Richard E. Baird

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.

Comparison of Aflatoxigenic and Nonaflatoxigenic Isolates of Aspergillus flavus using DNA Amplification Fingerprinting Techniques

Aspergillus flavus is a filamentous fungus that produces mycotoxins in many food and feed crops, such as maize (Zea mays L.). Isolates were analyzed for toxin production by nucleic acid profiles in an attempt to differentiate aflatoxigenic from nonaflatoxigenic isolates. A total of 41 aflatoxigenic and 34 nonalfatoxigenic isolates were included in the study. The isolates were evaluated initially using DNA amplification fingerprinting (DAF) without clear resolution of the groups. A weak association of aflatoxigenic isolates was observed, as evidenced by their clustering in 18 of 81 trees recovered from maximum parsimony analysis of binary characters derived from arbitrary signatures from amplification profiles (ASAP) data; nonaflatoxigenic isolates exhibited a pattern of paraphyletic laddering. Up to five markers unambiguously supported the aflatoxigenic isolate grouping, but the presence of alternative conflicting topologies in equally parsimonious trees precluded the observation of meaningful statistical support. With additional markers for genome of A. flavus, this method could be used to resolve toxigenic from nontoxigenic strains. This additional work could resolve aflatoxigenic isolates of A. flavus present on maize plants using ASAP, which would reduce labor intense costs and potentially lead to faster determination of resistant cultivars in breeding efforts.

A review of soybean (Glycine max) seed, pod, and flower mycofloras in North America, with methods and a key for identification of selected fungi.

A review of the fungi associated with soybean seeds, pods, or flowers was conducted in North America. Species of Deuteromycetes are the most common fungi in each of the soybean flower organs followed by the Ascomycetes and Phycomycetes which comprise about one-fourth of the total mycoflora. Eighty genera and about 135 or more species occur in seeds, pods, or flowers. With regard to numbers of taxa from separate mycofloras, 63 genera and about 108 or more species occur in seeds, 65 genera and about 88 or more species occur in pods, and 36 genera and approximately 47 or more species occur in flowers. Most of the fungi which occur in flowers can be cultured from pods, and the majority of those fungi occur in seeds. Methods for and a key are provided for the identification of the 30 most important selected fungi.

Variability of United States Isolates of Macrophomina phaseolina Based on Simple Sequence Repeats and Cross Genus Transferability to Related Genera Within Botryosphaeriaceae

Twelve simple sequence repeat (SSRs) loci were used to evaluate genetic diversity of 109 isolates of Macrophomina phaseolina collected from different geographical regions and host species throughout the United States (US). Genetic diversity was assessed using Nei’s minimum genetic distance, and the usefulness of each locus was determined by calculating the polymorphism information content (PIC). A total of 98 alleles were detected and of these 31 were unique to individual genotypes. Eight of twelve loci were highly informative with PIC values greater than 0.50. The majority of pairwise comparisons of genetic distance were greater than 0.60 indicating moderate to high genetic diversity. Dendrograms based on the genetic dissimilarities were created for the 109 isolates of which 79 were from soybean. Some clustering by host and geography was noted, but, the dendrograms generally grouped isolates independent of host or geography. Additionally, sequencing of the internal transcribed spacer region (ITS) for 10 isolates revealed that all of these isolates were 99% similar. Three SSR loci from M. phaseolina were cross amplified in other genera in the Botryosphaeriaceae. This was the first study of genotyping and assessing genetic diversity of M. phaseolina isolates collected from a widespread host and geographic range across the US with SSRs. With an additional 34 loci publically available for M. phaseolina, the results indicate that previously developed SSRs from one species can be used in future population, ecological, and genetic studies of M. phaseolina and other genera within the Botryosphaeriaceae.

Survey of Bacterial and Fungal Associates of Black/Hybrid Imported Fire Ants from Mounds in Mississippi

