Plant rhizosphere specificity and variability in the insect and plant adhesins, Madl and Mad2, within the genus Metarhizium suggest plant adaptation as an evolutionary force

Metarhizium is a soil-inhabiting fungus currently used as a biological control agent against various insect species, and research efforts are typically focused on its ability to kill insects. In section 1, we tested the hypothesis that species of Metarhizium are not randomly distributed in soils but show plant rhizosphere-specific associations. Results indicated an association of three Metarhizium species (Metarhizium robertsii, M. brunneum and M. guizhouense) with the rhizosphere of certain types of plant species. M. robertsii was the only species that was found associated with grass roots, suggesting a possible exclusion of M. brunneum and M. guizhouense, which was supported by in vitro experiments with grass root exudate. M. guizhouense and M. brunneum only associated with wildflower rhizosphere when co-occurring with M. robertsii. With the exception of these co-occurrences, M. guizhouense was found to associate exclusively with the rhizosphere of tree species, while M. brunneum was found to associate exclusively with the rhizosphere of shrubs and trees. These associations demonstrate that different species of Metarhizium associate with specific plant types. In section 2, we explored the variation in the insect adhesin, Madl, and the plant adhesin, Mad2, in fourteen isolates of Metarhizium representing seven different species. Analysis of the transcriptional elements within the Mad2 promoter region revealed variable STRE, PDS, degenerative TATA box, and TATA box-like regions. Phylogenetic analysis of 5' EF-Ia, which is used for species identification, as well as Madl and Mad2 sequences demonstrated that the Mad2 phylogeny is more congruent with 5' EF-1a than Madl. This suggests Mad2 has diverged among Metarhizium lineages, contributing to clade- and species-specific variation. While other abiotic and biotic factors cannot be excluded in contributing to divergence, it appears that plant associations have been the driving factor causing divergence among Metarhizium species.

Trading nitrogen for carbon: Nitrogen and carbon translocation in a plant/fungal (Metarhizium spp.) symbiosis

While nitrogen is critical for all plants, they are unable to utilize organically bound nitrogen in soils. Therefore, the majority of plants obtain useable nitrogen through nitrogen fixing bacteria and the microbial decomposition of organic matter. In the majority of cases, symbiotic microorganisms directly furnish plant roots with inorganic forms of nitrogen. More than 80% of all land plants form intimate symbiotic relationships with root colonizing fungi. These common plant/fungal interactions have been defined largely through nutrient exchange, where the plant receives limiting soil nutrients, such as nitrogen, in exchange for plant derived carbon. Fungal endophytes are common plant colonizers. A number of these fungal species have a dual life cycle, meaning that they are not solely plant colonizers, but also saprophytes, insect pathogens, or plant pathogens. By using 15N labeled, Metarhizium infected, wax moth larvae (Galleria mellonella) in soil microcosms, I demonstrated that the common endophytic, insect pathogenic fungi Metarhizium spp. are able to infect living soil borne insects, and subsequently colonize plant roots and furnish ts plant host with useable, insect-derived nitrogen. In addition, I showed that another ecologically important, endophytic, insect pathogenic fungi, Beauveria bassiana, is able to transfer insect-derived nitrogen to its plant host. I demonstrated that these relationships between various plant species and endophytic, insect pathogenic fungi help to improve overall plant health. By using 13C-labeled CO2, added to airtight plant growth chambers, coupled with nuclear magnetic resosnance spectroscopy, I was able to track the movement of carbon from the atmosphere, into the plant, and finally into the root colonized fungal biomass. This indicates that Metarhizium exists in a symbiotic partnership with plants, where insect nitrogen is exchanged for plant carbon. Overall these studies provide the first evidence of nutrient exchange between an insect pathogenic fungus and plants, a relationship that has potentially useful implications on plant primary production, soil health, and overall ecosystem stability.

The analysis of Metarhizium robertsii potential as endophytic plant root coloniser, plant growth enhancer and antagonist to bean root pathogen Fusarium solani f. sp. phaseolis.

The soil-inhabiting insect-pathogenic fungus Metarhizium robertsii also colonizes plant roots endophytically, thus showing potential as a plant symbiont. M robertsii is not randomly distributed in soils but preferentially associates with the plant rhizosphere when applied in agricultural settings. Root surface and endophytic colonization of switchgrass (Panicum virgatum) and haricot beans (Phaseolus vulgaris) by M robertsii were examined after inoculation with fungal conidia. Light and confocal microscopies were used to ascertain this rhizosphere association. Root lengths, root hair density and emergence of lateral roots were also measured. Initially, M robertsii conidia adhered to, germinated on, and colonized, roots. Furthermore, plant roots treated with Metarhizium grew faster and the density of plant root hairs increased when compared with control plants. The onset of plant root hair proliferation was initiated before germination of M robertsii on the root (within 1-2 days). Plants inoculated with M robertsii AMAD2 (plant adhesin gene) took significantly longer to show root hair proliferation than the wild type. Cell free extracts of M robertsii did not stimulate root hair proliferation. Longer term (60 days) associations showed that M robertsii endophytically colonized individual cortical cells within bean roots. Metarhizium appeared as an amorphous mycelial aggregate within root cortical cells as well as between the intercellular spaces with no apparent damage to the plant. These results suggested that not only is M robertsii rhizosphere competent but displays a beneficial endophytic association with plant roots that results in the proliferation of root hairs. The biocontrol of bean (Phaseolis vulgaris) root rot fungus Fusarium solani f. sp. phaseolis by Metarhizium robertsii was investigated in vitro and in vivo. Dual cultures on Petri dishes showed antagonism of M robertsii against F. solani. A relative inhibition of ca. 60% of F. solani growth was observed in these assays. Cell free culture filtrates of M robertsii inhibited the germination of F. solani conidia by 83% and the inhibitory metabolite was heat stable. Beans plants colonized by M robertsii then exposed to F. solani showed healthier plant profiles and lower disease indices compared to plants not colonized by M robertsii. These results suggested that the insect pathogenic/endophytic fungus M robertsii could also be utilized as a biocontrol agent against certain plant pathogens occurring in the rhizosphere.

The identification, distribution and persistence of oxamyl and its degradation products in planted corn seed, seedling root and soil from oxamyl-treated corn seeds

A simple method was developed for treating corn seeds with oxamyl. It involved soaking the seeds to ensure oxamyl uptake, centrifugation to draw off excess solution, and drying under a stream of air to prevent the formation of fungus. The seeds were found to have an even distribution of oxamyl. Seeds remained fungus-free even 12 months after treatment. The highest nonphytotoxic treatment level was obtained by using a 4.00 mg/mL oxamyl solution. Extraction methods for the determination of oxamyl (methyl-N'N'-dimethyl-N-[(methylcarbamoyl)oxy]-l-thiooxamimidate), its oxime (methyl-N',N'-dimethyl-N-hydroxy-1-thiooxamimidate), and DMCF (N,N-dimethyl-1-cyanoformanade) in seed" root, and soil were developed. Seeds were processed by homogenizing, then shaking in methanol. Significantly more oxamyl was extracted from hydrated seeds as opposed to dry seeds. Soils were extracted by tumbling in methanol; recoveries range~ from 86 - 87% for oxamyl. Root was extracted to 93% efficiency for oxamyl by homogenizing the tissue in methanol. NucharAttaclay column cleanup afforded suitable extracts for analysis by RP-HPLC on a C18 column and UV detection at 254 nm. In the degradation study, oxamyl was found to dissipate from the seed down into the soil. It was also detected in the root. Oxime was detected in both the seed and soil, but not in the root. DMCF was detected in small amounts only in the seed.