Search

By attacking plants, herbivorous mammals, insects, and belowground pathogens are known to play an important role in maintaining biodiversity in grasslands. Foliar fungal pathogens are ubiquitous in grassland ecosystems, but little is known about their role as drivers of community composition and diversity. Here we excluded foliar fungal pathogens from perennial grassland by using fungicide to determine the effect of natural levels of disease on an otherwise undisturbed plant community. Importantly, we excluded foliar fungal pathogens along with rabbits, insects, and mollusks in a full factorial design, which allowed a comparison of pathogen effects along with those of better studied plant enemies. This revealed that fungal pathogens substantially reduced aboveground plant biomass and promoted plant diversity and that this especially benefited legumes. The scale of pathogen effects on productivity and biodiversity was similar to that of rabbits and insects, but different plant species responded to the exclusion of the three plant enemies. These results suggest that theories of plant coexistence and management of biodiversity in grasslands should consider foliar fungal pathogens as potentially important drivers of community composition. Key words: biodiversity; coexistence; fungal pathogens; fungi; grasslands; insect herbivores; mollusks; Silwood Park, Berkshire, UK.

The insect epicuticle or waxy layer comprises a heterogeneous mixture of lipids that include abundant levels of long-chain alkanes, alkenes, wax esters and fatty acids. This structure represents the first barrier against microbial attack and for broad-host-range insect pathogens, such as Beauveria bassiana, it is the initial interface mediating the host-pathogen interaction, since these organisms do not require any specialized mode of entry and infect target hosts via the cuticle. B. bassiana is able to grow on straight chain alkanes up to n-033 as a sole source of carbon and energy. The cDNA and genomic sequences, including putative regulatory elements, for eight cytochrome P450 enzymes, postulated to be involved in alkane and insect epicuticle degradation, were isolated and characterized. Expression studies using a range of alkanes as well as an insect-derived epicuticular extract from the blood-sucking bug Triatomas infestans revealed a differential expression pattern for the P450 genes examined, and suggest that B. bassiana contains a series of hydrocarbon-assimilating enzymes with overlapping specificity in order to target the surface lipids of insect hosts. Phylogenetic analysis of the translated ORFs of the sequences revealed that the enzyme which displayed the highest levels of induction on both alkanes and the insect epicuticular extract represents the founding member of a new cytochrome P450 family, with three of the other sequences assigned as the first members of new P450 subfamilies. The remaining four proteins clustered with known P450 families whose members include alkane monooxygenases. DOI 10.1099/mic.0.039735-0

Candida albicans is a ubiquitous opportunistic pathogen that is the most prevalent cause of hospital-acquired fungal infections. In mammalian hosts, C. albicans is engulfed by phagocytes that attack the pathogen with DNA-damaging reactive oxygen species (ROS). Acetylation of histone H3 lysine 56 (H3K56) by the fungal-specific histone acetyltransferase Rttl09 is important for yeast model organisms to survive DNA damage and maintain genome integrity. To assess the importance of Rttl09 for C. albicans pathogenicity, we deleted the predicted homolog of Rttl09 in the clinical C. albicans isolate, SC5314. C. albicans rtt109-/- mutant cells lack acetylated H3K56 (H3K56ac) and are hypersensitive to genotoxic agents. Additionally, [rtt109.sup.-/-] mutant cells constitutively display increased H2A $129 phosphorylation and elevated DNA repair gene expression, consistent with endogenous DNA damage. Importantly, C. albicans rtt109-/- cells are significantly less pathogenic in mice and more susceptible to killing by macrophages in vitro than are wild-type cells. Via pharmacological inhibition of the host NADPH oxidase enzyme, we show that the increased sensitivity of [rtt109.sup.-/-] cells to macrophages depends on the host's ability to generate ROS, providing a mechanistic link between the drug sensitivity, gene expression, and pathogenesis phenotypes. We conclude that Rttl09 is particularly important for fungal pathogenicity, suggesting a unique target for therapeutic antifungal compounds. acetylation | chromatin | fungal pathogenesis | DNA damage resistance | macrophage doi/ 10.1073/pnas.0912427107

