoxam fungicides and tomato varietal resistance for the control of this new P. infestans type.
Is Avr1a gene present in virulent isolates of Phytophthora sojae from Iowa?S. M. STEWART (1), A. E. Robertson (1)(1) Iowa State UniversityPhytophthora sojae races or pathotypes are defined by their ability to infect a set of differential soybean cultivars that carry particular Rps resistant genes. Avirulence gene Avr1a of P. sojae determines the outcome of the interaction with the resistance Rps-1a gene in soybean. Avr1a has been identified as encoding an RXLR effector that is expressed in avirulent pathotypes but not in virulent ones (1). Expression of the Avr1a was congruent with avirulence-virulence phenotype of 17 isolates. Genomic copies of the gene were present in all avirulent isolates, and were also present in 33% of the virulent isolates. Our objective was to explore the presence or absence of genomic copies of Avr1a in a wider range of isolates of P. sojae from Iowa, and determine the proportion of isolates that are virulent due to Avr1a gene deletion or some form of gene silencing.
Comparison of inoculation methods for evaluating root pathogens of wheatP. TAMANG (1), J. M. Stein (1)(1) South Dakota State UniversityGreenhouse experiment was conducted to compare inoculation methods for common root and crown rot, caused by the fungus Cochliobolus sativus and Giberella zea respectively against six varieties each for spring and winter wheat. Two inoculation methods used were: 1) mixing inoculated millet and potting mix in the ratio of 1:50 at planting, and 2) dispensing the 10 µl/plant of Cochliobolus (104/ml) and Giberella conidial suspension (105/ml) directly at plant roots, 15 days after planting in spring wheat and 57 days (after vernalization) in winter wheat. The winter wheat was vernalized for 42 days at two-leaf stage. Plant height and percent severity of the sub crown internode (SCI) were assessed at 35 days after inoculation in both spring and winter wheat for five plants/pot and dry weight was taken. Disease data was analyzed using analysis of variance (ANOVA). Results indicate that height and dry matter weight were significantly higher in inoculated plants than those in un-inoculated plants for both inoculation methods and fungi (P-value < 0.001). It should be noted that no sterile millet was added to the control pots. However, mixing of inoculum with potting mix had significantly higher SCI symptoms than applying suspension inoculum on roots (P-value < 0.001).
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