重要虫生真菌莱氏野村菌芽生孢子的形态发生

经对比,萨氏麦芽糖-酵母浸粉培养液(SMY)较适合制备莱氏野村菌Nomuraea rileyi芽生孢子。以菌株Nr09接种该培养基,在130r/min、25℃全光照下震荡培养,观察芽生孢子在不同时期发育的形态变化。结果表明,分生孢子萌发产生芽管;24h萌发率为42%,36h时达84%。芽管迅速伸长形成菌丝。在48h前芽生孢子通过菌丝顶端及分枝末端缢缩的方式形成,后期则主要通过芽生孢子继续出芽的方式大量形成,78h芽生孢子数量达到最大值。芽生孢子的形成方式可为一端、两端或多端芽殖。芽生孢子的形成过程可分为5个阶段:(I)分生孢子膨大期;(II)芽管萌发期;(III)芽管延长期;(IV)芽生孢子形成初期;(V)芽生孢子指数生长期。30h后培养液中有少量草酸钙结晶出现并逐渐增多。到84h时有31%的芽生孢子细胞内液泡聚集增大,表明芽生孢子已开始进入衰老阶段。使用指数生长期制备的Nr09芽生孢子进行几丁质酶基因转化,转化效率达79个转化子/μgDNA。     

西花蓟马高毒力虫生真菌筛选及其生防应用潜力的研究进展

西花蓟马是一种外来入侵的世界性害虫,对农林业危害巨大。查阅国内外相关文献,综述了当前防治西花蓟马的虫生真菌的种类、高毒力菌株的筛选及防治现状。现已知西花蓟马的寄生病原真菌有5种,包括蜡蚧轮枝菌(半知菌:丝孢目)、球孢白僵菌(半知菌:丝孢目)、金龟子绿僵菌(半知菌:丝孢目)、玫烟色棒束孢(半知菌:束梗孢目)和小孢新接霉。其中,球孢白僵菌、金龟子绿僵菌在西花蓟马的生物防治中应用最广,具有良好的开发应用潜力,部分防效好的虫生真菌已申请专利及实现工厂化生产。     

几种虫生真菌附着胞的荧光显微及扫描电镜观察

本研究通过对细脚拟青霉、蝉拟青霉、玫烟色拟青霉、金龟子绿僵菌和莱氏野村菌在疏水表面产生附着胞的荧光显微镜和扫描电镜电镜观察,明确五种虫生真菌均可产生附着胞,细脚拟青霉和金龟子绿僵菌产生单附着胞和复合附着胞两种形态,均呈椭圆至长椭圆形,如遇到不合适侵染的部位,则重新产生芽管向前延伸直至找到适合入侵的部位,蝉拟青霉分生孢子多在顶端恨芽成人字形,末端椭圆形附着胞,该拟青霉再生附着胞能力强。金龟子绿僵菌     

几种虫生真菌附着胞的荧光显微及扫描电镜观察

本研究通过对细脚拟青霉、蝉拟青霉、玫烟色拟青霉、金龟子绿僵菌和莱氏野村菌在疏水表面产生附着胞的荧光显微镜观察和扫描电镜观察,明确五种虫生真菌均可产生附着胞,细脚拟青霉和金龟子绿僵菌产生单附着胞和复合附着胞两种形态,均呈椭圆至长椭圆形,如遇到不适合浸染的部位,则重新产生芽管向前延伸直至找到适合入侵的部位。蝉拟青霉分生孢子多在顶端发芽成人字形,末端形成椭圆形附着胞,该拟青霉再生附着胞能力强。金龟子绿僵菌和莱氏野村菌附着胞表面有较厚的粘液层,而三种拟青霉附着胞表面均未见有粘液层。     