The fungal pathogen Entomophaga maimaiga Humber, Shimazu et Soper has become an important biocontrol for gypsy moth (Lymantria dispar (L.)) in the northeastern United States and is commonly introduced into new areas with established gypsy moth populations. Germination of the fungus is dependent on spring temperature and moisture, but specific conditions associated with epizootics have not been determined. Whether E. maimaiga will be as effective in other regions that experience different weather conditions is not yet known. We examined similarity of weather conditions associated with 16 documented E. maimaiga epizootics with conditions at 1351 North American locations using the climate-matching software CLIMEX. Based on CLIMEX's overall index of climatic similarity, long-term annual climatic patterns across much of the eastern United States were 60%-80% similar to the conditions associated with epizootics. Monthly weather records from 1971 to 2000 in nine North Central states were examined to compare precipitation and temperature with conditions observed during epizootics. Based on climatic averages identified with the documented epizootics, temperature and precipitation conditions in Illinois, Indiana, Iowa, Kentucky, Missouri, and Ohio were more conducive for epizootics than conditions in Minnesota, Wisconsin, and Michigan, which were likely to support E. maimaiga epizootics in fewer than 6 of the 30 years considered. Le champignon pathogene Entomophaga maimaiga Humber, Shimazu et Soper est devenu un important moyen de lutte biologique contre la spongieuse (Lymantria dispar (L.)) dans le nord-est des Etats-Unis et est couramment introduit dans de nouvelles regions ou sont etablies des populations de spongieuse. La germination du champignon depend des conditions printanieres de temperature et d'humidite mais les conditions specifiquement associees aux epizooties n'ont pas ete determinees. On ne sait pas encore si E. maimaiga sera efficace dans d'autres regions ou les conditions meteorologiques sont differentes. Nous avons etudie la similitude entre les conditions meteorologiques associees a 16 epizooties documentees d'E. maimaiga et les conditions meteorologiques dans 1351 endroits en Amerique du Nord a l'aide du logiciel de comparaison du climat CLIMEX. Sur la base de l'indice general de similitude du climat de CLIMEX, les patrons annuels a long terme du climat a travers presque tout l'est des Etats-Unis sont de 60 a 80 % similaires aux conditions associees aux epizooties. Les donnees meteorologiques mensuelles de 1971 a 2000 dans neuf Etats du centre-nord ont ete examinees en comparant la temperature et les precipitations aux conditions observees lors des epizooties. Sur la base des moyennes observees lors des epizooties documentees, les conditions de temperature et les precipitations en Illinois, en Indiana, en Iowa, au Kentucky, au Missouri et en Ohio etaient plus propices aux epizooties que les conditions au Minnesota, au Wisconsin et au Michigan ou moins de six des 30 annees etudiees ont connu des conditions qui auraient pu entrainer une epizootie d'E. maimaiga. [Traduit par la Redaction], Introduction Substantial reductions in gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), defoliation in the northeastern United States in the last 10-15 years have been largely attributed to epizootics of Entomophaga [...]

The entomopathogenic fungus Beauveria bassiana is under intensive study as a pest biological control agent. B. bassiana produces several distinct single-cell types that include aerial conidia, in vitro blastospores and submerged conidia. Under appropriate nutrient conditions these cells can elaborate germ tubes that form hyphae, which in turn lead to the formation of a fungal mycelium. In addition, B. bassiana displays a dimorphic transition, producing in vivo specific yeast-like hyphal bodies during growth in the arthropod haemolymph. The amphiphilic styryl dye FM4-64 was used to investigate internalization and morphological features of in vitro and in vivo insect haemolymph-derived B. bassiana cells. In vitro blastospores and submerged conidia displayed a punctate pattern of internal labelling, whereas aerial conidia failed to internalize the dye under the conditions tested. FM4-64 was also taken up into both apical and subapical compartments of living hyphae in a time-dependent manner, with clearly observable vesicle labelling. Internalization, where occurring, was reversibly disrupted by lowering the temperature of the assay or by treatment with azide/fluoride and latrunculin A. Treatment with cytochalasin D and monensin also caused abnormal vesicle trafficking, although some staining of vesicles was noted. Fungal cells derived from infected Heliothis virescens haemolymph (in vivo cells) actively internalized FM4-64. The in vivo blastospores or hyphal bodies displayed bright membrane and internal vesicle staining, although diffuse staining of internal structures was also visible. These results suggest active uptake by different developmental stages of B. bassiana, including haemolymph-derived cells that can evade the insect immune system.

