Ultrastructural studies of the patterns of conidiogenesis in Dacrymyces stillatus nees: Fries (basidiomycete)

New patterns of conidial formation on basidiospores and vegetative hyphae are identified, illustrated and described. The mechanisms of conidial ontogeny so far unsatisfactorilly studied are elucidated and now better understood. Several types of conidiogenous cells are described and their ontogeny better illustrated and explained. Arthroconidia of the fungi are for the first time studied at ultrastructural level and their mode of formation and secession as well better understood. Likewise, chlamydospores of D. stillatus are for the first time investigated at ultrastructural level, what enables a better observation of their structure and morphology and as well a better understanding of their origin and various modes of formation.     

裴氏着色霉的扫描电镜研究

用扫描电镜研究了引起着色芽生菌病(chromoblastomycosis)的致病菌之一——裴氏着色霉(Fonsecaea pedrosoi)孢子的个体发生,除一级产孢细胞外,还有二级产孢细胞,喙枝孢型的分生孢子梗也可发育成为喙枝孢型的二级产孢细胞。     

Polyancora globosa gen. sp. nov., an aeroaquatic fungus from Malaysian peat swamp forests

During an investigation of submerged leaves and twigs sampled from tropical peat swamp forests located in Peninsular Malaysia, an anamorphic fungus not attributable to a described genus was detected and isolated in pure culture. Conidial ontogeny was thoroughly studied and illustrated using both light and SEM, which revealed a unique conidial morphology. Analysis of partial nuLSU rDNA and ITS data revealed a phylogenetic position within the Xylariales (Ascomycota), but family affiliation remained unclear.     

Basidiomycetes with branched, water-borne conidia

A number of water-borne fungi with branched conidia have been shown to be basidiomycetes. These fungi resemble aquatic hyphomyectes in their habitat, conidial morphology and ontogeny. Their conidia are typically tetraradiate or elaborately branched. Ingoldiella hamata and Taeniospora gracilis, which produce clamped, tetraradiate conidia, are anamorphs connected to the teleomorphs Sistotrema hamalum and Leptosporomyces galzinii, respectively. Both teleomorphs are members of the Corticiaceae. Dendrosporomyces prolifer and D. splendens, which produce non-clamped conidia resembling the aquatic hyphomycete Dendrospora, have binucleate conidia and typical dolipore septa are present. Other water-borne fungi with basidiomycete affinities include Ingoldiella fibulata and Tricladiomyces malaysianum.     

Taxonomy and morphology of dematiaceous fungi isolated from nature

The Hughes system of classification was applied to the identification of dematiaceous fungi isolated from nature. The majority of the isolates encountered could be identified with this system using brightfield microscopy. However, phase contrast microscopy was needed to determine the mode of conidial ontogeny with some species of Phialophora, Exophiala and Wangiella. The identifications based upon phase contrast microscopy were confirmed using the scanning electron microscope (SEM). Scanning electron photomicrographs are presented for Bispora betulina, Cladosporium trichoides, Exophiala jeanselmei, Phialophora revens, P. verrucosa and Wangiella dermatitidis. SEM observations of W. dermatitidis support the present classification of this organism as proposed by other workers. It is suggested that the Hughes system of classification and phase contrast microscopy be used to facilitate identification of isolates of dematiaceous fungi encountered in the clinical laboratory.     

Evidence for the involvement of serine protease in the conidial discharge of Conidiobolus coronatus

The involvement of serine protease(s) in the conidial discharge of Conidiobolus coronatus was investigated using the parent strain and a variant strain with reduced conidial discharge. Time course profiles of protease levels and conidial discharge showed that maximum protease levels coincided with maximum conidial discharge in both the parent and variant strains. Inhibition of serine protease(s) by phenyl methyl sulfonyl fluoride showed that low protease levels resulted in inhibition of the conidial discharge and a minimum activity of 1.0 U/mg protein is essential for triggering the conidial discharge. Using casein to induce proteases, it was further observed that early gain in the protease level (1.0 U/mg protein) leads to early onset of conidial discharge. The above evidence suggests the involvement of protease(s) in the conidial discharge of C. coronatus.     

