Modification of culture conditions for production of the anti-tubercular hirsutellones by the insect pathogenic fungus Hirsutella nivea BCC 2594

Aims:  This work aimed to improve the production of anti-tubercular hirsutellones by the insect pathogenic fungus Hirsutella nivea BCC 2594.
Methods and Results:  The fungus was cultivated under different carbon/nitrogen sources and aerations (shake vs static flasks) to improve the production of the anti-tubercular alkaloids, hirsutellones A–D. Under the basal conditions, static cultivation at 25°C in minimum salt medium, only hirsutellone B and C were detected with maximum concentrations of 139·00 and 18·27 mg l⁻¹. Substitution of fructose for glucose and peptone for yeast extract increased the titres of hirsutellones A, B and C about two- to threefold. However, hirsutellone D was not detected in this medium. Culture agitation induced the production of hirsutellone D. As a result, the significant amounts of hirsutellones A–D were obtained with the concentration of 29·93, 169·63, 22·65 and 15·71 mg l⁻¹ within 15 days.
Conclusions:  Improved titres of hirsutellones in H. nivea BCC 2549 were achieved with an agitated (200 rev min⁻¹) fructose–peptone medium at 25°C.
Significance and Impact of the Study:  Improved yields of hirsutellones B–D will enable medicinal chemistry modifications leading to a development of a potential candidate for tuberculosis therapy.     

Modulation by eicosanoid biosynthesis inhibitors of immune responses by the insect Manduca sexta to the pathogenic fungus Metarhizium anisopliae

Metarhizium anisopliae conidia (spores) reduced weight gain and caused death when injected into Manduca sexta larvae. When the fungus was co-injected with the eicosanoid biosynthesis inhibitor dexamethasone, larval weight gain was further reduced and mortality increased. These effects were reversed when dexamethasone was given together with the eicosanoid precursor arachidonic acid (AA). Similarly, treatment with other eicosanoid biosynthesis inhibitors (esculetin, phenidone, ibuprofen, and indomethacin) with differing modes of action enhanced the reduction in weight gain caused by mycosis. Injection of M. anisopliae conidia induced nodule formation in vivo; nodule numbers were reduced by dexamethasone, and restored by AA. Incubation of hemocytes with conidia caused microaggregation of hemocytes (indicative of nodule formation) in vitro and this was inhibited by dexamethasone, suggesting that dexamethasone acts directly on hemocytes, although inhibition was only partially reversed by AA. We suggest that the M. sexta immune response to fungal pathogens is normally modulated by physiological systems that include eicosanoid biosynthesis. This is the first demonstration that the virulence of a fungal entomopathogen can be enhanced by compromising the insect host's immune system.     

拮抗植物病原真菌的纤维素/木质素降解菌株的筛选及鉴定

【背景】秸秆还田在改善土壤肥力和丰富营养等方面有重要作用,但是也存在秸秆难以快速降解利用和病原真菌病害威胁的问题。【目的】为解决还田秸秆的降解和病原真菌病害问题,从长期秸秆还田地区采集样品,从中筛选出具有降解秸秆和抑菌功能的菌株。【方法】采用稀释分离法、苯胺蓝染色法和刚果红染色法等对秸秆高效降解菌株进行筛选,通过16S rRNA基因测序及构建系统发育树进行菌株鉴定。采用对峙培养法测定筛选到的秸秆降解菌株对玉米大斑病菌(Setostphaeria turcica)、梨黑斑病菌(Alternaria kikuchiana)、马铃薯早疫病菌(Alternaria solani)、链格孢属真菌(Alternaria alternata) ACCC38230和ACCC38231等5种供试植物病原真菌的抑制作用。以玉米大斑病菌(Setostphaeria turcica)为后期供试植物病原真菌,测定拮抗菌株代谢产物的抑菌能力;通过观察拮抗菌株粗提液对玉米大斑病菌分生孢子萌发和菌丝生长的影响,从而测定菌株对病原真菌的抑制作用。【结果】从秸秆还田土壤中分离筛选获得3株高效降解纤维素及木质素菌株并命名为JY122、ZY133和JY215。通过形态学观察和16S rRNA基因测序鉴定上述菌株全部为芽孢杆菌属(Bacillus)。通过系统发育树分析结果表明,JY122与蜡样芽孢杆菌(Bacillus cereus)相似性为99.4%,ZY133与枯草芽孢杆菌(Bacillus subtilis)相似性为100%,JY215与贝莱斯芽孢杆菌(Bacillus velezensis)相似性为99.1%。平板对峙实验结果显示,JY122、ZY133和JY215菌株对不同种属的植物病原真菌均有较强的抑制作用,抑制率高达43.74%-67.54%。此外,上述芽孢杆菌代谢产物具有抑菌活性及很强的热稳定性,经95℃处理后依然具有良好的抑菌效果。【结论】筛选获得的JY122、JY133和JY215菌株具有高效降解纤维素/木质素能力,抑制多种植物病原真菌生长,代谢产物抑菌能力强且热稳定性高。这为玉米秸秆还田提供菌株资源,也为进一步解决秸秆还田难点提供了新方法和思路。     