The entomopathogenic fungus Beauveria bassiana and its insect host target represent a model system with which to examine host-pathogen interactions. Carbohydrate epitopes on the surfaces of fungal cells play diverse roles in processes that include adhesion, non-self recognition and immune invasion with respect to invertebrate hosts. B. bassiana produces a number of distinct cell types that include aerial conidia, submerged conidia, blastospores and haemolymph-derived cells termed in vivo blastospores or hyphal bodies. In order to characterize variations in the surface carbohydrate epitopes among these cells, a series of fluorescently labelled lectins, combined with confocal microscopy and flow cytometry to quantify the response, was used. Aerial conidia displayed the most diverse lectin binding characteristics, showing reactivity against concanavalin A (ConA), Galanthus nivalis (GNL), Griffonia simpficifolia (GSII), Helix pomatia (HPA), Griffonia simplicifolia isolectin (GSI), peanut agglutinin (PNA), Ulex europaeus agglutinin I (UEAI) and wheatgerm agglutinin (WGA), and weak reactivity against Ricinus communis I (RCA), Sambucus nigra (SNA), Limax flavus (LFA) and Sophora japonica (SJA) lectins. Lectin binding to submerged conidia was similar to that to aerial conidia, except that no reactivity against UEAI, HPA and SJA was noted, and WGA appeared to bind strongly at specific polar spots. In contrast, the majority of in vitro blastospores were not bound by ConA, GNL, GSII, GSI, SNA, UEAI, LFA or SJA, with PNA binding in large patches, and some polarity in WGA binding noted. Significant changes in lectin binding also occurred after aerial conidial germination and in cells grown on either lactose or trehalose. For germinated conidia, differential lectin binding was noted between the conidial base, the germ tube and the hyphal tip. Fungal cells isolated from the haemolymph of the infected insect hosts Manduca sexta and Heliothis virescens appeared to shed most carbohydrate epitopes, displaying binding only to the GNL, PNA and WGA lectins. Ultrastructural examination of the haemolymph-derived cells revealed the presence of a highly ordered outermost brush-like structure not present on any of the in vitro cells. Haemolymph-derived hyphal bodies placed into rich broth medium showed expression of several surface carbohydrate epitopes, most notably showing increased PNA binding and strong binding by the RCA lectin. These data indicate robust and diverse production of carbohydrate epitopes on different developmental stages of fungal cells and provide evidence that surface carbohydrates are elaborated in infection-specific patterns.

Cercosporin is a non-host-selective, photoactivated polyketide toxin produced by many phytopathogenic Cercospora species, which plays a crucial role during pathogenesis on host plants. Upon illumination, cercosporin converts oxygen molecules to toxic superoxide and singlet oxygen that damage various cellular components and induce lipid peroxidation and electrolyte leakage. Three genes (CTB5, CTB6 and CTB7) encoding putative FAD/FMN- or NADPH-dependent oxidoreductases in the cercosporin toxin biosynthetic pathway of C. nicotianae were functionally analysed. Replacement of. each gene via double recombination was utilized to create null mutant strains that were completely impaired in cercosporin production as a consequence of specific interruption at the CTB5, CTB6 or CTB7 locus. Expression of CTB1, CTB5, CTB6, CTB7 and CTB8 was drastically reduced or nearly abolished when CTB5, CTB6 or CTB7 was disrupted. Production of cercosporin was revived when a functional gene cassette was introduced into the respective mutants. All ctb5, ctb6 and ctb7 null mutants retained wild-type levels of resistance against toxicity of cercosporin or singlet-oxygen-generating compounds, indicating that none of the genes plays a role in self-protection.

Malic enzyme (ME; [NADP.sup.+]-dependent; EC 1 . 1 . 1 . 40) has been postulated to be the rate-limiting step for fatty acid biosynthesis in oleaginous fungi in which the extent of lipid accumulation is below the maximum possible. The genes encoding the isoform of ME involved in fatty acid synthesis were identified in Mucor circinelloides and Mortierella alpina, two commercially useful oil-producing fungi, using degenerate primers. Both showed high similarity with ME genes from other micro-organisms. The whole-length ME gene from each source was cloned into a leucine auxotroph of Mc. circinelloides and placed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase gene (gpd1) promoter. After confirming correct expression of the ME genes, the two recombinant strains were grown in fully controlled, submerged-culture bioreactors using a high C:N ratio medium for lipid accumulation. Activities of ME were increased by two- to threefold and the lipid contents of the cells, in both recombinants, were increased from 12% of the biomass to 30%. Simultaneously, the degree of fatty acid desaturation increased slightly. Thus, increased expression of the ME gene leads to both increased biosynthesis of fatty acids and formation of unsaturated fatty acids, including [gamma]-linolenic acid (18 : 3 n-6). At the end of lipid accumulation (96 h), ME activity in the recombinant strains had ceased, as it had done in the parent wild-type cells, indicating that additional, but unknown, controls over its activity must be in place to account for this loss of activity: this may be due to the presence of a specific ME-cleaving enzyme. The hypothesis that the rate-limiting step of fatty acid biosynthesis is therefore the supply of NADPH, as generated specifically and solely by ME, is therefore considerably strengthened by these results.

