An investigation into fungal metabolism of halogenated and related steroids

Fungal metabolism of halogenated and related steroids was investigated. The fungi Aspergillus niger ATCC 9142, Curvularia lunata NRRL 2380 and Rhizopus stolonifer ATCC6227b were studied in this regard. 2l-Fluoro-, 2l-chloro, 2l-bromo- and 2l-methyl-pregn-4-ene-3,20diones were prepared and incubated with ~ niger (a C-2l-hydroxylator) in order to observe the effect of the C-2l substituent on the metabolism of these substrates. In all four cases, the C-2l substituent prevented any significant metabolism of these substrates. llB-Fluoropregn-4-ene-3,20-dione was prepared and incubated with C. lunata (an llB-hydroxylator) and ~ stolonifer (an lla-hydroxylator). With ~ lunata, the ll-fluoro- substituent prevent hydroxylation at the 11 position, but diverted it to a site remote from the fluorine atom. In contrast, with ~ stolonifer the llB-fluoro- substituent, although slowing the apparent rate of hydroxylation, did not prevent its occurrence at the 11a- position. llB-Hydroxypregn-4-ene-3,20-dione was also incubated with R. stolonifer. The llB-hydroxy-;group did not appear to have any significant effect on hydroxylation at the lla- position. The incubation of a substrate, unsaturated at a favoured site of hydroxylation with Rhizopus arrhizus ATCC 11145 provided a complex mixture of products; among them were both the a and S epoxides. The formation of these products is rationalized as arising because of the lack of regio- and stereospecificity of the hydroxylase enzyme(s) involved.

The 21-dehydroxylation of corticosteroids: evidence for an enol intermediate and the hydroxylation of steroids by fungal cyto-chrome P450: evidence for a stepwise mechanism

Two enzyme mechanisms were examined: the 21-dehydroxylation of corticosteroids by the anaerobe Eubacterium l en tum, and the hydroxylation of steroids by fungal cytochrome P450. Deuterium labelling techniques were used to study the enzymic dehydroxylation. Corticosteroids doubly labelled (2H) at the C-21 position were incubated with a culture of Eubacterium lentum. It was found that t he enzymic dehydroxylation proceeded with the loss of one 2H f rom C-21 per molecule of substrate. The kinetic isotope ef fect f or the reaction was found to be k~kD = 2. 28. These results suggest that enzyme/substr ate binding in this case may proceed via t he enol form of the substrate. Also , it appears that this binding is, at least in part, the rate determining step of t he reaction. The hydroxylation of steroids by fungal cytochrome P450 was examined by means of a product study. Steroids with a double bond at the A8 (9), ~( lO ), or ~ (ll) position were synthesized. These steroids were then incubated with fungal strains known to use a cytochrome P450 monooxygenase to hydroxylate at positions allylic to these doubl e bonds. The products formed in these incubations indicated that the double bonds had migrated during allylic hydroxylat ion. This suggests that a carbon centred radical or ion may be an intermediate i n the cytochrome P450 cat alytic cycle.

Fungal biotransformation of morphine alkaloids

The purpose of the study was to determine the ability of certain fungi to biotransform morphine alkaloids into medicinally relevant intermediates. Fungal strains screened for their ability to affect biotransformation of morphine alkaloids include Cunninghamella echinulata, Helicostylum pirijorme, Pycnoporus sanguinea, Pycnoporus cinnabarina, Curvularia lunata and Sporotrichum sulfurescens. The research demonstrated that Cunninghamella echinulata N-demethylated thebaine, hydrocodone, codeine, oripavine and oxycodone into corresponding nor-compounds in varying yields. The study further focused on the characterization of the enzyme responsible for the biotransformation of thebaine into northebaine by Cunninghamella echinulata. The study clearly showed that incubation of the fungal culture with thebaine over a period of 48 hours was required to activate the biotransformation process. The biotransformation studies with [14C] labeled thebaine showed that Ndemethylation by Cunningham ella echinulata does not involve O-demethylation followed by methyl group transfer as suggested in previous studies.