1株人工栽培蛹虫草病原真菌的分离鉴定及生物学特性研究

为明确蛹虫草（Cordyceps militaris）栽培过程中发现的一种病原真菌及其生长特性,采用组织分离法自发病部位分离获得1株真菌CCBH-L,经柯赫法则确定其致病性,通过形态学特征及ITS序列分析,确定菌株CCBH-L为轮枝样镰刀菌(Fusarium verticillioides)。该病原菌在感染初期,通过竞争生长空间和营养物质,影响蛹虫草菌丝体生长和原基分化,导致子实体畸形,后期菌丝体蔓延至已经形成的子实体,导致子实体生长受阻。该病原菌生长的适宜温度为25~30 ℃,可见,高温利于该病原菌的生长,其生长的适宜pH值为6.0~8.0,适宜含水量为60%~75%。因此,在蛹虫草栽培过程中,将温度控制在20 ℃以下,可抑制该病原菌的生长,降低感染率。     

Production of red pigments byMonascus purpureus in solid-state culture

To maximize and sustain the productivity ofMonascus pigments, various environmental and nutritional parameters, such as the initial moisture content, pH, inoculum size, sample size, and nutrient supplement, that influence pigment production were evaluated in solid-state cultures as follows: initial moisture content, 50%; pH, 6.0; inoculum size 1 x 10⁴ spore cells (grams of dry solid substrate)⁻¹; sample size, 300 g. All supplementary nutrients (carbon, nitrogen, and mineral sources) added has inhibitory effects on the cell growth and red pigment production. In open tray culture the maximum biomass yield and specific productivity of red pigments were 223 mg DCW (grams of initial dry substrate)⁻¹ and, 47.6 OD₅₀₀ (DCW grams)⁻¹ h⁻¹, respectively.     

Production of red pigments byMonascus purpureus in submerged culture

For the purpose of mass producingMonascus red pigments optimum medium composition and environmental conditions were investigated in submerged flask cultures. The optimum carbon and nitrogen sources were determined to be 30 g/L of glucose and 1.5 g/L of monosodium glutamate (MSG). Of the three metals examined, Fe²⁺ showed the stronges stimulatory effect on pigment production and some stimulatory effect was also found in Mn²⁺. Optimum pH and agitation speed were determined to be 6.5 and 700 rpm, respectively. Under the optimum culture conditions batch fermentation showed that the maximum biomass yield and specific productivity of red pigments were 0.20 g DCW/g glucose and, 32.5 OD₅₀₀ g DCW⁻¹ h⁻¹, respectively.     

