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.

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.

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.

Contamination, error, and nonspecific molecular tools.

ABSTRACT Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) are widely used in studies of genetic variation. Although it is recognized that contamination should be avoided in DNA samples, little is known about the potential hazards of low level bacterial contamination of samples from which DNA is extracted for RAPD or AFLP analyses. We found that contamination of Aphanomyces cochlioides cultures with a prokaryote at visibly undetectable levels markedly altered the results of RAPD and AFLP analyses. The contamination resulted in seven contaminant-specific RAPD products and in the suppression of eight products characteristic of uncontaminated A. cochlioides cultures. Prokaryote contamination resulted in 39 contaminant-specific AFLP products, but did not cause suppression of AFLP products. Comparing A. cochlioides samples with outgroup A. euteiches did not clearly indicate the presence of contaminant DNA, because uneven product suppression in RAPD analysis increased the apparent similarity between contaminated samples and A. euteiches and because a high proportion of the contaminant-specific amplified products comigrated with products from A. euteiches in both RAPD and AFLP analyses. Work with organisms that are prone to contamination should employ techniques such as restriction fragment length polymorphism or DNA sequence comparisons rather than relying solely on RAPD or AFLP analyses.