Incidence of resistance to benzimidazole fungicides in a field population of Venturia inaequalis /

The allele-specific polymerase chain reaction (PCR) was used to screen for the presence of benomyl resistance, and to characterize their levels and frequencies in field populations of Venturia inaequalis during two seasons. Three hundred isolates of V. inaequalis were collected each season from infected leaves of MalusX domestica. Borkh c.v. Mcintosh. The trees used were sprayed in the year prior to collection with five applications of benomyl, its homologue Azindoyle, or water. Monoconidial isolates of V. inaequalis were grown on 2% potato dextrose agar (PDA) for four weeks. Each isolate was taken from a single lesion from a single leaf. Total genomic DNA was extracted from the four week old colonies of V. inaequalis, prepared and used as a template in PCR reactions. PCR reactions were achieved by utilizing allele-specific primers. Each primer was designed to amplify fragments from a specific allele. Primer Vin was specific for mutations conferring the ben^^"^ phenotype. It was expected to amplify a 171 bp. DNA fragment from the ben^"^ alleles only. Primers BenHR and BenMR were specific for mutations conferring the ben"" and ben'^'' phenotypes, respectively. They were expected to amplify 172 bp. and 165 bp. DNA fragments from the ben"" and ben"^" alleles, respectively. Of the 953 isolates tested, 414 (69.9%) were benomyl sensitive (ben^) and 179 (30.1%) were benomyl resistant. All the benomyl resistant alleles were ben^"", since neither the ben"" nor the ben"" alleles were detected. Frequencies of benomyl resistance were 23%, 24%, and 23% for the 1997 collections, and were 46%, 26% and 38% for the 1998 collections for benomyl, Azindoyle and water treatments, respectively. Growth assay was performed to evaluate the applicability of using PCR in monitoring benomyl resistance in fungal field populations. Tests were performed on 14 isolates representing the two phenotypes (ben^ and ben^"'' alleles) characterized by PCR. Results of those tests were in agreement with PCR results. Enzyme digestion was also used to evaluate the accuracy and reliability of PCR products. The mutation associated with the ben^"'' phenotype creates a unique site for the endonuclease enzyme Bsh^236^ allowing the use of enzyme digestion. Isolates characterized by PCR as ben^'^'^ alleles had this restriction site for the SsA7l2361 enzyme. The most time consuming aspect of this study was growing fungal isolates on culture media for DNA extraction. In addition, the risk of contamination or losing the fungus during growth processes was relatively high. A technique for extracting DNA directly from lesions on leaves has been used (Luck and Gillings 1 995). In order to apply this technique in experiments designed to monitor fungicide resistance, a lesion has to be homogeneous for fungicide sensitivity. For this purpose, PCR protocol was used to determine lesion homogeneity. One hundred monoconidial isolates of V. inaequalis from 10 lesions (10-conidia/ lesion) were tested for their phenotypes with respect to benomyl sensitivity. Conidia of six lesions were homogeneous, while conidia of the remaining lesions were mixtures of ben^ and ben^ phenotypes. Neither the ben" nor the ben' phenotype was detected.

The investigation of the reduction mechanism of the 5a-reductase enzyme of Penicillium decumbens /