Regulators of the G protein signalling (RGS) pathway have been implicated in the control of a diverse array of cellular functions, including conidiation in filamentous fungi. However, the regulatory processes involved in conidiation in insect-pathogenic fungi are poorly understood. Since conidia are the infective propagules in these fungi, an understanding of the regulatory processes involved in conidiation is essential to the development of an effective biocontrol fungus. Here, the cloning and characterization of an RGS protein gene, cag8 (conidiation-associated gene), from the insect-pathogenic fungus Metarhizium anisopliae is reported. Phylogenetic analysis showed that CAG8 was orthologous to the RGS protein FlbA from Aspergillus nidulans. Complementation of A. nidulans [DELTA]flbA, which cannot conidiate, with M. anisopliae cag8 restored conidiation. Gene disruption of cag8 in M. anisopliae resulted in the lack of conidia on agar plates and on infected insects, reduced mycelial growth, decreased virulence, lysis during growth in liquid medium as well as lack of pigmentation and irregularly shaped blastospores. Transcript levels of ssgA (hydrophobin-encoding gene) were markedly reduced in a [DELTA]cag8 strain, while pr1A (subtilisin-like protease) transcription was unaffected. These results suggest that cag8 is involved in the modulation of conidiation, virulence and hydrophobin synthesis in M. anisopliae.

Fusicoccins are a class of diterpene glucosides produced by the plant-pathogenic fungus Phomopsis amygdali. As modulators of 14-3-3 proteins, fusicoccins function as potent activators of plasma membrane [H.sup.+]-ATPase in plants and also exhibit unique biological activity in animal cells. Despite their well studied biological activities, no genes encoding fusicoccin biosynthetic enzymes have been identified. Cyclic diterpenes are commonly synthesized via cyclization of a [C.sub.20] precursor, geranylgeranyl diphosphate (GGDP), which is produced through condensation of the universal [C.sub.5] isoprene units dimethylallyl diphosphate and isopentenyl diphosphate by prenyltransferases. We found that (+)-fusicocca-2,10 (14)-diene, a tricyclic hydrocarbon precursor for fusicoccins, is biosynthesized from the [C.sub.5] isoprene units by an unusual multifunctional enzyme, P. amygdali fusicoccadiene synthase (PaFS), which shows both prenyltransferase and terpene cydase activities. The functional analysis of truncated mutants and site-directed mutagenesis demonstrated that PaFS consists of two domains: a terpene cyclase domain at the N terminus and a prenyltransferase domain at the C terminus. These findings suggest that fusicoccadiene can be produced efficiently in the fungus by using the [C.sub.5] precursors, irrespective of GGDP availability. In fact, heterologous expression of PaFS alone resulted in the accumulation of fusicocca2,10 (14)-diene in Escherichia coil cells, whereas no product was detected in E. coli cells expressing Gibberella fujikuroi ent-kaurene synthase, another fungal diterpene cyclase that also uses GGDP as a substrate but does not contain a prenyltransferase domain. Genome walking suggested that fusicoccin biosynthetic enzymes are encoded as a gene cluster near the PaFS gene. biosynthesis | cyclase | prenyltransferase

Abstract: The causative organism of crown rust in ryegrasses (Puccinia coronata f.sp. lolii) is an obligate biotroph that causes significant economic losses within the temperate grazing industries of dairy, meat, [...]

Byline: D. Pilarska (1), M. McManus (2), P. Pilarski (3), G. Georgiev (4), P. Mirchev (4), A. Linde (5) Keywords: Bulgaria; Entomophaga maimaiga; Introduction; Lymantria dispar; Microsporidia Abstract: The establishment and prevalence of the entomopathogenic fungus Entomophaga maimaiga, (Zygomycetes, Entomophthorales), introduced into two gypsy moth populations in Bulgaria, was monitored during 2000--2004. In the Karlovo Region population, where E. maimaiga was introduced in 1999, the fungus was recovered every year and the prevalence of infection varied from 6.1% to 15.9%. A microsporidian infection caused by an Endoreticulatus sp. (Protista, Microsporidia) was also recorded every year and the prevalence varied from 2.1% and 5.0%. In the Svoge Region population, where E. maimaiga was introduced in late 2000 and first found in 2002, the prevalence of infection varied from 8.8% to 13.8%. Larval parasitism caused by Cotesia melanoscela, Protapanteles liparidis (Hymenoptera, Braconidae) and species of tachinids (Diptera, Tachinidae) was also recorded in the larvae of both populations. We provide a rationale as to why other countries should consider introducing E. maimaiga for biological control of Lymantria dispar populations. Author Affiliation: (1) Institute of Zoology, Bulgarian Academy of Sciences, Sofia, Bulgaria (2) USDA Forest Service, Northeastern Center for Forest Health Research, Hamden, CT, USA (3) Institute of Plant Physiology, Bulgarian Academy of Sciences, Sofia, Bulgaria (4) Forest Research Institute, Bulgarian Academy of Sciences, Sofia, Bulgaria (5) University of Applied Science, Eberswalde, Germany Article History: Registration Date: 11/08/2005 Received Date: 04/04/2005 Accepted Date: 07/08/2005 Online Date: 08/12/2005 Article note: Communicated by Andreas Vilcinskas