虫生真菌深层培养分生孢子的喷雾干燥技术

虫生真菌制剂田间应用的一个关键问题是液体深层培养中产生的分生孢子（下简称液生孢子）或芽孢子的稳定性。最近,Ｓｔｅｐｈｅｎ等（见Ｂｉｏｃｏｎｔｒｏｌ　Ｓｃｉｅｎｃｅ　ａｎｄＴｅｃｈｎｏｌｅｇｙ,１９９８,８：３－１１）发展了一种喷雾干燥技术。采用这一技术可使芽抱子和／或液生抱子干燥,又不显著降低孢子的活力。这项研究是在实验室用小型喷雾干燥器进行的。１保护剂的筛选测定了６种保护剂对金龟子绿僵菌Ｍｅｔａｒ－ｈｉｚｉｕｍ　ａｎｉｓｏｐｌｉａｅ　（Ｍｅｔｓｃｈｎｉｋｏｖ）ＳｏｒｏｋｉｎＭａ９７株的存活力的…     

爪哇棒束孢侵染美国白蛾过程及抗氧化酶活性变化

[目的]爪哇棒束孢Isaria javanica是一种重要的虫生真菌,已广泛应用于多种害虫的防治.本研究从美国白蛾Hyphantria cunea僵虫尸体中分离出对美国白蛾具有致病作用的爪哇棒束孢BE01菌株,为了解该菌株对美国白蛾的侵染过程,并通过研究宿主体内抗氧化酶系对爪哇棒束孢BE01菌株入侵的响应,评价该菌的杀虫作用,为爪哇棒束孢BE01防治美国白蛾提供依据.[方法]采用扫描电镜技术,观察爪哇棒束孢BE01分生孢子侵染的美国白蛾3龄幼虫,并测定美国白蛾接菌后体内超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)含量的变化.[结果]爪哇棒束孢对美国白蛾侵染过程包括:分生孢子粘附在美国白蛾的角质层上,接种后6h孢子开始萌发,24h产生附着胞,48 h菌丝在体表继续生长,96h菌丝从虫体内长出并产分身孢子,120 h菌丝覆盖整个虫体.在受到爪哇棒束孢BE01侵染后,美国白蛾幼虫体内的3种抗氧化酶SOD、POD、CAT酶活性明显升高,并在侵染48 h时达到峰值.随着侵染时间的增加,48 h后3种酶的活性开始下降, 60 h后酶活性均低于对照组.[结论]爪哇棒束孢BE01孢子活力高、萌发速率快,导致美国白蛾体内的保护酶系难以发挥保护作用.爪哇棒束孢BE01菌株能够快速侵染美国白蛾幼虫,具有开发成新的高效生物防治菌株的潜能.     

蝉棒束孢显微形态变异式样

【目的】揭示蝉棒束孢居群形态性状的变异式样。【方法】通过对15个居群,75个蝉棒束孢菌株17个显微形态性状的观察和测量,采用SAS 8.1软件对蝉棒束孢显微形态性状数据进行描述性、巢式方差和Q聚类分析,获得蝉棒束孢显微形态变异数据。【结果】蝉棒束孢具有大小两型分生孢子,产孢细胞的形态可分为葫芦状和瓶状2种类型;蝉棒束孢在PDA培养基中易形成数量众多的厚垣孢子,菌丝间多能形成融合菌丝,并可观察到少量的融合菌丝在厚垣孢子与菌丝间、产孢细胞间形成。描述性分析表明,蝉棒束孢居群17个形态性状的变异系数(CV)在13.07%–104.09%之间,在物种水平上表现出丰富的变异;巢式方差分析表明,蝉棒束孢显微形态性状在居群间平均方差分量占总变异的11.29%,居群内个体间平均方差分量占总变异的15.27%,菌株内观察值间平均方差分量占总变异的73.44%。【结论】蝉棒束孢菌株形态性状变异丰富,是形态性状变异的主要来源,蝉棒束孢居群显微形态性状不存在明显的地理相关性。     