The 5a-reductase of Penicillium decumbens ATCC 10436 was used as a model for the mammalian enzyme to investigate the mechanism of reduction of testosterone to 5adihydrotestosterone . The purpose of this study was to search for specific 5a-reductase inhibitors which antagonize prostate cancer . In a whole-cell biotransformation mode, this organism reduced testosterone (1) to 5a-dihydrosteroids (8) and 5aandrostane- 3, 17-dione (9) in yields of 28% and 37% respectively. Control experiments have shown that 5aandrostane- 3, 17-dione (9) can be produced from the corresponding alcohol (8) in a subsequent reaction separate from that catalysed by the 5a-reductase enzyme . Androst-4- ene-3, 17-dione (2) is reduced to give only (9) with a recovery of 80% The stereochemistry of the reduction was determined by 500 MHz ^H NMR analysis of the products resulting from the deuterium labelled substrates. The results were obtained by an analysis of the NOE difference spectra, double-quantum filtered phase sensitive COSY 2-D spectra, and ^^c-Ir 2-D shift correlation spectra of deuterium labelled products. According to the unambiguous assignment of the signals due to H-4a and H-4Ii in 5a-dihydro steroids, the NMR data show clearly that addition of hydrogen to the 4{5)K bond has occurred in a trans manner at positions 413 and 5a. To Study the reduction mechanism of this enzyme, several substrates were prepared as following; 3-methyleneandrost-4-en- 17fi-ol(3), androst-4-en-17i5-ol(5) , androst-4-en-3ii, 17fi-diol (6) and 4, 5ii-epoxyandrostane-3, 17-dione (7) . Results suggest that this enzyme system requires an oxygen atom at the 3-position of the steroid in order to bind the substrate. Furthermore, the mechanism of this 5a-reductase may proceed via direct addition of hydrogen at the 4,5 position without involvement of a carbonyl group as an intermediate.

Host-parasite relationships in mycoparasite /

A mycoparasite, Piptocephalis virginiana ^ shows a resemblance to fungal parasites of higher plants in the fine structure of hyphae and haustoria. The morphology and fine structure of host and parasitic fungi have been described. The mode of penetration of the host cell, Choanephora cucurbitarum , probably involves mechanical forces. Although the presence of cell wall degrading enzyme was not detected by conventional techniques, its role in penetration can't be ruled out. A collar around the haustorial neck is formed as an extension of the host cell wall. No papilla was detected although appressorixim was seen during penetration. The young haustorium is enclosed in highly invaginating plasmalemma of the host cell and n\imerous cisternae of endoplasmic reticulum. Appearance of an electron—dense sheath around the mature haustorium seems to coincide with the disappearance of cisternae of endoplasmic reticulum from the host cystoplasm in the vicinity of the haustorium. The role of host cytoplasm particularly of endoplasmic reticulum in the development of the sheath is discussed. Extensive accumulation of spherosomes-like bodies, containing lipids, is found in haustorium, parasite and host hypha. Electron microscope revealed the parasiticculture spore has more lipid content than the axenic culture spore of P. virginiana . The biochemical and cytochemical tests also support these results. The mature spore of C. cucurbitarum possesses a thick three-layered cell wall, different from the hyphal wall. Its germination is accompanied by the formation of an elastic thin inner layer which surrounds the emerging germ tube and the growing hypha. High resolution autoradiography showed that H N-acetyl-glucosamine , a precursor of chitin, was incorporated preferentially in the thin inner layer of the spore wall and also in the cell wall of the growing hypha. When the label was fed to the infected cells, at different intervals after inoculation, grains were observed on the sheath which developed around the haustorium of P. virginiana , 30 hours after inoculation. The significance of these results in relation to the origin and composition of the sheath is discussed.

A comparative study of in vitro chitin synthase activity in mucoraceous hosts of a mycoparasite

A comparative study of in vitro chitin synthase activity in mucoraceous hosts of a mycoparasite: Chitin synthase, the enzyme responsible for the synthesis of chitin in fungal cell wall was extracted from young hyphae of Choanephora cucurbitarum and Phascolomyces articulosus, susceptible and resistant hosts, respectively, to the mycoparasite, Piptocephalis virginiana. Crude enzyme was identified and characterized by measuring the incorporation of the substrate [14C]-UDP-N-acetylglucosamine, into chitin. Most activity occurred in mixed membrane fraction. Inhibition of activity with Polyoxin D and activation with proteases, N-acetyl-glucosamine and magnesium and other ions was observed. Properties of the crude enzyme preparation such as cofactor requirement, Vmax , apparent Km value for UDP-GlcNAc, inhibition by Polyoxin D, response to pH and to temperature, and stability at 4°C were determined. Enzyme activity from both fungi displayed basically the same features as the corresponding enzymes reported from other mucoraceous fungi. However, the two preparations from P. articulosus and C. cucurbitarum differed from each other in their expressed activity (i.e., the preparations from ~ articulosus exhibited higher latency and higher specific chitin synthase activity than the corresponding preparations from ~ cucurbitarum). Trypsin was effective in activation only over a narrow concentration range. Acid protease was the most effec.tive activator. En.dogenous protease estimation indicated higher protease activity in C. cucurbitarum than in P. articulosus. The suggestion is made that regulation of chitin synthase activities may be related to host resistance in the mycoparasitic system.

