Alan T. Dyer

Applying Real-Time Quantitative PCR to Fusarium Crown Rot of Wheat

Fusarium crown rot (FCR) of wheat is a persistent problem that causes significant losses worldwide. In Montana, FCR is caused primarily by Fusarium culmorum and F. pseudograminearum. Recently, a real-time quantitative PCR (QPCR) assay was developed for FCR using primers and probes specific for a segment of the trichodiene synthase (tri5) gene. The purpose of this study was to determine the utility of QPCR for accessing FCR severity on wheat in field experiments. In 2004 and 2005, plots of spring and durum wheat were inoculated with varying levels of F. pseudograminearum oat inoculum and grown under rain-fed conditions. Two weeks prior to harvest, plants were collected from the plots and assessed for FCR severity and analyzed by QPCR for Fusarium DNA quantities. Disease severity scores (DSS) and Fusarium DNA quantities were positively correlated with each other for all three cultivars in 2004 but for only the durum cultivar in 2005 (P < 0.05). In 2004, grain yields for both spring wheat cultivars were negatively correlated with Fusarium DNA quantities (P > 0.05). When DSS and Fusarium DNA quantities negatively correlated with yield, both measurements were comparable in predicting yield reduction (R = -0.64 and -0.77, respectively). Results indicate that this QPCR assay is effective in measuring FCR severity in wheat.

Monitoring Fusarium Crown Rot Populations in Spring Wheat Residues Using Quantitative Real-Time Polymerase Chain Reaction

Caused by a complex of Fusarium species including F. culmorum, F. graminearum, and F. pseudograminearum, Fusarium crown rot (FCR) is an important cereal disease worldwide. For this study, Fusarium population dynamics were examined in spring wheat residues sampled from dryland field locations near Bozeman and Huntley, MT, using a quantitative real-time polymerase chain reaction (qPCR) Taqman assay that detects F. culmorum, F. graminearum, and F. pseudograminearum. Between August 2005 and June 2007, Fusarium populations and residue decomposition were measured eight times for standing stubble (0 to 20 cm above the soil surface), lower stem (20 to 38 cm), middle stem (38 to 66 cm), and chaff residues. Large Fusarium populations were found in stubble collected in August 2005 from F. pseudograminearum-inoculated plots. These populations declined rapidly over the next 8 months. Remnant Fusarium populations in inoculated stubble were stable relative to residue biomass from April 2006 until June 2007. These two phases of population dynamics were observed at both locations. Relative to inoculated stubble populations, Fusarium populations in other residue fractions and from noninoculated plots were small. In no case were FCR species observed aggressively colonizing noninfested residues based on qPCR data. These results suggest that Fusarium populations are unstable in the first few months after harvest and do not expand into noninfested wheat residues. Fusarium populations remaining after 8 months were stable for at least another 14 months in standing stubble providing significant inoculums for newly sown crops.

Distribution and Prevalence of Fusarium Crown Rot and Common Root Rot Pathogens of Wheat in Montana

Distribution of Fusarium crown rot (FCR) and common root rot (CRR) pathogens associated with wheat (Triticum aestivum) in 91 fields in Montana were determined during the 2008 and 2009 crop seasons using real-time quantitative polymerase chain reaction (qPCR) and conventional isolation methods. Correlations (P < 0.001) were found between detection methods for both diseases. FCR was detected in 57% of the fields and CRR was detected in 93% of the fields surveyed. Percent incidence based on isolation from individual tillers was Bipolaris sorokiniana (15%), F. culmorum (13%), and F. pseudograminearum (8%). FCR populations were highly variable across the regions and were not detected in any fields from the Gb5 soil types of Judith Basin and Fergus counties. The spatial distributions of FCR and CRR were affected by elevation, soil type, and temperature. High FCR populations were associated with spring wheat crops rather than winter wheat based on qPCR (P < 0.001). FCR and CRR could produce yield losses in a range of 3 to 35%. This study is the first time that qPCR was used to survey these two pathogen groups, and the merits and weakness of qPCR relative to traditional isolation methods are discussed.

Comparison of pathogenicity of the Fusarium crown rot (FCR) complex (F. culmorum, F. pseudograminearum and F. graminearum) on hard red spring and durum wheat.

Fusarium species involved in the Fusarium crown rot (FCR) complex affect wheat in every stage of development from seedling to grain fill. This study was designed to compare the aggressiveness of the FCR complex members including F. culmorum, F. pseudograminearum and F. graminearum in causing seedling blight, decreased plant vigour and crown rot. To assess their relative pathogenicity, two hard red spring wheat cultivars and two durum wheat cultivars were inoculated in the field with five isolates from each of the three species for two years. Significant differences in patterns of pathogenicity were identified. In particular, F. culmorum caused greater seedling blight while F. pseudograminearum and F. graminearum caused greater crown rot. Greatest yield reductions were caused by F. pseudograminearum. Cultivar differences were identified with respect to seedling disease and late season crown rot. No interactions were identified between cultivar performance and isolates or species with which they were challenged.