不同基质对指状节丛孢孢子捕食器官形态建成的影响*

利用牛粪、土壤等基质诱导捕食线虫真菌指状节丛孢产生孢子捕食器官。孢子捕食器官形成过程与正常情况下捕食器官的形成基本相似;不同基质诱导孢子捕食器官的能力不同, 同种内不同菌株间形成孢子捕食器官的能力也有异别。预培养实验,温度处理实验的结果表明基质中诱导孢子捕食器官的主要诱导因子可能是某些化合物,这些物质在100℃下基本上丧失诱导活性;在诱导孢子捕食器官的过程中,基质中的微生物所起的作用不大。     

Detection and Identification of Fungi Intimately Associated with the Brown Seaweed Fucus serratus

The filamentous fungi associated with healthy and decaying Fucus serratus thalli were studied over a 1-year period using isolation methods and molecular techniques such as 28S rRNA gene PCR-denaturing gradient gel electrophoresis (DGGE) and phylogenetic and real-time PCR analyses. The predominant DGGE bands obtained from healthy algal thalli belonged to the Lindra, Lulworthia, Engyodontium, Sigmoidea/Corollospora complex, and Emericellopsis/Acremonium-like ribotypes. In the culture-based analysis the incidence of recovery was highest for Sigmoidea marina isolates. In general, the environmental sequences retrieved could be matched unambiguously to isolates recovered from the seaweed except for the Emericellopsis/Acremonium-like ribotype, which showed 99% homology with the sequences of four different isolates, including that of Acremonium fuci. To estimate the extent of colonization of A. fuci, we used a TaqMan real-time quantitative PCR assay for intron 3 of the beta-tubulin gene, the probe for which proved to be species specific even when it was used in amplifications with high background concentrations of other eukaryotic DNAs. The A. fuci sequence was detected with both healthy and decaying thalli, but the signal was stronger for the latter. Additional sequence types, representing members from the Dothideomycetes, were recovered from the decaying thallus DNA, which suggested that a change in fungal community structure had occurred. Phylogenetic analysis of these environmental sequences and the sequences of isolates and type species indicated that the environmental sequences were novel in the Dothideomycetes.     

A new non-hydrophobic cell wall protein (CWP10) of Metarhizium anisopliae enhances conidial hydrophobicity when expressed in Beauveria bassiana

A cell wall protein, CWP10, resolved from the conidial formic acid extract of a Metarhizium anisopliae isolate, was characterized as a new 9.9-kDa protein with a 32-aa signal peptide with a central hydrophobic region (ca. 10 residues) at its N-terminus. This protein was proven neither to be hydrophobic nor glycosylated and encoded by a 363-bp, single-copy gene with three introns. CWP10 was existent in the conidial extracts of seven of 18 tested M. anisopliae isolates and much more abundant (immunogold-labeled) on conidial walls than in cytoplasm. Integrating the gene into a CWP10-absent strain of Beauveria bassiana led to excellent expression of CWP10 in aerial conidia, increasing net conidial hydrophobicity by 10.8% or adhesion to hydrophobic Teflon by 1.3-fold. However, the expressed protein had no effect on conidial tolerance to thermal and ultraviolet stresses. This is the first report on a non-hydrophobic cell-wall protein enhancing conidial hydrophobicity and adhesion of the fungal species.     