采用EF-1α序列及气生菌丝上的孢子形态特征对九州镰孢菌、拟枝孢镰孢菌和厚垣镰孢菌的鉴定

对九州镰孢菌Fusarium kyushuense、厚垣镰孢菌F. chlamydosporum 和拟枝孢镰孢菌F. sporotrichioides在气生菌丝上产生的孢子进行了比较。九州镰孢菌在气生菌丝上产生多隔孢子（即中型分生孢子）;厚垣镰孢菌在气生菌丝上产生的主要是0隔针叶状分生孢子;拟枝孢镰孢菌在气生菌丝上产生两种类型的分生孢子：芜菁形、单胞分生孢子以及椭圆形、0－1隔的分生孢子。多隔的气生孢子（中型分生孢子）在厚垣镰孢菌和拟枝孢镰孢菌这两个种中偶尔可以观察到,但是不应作为对这两个种进行鉴定的主要     

绿僵菌属的三个新种

作者从广东、贵州、北京等地收集的虫生真菌中,发现在我国具有绿僵菌属特征的真菌可分为四个种。根据菌落颜色、分生孢子团块大小、紧密和牢固程度、孢子链连接方式、分生孢子形状等,将我们收集的属于本属的菌株分为四个种。本文报道绿僵菌属三个新种的描述。金龟子绿僵菌Metarhizium anisopliae(Metsch.)Sorokin为最常见的甘蔗金龟子致病菌,是本属的模式种。平沙绿僵菌Metarhizium pingshaense Chen et Guo sp.nov.、柱孢绿僵菌Metarhizium cylindrosporae Chen et Guo sp.nov.和贵州绿僵菌Metarhizium guizhouense Chen et Guo sp.nov.是三个新种。     

糖类对冬虫夏草菌Ophiocordyceps sinensis芽生孢子产量及毒力的影响

冬虫夏草Chinese cordyceps是食药两用的传统名贵资源。其人工培养需要冬虫夏草菌Ophiocordyceps sinensis的优质芽生孢子。为获得高产量及高感染力芽生孢子,本研究测定不同糖类培养基对冬虫夏草菌芽生孢子产量及毒力（对小金蝠蛾Thitarodes xiaojinensis幼虫的存活率、携菌率和僵虫率）的影响。两个冬虫夏草菌株于含6种糖（葡萄糖、果糖、阿拉伯糖、麦芽糖、蔗糖和海藻糖）的液体培养基中分别培养30、45和60 d,计数芽生孢子产量并将收集的孢子注射感染两个品系的小金蝠蛾6龄幼虫,置于不同温度下观察幼虫的存活率、携菌率及僵虫率。结果显示：不同糖源培养基中芽生孢子的产量差异显著,其中含麦芽糖培养基中芽生孢子产量最高;注射菌株和幼虫品系对被注射幼虫的存活率无显著差异;不同糖源培养基获得的芽生孢子对蝠蛾幼虫的存活率无显著差异,对携菌率和僵虫率产生影响;以含麦芽糖为糖源的培养基、芽生孢子培养时间显著影响被注射幼虫的存活率与僵虫率,其中培养30 d的冬虫夏草菌所注射的幼虫的存活率、僵虫率显著高于60 d的;温度也显著影响被注射幼虫的存活率、僵虫率,10℃的幼虫存活率和僵虫率显著高于14℃、18℃的。研究结果为培养优质冬虫夏草芽生孢子,提高被侵染蝙蝠蛾幼虫僵虫率提供了参考。     

Drying and formulation of blastospores of Paecilomyces fumosoroseus (Hyphomycetes) produced in two different liquid media

Formulation matrices can play an important role in improving the storage survival and biocontrol efficacy of microorganisms used for the control of pest insects. In this study, liquid culture-produced blastospores of the entomopathogenic fungus Paecilomyces fumosoroseus were formulated with different inert and organic materials prior to air-drying. Paecilomyces fumosoroseus blastospores were produced in two different liquid media, a basal salts medium supplemented with Casamino acids and glucose (LM1) and a medium containing peptone of collagen and glucose (LM2). Blastospores produced in the two test media were formulated with various supports. The formulation supports were cornstarch, rice flour, talc powders, Mexican lime, calcined kaolin clay, and diatomaceous earth. Several of the supports were tested at different concentrations. The initial and long-term (after storage at 4 and 28 °C) survival of the formulated, air-dried blastospores were evaluated. Initial blastospore viabilities were affected by the formulation material and by the blastospore production medium. Medium composition, drying support and storage temperature had an impact on the long-term survival of the blastospores. Under the conditions of the study, LM1 produced higher concentrations of blastospores that not only survived drying better than blastospores produced in LM2 but also maintained viability longer during storage in the formulation supports tested. The nature of the drying supports was shown to have a significant impact on the storage stability of all blastospores, particularly those produced in LM1. Under the production, drying and storage conditions used in the study, calcined kaolin clay formulations stored at 4 °C had the best storage stability. In all formulations tested, spore survival over time was reduced for blastospore formulations stored at 28 °C rather than 4 °C.     