Population structure, races, and host range of Aphanomyces euteiches from alfalfa production fields in the central USA

Aphanomyces euteiches (races 1 and 2) causes root rot of alfalfa; however, its population biology and distribution are poorly understood where alfalfa is a major crop. The objectives of this study were to (1) characterise the distribution and frequency of races of A. euteiches in Illinois alfalfa fields, (2) determine host range of A. euteiches on cultivated and native legumes, and (iii) to describe genetic diversity and population genetic structure of A. euteiches in alfalfa fields. To accomplish this, soil samples (n = 103) were collected from 30 alfalfa fields in 18 Illinois counties. Using the susceptible cv. ‘Saranac’, 148 isolates of A. euteiches were baited from the soil. The virulence phenotype of isolates representing all 18 counties was tested, and 54% were R1 and 46% were R2. Both races were detected in 61% of the counties, whereas only R1 was detected in 22% and R2 in 17%. Thirteen legume hosts for isolates from alfalfa fields were identified based on symptoms and/or production of oospores in roots. In addition to six previously known hosts, seven species were susceptible to infection: kura clover, purple prairie clover, white prairie clover, ladino clover, hairy vetch, Canadian milk vetch, and Illinois tick trefoil. AFLP analysis revealed high levels of genetic diversity among the isolates from different fields and counties and a lack of genetic structuring of populations based on race or geographical origin. The results suggest that populations of A. euteiches in alfalfa fields are diverse, often composed of races 1 and 2, and create risk for alfalfa and to multiple cultivated and native legume species.

Survival Dynamics of Aphanomyces cochlioides Oospores Exposed to Heat Stress.

ABSTRACT To determine how exposure to heat effects their survival, oospores of Aphanomyces cochlioides isolate C22 were exposed in water to 35, 40, 45, or 50 degrees C for prescribed times and then examined for viability. The Weibull model was modified to represent the effects of temperature on survival of oospores. The final fitted model gave lethal doses for 50% of the oospores of 251, 49.8, 9.8, and 1.9 h at 35, 40, 45, and 50 degrees C, respectively. To determine if alternating high and low temperatures resulted in (i) recovery from heat damage during low temperature periods, (ii) increased susceptibility to heat damage, or (iii) if effects of heat damage were cumulative, oospores were examined after each of four 24-h cycles at 45 degrees C for 4 h and 21 degrees C for 20 h. Survival of oospores exposed to alternating high and low temperatures fit the cumulative effects model. Significant variability in heat tolerance among five isolates was observed (P< 0.001) but model parameters successfully accommodated this variability (R(2) = 0.96, P < 0.001). This research shows that under wet conditions, there are predictable patterns to mortality for A. cochlioides oospores exposed to continuous or fluctuating high temperatures.

Competition Between Fusarium pseudograminearum and Cochliobolus sativus Observed in Field and Greenhouse Studies

Among root pathogens, one of the most documented antagonisms is the suppression of Cochliobolus sativus by Fusarium (roseum) species. Unfortunately, previous studies involved single isolates of each pathogen and thus, provided no indication of the spectrum of responses that occur across the respective species. To investigate the variability in interactions between Cochliobolus sativus and Fusarium pseudograminearum, field and greenhouse trials were conducted that included monitoring of spring wheat plant health and monitoring of pathogen populations via quantitative real-time polymerase chain reaction. The interactions between two isolates of C. sativus and four isolates of F. pseudograminearum were explored in three geographically distinct wheat fields. To complement field trials and to limit potentially confounding environmental variables that are often associated with field studies, greenhouse trials were performed that investigated the interactions among and between three isolates of C. sativus and four isolates of F. pseudograminearum. Across field locations, C. sativus isolate Cs2344 consistently and significantly reduced Fusarium populations by an average of 20.1%. Similarly, F. pseudograminearum isolate Fp2228 consistently and significantly reduced C. sativus field populations by an average of 30.9%. No interaction was detected in the field between pathogen species with regards to disease or crop losses. Greenhouse results confirmed a powerful (>99%), broadly effective suppression of Fusarium populations by isolate Cs2344. Among greenhouse trials, additional isolate-isolate interactions were observed affecting Fusarium populations. Due to lower C. sativus population sizes in greenhouse trials, significant Fusarium suppression of C. sativus was only detected in one isolate-isolate interaction. This study is the first to demonstrate suppression of Fusarium spp. by C. sativus in field and greenhouse settings. These findings also reveal a complex competitive interaction between these two pathogen species that was previously unknown.