Relationship between number and type of adhesions of Penicillium oxalicum conidia to tomato roots and biological control of tomato wilt

We studied (a) the extent adhesion of Penicillium oxalicum conidia to tomato roots after application of P. oxalicum conidial formulations with or without stickers, (b) the relationship between the extent of conidial adhesion to roots and biocontrol of the conidial formulations against tomato wilt, and (c) colonisation of roots by P. oxalicum. Adhesion of P. oxalicum conidia to tomato roots occurred within the first minute of contact between the root and the conidial formulation and the bonding strength was sufficiently strong to prevent conidial removal from the roots. In addition, some formulations with stickers that increased conidial adhesion to roots improved the biocontrol of tomato wilt, when compared to that of formulations without stickers. A “dried conidia without stickers” with 0.025% Nu-Film 17 had no effect on the biocontrol of tomato wilt, despite good adherence of the conidia to the roots. The numbers of P. oxalicum conidia that adhered to the roots was constant for 60 days after application of a “dried conidia without stickers” conidial formulation. The significance of these results (speed of adhesion, number of adhered conidia, and variability of conidial external surface) are discussed in relation to the biocontrol success of tomato wilt using different types of conidial formulations with and without stickers.     

Persistence of <Emphasis Type="Italic">Isaria fumosorosea</Emphasis> (Hypocreales: Cordycipitaceae) SFP-198 Conidia in Corn Oil-Based Suspension

Long-term persistence of entomopathogenic fungi as biopesticides is a major requirement for successful industrialization. Corn oil carrier was superior in maintaining germination rates of Isaria fumosorosea SFP-198 conidia during exposure to 50°C for 2 h, when compared with other oils, such as soybean oil, cottonseed oil, paraffin oil, and methyl oleate. The corn oil-based conidial suspension (91.6% germination) was also better in this regard than conidial powder (28.4% germination) after 50°C for 8 h. Long-term storage stabilities of corn oil-based conidial suspension and conidial powder at 4 and 25°C for 24 months were investigated, based on the correlation of germination rate with insecticidal activity against greenhouse whiteflies, Trialeurodes vaporariorum. Viability of conidia in corn oil was more than 98.4% for up to 9 months of storage at 25°C, and followed by 23% at 21 months. However, conidial powder had only 34% viability after 3 months of storage at 25°C, after which its viability rapidly decreased. The two conidial preparations stored at 4°C had better viabilities than those at 25°C, showing the same pattern as above. These results indicate that corn oil-based conidial suspension can be used to improve conidial persistence in long-term storage and be further applied to the formulation of other thermo-susceptible biological control agents.     

分光光度法测定白僵菌孢子粉的含孢量

研究了用分光光度计法测定白僵菌孢悬液的含孢量。首先用筛选出的适宜稀释液SF1将孢子粉作不同浓度的稀释,使用分光光度计在470nm波长下测出孢悬液的吸光度值（x）,再用血球计数板测出对应合孢量（y）,得到回归方程y＝10^{0.8895x＋6.5869}（r＝0．9678）。经检验,方程回归显著,并且不存在菌株间的差异。孢悬液的适宜稀释浓度范围为1500—2000倍。     

Impact of moisture and UV degradation on Beauveria bassiana (Balsamo) Vuillemin conidial viability in turfgrass

Beauveria bassiana conidial viability in turfgrass was evaluated using a two-component nucleic acid stain and fluorescence microscopy. Turfgrass samples along with the top 5 cm of soil were used for conidial extraction and viability evaluation on 1, 2, 3, 7, 14 and 21 days after treatment. There were no differences in conidial viability between two Orthoptera strains, 3622 and 5977, and both strains were able to persist in a sandy loam soil for up to three weeks after application. High and low irrigation levels were applied to each of the two strains and results show that higher irrigation (5.1 cm/week) maintains conidial viability better than a low irrigation level (2.5 cm/week). Mean conidial viability was approximately 8–12% greater in plots with the high irrigation regime. Rather than significantly increase soil moisture levels, it is hypothesized that the greater amount of irrigation helps to move the conidia deeper into the thatch layer and soil profile, an area that provides protection from damaging surface temperatures and UV exposure. Rainfall that occurred during the beginning of the 2005 test minimized the irrigation effect, and the irrigation treatment differences were more pronounced after 48 h. Four different UV protectants were evaluated for an impact on conidial viability of strain 3622. Two protectants, an optical brightener and magnesium silicate clay, when added to an emulsifiable oil formulation, significantly increased conidial viability on all evaluation dates. The clay particles act as a sunlight blocker while the optical brightener absorbs UV light. The combined protection from the oil and the additive increased conidial viability by approximately 10% on all evaluation dates. Results from this study provide insight into ways to increase entomopathogenic fungal viability under field conditions.     