An investigation into some mechanistic aspects of estrogen biosynthesis

The mechanistic aspects of the 19-hydroxy1ation and aromatization of androgens were investigated. Fungal, bacterial and mammalian enzymatic activities were studied in this regard . The fungus Pell i cular~ fi1amentosa metabolized androst-4-ene-3 , 17-dione to the corresponding 110<' , 11 f and 14 0( hydroxylated derivatives. No ~19- hydroxylated products were isolated, although this transformation was previously observed for the C21-steroids . The intestinal bacterium Clostridi um paraputrific~ had been reported to aromatize androsten-4-ene-3,17-dione. In the present study, however, only the ring A reduced products , 17(3 - hydroxy-5f -andro8tane- 3-one and 5f-androstane-3,17-dione , were recovered . Human placental microsomes contain substantial aromatase activity and were employed in an effort to elucidate some of the mechanistic details of aromatization. Selectively deuterated steroidal substrates were employed as a probe in order to distinguish b'!tween certain of the mechanisms proposed for aromatization . Retention of deuterium at C4 and C6 was observed. It was concluded that no free intermediates allowing for loss of hydrogen from either of these two positions are implicated in this process . The involvement of a Schiff base enzyme-sup strate complex in aromatization was examined using the substrate 17f - hydroxyandrost-4-ene-3-one- 3_ 1BO. Since no loss of label was ob~erved, the implication of a Schiff base was discounted . Mixed label1ir~ studies were performed in order to determine if hydroxylation at C19 is a rate-determining process in aromatization . Isotope effects of 2 .1 and 1.7 were determined for the conversion of 17f - hydroxyandrost-4-ene-J-one-19,19,19-dJ and -19-dl respectively to estrogens. It was concluded from this that 19-hydroxylation is at l east a partially rate-determinjng process in aromatization. A homoenb~ation mechanism for 19-hydroxylation was not supported by the data obtained in this s tudy. In vitro 1JC NMR monitoring using l7f-hydroxyandrost-4-ene-Jone- 19-l3C was found not to be a successful approach in the study of steroid transformations, owing in part t o their low solubility in the incubation medium.

Molecular cloning restriction enzyme analysis, bovine adenovirus type 2 genome

Adenoviruses are non-enveloped icosahedral-shaped particles which possess a double-stranded DNA genome. Currently, nearly 100 serotypes of adenoviruses have been identified, 48 of which are of human origin. Bovine adenoviruses (BAVs), causing both mild respiratory and/or enteral diseases in cattle, have been reported in many countries all over the world. Currently, nine serotypes of SAVs have been isolated which have been placed into two subgroups based on a number of characteristics which include complement fixation tests as well as the ability to replicate in various cell lines. Bovine adenovirus type 2 (BAV2), belonging to subgroup I, is able to cause pneumonia as well as pneumonic-like symptoms in calves. In this study, the genome of BAV2 (strain No. 19) was subcloned into the plasmid vector pUC19. In total, 16 plasmids were constructed; three carry internal San fragments (spanning 3.1 to 65.2% ), and 10 carry internal Pstl fragments (spanning 4.9 to 97.4%), of the viral genome. Each of these plasmids was analyzed using twelve restriction endonucleases; BamHI, CiaI, EcoRl, HiOOlll, Kpnl, Noll, NS(N, Ps~, Pvul, Saj, Xbal, and Xhol. Terminal end fragments were also cloned and analyzed, sUbsequent to the removal of the 5' terminal protein, in the form of 2 BamHI B fragments, cloned in opposite orientations (spanning 0 to 18.1°k), and one Pstll fragment (spanning 97.4 to 1000/0). These cloned fragments, along with two other plasmids previously constructed carrying internal EcoRI fragments (spanning 20.6 to 90.5%), were then used to construct a detailed physical restriction map using the twelve restriction endonucleases, as well as to estimate the size of the genome for BAV2(32.5 Kbp). The DNA sequences of the early region 1 (E1) and hexon-associated gene (protein IX) have also been determined. The amino acid sequences of four open reading frames (ORFs) have been compared to those of the E1 proteins and protein IX from other Ads.

