Clarence Watson

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.

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.

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.

Microfungi of forest litter from healthy American Beech, Fraser Fir, and Eastern Hemlock stands in Great Smoky Mountains National Park.

Abstract n As part of an All Taxa Biodiversity Inventory of the Great Smoky Mountains National Park, an assemblage of microfungi associated in litter samples from healthy Fagus grandifolia (American Beech), Abies fraseri (Fraser Fir), and Tsuga canadensis (Eastern Hemlock) trees was determined in 2005 and 2006. Additionally, litter samples from the collection sites were assayed for pH, nutrient content, ash, crude proteins, and levels of organic matter to determine their impact on the mycobiota. Species richness, diversity, and evenness patterns were evaluated from the litter samples collected in May, July, and September of each year. A total of 6249 isolates of fungi were obtained, with greater than 90% belonging to the Deuteromycota. Over 100 species of fungi were identified from litter of the three tree species, with 55 being new records from the Park. As in previous studies, the most common fungi isolated from the three tree species were 13 species of Trichoderma during the two-year study. Other common fungi included Virgaria nigra and Penicillium spp. Species richness and diversity values pooled across sampling dates and years were significantly greater from American Beech litter, followed by Eastern Hemlock and lowest for Fraser Fir. Species richness and diversity values compared by sampling dates for each year were generally greater in May than July or September, but evenness values showed a reverse trend for each year. When species richness and diversity were compared between sampling dates per year and among or by tree species, significant differences often occurred, but no trends were determined. Data from the litter tissue assay showed that Fraser Fir, which had the lowest species richness and diversity, may have been impacted by having significantly lower pH and percent litter chemical compositions of ash, crude protein, and N than the other tree species. All other comparisons of species richness were similar.

Bacteria Associated with Red Imported Fire Ants (Solenopsis invicta) from Mounds in Mississippi

Abstract A study was conducted to determine microbial community structure and baseline information of cultural bacteria taxa within Solenopsis invicta (Red Imported Fire Ant) mounds from 3 locations along the roadside of Natchez Trace Parkway in Mississippi. At each location, samples consisting of mound soils, plant debris of primarily grass stem and leaves (control), and ant body tissues were obtained from replicate mounds during March, July, and November 2004. Bacteria isolate frequencies from soil were significantly greater than from plant or ant body tissues. Using 16S sequence data, 68 taxa from 2324 isolates were obtained from the 3 substrate types. The 7 most common bacteria following in order of greatest isolation frequencies were Bacillus sp. (5) (species complex), Achromobacter xylosoxidans, Bacillus cereus (complex), Lysininibacillus boronitolerans, Serratia liquefaciens, Pseudomonas protegens, and Lysinibacillus sphaericus. Richness, diversity, and evenness values varied between the locations, sampling dates, and the 3 isolation substrates. Total community-coefficient values were 0.74 to 0.84 across sampling dates. Overall these values indicated uniform communities across the different locations, isolation substrates, and across 3 sampling dates. Furthermore, no consistent trends in frequencies were observed by comparing ant tissues, location, and sampling dates to occurrences of bacterial taxa. Isolates and data obtained from this survey will allow for further testing to determine their role as food sources, saprophytes, or pathogens in Red Imported Fire Ant mound ecosystems.

A Molecular Clone and Culture Inventory of the Root Fungal Community Associated with Eastern Hemlock in Great Smoky Mountains National Park

Abstract n Below- and above-ground microbial communities are expected to dramatically change following the loss of healthy Tsuga canadensis (Eastern Hemlock) ecosystems due to the effects of Adelges tsugae (Hemlock Woolly Adelgid [HWA]) infestation. In 2006, few healthy Eastern Hemlock stands remained in Great Smoky Mountains National Park (GRSM), and we sought to obtain baseline soil microbial data from these undisturbed areas to better understand them. This study surveyed root fungal communities (e.g., mycorrhizae) using molecular cloning and cultural methods at 2 remaining healthy Eastern Hemlock stands in GRSM. We sampled roots from 40 trees representing 2 age classes (<75 and >150 years old) and 2 elevations (486 m and 1158 m) to obtain molecular clones and isolated cultures. Molecular clones from the ITS region indicated the presence of 124 taxa in 23 genera. The dominant genera were Piloderma (8.2%), Russula (6.8%), Tomentella (4.0%), Meliniomyces (3.4%), and Clavulina (2.9%). Ninety-seven of the taxa identified occurred at <1.0% relative abundance. The most frequent fungal species were Meliniomyces variabilis (22.6%), Russula granulata (16.1%), Phialocephala fortinii (9.7%), and Cladophialophora chaetospira (16.1%). Species richness, diversity, and evenness for total fungi, ectomycorrhizal species, and saprobic species were similar at different elevations and stand ages. Using ITS sequence data, we identified 9 species from 1800 fungal isolates from Eastern Hemlock root tissue, the most common of which were Trichoderma harzianum (24.4%), Trichoderma viride (24.4%), Trichoderma asperellum (17.1%), and Ilyonectria radicicola (12.2%). Species richness, diversity, and evenness were not significantly different across locations for the molecular data. Baseline data for the fungal taxa identified in the study may be important for understanding ecological changes that can occur post-HWA damage and HWA-caused stand losses. These data could also be used to support Eastern Hemlock reforestation efforts within current or former habitats in GRSM and surrounding ecosystems.