Application of flow cytometry for exploring the evolution of Geosmithia fungi living in association with bark beetles: the role of conidial DNA content

Geosmithia belongs among fungi living in symbiosis with phloem-feeding bark beetles. Several species have altered their ecology to that of obligatory symbiosis with ambrosia beetles, which has led to a shift in their phenotype and caused formation of large spherical conidia. In this study, we pose the following questions; (1) Is the conidial DNA content of Geosmithia correlated with conidial volume?; (2) Is the DNA content of Geosmithia related to the degree of mutual dependence between Geosmithia and their vector? There was a positive and strong correlation between conidial DNA content and conidial volume in Geosmithia. Also species more narrowly associated with the vector tend to have a larger conidial DNA content and volume than less narrowly associated species. Ambrosia fungi achieved the biggest conidial DNA content and volume compared to other species. We suppose that polyploidisation occurred during the evolution of ambrosia species in the genus Geosmithia.     

Consecutive monitoring of lifelong production of conidia by individual conidiophores of Blumeria graminis f. sp. hordei on barley leaves by digital microscopic techniques with electrostatic micromanipulation

Conidial formation and secession by living conidiophores of Blumeria graminis f. sp. hordei on barley leaves were consecutively monitored using a high-fidelity digital microscopic technique combined with electrostatic micromanipulation to trap the released conidia. Conidial chains formed on conidiophores through a series of septum-mediated division and growth of generative cells. Apical conidial cells on the conidiophores were abstricted after the conidial chains developed ten conidial cells. The conidia were electrically conductive, and a positive charge was induced in the cells by a negatively polarized insulator probe (ebonite). The electrostatic force between the conidia and the insulator was used to attract the abstricted conidia from the conidiophores on leaves. This conidium movement from the targeted conidiophore to the rod was directly viewed under the digital microscope, and the length of the interval between conidial septation and secession, the total number of the conidia produced by a single conidiophore, and the modes of conidiogenesis were clarified. During the stage of conidial secession, the generative cells pushed new conidial cells upwards by repeated division and growth. The successive release of two apical conidia was synchronized with the successive septation and growth of a generative cell. The release ceased after 4-5 conidia were released without division and growth of the generative cell. Thus, the life of an individual conidiophore (from the erection of the conidiophore to the release of the final conidium) was shown to be 107 h and to produce an average of 33 conidia. To our knowledge, this is the first report on the direct estimation of life-long conidial production by a powdery mildew on host leaves.     

Gamma-ray inactivation of conidia from heterokaryons of Neurospora crassa containing UV-sensitive mutations.

Two-component heterokaryons were formed with the fungus Neurospora crassa. The UV-sensitive mutations upr-I, uvs-4, and uvs-6 were utilized. Conidia produced by these heterokaryons were exposed to gamma-rays and survival curves were established for the three conidial fractions produced by each heterokaryon. Results showed that upr-I, when included in only one nuclear component, did not affect the sensitivity of any conidial fraction; however, when included in both components, all three conidial fractions exhibited two- to four-fold decreases in survival at the 30 krad exposure. The uvs-4 mutation, when included in one or both components, did not increase the sensitivity of any conidial fraction and appeared, in contrast, to impart a small increase in resistance to inactivation by gamma-rays. When included in only one component, uvs-6 increased the sensitivity of homokaryotic uvs-6 conidia but had no affect on the other two conidial fractions. When included in both components, uvs-6 resulted in exponential inactivation curves and at the 30 krad exposure, 100-fold decreases in survival for all three conidial fractions.     

