Impact of nutritional conditions on yields,germination rate and shelf-life of Plectosporium alismatis conidia and chlamydospores as potential candidates for the development of a mycoherbicide of weeds in rice crops

The effect of nutritional conditions on spore qualities was investigated in order to select which propagules, conidia or chlamydospores, would be most suitable for mycoherbicide development. Plectosporium alismatis was grown in a liquid basal medium supplemented with glucose and a mineral nitrogen source (sodium nitrate) or an organic nitrogen source (casamino acids). Conidial and chlamydospore yields, germination rate and shelf-life were compared. Two growth models were developed: on one hand, sodium nitrate added as the sole nitrogen source was partially utilised (8%), resulting in poor growth (1.77±0.02 mg mL^?1; 6±1.7×10⁵ conidia mL^?1). Under these conditions, P. alismatis produced dense, melanised-like aggregates that contained chlamydospores (12.4±0.7×10⁴ chlamydospores mL^?1). Germination rates of chlamydospores and conidia produced under these conditions was high (80%). Twenty percent of chlamydospores were able to germinate after 4 months storage at 25°C, while survival of conidia declined rapidly (<2%). When casamino acids were added to the liquid medium as the sole nitrogen source, P. alismatis produced sparser pellets resulting in high dry weights (5.37±0.09 mg mL^?1 and high conidia numbers (9.6±1.5×10⁶ conidia mL^?1), while no chlamydospore were observed. The germination rate of conidia produced in casamino acids was low (33±13%) after 8 h incubation and microcycle conidiation occurred. Five percent of these conidia germinated after 4 months storage. These data indicate that chlamydospores may be suitable for mycoherbicide development, provided further optimisation of yields is achieved.     

Culture age impacts Plectosporium alismatis propagule yields and subsequent desiccation and UV-radiation tolerance

The effect of culture age on yields, desiccation tolerance and resistance to ultraviolet radiation of Plectosporium alismatis, a potential mycoherbistat of aquatic weeds in Australian rice fields, was studied. P. alismatis was grown in a liquid basal medium supplemented with malt extract and sodium nitrate and harvested after 7, 14 or 21 days incubation. Although chlamydospore yields harvested from 14-day-old liquid cultures were significantly higher (29.2×10⁵ chlamydospores mL^?1) than chlamydospore yields harvested from 7-day-old liquid cultures (1.07×10⁵ chlamydospores mL^?1) or from 21-day-old liquid cultures, the germination of freshly-harvested chlamydospores from 7-day-old cultures (72.7%) was significantly reduced when propagules were grown for 14 days (55.3%). When exposed to UV-radiation, conidia and chlamydospores harvested from 14-day-old cultures germinated at a lower rate (<20%) than conidia and chlamydospores harvested from 7-day-old cultures (>40%). When conidia and chlamydospores were dried and subsequently exposed to UV, less than 30% of propagules harvested from 7-day-old cultures germinated, whereas less than 10% of propagules harvested from 14-day-old cultures germinated. A three-way analysis of variance including culture age, UV exposure and type of propagules confirmed that the culture age had more impact on the germination of fresh or dry propagules (P=0.00001 and P=0.0004, respectively) than the type of propagules considered (P=0.5). These results demonstrate that the culture age impacts significantly propagule yields and germination of P. alismatis conidia and chlamydospores, particularly after stress caused by dehydration and/or exposure to UV-B radiation.     

Successful induction and recognition of conidiation,conidial germination and chlamydospore formation in pure culture of Morchella

Morels, fungi from the genus Morchella, are popular edible mushrooms. However, little knowledge of their asexual reproduction and inaccessible pure mitospores hamper illumination of their life cycle. Herein, we successfully induced conidiation, conidial germination and chlamydospore formation in pure culture of Morchella sextelata. Conidiation proceeded via four morphologically distinct stages: development of the conidiophore stalk, stalk branching, phialide differentiation, and conidium production. Terminal and intercalary chlamydospores were formed on conidial hyphae. The development of conidiophores occurred earlier, with more conidia produced, in cross-mating cultures than in single-spore cultures. Mature conidia were spherical and 2.5–8 μm in diameter, with a vast majority (nearly 99%) 2.5–5 μm in diameter. Each conidium contained one to three nuclei (80.2% conidia contained one nucleus, 19.1% contained two nuclei, and 0.7% contained three nuclei). The conidial nucleus diameter was 1–2 μm. The nuclear mitosis in detached conidia that was observed may benefit their colony initiation. Additionally, morel conidia formed conidial anastomosis tubes. Conidia (mitospores) likely not only function as spermatia, but also as reproductive propagules in Morchella. Further research is imperative to elucidate the relationship between the conidia and chlamydospores, and their unique function in the morel life cycle.     