Candida guilliermondii is a haploid opportunistic pathogen accounting for about 2% of human blood yeast infections. Recent analyses using multilocus enzyme electrophoresis and karyotyping suggest that strains from human sources traditionally designated C. guilliermondii in fact include at least two species, C. guilliermondii and Candida fermentati. However, the patterns of molecular variation within and between these two species remain largely unknown. In this study, DNA fragments were sequenced from five genes for each of 37 strains collected from Canada, China, the Philippines and Tanzania. The analyses identified significant sequence differences between C. guilliermondii and C. fermentati. The five gene genealogies showed no apparent incongruence, suggesting a predominantly clonal reproductive structure for both species in nature. Indeed, two large clones of C. guilliermondii were identified, with one from Ontario, Canada, and the other from China. Interestingly, the results indicate that strains currently designated C. guilliermondii may contain additional divergent lineages. On the practical side, the results revealed several diagnostic molecular markers that can be used in clinical microbiology laboratories to distinguish C. guilliermondii and C. fermentati. The multiple gene genealogical analyses conducted here revealed significant divergence and clonal dispersal in this important pathogenic yeast complex.

Some pathogenesis-related genes are expressed in fungi only when the pathogen is in the host, but the host signals that trigger these gene expressions have not been identified. Virulent Nectria haematococca infects pea plants and requires either pelA, which is induced by pectin, or pelD, which is induced only in planta. However, the host signal(s) that trigger pelD expression was unknown. Here we report the isolation of the host signals and identify homoserine and asparagine, two free amino acids found in uniquely high levels in pea seedlings, as the pelD-inducing signals. N, haematococca has evolved a mechanism to sense the host tissue environment by using the high levels of two free amino acids in this plant, thereby triggering the expression of pelD to assist the pathogenic process. fungal infection | pectate lyase | Pisum sativum | host-pathogen interactions | pathogen-host coevolution

Ecologists increasingly recognize pathogens as playing significant roles in community dynamics, yet few experimental studies have documented patterns of disease impacts on natural systems. We studied the effects of a pathogenic fungus (Batrachochytrium dendrobatidis) on community interactions between two sympatric North American anurans: Fowler's toad, Bufo fowleri, and the gray treefrog, Hyla chrysoscelis. Recent studies have identified chytridiomycosis as an emerging infectious disease, whose widespread occurrence poses a significant risk for amphibian populations. We manipulated the presence of B. dendrobatidis in. replicated experimental tanks and examined if exposure to this fungus during development could influence recruitment and competitive interactions between larval Bufo and Hyla. Tanks were followed throughout larval development, and we recorded survival, time to metamorphosis, and body mass at metamorphosis. We also tested the effects of competitive and pathogen-mediated stress on developmental stability by measuring fluctuating asymmetry (FA) of surviving metamorphs. B. dendrobatidis had no significant effect on larval survival, but the pathogen reduced metamorphic body mass and increased larval-period length. B. dendrobatidis had different effects on larval growth depending on the community structure; in the presence of the pathogen, both Bufo and Hyla metamorphosed at smaller body masses when reared together compared to when reared separately. Bufo also had strong negative effects on Hyla development, but only in the presence of B. dendrobatidis. FA of hind-limb length was significantly higher for metamorphs from pathogen tanks, but was not associated with density or competitive environment. Our results suggest that chytridiomycosis may impact Bufo and Hyla directly by reducing life-history performance and increasing developmental instability, and indirectly by inducing interspecific competitive effects in mixed-species environments. Key words: amphibian larvae; Batrachochytriurn dendrobatidis; Bufo; chytridiomycosis; community ecology; ecological ethics and disease experiments; fluctuating asymmetry; Hyla; interspecific competition; mesocosm experiments; pathogen role in community dynamics.

A cell-wall-associated phosphatase in hyphae of Fonsecaea pedrosoi, a fungal pathogen causing chrornoblastomycosis, was previously characterized by the authors. In the present work, the expression of an acidic ectophosphatase activity in F. pedrosoi conidial forms was investigated. The surface phosphatase activity in F. pedrosoi is associated with the cell wall, as demonstrated by transmission electron microscopy. This enzyme activity was strongly inhibited by exogenous inorganic phosphate ([P.sub.i]). Accordingly, removal of [P.sub.i] from the culture medium of F. pedrosoi resulted in a marked (130-fold) increase of ectophosphatase activity. With the artificial phosphatase substrate p-nitrephenyl phosphate, a [K.sub.m] value of 0.63 [+ or -] 0.04 mM was estimated for the phosphatase activity of fungal cells strongly expressing the enyzme activity. This enzyme activity was not modulated by cations. Conidia with greater ectophosphatase activity showed greater adherence to mammalian cells than did fungi cultivated in the presence of [P.sub.i] (low phosphatase activity). Surface phosphatase activity was apparently involved in the adhesion to host cells, since the enhanced attachment of F. pedrosoi to host cells was reversed by pre-treatment of conidia with phosphatase inhibitor. Since conidial forms are the putative infectious propagules in chromoblastomycosis, the expression and activity of acidic surface phosphatases in these cells may contribute to the early mechanisms required for disease establishment.

