双孢蘑菇上的丝枝霉新种

1980—1981年先后自北京、昆明、福州等地双孢蘑菇[Agaricus bisporus (Lange)Sing.]上分离到一种真菌,在PDA上的培养性状与蘑菇轮枝菌(Vertieillium psalliotae Tresch．)和蛛网丝枝霉中国变种[Aphanocladium aranearum(Petch) Gams var sincase J．D．Chen]近 似。镜检可见其兼具上述两种菌的形态。经反复多次纯化,并挑取单孢子培养,肯定了这种真菌是纯菌。在培养基上已保存4年,形态稳定。它具典型蘑菇轮枝菌形态,而其丝枝霉型瓶梗的形成和瘪缩时间、长度、原瓶梗数量、末端瓶梗数及排列密度等,都与蛛网丝枝霉中国变种有一定差异,与丝枝霉属其它种亦不同,故定为两型丝枝霉新种(Aphanocladium dimorphum sp-nov.)。本文详细描述了该新种的形态,井对文甲所提的五种真菌及其与Engyodontium属间的关系进行了讨论。     

寄生双孢蘑菇的蛛网丝枝霉中国变种

1980—1981年自北京、福州、三明、泉州、漳州、厦门和昆明等地双孢蘑菇[Agaricusbisporus(Lange)Sing]病菇上和不出蘑菇的培养料内,分离到一种真菌。在 PDA 培养基上的培养性状,肉眼观察与蘑菇轮枝菌(Verticillium psalliotae Tresch.)极难区别。根据其分生孢子和瓶梗的形态及其在匐匍气丝上的排列情况等,鉴定为珠网丝枝霉。因具短棒状原瓶梗,认为是一新变种,定名为珠网丝枝霉中国变种 Aphanocladium aranearum(Petch)W.Gams var.sinense J.D.Chen var.nov.它是双孢蘑菇的重要害菌之一。这个“属”和“种”都是我国的新记录。在猴头(Hericium erinaceus)、香菇(Lentinus edo-des),糙皮侧耳(Pleurotus ostreatus)上和水稻土中也有少量分布。     

Insecticidal and insect growth regulatory activities of secondary metabolites from entomopathogenic fungi,Lecanicillium attenuatum

Insect growth regulators (IGRs) are effective alternatives to chemical insecticides because of their specificity and low environmental toxicity. Entomopathogenic fungi are an important natural pathogen of insects and have been developed as biological control agents. They produce a wide range of secondary metabolites such as antibiotics, pesticides, growth-promoting or inhibiting compounds and insect attracting agents. In this study, to explore novel IGR substances from entomopathogenic fungi, culture extracts of 189 entomopathogenic fungi isolated from Korean soil samples were investigated for their juvenile hormone (JH)-based IGR activities. Whereas none of the culture extracts exhibited JH agonist (JHA) activity, 14 extracts showed high levels of JH antagonist (JHAN) activity. Among them, culture extract of JEF-145 strain, which was identified as Lecanicillium attenuatum, showed the highest insecticidal against Aedes albopictus and Plutella xylostella. At liquid culture condition, JHAN activity was observed in culture soup rather than mycelial cake, indicating that substances with JHAN activity are released from the JEF-145 strain during culture. Furthermore, while extract from solid cultured JEF-145 strain showed insecticidal activities against both A. albopictus and P. xylostella, that from liquid cultured fungi showed insecticidal activity only against A. albopictus, indicating that L. attenuatum JEF-145 strain produces different kinds of secondary metabolites with JHAN activity depending on culture conditions. These results suggested that JHAN substances derived from entomopathogenic fungi could be usefully exploited to develop novel eco-friendly IGR insecticides.     