Deletion and Complementation of the Mating Type (MAT) Locus of the Wheat Head Blight Pathogen Gibberella zeae

Gibberella zeae, a self-fertile, haploid filamentous ascomycete, causes serious epidemics of wheat (Triticum aestivum) head blight worldwide and contaminates grain with trichothecene mycotoxins. Anecdotal evidence dating back to the late 19th century indicates that G. zeae ascospores (sexual spores) are a more important inoculum source than are macroconidia (asexual spores), although the fungus can produce both during wheat head blight epidemics. To develop fungal strains to test this hypothesis, the entire mating type (MAT1) locus was deleted from a self-fertile (MAT1-1/MAT1-2), virulent, trichothecene-producing wild-type strain of G. zeae. The resulting MAT deletion (mat1-1/mat1-2) strains were unable to produce perithecia or ascospores and appeared to be unable to mate with the fertile strain from which they were derived. Complementation of a MAT deletion strain by transformation with a copy of the entire MAT locus resulted in recovery of production of perithecia and ascospores. MAT deletion strains and MAT-complemented strains retained the ability to produce macroconidia that could cause head blight, as assessed by direct injection into wheat heads in greenhouse tests. Availability of MAT-null and MAT-complemented strains provides a means to determine the importance of ascospores in the biology of G. zeae and perhaps to identify novel approaches to control wheat head blight.     

Trichoderma koningii as a potential parasite of sclerotia of Sclerotium rolfsii

Three different inoculum forms of Trichoderma koningii were tested in vitro for their ability to parasitize the sclerotia of Sclerotium rolfsii. Tests were conducted under two temperature regimes and three incubation periods. Wheat bran was proved to be the most potent inoculum form of the antagonist in reducing 0the viability of the sclerotia. Microscopical observations revealed the presence of hyphae, chlamydospores and conidia of T. koningii in the medullar tissues of the sclerotia. This is the first report of the effect of different inoculum forms of T. koningii on the sclerotia of S. rolfsii and of propagule (chlamydospores and conidia) formation of the antagonist inside the sclerotia of S. rolfsii.     

Description of Gibellulopsis chrysanthemi sp. nov. from leaves of garland chrysanthemum

Gibellulopsis chrysanthemi sp. nov. is described and illustrated from specimens on rotten leaves of garland chrysanthemum (Chrysanthemum coronarium L. var. spatiosum L.H. Bailey), collected in three different sites of Osaka Prefecture, Japan. This species is characterized by having short and long verticillium-like conidiophores, 1-septate, long-cylindrical conidia with tapering ends and slightly brownish chlamydospores. Compared morphologically with other species in the Plectosphaerellaceae (sister to the Glomerellales), G. chrysanthemi is similar to Gibellulopsis nigrescens, from which it differs in the formation of long conidiophores and 1-septate, long-cylindrical conidia with tapering ends. In addition, the molecular analyses of G. chrysanthemi and other members of the Plectosphaerellaceae based on a phylogenetic tree with three-loci (ITS, D1/D2, tef1-α) sequences reveal that G. chrysanthemi is located as a sister group of G. nigrescens.     

Putative Polyketide Synthase and Laccase Genes for Biosynthesis of Aurofusarin in Gibberella zeae

Mycelia of Gibberella zeae (anamorph, Fusarium graminearum), an important pathogen of cereal crops, are yellow to tan with white to carmine red margins. We isolated genes encoding the following two proteins that are required for aurofusarin biosynthesis from G. zeae: a type I polyketide synthase (PKS) and a putative laccase. Screening of insertional mutants of G. zeae, which were generated by using a restriction enzyme-mediated integration procedure, resulted in the isolation of mutant S4B3076, which is a pigment mutant. In a sexual cross of the mutant with a strain with normal pigmentation, the pigment mutation was linked to the inserted vector. The vector insertion site in S4B3076 was a HindIII site 38 bp upstream from an open reading frame (ORF) on contig 1.116 in the F. graminearum genome database. The ORF, designated Gip1 (for Gibberella zeae pigment mutation 1), encodes a putative laccase. A 30-kb region surrounding the insertion site and Gip1 contains 10 additional ORFs, including a putative ORF identified as PKS12 whose product exhibits about 40% amino acid identity to the products of type I fungal PKS genes, which are involved in pigment biosynthesis. Targeted gene deletion and complementation analyses confirmed that both Gip1 and PKS12 are required for aurofusarin production in G. zeae. This information is the first information concerning the biosynthesis of these pigments by G. zeae and could help in studies of their toxicity in domesticated animals.     