Abstract The assemblage of bacteria and fungi from black (Solenopsis richteri)/hybrid imported fire ant (BIFA/HIFA) mounds were obtained from four counties in northeast Mississippi. These locations were selected due to high concentrations of BIFA/HIFA that were free from red imported fire ants (RIFA). Mound samples were obtained during October, November, and December in 2003 and January 2004. Patterns of species composition and diversity (species richness) were evaluated from mound soil, mound plant debris, and ant bodies. A total of 5742 isolates consisting of 58 bacterial and 35 fungal taxa were obtained. The most common bacteria identified included Chryseobacterium indolegenes, Stenotrophomonas maltophilia, Actinomadura yumaensis, and Arcanobacterium haemolyticum. Approximately 66% of the fungi cultured belonged to the artificial assemblage Fungi Imperfecti, including Curvularia geniculata, Penicillium spp., Nigrospora sphaerica, and Monoacrosporium leptosporium. The insect pathogen Beauveria bassiana was obtained from mound soil, mound plant debris, and ant bodies, with the greatest percentage from ant bodies. Species richness for bacteria and fungi were both highest from mound soil at 53 and 30 taxa, respectively, and lowest, with 8 bacteria and 25 fungal taxa, from mound plant debris. Species diversity for bacteria was also highest from mound soil, and highest for fungi from ant bodies than the other two isolation conditions. Evenness values for bacteria (0.72–0.80) and fungi (0.74–0.77) during each sampling date had moderate to high relative abundance (1.0 = highest level possible), indicating similarity of taxa among bacteria and among fungi from the four sampling dates. Coefficient of community values comparing sampling dates for bacteria and fungi were greatest between the first and last sampling date (October and January). Temperatures during those dates ranged from 14.4 °C to 28.9 °C in October and −2.8 °C to 10.0 °C in January. As a continuation of this research, cultures of the different bacteria and fungi obtained in this study are currently being evaluated for their potential as biological control agents of BIFA/HIFA and RIFA that occur in Mississippi.

Pod and Seed Mycoflora on Transgenic and Conventional Soybean [Glycine max (L.) Merrill] cultivars in Mississippi

A 2-year (1999–2000) study was conducted at Starkville and Stoneville, MS to determine if the occurrence of the mycoflora varied on Roundup® Ready (transgenic) compared to conventional soybean (Glycine max) cultivars. A total of 7,658 fungal isolates were identified from the pod and seed tissues of four cultivars compared at growth stages R6 and R8. Ninety-nine percent of all fungi isolated were mitosporic fungi and ascomycetes. In both years, total fungal isolates from the two locations were greater from the pod (65%) than from seed (33%) tissues. Isolation frequency from conventional cultivars was 54% compared to 46% for the transgenic cultivars. The most common fungi identified that are reported pathogens of soybean included Alternaria, Cercospora, Cladosporium, Diaporthe, Fusarium and Verticillium spp. When main effects and interactions were compared among the frequency data for the fungal genera, significant differences occurred, but consistent trends were not noted. Isolation frequencies of Diaporthe spp. during the R6 growth stage, were significantly greater on the conventional than on the transgenic cultivars in both years of the study, but only at Starkville. Isolation frequencies from samples taken during the R8 growth stage were similar at both locations in 1999 and 2000.Fusarium spp. isolated at R6 and R8 growth stages from pod and seed tissues were significantly greater on conventional than on transgenic cultivars in 2000. Even though frequencies were often significantly different between the transgenic and conventional cultivars, the data was not consistent between locations, pod and seed tissues, or growth stages. The pod and seed mycoflora of transgenic and conventional soybean cultivars was, therefore, similar in Mississippi.

Microsatellites from the charcoal rot fungus (Macrophomina phaseolina)

Microsatellite loci were identified from the charcoal rot fungus (Macrophomina phaseolina). Primer pairs for 46 loci were developed, and of these, 13 were optimized and screened using genomic DNA from 55 fungal isolates collected predominantly from two soybean fields in Mississippi. Twelve of the optimized loci were polymorphic and the number of alleles per locus ranged from 6 to 22. These microsatellites will be useful in population and pathogenicity studies to correspond with development of potential disease‐resistant soybean and other susceptible crops.