独蒜兰提取物对植物病原真菌的抑菌活性

【目的】研究独蒜兰假鳞茎乙醇提取物对植物病原真菌的抑菌作用,为植物源杀菌剂的开发提供依据。【方法】采用菌丝生长速率法,研究独蒜兰假鳞茎乙醇提取物对15种植物病原菌的抑制活性;以西瓜尖孢镰刀菌作为供试菌,进一步研究该提取物对病原真菌的菌丝干重、细胞膜、过氧化氢酶（CAT）、过氧化物酶（POD）和超氧化物歧化酶（SOD）等的影响。【结果】独蒜兰假鳞茎提取物对辣椒疫霉病菌、西瓜尖孢镰刀菌、番茄灰霉病菌和非洲隐地疫霉菌的抑菌效果明显,其EC₅₀值分别为0.849、0.782、0.813和1.161 mg·mL^-1;经独蒜兰提取物处理后的西瓜尖孢镰刀菌菌丝干重随着药剂浓度的增加而减少;细胞膜丙二醛含量和相对电导率增加;菌丝体细胞内CAT、POD和SOD 3种保护酶活性增加。【结论】独蒜兰假鳞茎提取物对植物病原真菌具有较好的抑菌活性,其抑菌作用可能与其干扰菌丝生长、使菌丝细胞膜正常功能受损等有关。     

Effect of pH on citrinin and red pigments production by Monascus purpureus CCT3802

The production of red pigments and citrinin by Monascus purpureus CCT3802 was investigated in submerged batch cultures performed in two phases: in the first phase, cells were grown on glucose, at pH 4.5, 5.5 or 6.5; after glucose depletion, pH was adjusted, when necessary, to 4.5, 5.5, 6.5, 7.0, 8.0 or 8.5, for a production phase. The highest total red pigments absorbance of 11.3 U was 16 times greater than the lowest absorbance and was achieved with growth at pH 5.5, followed by production at pH 8.5, which causes an immediate reduction of the intra cellular red pigments from 75% to 17% of the total absorbance. The lowest citrinin concentration, 5.5 mg L⁻¹, was verified in the same culture while the highest concentration, 55 mg L⁻¹, was verified in cultures entirely carried out at pH 5.5. An alkaline medium, besides promoting intra cellular red pigments excretion, strongly represses citrinin synthesis.     

Transcriptome analysis and molecular studies on sulfur metabolism in the human pathogenic fungus Paracoccidioides brasiliensis

The dimorphic pathogenic fungus Paracoccidioides brasiliensis can grow as a prototroph for organic sulfur as a mycelial (non-pathogenic) form, but it is unable to assimilate inorganic sulfur as a yeast (pathogenic) form. Temperature and the inability to assimilate inorganic sulfur are the single conditions known to affect P. brasiliensis mycelium-to-yeast (M-Y) dimorphic transition. For a comprehensive evaluation of genes that have their expression modulated during the M-Y transition in different culture media, we performed a large-scale analysis of gene expression using a microarray hybridization approach. The results of the present work demonstrate the use of microarray hybridization analysis to examine gene expression during the M-Y transition in minimal medium and compare these results with the M-Y transition in complete medium. Our results showed that about 95% of the genes in our microarray are mainly responding to the temperature trigger, independently of the media where the M-Y transition took place. As a preliminary step to understand the inorganic sulfur inability in P. brasiliensis yeast form, we decided to characterize the mRNA accumulation of several genes involved in different aspects of both organic and inorganic sulfur assimilation. Our results suggest that although P. brasiliensis cannot use inorganic sulfur as a single sulfur source to initiate both M-Y transition and Y growth, the fungus can somehow use both organic and inorganic pathways during these growth processes.     

Effect of brassicaceous plants on the survival and infectivity of insect pathogenic fungi

Studies were conducted to investigate potentialinteractions between brassicaceous plants, theisothiocyanates they produce and insectpathogenic fungi. Studies in vitro showedthat 100 ppm of 2-phenylethyl isothiocyanatecompletely inhibited growth of Metarhiziumanisopliae and Tolypocladiumcylindrosporum. T. cylindrosporum wassignificantly inhibited by 10 ppm 2-phenylethylisothiocyanate, but not by 1 ppm. M.anisopliae was not inhibited by 10 or 1 ppm2-phenylethyl isothiocyanate. The effect offreshly grated rutabaga on virulence of M.anisopliae to Galleria mellonella larvaewas also tested, resulting in reduced infectionby M. anisopliae. To study the effect ofplants in the Brassicaceae under morefield-like conditions fungi were added to soilwithout plants or at the bases of two differentplant species, Barbarea vulgaris and Eruca vesicaria sativa. Soil was sampled at 5,10 and 15 days to determine the concentrationof colony forming units (CFUs). Treatments withplants did not have fewer CFUs than thecontrol. Instead significantly fewer CFUsresulted when M. anisopliae was added topots with soil only than to pots with plants.There were no significant differences betweenthe two plant species in the number of CFUs atday 5. For days 10 and 15, however,significantly more M. anisopliae CFUswere found in pots containing E. vesicariasativa than B. vulgaris. T.cylindrosporum was less affected by thedifferent treatments than M. anisopliae.Our studies demonstrated that whileisothiocyanates can inhibit insect pathogenicfungi in Petri dishes, when using a morerealistic fungus/plant/soil microcosm no fungalinhibition was found.     