The oxygen concentration in cultures modulates protein expression and enzymatic antioxidant responses in Metarhizium lepidiotae conidia

Conidia from Metarhizium spp. are used for integrated pest control; however, environmental factors diminish the effectivity of these programs. Several approaches tried to improve conidia resistance to overcome this limitation, although little is known about the mechanisms involved in this effect. Here we measured the activity of antioxidant enzymes and conidia virulence, comparing the proteomic profiles of Metarhiziumlepidiotae CP-OAX conidia produced under normal (21% O₂) and high oxygen atmospheres (pulses with 30% O₂). We detected a higher virulence against Tenebrio molitor larvae, in addition to an increase in ultraviolet light tolerance in conidia produced under 30% O₂, which correlates with increased glutathione reductase activity. Two-dimensional gel electrophoresis (2D SDS–PAGE) of proteins extracted in conidia harvested from both experimental conditions revealed a group of proteins that was observed only in conidia from oxidant atmospheres. Some of those proteins were directly involved in oxidative stress responses, whereas others were involved in conidial virulence, thermo-tolerance, and the central metabolism. Thus, a high atmospheric oxygen concentration (30%) activates antioxidant defence and general stress response mechanisms involved in conidia resistance to adverse environmental factors, which can ultimately translate into higher effectivity for the use of entomopathogenic fungi conidia in pest control.     

A Novel Gene,ROA, Is Required for Normal Morphogenesis and Discharge of Ascospores in Gibberella zeae