Impact of moisture and UV degradation on Beauveria bassiana (Balsamo) Vuillemin conidial viability in turfgrass

Beauveria bassiana conidial viability in turfgrass was evaluated using a two-component nucleic acid stain and fluorescence microscopy. Turfgrass samples along with the top 5 cm of soil were used for conidial extraction and viability evaluation on 1, 2, 3, 7, 14 and 21 days after treatment. There were no differences in conidial viability between two Orthoptera strains, 3622 and 5977, and both strains were able to persist in a sandy loam soil for up to three weeks after application. High and low irrigation levels were applied to each of the two strains and results show that higher irrigation (5.1 cm/week) maintains conidial viability better than a low irrigation level (2.5 cm/week). Mean conidial viability was approximately 8–12% greater in plots with the high irrigation regime. Rather than significantly increase soil moisture levels, it is hypothesized that the greater amount of irrigation helps to move the conidia deeper into the thatch layer and soil profile, an area that provides protection from damaging surface temperatures and UV exposure. Rainfall that occurred during the beginning of the 2005 test minimized the irrigation effect, and the irrigation treatment differences were more pronounced after 48 h. Four different UV protectants were evaluated for an impact on conidial viability of strain 3622. Two protectants, an optical brightener and magnesium silicate clay, when added to an emulsifiable oil formulation, significantly increased conidial viability on all evaluation dates. The clay particles act as a sunlight blocker while the optical brightener absorbs UV light. The combined protection from the oil and the additive increased conidial viability by approximately 10% on all evaluation dates. Results from this study provide insight into ways to increase entomopathogenic fungal viability under field conditions.     

Effect of temperature and sources of carbon and nitrogen on the conidial germination and appresoria formation in Colletotrichum capsici

The effect of temperatures and exogenous supply of different carbon and nitrogen sources on the conidial germination and appresoria formation inC. capsici has been studied. 25 °C was observed to be the best temperature for conidial germination. At 18 °C, conidia germinated only in hanging drops and did not germinate on 2 % agar. Amongst carbon sources, 1-rhamnose supported maximum conidial germination and appresoria formation. Potassium nitrate supported very good conidial germination and appresoria formation. Ammonium nitrate, ammonium sulphate and ammonium chloride were found to be inhibitory. A few aminoacids stimulated conidial germination but dl-alanine, dl-methionine, tyrosine and l-glutamine were found to be inhibitory.     

Effects of culture media on hydrophobicity and thermotolerance of Bb and Ma conidia, with description of a novel surfactant based hydrophobicity assay

Hypocrealean entomopathogenic fungal conidia are made up of multi-aged groups given their chronological conidiogenesis. Most thermotolerance assays have been conducted using mixed-age conidia. The present work exploited a polysiloxane polyether copolymer (siloxane) (Silwet L-77®) mediated conidial collection method, validated by a hydrophobicity assay. This was done to divide mixed-age conidia into two groups based on hydrophobicity and test their thermotolerance, relying on the relationship of conidial age with hydrophobicity. Beauveria bassiana GHA and ERL1170 and Metarhizium anisopliae ERL1171 and ERL1540 conidia, produced on millet agar, whey permeate agar, and ¼SDAY were subjected to hydrophobicity assays that included data on yield of conidia/unit of surface area. Conidia were also collected using 0.01% siloxane, and those remaining with 0.08% siloxane. Hydrophobicity was correlated with percent conidia collected in the two siloxane solutions and yield, suggesting a relationship between percent conidia collected and conidial age (maturation). The conidial suspensions were exposed to 45 °C for 45 min, and conidial germination was examined. Overall, conidia which were collected in 0.08% siloxane had lower germination after heat exposure than those collected in the 0.01% solution. Conidia of both fungi produced by incubation on millet or whey permeate for 14 d were more hydrophobic and exhibited greater thermotolerance than those produced on ¼SDAY. These results suggest that conidia can be divided into two groups with different thermotolerance by using a siloxane-mediated conidial collection method based on hydrophobicity. This depends on the types of substrates used that could influence conidial maturation.