All Taxa Biodiversity Inventory Survey of Select Soil and Plant Ecological Parameters Associated with Rhododendron Decline in the Great Smoky Mountains and Surrounding Areas

Abstract n As part of the All Taxa Biodiversity Inventory (ATBI) of Great Smoky Mountains National Park (GRSM), select site parameters associated within upland sites were measured within variable sized pockets of stressed and dead Rhododendron maximum (Great Rhododendron). During the last 20 years, Great Rhododendron, an important shrub in the southern Appalachian Mountains, has been dying in small to larger areas from an unknown cause. With increased visibility of dieback, the study (2006–2009) was conducted at 10 sites in GRSM and 1 in Nantahala National Forest (NNF). Eleven nematode species were identified, with no specific trends across locations or by plot treatments in frequencies of occurrences. Exceptions were Criconemella xenoplax, which was found at all 11 locations and was generally significantly greater in healthy or control than dieback plots, whereas Helicotylenchus sp. was more frequent in dieback plots. Meloidogyne spp., known parasitic nematodes of woody plants and agricultural crops, occurred in over 50% of the locations; Hoploliamus sp. occurred at 40%; and Belomolaimus sp., which are very destructive to root systems, were found at low levels at the NNF site. Also, Heterodera sp. occurred in control and dieback plots in 10 of the locations. Dieback ratings were not significantly correlated to stem diameter; although the finding was not statistically significant, six of seven sites with dieback plots had numerically greater-sized stems than in the controls. No other parameters such as number of clonal units, site aspect, percent slope, or elevation showed any trends at both sites. Nutrient data did not indicate any specific relationships to plot damage or health. This study provides the first comprehensive reporting of nematode species that occur in Great Rhododendron and associated riparian and upland sites. With concerns of global climate impacts, this research provided additional baseline data for the ATBI of GRSM and NNF.

Temporal and Locational Variations of a Phytophthora spp. Community in an Urban Forested Water Drainage and Stream-Runoff System

Abstract Species richness and diversity of Phytophthora spp. (water molds) in urban riparian-forest ecosystems, which serve as primary drainage passageways for surface-water runoff, may be attributed to surrounding landscape management, associated vegetation, and environmental conditions. These riparian areas, although generally small, are always flooded during wet seasons and almost completely dry during the hottest parts of each year when there is limited precipitation. Little is known about Phytophthora spp. diversity within these heavily impacted sites. We sampled water, soil, and vegetation (phenology dependent) across 14 dates, over ~2 y at a site containing a drainage ditch that enters Hog Creek, in Rankin County, MS. We cultured all Phytophthora spp. using 4 published protocols to ensure maximum isolation potential. Across all sampling dates, 65 isolations were positive for Phytophthora spp., 12 of which were recovered from vegetation. We employed morphological and internal transcribed sequence (ITS) data to confirm taxa. We determined a total of 11 taxa on the basis of their phylogenetic clustering with known species of Phytophthora in a bayesian analysis. The most common taxa were P. chlamydospora, P. mississippiae, and P. cinnamomi at frequencies of 12.5%, 11.0%, and 10%, respectively. We verified morphologically and by sequence similarity an undescribed species, Phytophthora oaksoil taxon, which has been reported previously in the Western US, as well as other countries, such as Australia. Overall, the bottle-of-bait (BOB) intact-leaf and water-filtration methods had numerically greater frequencies (P ≤ 0.05) than BOB leaf disks, soil-baiting leaf disks, or vegetationsampling protocols. Overall frequency (14%) of Phytophthora spp. was significantly greater (P ≤ 0.05) for the 17 December 2014 sampling date. Even though several taxa identified in this study are reported to be pathogenic to riparian forest trees and vegetation at the Hog Creek site, symptoms on surrounding trees and vegetation was generally limited to foliar lesions, and we observed no visible damage or decline during the study period. It would be judicious to visit different, similar urban habitats to determine if common Phytophthora in this study are present in other central and southern Mississippi riparian habitats.

Fungi Associated with Solenopsis invicta Buren (Red Imported Fire Ant, Hymenoptera: Formicidae) from Mounds in Mississippi

Abstract n In 2004, we determined baseline data on fungal-community assemblages from Solenopsis invicta (Red Imported Fire Ant) mounds in 3 counties (Hinds, Leake, and Madison) within the Natchez Trace Parkway, MS. We assayed mound soil, plant debris within the mounds, and ants obtained from mounds on 3 sampling dates (March, July, and November). We processed samples based on standard microbiological protocols, and used traditional morphological and molecular techniques to identify fungal taxa. We documented a total of 1445 isolates consisting of 50 fungal taxa and calculated a diversity index value (H′) of 3.11 across all substrates, which was indicative of a variable fungal community within the mounds. The taxa with the highest percent isolation frequencies included Hypocrea lixii (12.8%), Fusarium sp. 1 (12.3%), Fusarium equiseti (7.9%), Purpureocillium lilacinum (= Paecilomyces lilacinus) (6.5%), Fusarium oxysporum 2 (5.8%), and Mortierella alpina (5.4%). We isolated 2 common parasitic (entomopathogenic) fungi, Purpureocillium lilacinum and Metarhizium anisopliae var. anisopliae (9.4%), from mound soil, plant debris, and ant external tissues. Hypocrea lixii, the teleomorphic reproductive stage of Trichoderma harzianum, is noted as a natural biological control of some soil-borne microbes, possibly limiting important natural entomopathogenic activity within the mounds. Species richness and diversity values from mound soils across locations were significantly greater (P ≤ 0.05) than those from the plant debris and ant body-tissue substrates. Species richness values between locations were similar. Species richness of samples collected in November (47) was significantly greater (P ≤ 0.05) than that of the March (41) and July (39) samples. Community coefficient values ranged from 0.79 to 0.87 between substrates, 0.85 to 0.91 between locations, and 0.85 to 0.86 between sampling dates, indicating that taxa were similar.