The findings on the transcriptional regulatory system in fungi that enables genes to be expressed only when the ambient pH is conductive to their ultimate functions is discussed. It is concluded that proper functioning of the regulatory system responding to ambient pH is essential for fungal pathogenicity of both animals and plants.

Strains of Periconia circinata that are not pathogenic appear to be deficient in the synthesis of peritoxins. This was the conclusion of researchers who found intermediates of peritoxin synthesis only in pathogenic, toxin-producing strains but not in non-pathogenic, non-toxin-producing strains.

Researchers have discovered two versions of the insect-pathogenic fungus Metarhizium anisopliae that are genetically distinct and are found in two different habitats. This indicates that the habitat drives the selection of these two strains, not the insect host.

The fungus Beauveria bassiana could be used as an natural insecticide on corn plants. When the fungus invades the corn leaf, it does not lose its virulence toward the European corn borer, Ostrinia nubilalis.

The study investigates therapeutic agents active against pathogenic fungus Cryptococcus neoformans. New targets for discovery of antifungal drug are discussed.

Sixteen strains of the indoor air, toxigenic fungus Stachybotrys chartarum isolated from case or control homes in Cleveland, OH, and 12 non-Cleveland strains from diverse geographic locations were examined for hemolytic activity, conidial toxicity and randomly amplified polymorphic DNA banding patterns. Only three of the 28 strains studied were both consistently hemolytic and produced highly toxic conidia. Each of these strains was isolated from a house in Cleveland, OH, where an infant had idiopathic pulmonary hemorrhage.

The cloning and partial characterization of endopolygalactorunase genes from the plant-pathogenic fungus Botrytis cinerea are described. Five monophyletic groups of homologous proteins have been identified from phylogenetic analysis of the fungus. In addition, the identity at the amino acid level between the six endopolygalacturonase genes was found to differ from 34% to 73%. Other strains of Botrytis cinerea and related species also revealed the complete endopolygalacturonase gene family.

The relationship between the host plant Silene latifolia and its fungal pathogen Microbotryum violaceum, both of the Rhine Valley, has been investigated. The hosts in this system have been shown to be obligate outcrossers, migrating by seeds and pollen, while the parasites can self-fertilize and migrate only through pollinating insects. These parasite populations are 12 times more differentiated than their hosts.

Research was conducted to examine the composition and enzymatic activity of the extracellular matrix secreted by germlings of Botrytis cinerea. Samples derived by growing germlings on a glass surface were used to ascertain the composition of the ensheathing film. Results reveal cellulase, pectin lyase and pectin methyl esterase activities with both unheated and heated extracellular matrix preparations while no proteinase activity was noted.

High-frequency reversible changes in colony morphology were observed in three strains of Cryptococcus neoformans. For one strain (SB4, serotype A), this process produced three colony types: smooth (S), wrinkled (W), and serrated (C). The frequency of switching between colony types varied for the individual colony transitions and was as high as [10.sup.-3]. Mice infected with colony type W died faster than those infected with other colony types. The rat inflammatory response to infection with colony types S, W, and C was C > S > W and ranged from intense granulomatous inflammation with caseous necrosis for infection with type C to minimal inflammation for infection with type W. Infection with the various colony types was associated with different antibody responses to cryptococcal proteins in rats. Analysis of cellular characteristics revealed differences between the three colony types. High-frequency changes in colony morphology were also observed in two additional strains of C. neoformans. For one strain (24067A, serotype D) the switching occurred between smooth and wrinkled colonies. For the other strain (J32A, serotype A), the switching occurred between mucoid and nonmucoid colonies. The findings indicate that C. neoformans undergoes phenotypic switching and that this process can affect virulence and host inflammatory and immune responses. Phenotypic switching may play a role in the ability of this fungus to escape host defenses and establish chronic infections.

A method to immobilize the Erynia neoaphidis hyphae in alginate matrix was developed to determine whether conidia ejected from aliginate beads still infect aphid hosts. Results show that a 40% concentration of fungal biomass produced more conidia than in 20% and 10% concentration and persisted even after the beads were dried overnight and stored at four degree centigrade for four days. Moreover, algination seems to be a potential tool for using E. neoaphidis in aphid pests biocontrol.