Head blight, caused by Gibberella zeae, is a significant disease among cereal crops, including wheat, barley, and rice, due to contamination of grain with mycotoxins. G. zeae is spread by ascospores forcibly discharged from sexual fruiting bodies forming on crop residues. In this study, we characterized a novel gene, ROA, which is required for normal sexual development. Deletion of ROA (Δroa) resulted in an abnormal size and shape of asci and ascospores but did not affect vegetative growth. The Δroa mutation triggered round ascospores and insufficient cell division after spore delimitation. The asci of the Δroa strain discharged fewer ascospores from the perithecia but achieved a greater dispersal distance than those of the wild-type strain. Turgor pressure within the asci was calculated through the analysis of osmolytes in the epiplasmic fluid. Deletion of the ROA gene appeared to increase turgor pressure in the mutant asci. The higher turgor pressure of the Δroa mutant asci and the mutant spore shape contributed to the longer distance dispersal. When the Δroa mutant was outcrossed with a Δmat1-2 mutant, a strain that contains a green fluorescence protein (GFP) marker in place of the MAT1-2 gene, unusual phenotypic segregation occurred. The ratio of GFP to non-GFP segregation was 1:1; however, all eight spores had the same shape. Taken together, the results of this study suggest that ROA plays multiple roles in maintaining the proper morphology and discharge of ascospores in G. zeae.Gibberella zeae (anamorph: Fusarium graminearum) causes Fusarium head blight in wheat, barley, and rice, as well as ear rot and stalk rot in maize (20, 23). The infected grains are frequently contaminated by mycotoxins, such as trichothecenes and zearalenone, which are harmful to humans and animals (6). The fungus overwinters in crop debris in the form of storage hyphae and develops ephemeral fruiting bodies (perithecia) at warmer temperatures. Ascospores formed within the perithecia are forcibly discharged into the air and are believed to serve as the primary inoculum of the disease (7, 27, 37, 39,–42). Therefore, sexual development and ascospore discharge are important factors in fungal survival and disease initiation.In fungi of the phylum Ascomycota, the sexual cycle is initiated when two genetically distinct nuclei combine to form a binucleate cell (31). As a homothallic fungus, G. zeae possesses the two mating type genes MAT1-1 and MAT1-2 in the haploid genome and therefore does not require a mating partner for sexual development (22, 46). Perithecium initials give rise to small, coiled initials that develop into perithecia filled with asci, tubular sacs of ascospores, which are the products of meiosis. Mature asci extend through the ostiole of perithecia and discharge their ascospores (40).Unique features of cell differentiation are involved in ascus and ascospore morphogenesis. Ascospore delimitation within the ascus and the development of a cell wall between the ascus and ascospore membranes are unique features of the process (31). Most studies of morphogenesis have described these changes in detail; however, much of these data have been limited to microscopic observations. Several genes involved in ascospore morphogenesis have been identified in Neurospora crassa (30), but the detailed mechanisms and genes involved in ascus and ascospore morphogenesis remain to be elucidated. The Round spore (R) mutant of N. crassa was shown to have round ascospores (24), and the gene responsible for this phenotype, rsp, was subsequently cloned (28). However, in G. zeae, no genes have been identified that are involved in ascus and ascospore morphogenesis.Although recent research has shed light on the physiological basis of ascospore discharge, the genetic basis remains largely unknown (38). The main force responsible for the observed shooting is turgor pressure within the extended asci. In G. zeae, a buildup of K⁺ and Cl⁻ ions drives the influx of water and causes turgor pressure that stretches the asci (41). Asci can accumulate polyols as well as ions. In a previous study, it was shown that the polyols are comprised mainly of mannitol and glucose; however, the concentration of these polyols is too low to make a significant contribution to turgor pressure (42). When the turgor pressure exceeds the threshold of the asci, apical pores rupture and ascospores are forcibly discharged (38). Trail et al. (41) estimated that the acceleration of ascospores in G. zeae is 8,500,000 m s⁻² using an iterative model to predict initial velocity. Recently, Yafetto et al. (44) used high-speed video photography to examine several large-spore fungi, including Ascobolus immerses, and to predict acceleration during dispersal. The asci of A. immerses are more than 12-fold larger in diameter than the asci of G. zeae (38). The size difference between these fungi greatly affects the behavior of their projectiles and results in an initial speed for G. zeae that is too great for application of the video photography method (for further discussion, see the supplemental material).To date, only one gene from G. zeae, the calcium ion channel gene cch1, has been shown to be involved in ascospore discharge (12). Deletion of this gene was shown to arrest ascospore discharge without affecting spore and ascus morphology. Since the genomic sequence of G. zeae is now available, the functional analysis of genes involved in sexual development has been accelerated. Random insertional mutagenesis is one strategy that has been used to identify novel genes associated with sexual development (13, 34). Previously, we produced a collection of more than 20,000 mutants from G. zeae by using the restriction enzyme-mediated integration (REMI) transformation procedure (13). In this study, the G. zeae mutant Z43R9901, which was isolated from a screening of REMI transformants, showed an unusual phenotype during sexual development. Further analysis demonstrated that the novel gene ROA is involved in ascospore morphogenesis and discharge in G. zeae. The results of this study increase our understanding of sexual development in the fungus.     

Comparative Mycotoxin Profiles of Gibberella zeae Populations from Barley, Wheat, Potatoes, and Sugar Beets

Gibberella zeae is one of the most devastating pathogens of barley and wheat in the United States. The fungus also infects noncereal crops, such as potatoes and sugar beets, and the genetic relationships among barley, wheat, potato, and sugar beet isolates indicate high levels of similarity. However, little is known about the toxigenic potential of G. zeae isolates from potatoes and sugar beets. A total of 336 isolates of G. zeae from barley, wheat, potatoes, and sugar beets were collected and analyzed by TRI (trichothecene biosynthesis gene)-based PCR assays. To verify the TRI-based PCR detection of genetic markers by chemical analysis, 45 representative isolates were grown in rice cultures for 28 days and 15 trichothecenes and 2 zearalenone (ZEA) analogs were quantified using gas chromatography-mass spectrometry. TRI-based PCR assays revealed that all isolates had the deoxynivalenol (DON) marker. The frequencies of isolates with the 15-acetyl-deoxynivalenol (15-ADON) marker were higher than those of isolates with the 3-acetyl-deoxynivalenol (3-ADON) marker among isolates from all four crops. Fusarium head blight (FHB)-resistant wheat cultivars had little or no influence on the diversity of isolates associated with the 3-ADON and 15-ADON markers. However, the frequency of isolates with the 3-ADON marker among isolates from the Langdon, ND, sampling site was higher than those among isolates from the Carrington and Minot, ND, sites. In chemical analyses, DON, 3-ADON, 15-ADON, b-ZEA, and ZEA were detected. All isolates produced DON (1 to 782 μg/g) and ZEA (1 to 623 μg/g). These findings may be useful for monitoring mycotoxin contamination and for formulating FHB management strategies for these crops.     