The regulation of oat coleoptile phosphoenolpyruvate carboxylase and malic enzyme by Hâ ½ and metabolites : kinetic evidence for and against a cytosolic pH-stat

Phosphoenolpyruvate carboxylase (PEPC) and malic enzyme activities in soluble protein extracts of Avena coleoptiles were investigated to determine whether their kinetics were consistent with a role in cytosol pH regulation. Malic enzyme activity was specific for NADP+ and Mn2+. Maximal labelled product formation from [14C]-substrates required the presence of all coenzymes, cofactors and substrates. Plots of rate versus malate concentration, and linear transformations there- 2 of, indicated typical Michaelis-Menten kinetics at non-saturating malate levels and substrate inhibition at higher malate levels. pH increases between 6.5 and 7.25 increased near-optimal activity, decreased the degree of substrate inhibition and the Kmapp(Mn2+) but did not affect the Vmax or Kmapp(malate). Transformed data of PEPC activity demonstrated non-linear plots indicative of non-Michaelian kinetics. pH increases between 7.0 and 7.6 increased the Vmax and decreased the Km app (Mg2+) but did not affect the Kmapp(PEP). Various carboxylic acids and phosphorylated sugars inhibited PEPC and malic enzyme activities, and these effects decreased with pH increases. Metabolite inhibited malic enzyme activity was non-competitive and resulted mainly from Mn2+ chelation. In contrast, metabolite inhibited PEPC activity was unique for each compound tested, being variously dependent on the PEP concentration and the pH employed. These results indicate that fluctuations in pH and metabolite levels affect PEPC and malic enzyme activities similarly and that 3 the in vitro properties of PEPC are consistent with its proposed role in a pH-stat, whereas the in vitro properties of the malic enzyme cannot be interpreted in terms of a role in pH regulation.

Regulation of Systemic Acquired Resistance through the Interaction of Arabidopsis thaliana Transcription factors TGAI and TGA2 with NPRI

Arabidopsis thaliana is an established model plant system for studying plantpathogen interactions. The knowledge garnered from examining the mechanism of induced disease resistance in this model system can be applied to eliminate the cost and danger associated with current means of crop protection. A specific defense pathway, known as systemic acquired resistance (SAR), involves whole plant protection from a wide variety of bacterial, viral and fungal pathogens and remains induced weeks to months after being triggered. The ability of Arabidopsis to mount SAR depends on the accumulation of salicylic acid (SA), the NPRI (non-expressor of pathogenesis related gene 1) protein and the expression of a subset of pathogenesis related (PR) genes. NPRI exerts its effect in this pathway through interaction with a closely related class of bZIP transcription factors known as TGA factors, which are named for their recognition of the cognate DNA motif TGACG. We have discovered that one of these transcription factors, TGA2, behaves as a repressor in unchallenged Arabidopsis and acts to repress NPRI-dependent activation of PRJ. TGA1, which bears moderate sequence similarity to TGA2, acts as a transcriptional activator in unchallenged Arabidopsis, however the significance of this activity is J unclear. Once SAR has been induced, TGAI and TGA2 interact with NPRI to form complexes that are capable of activating transcription. Curiously, although TGAI is capable of transactivating, the ability of the TGAI-NPRI complex to activate transcription results from a novel transactivation domain in NPRI. This transactivation domain, which depends on the oxidation of cysteines 521 and 529, is also responsible for the transactivation ability of the TGA2-NPRI complex. Although the exact mechanism preventing TGA2-NPRI interaction in unchallenged Arabidopsis is unclear, the regulation of TGAI-NPRI interaction is based on the redox status of cysteines 260 and 266 in TGAl. We determined that a glutaredoxin, which is an enzyme capable of regulating a protein's redox status, interacts with the reduced form of TGAI and this interaction results .in the glutathionylation of TGAI and a loss of interaction with NPRl. Taken together, these results expand our understanding of how TGA transcription factors and NPRI behave to regulate events and gene expression during SAR. Furthermore, the regulation of the behavior of both TGAI and NPRI by their redox status and the involvement of a glutaredoxin in modulating TGAI-NPRI interaction suggests the redox regulation of proteins is a general mechanism implemented in SAR.