Pathogenicity and specificity of Neozygites tanajoae and Neozygites floridana (Zygomycetes: Entomophthorales) isolates pathogenic to the cassava green mite

The cassava green mite (CGM), Mononychellus tanajoa, a native of South America was accidentally introduced into Africa where it causes serious crop losses. The possibility of introducing classical biological agents from the native home of CGM into Africa was investigated. Thus, we conducted a series of laboratory assays of the native fungal pathogens, Neozygites tanajoae from Brazil and Neozygites floridana from Colombia and Brazil, and compared them with N. tanajoae isolates from Benin. Infectivity of both fungal species, was assayed against the twospotted spider mite, Tetranychus urticae, and against the red mite, Oligonychus gossypii. Pathogenicity against CGM and host range studies were conducted by transferring adult females of each mite species to leaf discs containing sporulated cadavers with a halo of conidia of each fungal isolate. All isolates caused some degree of infectivity to CGM. None of the isolates of N. floridana and N. tanajoae tested were pathogenic to O. gossypii, and only two isolates infected T. urticae. Most isolates from Brazil were highly virulent and infected only CGM. Sixteen N. tanajoae isolates caused more than 89% mortality and more than 62% of the CGM became mummified. A mummified CGM is characteristically a swollen, brown fungus-killed mite that has great potential to produce conidia. However, high mortality was not always associated with high mummification. The median mummification time ranged from 4.4 to 6.7 days. Five Brazilian isolates caused >75% mummification with a median mummification time <5 days. Isolates that cause high mummification in a short period of time would be more likely to cause epizootics and to establish in the new environment. Therefore, these isolates would be the best candidates for introduction to Africa.     

Towards a molecular genetic system for the pathogenic fungus Paracoccidioides brasiliensis

We herein report the development of a molecular toolbox for the dimorphic fungus Paracoccidioides brasiliensis, specifically a more efficient transformation and a gene expression system. We evaluated several parameters that influence Agrobacterium tumefaciens-mediated transformation (ATMT), such as co-cultivation conditions and host cell susceptibility. Our results show that cellular recovery and air drying of A. tumefaciens:P. brasiliensis mixtures are essential for ATMT. Overall, our data indicate a transformation efficiency of 78+/-9 transformants/co-cultivation (5+/-1 transformants/10(6) target cells). P. brasiliensis GFP-expressing isolates were also constructed by insertion of the GFP gene under the control of several fungal promoters. RT-PCR, epifluorescence microscopy and flow cytometry analysis revealed Gfp visualization for all studied promoters but without significant differences in fluorescence and gene expression levels. Moreover, we present evidence for the occurrence of random single gene copy integration per haploid nuclei and the generation of homokaryon progeny, relevant for the future use in targeted mutagenesis and linking mutations to phenotypes.     

Insecticidal properties of Sclerotinia sclerotiorum agglutinin and its interaction with insect tissues and cells