The molecular structures of hybrids of Beauveria bassiana and Beauveria sulfurescens were studied using six nuclear genes and a telomeric fingerprint probe. The hybrids, which resulted from protoplast fusion, are widely different from their parents in pathogenicity and appeared to be diploid or aneuploid with some portions of their genome exhibiting heterozygous characteristics. The study proved that somatic hybridization is ideal for the genetic improvement of biocontrol efficiency in the genus Beauveria.

Examination of the structure of secreted aspartic proteinase from Candida tropicalis yeast (SAPT) found that the substrate binding cleft is more spacious and has more charged amino acids D/E and R when compared to pepsin. Results indicate that modifications in the primary sequence change SAPT structure to create a more spacious binding cleft with altered specificity. A tetrapeptide in binding sites S1'-S3' suggests the order of release of peptide products in the catalytic processes of the enzymes.

A comparative anlysis of depolymerases produced by saprophytes and opportunists and pathogens of plants, insects and fungi was conducted to determine the adaptation of proteases and carbohydrases to different ecological niches. Results indicate that the pathogens showed enzymic adaptation to the polymers present in the integuments of their particular hosts. The plant pathogens produced high levels of polysaccharide degrading enzymes while the entomopathogens degraded a broad spectrum of proteins. However, the saprophytes and opportunistic pathogens produced the broadest spectrum of protein and polysaccharide degrading enzymes.

1 Soil-borne fungi are a major group of economically important plant pathogens, yet they have rarely been studied in the context of host-pathogen population biology. We develop general models of annual hosts and soil-borne fungal pathogens to explore the conditions for host-pathogen coexistence in both agricultural and natural plant populations. We use empirical data from the literature to parameterize and simulate dynamics with these models. 2 Initially we consider a simple system in which host density is assumed to be constant, as would be appropriate for agricultural systems. Model analysis shows that initial increase of the pathogen population requires that host (crop) density be above a threshold; this threshold decreases with increases in the pathogen's over-winter survival rate and ability to grow saprophytically. 3 A more complex model, in which both host and pathogen populations can vary, is needed for natural populations. Results from this model show that stable coexistence is possible even when the pathogen has a positive intrinsic growth rate (and therefore it is also possible for the pathogen to persist in the complete absence of the host). 4 Model parameter estimates were obtained from the empirical literature for two common and important soil pathogens: Phytophthora spp. and Fusarium oxysporum; these pathogens differ in several life-history features. Computer simulation showed that for Fusarium, there were substantial ranges for which coexistence or loss of the pathogen were predicted, while for Phytophthora most parameter estimates resulted in complete extinction when linear disease transmission was assumed; under the assumption of exponential disease transmission, predicted dynamics were most likely to lead to host persistence. 5 For both pathogens, within biologically realistic regions of parameter space, small changes in parameter values could lead to qualitatively different outcomes, including deterministic chaos, suggesting that long-term dynamics may be difficult to predict. Keywords: Fusarium, host-pathogen coexistence, Phytophthora, saprophytic growth

A method using morphology index (Mi) which was originally designed for the quantitative determination of Candida albicans populations was applied to Paracoccidioides brasiliensis. The method was intended to make the definition of cellular and molecular markers specific for cell development easy. It also aimed to determine the distribution of morphological types in different environmental conditions. The results of the study comparing wild and mutant strains of P. brasiliensis revealed that the Mi is inversely related to the content of both cell wall chitin and 1,3-alpha-glucan.

Electrophoretic karyotypes (EK) of eight to ten chromosomesized DNAs of the pathogenic fungi Sclerotinia sclerotiorum, S. minor and S. trifoliorum are found in agricultural and natural populations. Six mitotic and three meiotic lineages of the isolates do not show variation in EKs. The experiment reveals that changes in karyotype occur over many generations of nuclear division. Homologous and heterologous probes hybridize to four linkage groups.

A nested multiplex PCR approach allows the direct determination of root rot fungi in infected seedlings by amplifying two different species specific PCR fragments with individual primer combinations from genomic DNA. The first amplification with fungus specified primers increases the population of fungal internal transcribed tracer (ITS). This increases the ratio of fungal:host ITS molecules. In the second round of amplification, the ITS molecules acted as a template for the species-specific primers. The problems with this method are the danger of carry over contamination and high costs.

The level of polyols in the conidia of the entomopathogenic fungi Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosus decreases with an increase in culture age. The polyol and trehalose levels are highest at 20 or 25 degree celsius. The trehalose level fails to increase with an increase in temperature. The polyol and trehalose levels are unaltered between the pH range of 5 to 10. Low pH causes accumulation of the polyols, glycerol and erythritol in the conidia of P. farinosus, but causes no effect in B. bassiana and M. anisopliae.