Molecular and Morphological Characterization of the Corn Cyst Nematode,Heterodera zeae,from Greece

The corn cyst nematode Heterodera zeae was detected in soil from an organic maize field in northern Greece. In greenhouse studies, reproduction of H. zeae was detected on maize plants (Zeae mays) using soil high in organic matter; the field was under winter fallow at the time of sampling. Maize plants were grown in a greenhouse with soil from the affected field used as inoculum. Females appeared after six weeks incubation, and abundant cysts were present after 12 weeks. Morphological and molecular diagnosis confirmed the presence of H. zeae in the field. Cysts were identified on the basis of cyst shape and characteristics of the cyst terminal cone, including nature of fenestration, presence of bullae, cyst wall pattern, and fenestral diameter. Second-stage juveniles were identified by body and stylet length, the shape of stylet knobs, shape and length of the tail and hyaline tail terminus, and by the number of lateral lines. Molecular analysis included amplification of the ribosomal internal transcribed spacer regions (ITS 1&2 rDNA) 28S large ribosomal subunit (LSU) D2-D3 expansion segment, and partial 18S small ribosomal subunit (SSU). Restriction fragment length polymorphism (RFLP) of ITS rDNA exhibited several unique enzyme patterns that may be diagnostically useful for H. zeae. These findings are in agreement with prior analysis of H. zeae populations from the U.S. and India. Phylogenetic relationships inferred from ITS rDNA are congruent with previous analyses that placed H. zeae in a clade with H. turcomanica, H. salixophila and species of the Humuli group. Phylogenetic trees based upon heat shock protein (Hsp90) coding sequence were in general agreement with a prior study using the same marker. This study represents the first record of H. zeae in Greece and the second report of this nematode in Europe.     

New species ofAcremonium, Cylindrocarpon andVerticillium from soil in the Bonin (Ogasawara) Islands, Japan

Three new soil fungi from the Bonin (Ogasawara) Islands, Japan are described:Acremonium macroclavatum, characterized by large clavate guttulate conidia;Cylindrocarpon boninense, characterized by 3–7-septate clavate macroconidia, terminal or intercalary chlamydospores, rarely produced unicellular clavate microconidia; andVerticillium hahajimaense, characterized by conidial heads bearing cylindrical conidia, and catenulate chlamydospores.     

Glycosylphosphatidylinositol-Anchored Ecm33p Influences Conidial Cell Wall Biosynthesis in Aspergillus fumigatus

ECM33 encodes a glycosylphosphatidylinositol-anchored protein whose orthologs in yeast are essential for sporulation. Aspergillus fumigatus Ecm33p is unique and has an apparent mass of 55 kDa. Disruption of A. fumigatus ECM33 results in a mutant with several morphogenetic aberrations, including the following: (i) a defect in conidial separation, (ii) an increase in the diameter of the conidia of the mutant associated with an increase in the concentration of the cell wall chitin, (iii) conidia that were sensitive to the absence of aeration during long-term storage, and (iv) conidia that were more resistant to killing by phagocytes, whereas the mycelium was more easily killed by neutrophils.     

Unraveling the Nanoscale Surface Properties of Chitin Synthase Mutants of?Aspergillus fumigatus and Their Biological Implications

Understanding the surface properties of the human opportunistic pathogen Aspergillus fumigatus conidia is essential given the important role they play during the fungal interactions with the human host. Although chitin synthases with myosin motor-like domain (CSM) play a major role in cell wall biosynthesis, the extent to which deletion of the CSM genes alter the surface structural and biophysical-biological properties of conidia is not fully characterized. We used three complementary atomic force microscopy techniques—i.e., structural imaging, chemical force microscopy with hydrophobic tips, and single-molecule force spectroscopy with lectin tips—to gain detailed insights into the nanoscale surface properties (ultrastructure, hydrophobicity) and polysaccharide composition of the wild-type and the chitin synthase mutant (ΔcsmA, ΔcsmB, and ΔcsmA/csmB) conidia of A. fumigatus. Wild-type conidia were covered with a highly hydrophobic layer of rodlet nanostructures. By contrast, the surface of the ΔcsmA mutant was almost completely devoid of rodlets, leading to loss of hydrophobicity and exposure of mannan and chitin polysaccharides. The ΔcsmB and ΔcsmA/csmB mutants showed a different behavior, i.e., the surfaces featured poorly organized rodlet layers, yet with a low hydrophobicity and substantial amounts of exposed mannan and chitin at the surface. As the rodlet layer is important for masking recognition of immunogenic fungal cell wall components by innate immune cells, disappearance of rodlet layers in all three chitin synthase mutant conidia was associated with an activation of human dendritic cells. These nanoscale analyses emphasize the important and distinct roles that the CSMA and CSMB genes play in modulating the surface properties and immune interactions of A. fumigatus and demonstrate the power of atomic force microscopy in fungal genetic studies for assessing the phenotypic characteristics of mutants altered in cell surface organization.     