一株侵染豌豆蚜的昆虫病原真菌的分离及鉴定

【目的】从采集到的自然染菌的豌豆蚜Acyrthosiphon pisum虫尸分离纯化得到一株病原真菌,定名为TF-2。本研究旨在确定该菌株的分类地位,为豌豆蚜生物防治提供真菌资源。【方法】对自然染菌的豌豆蚜虫尸上寄生真菌TF-2进行回接试验,分离纯化出致病菌株TF-2;在显微镜下配制TF-2菌株不同浓度孢子悬浮液,采用浸渍法和离体叶片饲养法测定其对豌豆蚜成虫的毒力;利用光学显微镜观察菌株形态学特征。PCR扩增TF-2的rDNA-ITS序列并测序,构建系统发育树对TF-2菌株进行分子鉴定。【结果】毒力测定结果表明,TF-2菌株对豌豆蚜成虫表现出很强的致病力,1×10^7孢子/mL处理6 d后豌豆蚜成虫校正死亡率达到100%。TF-2在PDA培养基上菌落呈圆形,白色或淡黄色毡状,菌落背面呈奶油色;菌株孢梗呈瓶状,在菌丝上单生或侧生2~3个,大小为(19-42)μm×(1.1-2.5)μm,基部较粗至尖端逐渐变细,分生孢子长椭圆形,大小为(4.2-11.8)μm×(1.6-2.6)μm。菌丝体产生晶体呈八面体。该菌株的rDNA-ITS序列与长孢蜡蚧菌Lecanicillium longisporum(GenBank登录号:KX426564)的rDNA-ITS核苷酸序列一致性达99%,位于系统发育树的同一分支。【结论】菌株TF-2被鉴定为豌豆蚜的病原真菌长孢蜡蚧菌L.longisporum,对豌豆蚜的生物防治具有潜在的应用价值。     

High production of cold-tolerant chitinases on shrimp wastes in bench-top bioreactor by the Antarctic fungus Lecanicillium muscarium CCFEE 5003: Bioprocess optimization and characterization of two main enzymes

The Antarctic fungus Lecanicillium muscarium CCFEE-5003 was preliminary cultivated in shaken flasks to check its chitinase production on rough shrimp and crab wastes. Production on shrimp shells was much higher than that on crab shells (104.6 ± 9.3 and 48.6 ± 3.1 U/L, respectively). For possible industrial applications, bioprocess optimization was studied on shrimp shells in bioreactor using RSM to state best conditions of pH and substrate concentration. Optimization improved the production by 137% (243.6 ± 17.3). Two chitinolytic enzymes (CHI1 and CHI2) were purified and characterized. CHI1 (MW ca. 61 kDa) showed optima at pH 5.5 and 45 °C while CHI2 (MW ca. 25 kDa) optima were at pH 4.5 and 40 °C. Both enzymes maintained high activity levels at 5 °C and were inhibited by Fe⁺⁺, Hg⁺⁺ and Cu⁺⁺. CHI2 was strongly allosamidin-sensitive. Both proteins were N-acetyl-hexosaminidases (E.C. 3.2.1.52) but showed different roles in chitin hydrolysis: CHI1 could be defined as “chitobiase” while CHI2 revealed a main “eso-chitinase” activity.     

Entomogenous fungi of arboreal Coleoptera from Worcestershire,England, including the new species <Emphasis Type="Italic">Harposporium bredonense</Emphasis>

During entomological surveys in ancient woodland and orchards in the English county of Worcestershire, dead arboreal beetles were found exhibiting external evidence of primary colonisation by anamorphic fungi belonging to the Clavicipitales. One of these proved to be an undescribed species and a new taxon is proposed: Harposporium bredonense on the larva of a longhorn beetle, Leiopus nebulosus (Cerambycidae), with a Hirsutella synanamorph. Hirsutella guignardii on the adult histerid beetle, Paromalus flavicornis, is a new host record, as well as a new fungal record for the British Isles. In addition, Lecanicillium psalliotae and Paecilomyces farinosus were isolated from mummified ecloded pupae of the Noble Chafer, Aleurostictus nobilis (Scarabaeidae), which is recognised as a rare or endangered species in Britain.     

The relationship between conidial dose,moulting and insect developmental stage on the susceptibility of cotton aphid,Aphis gossypii,to conidia of Lecanicillium attenuatum,an entomopathogenic fungus

The cotton aphid is one of the most serious pests of greenhouse vegetable crops worldwide. It is difficult to control because field populations usually include simultaneously several insect developmental stages. The current research evaluated an isolate (CS625) of Lecanicillium attenuatum, a fungal pathogen of aphids, as to its virulence against different developmental stages of cotton aphid, Aphis gossypii. The influence on mortality of several other factors also was examined: (a) insect moulting, (b) the number of conidia attached to insect cuticles and (c) germination rates of conidia on cuticles of aphids at various developmental stages. Mortality of cotton aphids treated with L. attenuatum conidia varied according to the developmental stage of the host, i.e. the LT_50s with third-instar nymphs and adults was shorter than with first-instar nymphs. The number of spores attached to the surface of first-instar nymphs was approximately one-half of that on third-instar nymphs and adults. Also, the level of spore germination on the surface of first-instar nymphs was lower than on the surface of other stages of the aphid. After moulting, the numbers of conidia attached to new insect cuticles were less than on exuviae. These results suggest that early nymphal stages of cotton aphids may escape fungal disease due, at least in part, to a combination of three factors: low numbers of conidia attached to their cuticles; low levels of conidial germination and rapid ecdyses, which removed conidia before their germ tubes penetrated the host hemolymph.     