Effects of Nematicides on Cotton Root Mycobiota

Baseline information on the diversity and population densities of fungi collected from soil debris and cotton (Gossypium hirsutum L.) roots was determined. Samples were collected from Tifton, GA, and Starkville, MS containing cotton field soil treated with the nematicides 1,3-dichloroproprene (fumigant) and aldicarb (granules). A total of 10,550 and 13,450 fungal isolates were collected from these two study sites, respectively. Of this total, 34 genera of plant pathogenic or saprophytic species were identified. Pathogenic root fungi included Fusarium spp. (40% of all isolations), Macrophomina, Pythium, Rhizoctonia, and Sclerotium. Fusarium and Rhizoctonia were the most common fungal species identified and included F. oxysporum, F. verticillioides and F. solani, the three Fusarium species pathogenic on cotton plants. Population densities of Fusarium were not significantly different among locations or tissue types sampled. Macrophomina was isolated at greater numbers near the end of the growing seasons. Anastomosis groups of R. solani isolated from roots and soil debris included AG-3, -4, -7, 2-2, and -13 and anastomosis groups of binucleate Rhizoctonia included CAG-2, -3, and -5. Occurrences and frequency of isolations among sampling dates were not consistent. Fluctuations in the frequency of isolation of Rhizoctonia did not correspond with changes in frequency of isolation of the biological control fungus, Trichoderma. When individual or pooled frequencies of the mycobiota were compared to nematicide treatments, no specific trends occurred between treatments, application methods or rates. Results from this study show that use of 1,3-D and aldicarb in cotton fields does not significantly impact plant pathogenic fungi or saprophytic fungal populations. Thus cotton producers need not adjust seedling disease control measures when these two nematicides are used.

Microfungi from Bark of Healthy and Damaged American Beech, Fraser Fir, and Eastern Hemlock Trees During an All Taxa Biodiversity Inventory in Forests of the Great Smoky Mountains National Park

Abstract The assemblage of microfungi associated with bark samples of healthy and damaged Fagus grandifolia (American beech), Abies fraseri (Fraser fir), and Tsuga canadensis (eastern hemlock) trees was evaluated during an All Taxa Biodiversity Inventory of the Great Smoky Mountains National Park in 2003 and 2004. Bark samples were collected from sampling points 0.3, 0.6, 0.9, and 1.2 m above the ground surface on the bole of each replicate tree. Patterns of species composition and diversity (species richness) were evaluated from bark samples over three sampling dates (May, July, and September) each year. A total of 4814 isolates were obtained, with greater than 95% belonging to the Deuteromycota. Over 94 species of fungi were identified from bark of the three tree species, which were either healthy or were damaged or under pressure from exotic pests. The most common genus was Trichoderma, for which a total of 13 species were identified during the two-year study. Frequencies of microfungi between healthy and damaged trees were similar across years, but when data was compared by year, frequencies were significantly greater in 2004 than 2003. Species richness was almost always significantly greater in September than in May and July. Frequencies of microfungi isolated from bark samples collected 1.2 m above the ground were significantly greater than in samples collected at 0.9, 0.6 and 0.3 m. Increased species richness at the higher bole positions was likely related to changes in microenvironment, as proposed by previous researchers. All other comparisons of species richness were similar.

Rhododendron Decline in the Great Smoky Mountains and Surrounding Areas: Intensive Site Study of Biotic and Abiotic Parameters Associated with the Decline

Abstract Rhododendron dieback was continuously observed with increasing frequency on Rhododendron maximum (Rosebay Rhododendron) during the last 20 years in the southern Appalachian Mountains. The dieback was especially evident following several years of drought from 2004 to 2008 recorded in Great Smoky Mountains National Park (GRSM). With the concern that a disease epidemic could occur, a holistic study evaluated site factors including tree health, number of clonal units, aspect, slope, depth to bedrock, and rhizosphere microbes. This study was conducted at two locations: Laurel Falls in GRSM and Albert Mountain in Nantahala National Forest (NNF). Yearly sampling for nematodes showed no differences in frequencies across or between years. A total of 11 species were identified from replicated healthy and dieback plots with no significant trends observed. Criconemella xenophus, Helicotylenchus sp., and Meloidogyne sp. were the species most commonly found. Belonolaimus sp. occurred at the NNF site at below 1% of the total nematode population identified, but this nematode species is considered damaging to crops and forest nursery seedlings even at low numbers. Fungal/Oomycota diversity and densities were determined from roots and rhzosphere soil samples using three identification methods. The results ranged from 110 species of fungi to 0 for Oomycota. Of 110 fungi isolated, one putative root pathogen was identified, and the saprophytic species Mycena silvae-nigrae (unknown Basidiomycota 1) was the most common match using the GenBank database. Elevation at NNF was significantly greater than at GRSM, with significantly greater dieback levels at the higher elevation. Furthermore, greater dieback ratings were associated with significantly greater tree diameters. No trends were observed for percent slope or nutrient levels when compared between healthy and dieback sites or locations. Site factors such as aspect, elevation, associated nematode species, and a putative root pathogen may form a disease complex resulting in Rosebay Rhododendron dieback.