Mapping quantitative trait loci (QTLs) for resistance to Gibberella zeae infection in maize

The basic prerequisite for an efficient breeding program to improve levels of resistance to pathogens in plants is the identification of genes controlling the resistance character. If the response to pathogens is under the control of a multilocus system, the utilization of molecular markers becomes essential. Stalk and ear rot caused by Gibberella zeae is a widespread disease of corn: resistance to G. zeae is quantitatively inherited. Our experimental approach to understanding the genetic basis of resistance to Gibberella is to estimate the genetic linkage between available molecular markers and the character, measured as the amount of diseased tissue 40 days after inoculation of a suspension of Fusarium graminearum, the conidial form of G. zeae, into the first stalk internode. Sensitive and resistant parental inbreds were crossed to obtain F₁ and F₂ populations: the analysis of the segregation of 95 RFLP (restriction fragment length polymorphism) clones and 10 RAPD (random amplified polymorphic DNA) markers was performed on a population of 150 F₂ individuals. Analysis of resistance was performed on the F₃ families obtained by selfing the F₂ plants. Quantitative trait loci (QTL) detection was based either on analysis of regression coefficients between family mean value and allele values in the F₂ population, or by means of interval mapping, using MAPMAKER-QTL. A linkage map of maize was obtained, in which four to five genomic regions are shown to carry factors involved in the resistance to G. zeae.     

Conidiation under illumination enhances conidial tolerance of insect-pathogenic fungi to environmental stresses

Light is an important signal for fungi in the environment and induces many genes with roles in stress and virulence responses. Conidia of the entomopathogenic fungi Aschersonia aleyrodis, Beauveria bassiana, Cordyceps fumosorosea, Lecanicillium aphanocladii, Metarhizium anisopliae, Metarhizium brunneum, Metarhizium robertsii, Simplicillium lanosoniveum, Tolypocladium cylindrosporum, and Tolypocladium inflatum were produced on potato dextrose agar (PDA) medium under continuous white light, on PDA medium in the dark, or under nutritional stress (= Czapek medium without sucrose = MM) in the dark. The conidial tolerance of these species produced under these different conditions were evaluated in relation to heat stress, oxidative stress (menadione), osmotic stress (KCl), UV radiation, and genotoxic stress caused by 4-nitroquinoline 1-oxide (4-NQO). Several fungal species demonstrated greater stress tolerance when conidia were produced under white light than in the dark; for instance white light induced higher tolerance of A. aleyrodis to KCl and 4-NQO; B. bassiana to KCl and 4-NQO; C. fumosorosea to UV radiation; M. anisopliae to heat and menadione; M. brunneum to menadione, KCl, UV radiation, and 4-NQO; M. robertsii to heat, menadione, KCl, and UV radiation; and T. cylindrosporum to menadione and KCl. However, conidia of L. aphanocladii, S. lanosoniveum, and T. inflatum produced under white light exhibited similar tolerance as conidia produced in the dark. When conidia were produced on MM, a much stronger stress tolerance was found for B. bassiana to menadione, KCl, UV radiation, and 4-NQO; C. fumosorosea to KCl and 4-NQO; Metarhizium species to heat, menadione, KCl, and UV radiation; T. cylindrosporum to menadione and UV radiation; and T. inflatum to heat and UV radiation. Again, conidia of L. aphanocladii and S. lanosoniveum produced on MM had similar tolerance to conidia produced on PDA medium in the dark. Therefore, white light is an important factor that induces higher stress tolerance in some insect-pathogenic fungi, but growth in nutritional stress always provides in conidia with stronger stress tolerance than conidia produced under white light.     