Variability in the Insect and Plant Adhesins, Mad1 and Mad2, within the Fungal Genus Metarhizium Suggest Plant Adaptation as an Evolutionary Force

Several species of the insect pathogenic fungus Metarhizium are associated with certain plant types and genome analyses suggested a bifunctional lifestyle; as an insect pathogen and as a plant symbiont. Here we wanted to explore whether there was more variation in genes devoted to plant association (Mad2) or to insect association (Mad1) overall in the genus Metarhizium. Greater divergence within the genus Metarhizium in one of these genes may provide evidence for whether host insect or plant is a driving force in adaptation and evolution in the genus Metarhizium. We compared differences in variation in the insect adhesin gene, Mad1, which enables attachment to insect cuticle, and the plant adhesin gene, Mad2, which enables attachment to plants. Overall variation for the Mad1 promoter region (7.1%), Mad1 open reading frame (6.7%), and Mad2 open reading frame (7.4%) were similar, while it was higher in the Mad2 promoter region (9.9%). Analysis of the transcriptional elements within the Mad2 promoter region revealed variable STRE, PDS, degenerative TATA box, and TATA box-like regions, while this level of variation was not found for Mad1. Sequences were also phylogenetically compared to EF-1a, which is used for species identification, in 14 isolates representing 7 different species in the genus Metarhizium. Phylogenetic analysis demonstrated that the Mad2 phylogeny is more congruent with 59 EF-1a than Mad1. This would suggest that Mad2 has diverged among Metarhizium lineages, contributing to clade- and species-specific variation, while it appears that Mad1 has been largely conserved. While other abiotic and biotic factors cannot be excluded in contributing to divergence, these results suggest that plant relationships, rather than insect host, have been a major driving factor in the divergence of the genus Metarhizium.

Variability in the Insect and Plant Adhesins, Mad1 and Mad2, within the Fungal Genus Metarhizium Suggest Plant Adaptation as an Evolutionary Force

Several species of the insect pathogenic fungus Metarhizium are associated with certain plant types and genome analyses suggested a bifunctional lifestyle; as an insect pathogen and as a plant symbiont. Here we wanted to explore whether there was more variation in genes devoted to plant association (Mad2) or to insect association (Mad1) overall in the genus Metarhizium. Greater divergence within the genus Metarhizium in one of these genes may provide evidence for whether host insect or plant is a driving force in adaptation and evolution in the genus Metarhizium. We compared differences in variation in the insect adhesin gene, Mad1, which enables attachment to insect cuticle, and the plant adhesin gene, Mad2, which enables attachment to plants. Overall variation for the Mad1 promoter region (7.1%), Mad1 open reading frame (6.7%), and Mad2 open reading frame (7.4%) were similar, while it was higher in the Mad2 promoter region (9.9%). Analysis of the transcriptional elements within the Mad2 promoter region revealed variable STRE, PDS, degenerative TATA box, and TATA box-like regions, while this level of variation was not found for Mad1. Sequences were also phylogenetically compared to EF-1a, which is used for species identification, in 14 isolates representing 7 different species in the genus Metarhizium. Phylogenetic analysis demonstrated that the Mad2 phylogeny is more congruent with 59 EF-1a than Mad1. This would suggest that Mad2 has diverged among Metarhizium lineages, contributing to clade- and species-specific variation, while it appears that Mad1 has been largely conserved. While other abiotic and biotic factors cannot be excluded in contributing to divergence, these results suggest that plant relationships, rather than insect host, have been a major driving factor in the divergence of the genus Metarhizium.

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.