This project studied in detail the insecticidal activity of a fungal lectin from the sclerotes of Sclerotinia sclerotiorum, referred to as S. sclerotiorum agglutinin or SSA. Feeding assays with the pea aphid (Acyrthosiphon pisum) on an artificial diet containing different concentrations of SSA demonstrated a high mortality caused by this fungal lectin with a median insect toxicity value (LC₅₀) of 66 (49–88) μg/ml. In an attempt to unravel the mode of action of SSA the binding and interaction of the lectin with insect tissues and cells were investigated. Histofluorescence studies on sections from aphids fed on an artificial liquid diet containing FITC-labeled SSA, indicated the insect midgut with its brush border zone as the primary target for SSA. In addition, exposure of insect midgut CF-203 cells to 25 μg/ml SSA resulted in a total loss of cell viability, the median cell toxicity value (EC₅₀) being 4.0 (2.4–6.7) μg/ml. Interestingly, cell death was accompanied with DNA fragmentation, but the effect was caspase-3 independent. Analyses using fluorescence confocal microscopy demonstrated that FITC-labeled SSA was not internalized in the insect midgut cells, but bound to the cell surface. Prior incubation of the cells with saponin to achieve a higher cell membrane permeation resulted in an increased internalization of SSA in the insect midgut cells, but no increase in cell toxicity. Furthermore, since the toxicity of SSA for CF-203 cells was significantly reduced when SSA was incubated with GalNAc and asialomucin prior to treatment of the cells, the data of this project provide strong evidence that SSA binds with specific carbohydrate moieties on the cell membrane proteins to start a signaling transduction cascade leading to death of the midgut epithelial cells, which in turn results in insect mortality. The potential use of SSA in insect control is discussed.     

Isariotins G–J from cultures of the Lepidoptera pathogenic fungus Isaria tenuipes

Four new alkaloids isariotins G–J (1–4), together with the known isariotins, were isolated from cultures of the Lepidoptera pathogenic fungus Isaria tenuipes. The structures were elucidated on the basis of NMR spectroscopic and mass spectrometry data. Compounds 1–4 exhibited antimalarial and cytotoxic activities.     

Development of transgenic sorghum for insect resistance against the spotted stem borer (Chilo partellus)

Transgenic sorghum plants expressing a synthetic cry1Ac gene from Bacillus thuringiensis (Bt) under the control of a wound-inducible promoter from the maize protease inhibitor gene (mpiC1) were produced via particle bombardment of shoot apices. Plants were regenerated from the transformed shoot apices via direct somatic embryogenesis with an intermittent three-step selection strategy using the herbicide Basta. Molecular characterisation based on polymerase chain reaction and Southern blot analysis revealed multiple insertions of the cry1Ac gene in five plants from three independent transformation events. Inheritance and expression of the Bt gene was confirmed in T₁ plants. Enzyme-linked immunosorbant assay indicated that Cry1Ac protein accumulated at levels of 1–8 ng per gram of fresh tissue in leaves that were mechanically wounded. Transgenic sorghum plants were evaluated for resistance against the spotted stem borer (Chilo partellus Swinhoe) in insect bioassays, which indicated partial resistance to damage by the neonate larvae of the spotted stem borer. Reduction in leaf damage 5 days after infestation was up to 60%; larval mortality was 40%, with the surviving larvae showing a 36% reduction in weight over those fed on control plants. Despite the low levels of expression of Bt -endotoxin under the control of the wound-inducible promoter, the transgenic plants showed partial tolerance against first instar larvae of the spotted stem borer.     

Fruit-body production of an ectomycorrhizal fungus in genus <Emphasis Type="Italic">Boletus</Emphasis> in pure culture

Mycelial growth and fruit-body production of an ectomycorrhizal Boletus sp. were examined in pure culture. Mycelia of the strain Bo1 grew well on a medium consisting of sawdust and barley grains. Mature fruit bodies bearing basidiospores were produced after incubation at 22°C for 90 days in the dark, followed by incubation at 26°C for 30–46 days under conditions of high humidity and illumination. The addition of porous stone as a casing on the medium increased fruit-body yield. Deposited spores germinated well on an agar medium and formed mycelial colonies, thus completing the life cycle of Bo1 without a host plant and under axenic conditions. The ability of Bo1 to form ectomycorrhizas was confirmed by axenic resynthesis of mycorrhizas on Quercus serrata. Cultured fruit bodies of Bo1 resembled Gyroporus castaneus and Boletus subcinnamomeus, but its taxonomic position was not elucidated at the species level.     

