Characterization of expressed sequence tag-derived simple sequence repeat markers for Aspergillus flavus: emphasis on variability of isolates from the southern United States.

Simple sequence repeat (SSR) markers were developed from Aspergillus flavus expressed sequence tag (EST) database to conduct an analysis of genetic relationships of Aspergillus isolates from numerous host species and geographical regions, but primarily from the United States. Twenty-nine primers were designed from 362 tri-nucleotide EST-SSR sequences. Eighteen polymorphic loci were used to genotype 96 Aspergillus species isolates. The number of alleles detected per locus ranged from 2 to 24 with a mean of 8.2 alleles. Haploid diversity ranged from 0.28 to 0.91. Genetic distance matrix was used to perform principal coordinates analysis (PCA) and to generate dendrograms using unweighted pair group method with arithmetic mean (UPGMA). Two principal coordinates explained more than 75 % of the total variation among the isolates. One clade was identified for A. flavus isolates (n = 87) with the other Aspergillus species (n = 7) using PCA, but five distinct clusters were present when the others taxa were excluded from the analysis. Six groups were noted when the EST-SSR data were compared using UPGMA. However, the latter PCA or UPGMA comparison resulted in no direct associations with host species, geographical region or aflatoxin production. Furthermore, there was no direct correlation to visible morphological features such as sclerotial types. The isolates from Mississippi Delta region, which contained the largest percentage of isolates, did not show any unusual clustering except for isolates K32, K55, and 199. Further studies of these three isolates are warranted to evaluate their pathogenicity, aflatoxin production potential, additional gene sequences (e.g., RPB2), and morphological comparisons.

Identification of select fumonisin forming Fusarium species using PCR applications of the polyketide synthase gene and its relationship to fumonisin production in vitro.

A polymerase chain reaction (PCR) based diagnostic assay was used to develop markers for detection of Fusarium verticillioides (=F. moniliforme), a fumonisin producing fungus in maize tissues. Species-specific primers were designed based on sequence data from the polyketide synthase (PKS) gene (FUM1- previously FUM5) responsible for fumonisin production in fungi. Four sets of oligonucleotide primers were tested for their specificity using 24 strains of F. verticillioides, 10 F. proliferatum, and 12 of other Fusarium species. In addition, 13 species of other fungal genera, from four phyla, were tested as negative controls. Among the four sets, primer set B consistently amplified a 419-bp fragment from the DNA 96% of all F. verticillioides strains and 83% of F. proliferatum. All other fungi tested were negative using primer set B. A total of 38% of the F. verticillioides strains grown on a selective liquid medium produced fumonisin and 92% formed the toxin on standard rice medium. When fumonisin formed in culture, PCR assay using primer set B detected every strain of F. verticillioides, but only amplified 80% of F. proliferatum strains that produced the toxin. PCR detection was consistent at 100 pg/microl concentration of genomic DNA from 4 F. verticillioides strains, but varied at 10 pg/microl. Two duplicate greenhouse tests using artificially inoculated maize plants, had greater levels of F. verticillioides detected after re-evaluting using primer set B than from culturing of the tissues. The molecular protocols described in this study requires only 1 day for completion compared to approximately 10 days for cultural work and morphological determination. In conclusion, conventional PCR assay using primer set B provides a sensitive and accurate detection assay that can be used as a primary or secondary confirmation method for identification and occurrence of F. verticillioides within the maize tissues. However, studies using primer set B for fumonisin production determined by strains of F. verticillioides and F. proliferatum will require further verification.

Relative Longevity of Macrophomina phaseolina and Associated Mycobiota on Residual Soybean Roots in Soil.

Survival of the charcoal rot pathogen (Macrophomina phaseolina) in soybean (Glycine max) on residual root systems was studied over a 2-year period. Root segments colonized by M. phaseolina were placed into fiberglass-mesh bags and buried at depths of 0, 7.6, and 25.4 cm in a Marietta fine sandy loam soil in field microplots. Samples for year 1 and year 2 were buried in October 1999 and 2000, respectively, and sampled every 2 months over a 14-month period. Mean percent frequencies from both years for M. phaseolina showed a decreased linear trend over time from 91% before burial to 24.7% 2 months later, 10.3% at 6 months, and 0.3% after 14 months. Tissues were degraded in the June samplings of both years and this degradation was believed to be partially responsible for the reduced survival of M. phaseolina. Mean isolation frequencies were significantly (P ≤ 0.05) greater at the 0-cm depth than at 7.6 and 25.4 cm across all sampling dates. Other fungi which were present in the preliminary assay, including Fusarium spp. and those in the Phomopsis/Diaporthe complex, also declined by the end of the study. Trichoderma spp. was isolated at significantly (P ≤ 0.05) greater frequencies and a significant positive linear trend over time. It is possible that Trichoderma spp. were involved in direct tissue degradation and nutrient depletion of the root segments, or may have acted as a mycoparasite, reducing the survival of the pathogen M. phaseolina and other associated fungi. Results from this study indicate that farm practices which increase residue destruction immediately after harvest or those that enhance Trichoderma spp. populations may directly or indirectly lower the relative longevity of soilborne pathogens, including M. phaseolina.