Histopathological changes of Ceroplastes japonicus infected by Lecanicillium lecanii

The infection process and pathological changes of Japanese wax scale, Ceroplastes japonicus Green, by the hyphomycete Lecanicillium lecanii (Zimmermann) Gams & Zare were investigated by light, scanning and transmission electron microscopy. The results showed that L. lecanii generally infected the wax scale by penetrating the integument. The anal area, the body margin, around the base of mouthparts and legs, over the stigmatic furrow and the area around the vulva were susceptible places, while the wax test had an inhibitory effect on L. lecanii. Within 24 h after inoculation, conidia became attached to the cuticle, and within 48 h, hyphae adhered to the integument of the scale and their tips differentiated into specialized infection pegs. Penetration of the cuticle occurred within 72 h of inoculation; the fungus caused the insect cuticle to rupture and hyphae entered the insect body through these openings. Within 72 h after inoculation, L. lecanii entered the hemocoele of the scale and formed blastospores. After 96 h, blastospores were dispersed throughout the hemolymph and completely disrupted the hemocytes, resulting in damage of the cell nucleus and agglutination of chromatin. Concomitant to colonization of the hemolymph, the internal organs and tissues, e.g., tracheae, malpighian tubules and muscle fibers, were also infected. As the infection progressed, the wax test and body changed color from white and red, respectively, to yellowish. After 144 h, the internal tissue structure was totally compromised and the insects died. After this time, new conidiophores bearing conidia were produced on the surface of the cadavers.     

Taxonomic status of putative Verticillum alboatrum isolates

Two fungal isolates, formerly classified as Verticillium alboatrum and proposed as forming the basis of a new sub-group (`Group 2') within the species, have been shown to be non-pathogenic to known hosts of V. alboatrum and, on the basis of molecular evidence, to be closely related to Verticillium psalliotae and Verticillium fungicola. We propose that the taxon V. alboatrum be confined to those closely related isolates, usually plant pathogenic and usually producing dark resting mycelium, referred to by other authors as Group 1. The only sub-specific groupings which appear valid (based on pathological and molecular evidence) comprise: (1) host-adapted isolates from lucerne; and (2) all other isolates.     

Responses of entomopathogenic fungi to the mutagen 4-nitroquinoline 1-oxide

Survival of entomopathogenic fungi under solar ultraviolet (UV) radiation is paramount to the success of biological control of insect pests and disease vectors. The mutagenic compound 4-nitroquinoline 1-oxide (4-NQO) is often used to mimic the biological effects of UV radiation on organisms. Therefore, we asked whether tolerance to 4-NQO could predict tolerance to UV radiation in thirty isolates of entomopathogenic fungi and one isolate of a xerophilic fungus. A dendrogram obtained from cluster analyses based on the 50 and 90 % inhibitory concentrations (IC₅₀ and IC₉₀, respectively) divided the fungal isolates into six clusters numbered consecutively based on their tolerance to 4-NQO. Cluster 6 contained species with highest tolerance to 4-NQO (IC₅₀ > 4.7 μM), including Mariannaea pruinosa, Lecanicillium aphanocladii, and Torrubiella homopterorum. Cluster 1 contained species least tolerant to 4-NQO (IC₅₀ < 0.2 μM), such as Metarhizium acridum (ARSEF 324), Tolypocladium geodes, and Metarhizium brunneum (ARSEF 7711). With few exceptions, the majority of Metarhizium species showed moderate to low tolerances (IC₅₀ between 0.4 and 0.9 μM) and were placed in cluster 2. Cluster 3 included species with moderate tolerance (IC₅₀ between 1.0 and 1.2 μM). In cluster 4 were species with moderate to high tolerance (IC₅₀ between 1.3 and 1.6 μM). Cluster 5 contained the species with high tolerance (IC₅₀ between 1.9 and 4.0 μM). The most UV tolerant isolate of M. acridum, ARSEF 324, was the least tolerant to 4-NQO. Also, L. aphanocladii, which is very susceptible to UV radiation, showed high tolerance to 4-NQO. Our results indicate that tolerance to 4-NQO does not correlate with tolerance to UV radiation. Therefore this chemical compound is not a predictor of UV tolerance in entomopathogenic fungi.