Optimization of Process Parameters for the Production of Inulinase from a Newly Isolated Aspergillus niger AUP19

Summary Fifty strains were isolated from different soil samples on synthetic medium containing inulin as a sole carbon source for the production of extracellular inulinase. Of them, five isolates showed high inulinase activity and one of them was selected for identification and medium optimization studies. The isolate was identified as Aspergillus niger. Various physical and chemical parameters were optimized for inulinase production. Maximum productivity of inulinase (176 U ml⁻¹) was achieved by employing medium containing 5% (w/v) inulin, galactose as additional carbon source, corn steep liquor and (NH₄)H₂PO₄ as nitrogen sources, incubation period of 72 h, incubation temperature of 28 °C, pH 6.5, inoculum load at 10% (v/v) level and medium volume to flask volume ratio of 1:20 (v/v) with indented flasks.     

Intraspecific heritable variation in life-history traits can alter the outcome of interspecific competition among insect herbivores

Competition is one of the most important biotic factors determining the structure of ecological communities. In this study, we show that there is variation in competitive ability between two clones of the pea aphid, Acyrthosiphon pisum, both of which out-compete a clone of the vetch aphid, Megoura viciae, in the laboratory. We tested whether this variation in competitive ability would alter the outcome of interspecific competition in the field. While one pea aphid clone followed the pattern set in the laboratory, out-competing the Megoura viciae clone, another showed the reverse effect with Megoura viciae dominating. These differences appear to be the result of variation in early population growth rate between the pea aphid clones, rather than predation, although predation did lead to the eventual extinction of colonies. We also questioned whether intra- and interspecific differences in predator escape behaviour could affect the outcome of competition in the field. All three clones responded similarly to the presence of foraging hoverfly larvae (Episyrphus balteatus), but the Megoura viciae clone dropped from the plant significantly less often in response to the presence of a foraging two-spot ladybird (Adalia bipunctata). This work provides evidence that intraspecific variation in competitive ability can alter the outcome of interspecific competitive interactions in nature and suggests that species–specific behavioural traits may have the potential to modify the outcome of these interactions.     

Differential resistance and the importance of antibiotic production in Acromyrmex echinatior leaf-cutting ant castes towards the entomopathogenic fungus Aspergillus nomius

Paired exocrine metapleural glands are present in almost all ants and produce compounds with antibiotic properties towards a variety of pathogenic fungi and bacteria. In Acromyrmex leaf-cutting ants, small workers have relatively large metapleural glands compared to large workers, and thus harbour approximately half the number of gland cells of large workers, despite being only one-fifteenth their body mass. Here we present results showing that when the two worker castes of A. echinatior are treated with spores of the pathogenic fungus Aspergillus nomius in doses that correspond to the difference in metapleural gland cell numbers they do not differ in survival. However, we also show, for the first time, that small workers survive significantly longer than large workers when both are challenged with a dose of spores that corresponds to their difference in body mass. Furthermore, the time until Aspergillus nomius hyphae and spores appear on the cadavers of workers dead from infection, is significantly increased in the small worker caste. In addition to supporting previous findings that the metapleural glands have an important defence function, the results of this study indicate that the relatively large glands in small workers makes this caste particularly well adapted to preventing pathogenic microorganisms from entering the colony. Received 23 January 2006; revised 7 April 2006; accepted 11 April 2006.     

Teleomorph-anamorph relationships and reclassification of <Emphasis Type="Italic">Cordyceps cuboidea</Emphasis> and its allied species

Based on morphological characteristics and molecular phylogeny, we reclassified Cordyceps cuboidea and allied species C. alboperitheciata, C. prolifica, and Ophiocordyceps ryogamiensis. We investigated their teleomorph-anamorph relationships and revealed that these four species have Hirsutella-like anamorphs with morphological differences between them. By analyzing their molecular phylogeny, inferred from DNA sequences of internal transcribed spacer (ITS) and large subunit (LSU) D1/D2 region of rDNA, they were separated into four close-knit clades. Although C. prolifica and O. ryogamiensis formed their own clades, isolates of C. cuboidea separated into two clades, i.e., a true C. cuboidea clade and one resembling a new species, the O. paracuboidea clade. The latter two species are distinguished by the fruiting region of the stroma. In addition, C. alboperitheciata is regarded as a synonym of C. cuboidea. From the morphology, teleomorph-anamorph relationships, and molecular phylogeny, we concluded these species should be assigned to the genus Ophiocordyceps.