Mycoinsecticides are being used for the control of many insect pests as an environmentally acceptable alternative to chemical insecticides. A key aim of much recent work has been to increase the speed of kill and so improve commercial efficacy of these biocontrol agents. This might be achieved by adding insecticidal genes to the fungus, an approach considered to have enormous potential for the improvement of biological pesticides. We report here the development of a genetically improved entomopathogenic fungus. Additional copies of the gene encoding a regulated cuticle-degrading protease (Pr1) from Metarhizium anisopliae were inserted into the genome of M. anisopliae such that Pr1 was constitutively overproduced in the hemolymph of Manduca sexta, activating the prophenoloxidase system. The combined toxic effects of Pr1 and the reaction products of phenoloxidase caused larvae challenged with the engineered fungus to exhibit a 25% reduction in time of death and reduced food consumption by 40% compared to infections by the wild-type fungus. In addition, infected insects were rapidly melanized, and the resulting cadavers were poor substrates for fungal sporulation. Thus, environmental persistence of the genetically engineered fungus is reduced, thereby providing biological containment.

The mycoparasitic fungus Trichoderma harzianum containing the T. reesei cellulase gene promoter cbh1 has greater endochitinase production and activity than the wild type fungus. T. harzianum grown on glucose or sucrose shows no extracellular endochitinase activity. This is probably due to a low endochitinase content as cbh1 is inactive under these conditions. The transformed cells overexpress endochitinase when grown in a cellulase-inducing culture medium or in the presence of sophorose. T. harzianum is a biological control for fungal plant pathogens as its endochitinase degrades fungal cell walls.

Research on cultures of the riboflavin-producing fungus Ashbya gossypii during growth on soybean oil showed a specific isocitrate lyase of 0.17 U (mg protein) super -1. Enzyme activity could not be discerned during growth on glucose. This indicates a regulation. A 108-fold purification of the enzyme was achieved via ammonium sulphate fractionation, gel filtration and cation-exchange chromatography. The enzyme's pH optimum for the isocitrate cleavage was 7.0, and the K sub m for threo-DL-isocitrate was 550 micro M.

Isocitrate lyase (ICL) activity and riboflavin formation were studied in an attempt to detect a possible correlation. An ICL mutant of Ashbya gossypii was isolated and characterized to show such correlation, which was observed on soybean oil. The use of glucose as a source of carbon showed that riboflavin overproduction commenced in the late growth phase upon the exhaustion of glucose. ICL activity was parallel, reaching a maximum of 0.41 U (mg protein) super -1. The synthesis of vitamin B2 from the intracellular reserve fat is suggested.

Primers from a cloned mitochondrial DNA fragment of Tilletia indica are useful in differentiating the Karnal bunt of wheat fungus, T. indica, from other Tilletia species using a polymerase chain reaction assay. The primer pair Ti1/Ti4 amplifies a 2.3 kb DNA fragment of T. indica isolates from India, Pakistan, and Mexico. The DNA isolates from other smut fungi such as T. barclayana, T. caries, and T. foetida are incapable of producing bands during the assay. The primers are useful in assays with DNA from teliospores infecting wheat seeds.

The Ca2+/calmodulin signal transduction pathway is necessary for the spore germination and appressorium development of Colletotrichum trifolii on the host plant during the early stages of pathogenesis. Appressorium development on a hard surface and spore germination are necessary steps in the process of fungal development. The disruption of calcium homeostasis by calcium channel inhibitors or ethylene-bis(oxy-ethylenenitrolo)tetraacetic acid blocks appressorium development. Calmodulin suppressors block germination and differentiation.

A tripartite interaction among plant pathogenic fungi, herbivorous insects and their host plants has both direct and indirect effects on each of the participants. Effects on the host-plant are grouped into four categories, namely, synergistic, additive, equivalent and inhibitory. Pathogenic fungal infection can decrease host-plant quantity, reduce growth, competitive ability and reproduction and increase mortality.

The degradation process of polychlorinated biphenyls (PCBs) in the white rot fungus Phanerochaete chrysosporium resembles that of the bacterial systems. As compared to the degradation of 3,3',4,4'-tetrachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl, degradation of 4,4'-dichlorobiphenyl (DCB) is more and also it mineralizes partially. The DCB degradation products are 4-chlorobenzoic acid and 4-chlorobenzyl alcohol.

Streptomyces lydicus WYEC108 can inhibit the growth of the fungi Pythium ultimum and Rhizoctonia solani and can stably colonize roots emerging from pea and cotton seeds. WYEC108 coating of pea seeds reduced P. ultimum infection from 100% to under 40% of the seeds after 48 hours. The interaction between WYEC108 and P. ultimum was examined using scanning electron microscopy, revealing damage to both the oospores and the hyphae of P. ultimum.