Protoplats isolation and regeneration and nuclear staining of mycoparasitic Gliocladium species

Protoplast isolation and regeneration from 24 h germinating conidia of Gliocladium catenulatum, G. roseum and G. virens have been optimized. The number of nuclei per cell of four cell types of the Gliocladium spp. was determined. The optimal enzyme combination for protoplast production contained chitinase, lyticase and cellulase onuzaku in 0.5 M mannitol osmoticum. The quantity of protoplasts produced was dependent on the duration of enzymatic digestion, the concentration of germinating condidia and the species of Gliocladium being assayed. A maximum of 11 to 55% of the protoplasts of wild-type Gliocladium spp. and of two double amino acid autoxotrophic mutants of G. roseum (both Met⁻ Leu⁻) regenerated to mature, conidiating colonies depending on the regeneration medium utilized and the strain assayed. Protoplasts of G. roseum and G. catenulatum were approximately 4% multinucleate, 56% uninucleate and 40% anucleate. Protoplasts of G. virens were approximately 81% multinucleate, 6% uninucleate and 13% anucleate. Regenerating protoplasts and germinating conidia of G. catenulatum and G. roseum were predominantly uninucleate; those of G. virens were predominantly multinucleate. Conidia of all three Gliocladium spp. were predominantly uninucleate.     

Direct Binding of a Plant LysM Receptor-like Kinase, LysM RLK1/CERK1, to Chitin in Vitro

Plants induce immune responses against fungal pathogens by recognition of chitin, which is a component of the fungal cell wall. Recent studies have revealed that LysM receptor-like kinase 1/chitin elicitor receptor kinase 1 (LysM RLK1/CERK1) is a critical component for the immune responses to chitin in Arabidopsis thaliana. However, the molecular mechanism of the chitin recognition by LysM RLK1 still remains unknown. Here, we present the first evidence for direct binding of LysM RLK1 to chitin. We expressed LysM RLK1 fused with yeast-enhanced green fluorescent protein (LysM RLK1-yEGFP) in yeast cells. Binding studies using the solubilized LysM RLK1-yEGFP and several insoluble polysaccharides having similar structures showed that LysM RLK1-yEGFP specifically binds to chitin. Subsequently, the fluorescence microscopic observation of the solubilized LysM RLK1-yEGFP binding to chitin beads revealed that the binding was saturable and had a high affinity, with a K_d of ∼82 nm. This binding was competed by the addition of soluble glycol chitin or high concentration of chitin oligosaccharides having 4–8 residues of N-acetyl glucosamine. However, the competition of these chitin oligosaccharides is weaker than that of glycol chitin. These data suggest that LysM RLK1 has a higher affinity for chitin having a longer residue of N-acetyl glucosamine. We also found that LysM RLK1-yEGFP was autophosphorylated in vitro and that chitin does not affect the phosphorylation of LysM RLK1-yEGFP. Our results provide a new dimension to chitin elicitor perception in plants.     

Tri13 and Tri7 Determine Deoxynivalenol- and Nivalenol-Producing Chemotypes of Gibberella zeae

Gibberella zeae, a major cause of cereal scab, can be divided into two chemotypes based on production of the 8-ketotrichothecenes deoxynivalenol (DON) and nivalenol (NIV). We cloned and sequenced a Tri13 homolog from each chemotype. The Tri13 from a NIV chemotype strain (88-1) is located in the trichothecene gene cluster and carries an open reading frame similar to that of Fusarium sporotrichioides, whereas the Tri13 from a DON chemotype strain (H-11) carries several mutations. To confirm the roles of the Tri13 and Tri7 genes in trichothecene production by G. zeae, we genetically altered toxin production in 88-1 and H-11. In transgenic strains, the targeted deletion of Tri13 from the genome of 88-1 caused production of DON rather than NIV. Heterologous expression of the 88-1 Tri13 gene alone or in combination with the 88-1 Tri7 gene conferred on H-11 the ability to synthesize NIV; in the latter case, 4-acetylnivalenol (4-ANIV) also was produced. These results suggest that Tri13 and Tri7 are required for oxygenation and acetylation of the oxygen at C-4 during synthesis of NIV and 4-ANIV in G. zeae. These functional analyses of the Tri13 and Tri7 genes provide the first clear evidence for the genetic basis of the DON and NIV chemotypes in G. zeae.