Modified Adamek's medium renders high yields of Metarhizium robertsii blastospores that are desiccation tolerant and infective to cattle-tick larvae

Blastospores are yeast-like cells produced by entomopathogenic fungi that are infective to arthropods. The economical feasible production of blastospores of the insect killing fungus Metarhizium spp. must be optimized to increase yields. Moreover, stabilization process is imperative for blastospore formulation as a final product. In this sense, our goal was to increase blastospore production of two Metarhizium isolates (ESALQ1426 and ESALQ4676) in submerged liquid cultures. A modified Adamek's medium was supplemented with increased glucose concentrations and the fermentation time was accelerated by using a blastospore pre-culture as inoculum. Virulence of air-dried stable blastospores was compared with conidia toward larvae of the cattle tick, Rhipicephalus microplus. Our results revealed that blastospore production of Metarhizium is isolate- and species-dependent. Glucose-enriched cultures (140 g glucose/L) inoculated with pre-cultures improved yields with optimal growth conditions attained for Metarhizium robertsii ESALQ1426 that rendered as high as 5.9 × 10⁸ blastospores/mL within 2 d. Resultant air-dried blastospores of ESALQ1426 were firstly proved to infect and quickly kill cattle tick larvae with comparable efficiency to conidia. Altogether, we argue that both osmotic pressure, induced by high glucose titers, and isolate selection are critical to produce high yields of blastospores that hold promise to control cattle-tick larvae.     

Development of the entomopathogenic fungus Beauveria bassiana in liquid cultures

Growth and development of B. bassiana was followed in four liquid media: peptone, peptone-glucose, glucose and glucose-peptone-yeast extract. Six developmental stages were defined: (I) the unswollen conidium, (II) the swollen conidium, (III) emergence of the germ tube, (IV) elongation of the germ tube and formation of the first septum, (V) polar and bipolar elongation (growth) of the resulting mycelium and initiation of a blastospore and, (VI) seccession of that blastospore. Conidia of B. bassiana produced germ tubes in all liquid media. Blastospores were produced in all liquid media except glucose. In peptone-glucose, the yield of blastospores was four-fold higher than in glucose-peptone-yeast extract. However, biomass production was highest in peptone-glucose-yeast extract.     

耐低水活度高毒力虫生真菌菌株选育*

以球孢白僵菌Ｂｅａｕｖｅｒｉａ　 ｂａｓｓｉａｎａ和玫烟色拟青霉Ｐａｅｃｉｌｏｍｙｃｅｓ　ｆｕｍｏｓｏｒｏｓｅｕｓ为研究对象,利用紫外线诱变芽生孢子和低水活度条件胁迫筛选,选育出突变株Ｂｂ０７２４０、Ｐｆ０１１２０和Ｐｆ０１１６０,它们在低水活度（或相对湿度）下的生长和萌发均明显优于其相应的初始菌株。生物测定表明,突变株对桃蚜的毒力明显高于其初始菌株。     

Dissolved oxygen levels affect dimorphic growth by the entomopathogenic fungus Isaria fumosorosea

The entomopathogenic fungus Isaria fumosorosea is capable of dimorphic growth (hyphal or yeast-like) in submerged culture. Using 250-mL baffled flasks, culture volumes of 50, 100, 150, and 200 mL were grown in a shaker incubator at 350 rpm and 28°C. Dissolved oxygen (DO) was continuously monitored using a non-invasive oxygen monitoring system. Culture volumes of 50 mL maintained DO concentrations above 10% throughout the 3-day growth period and accumulated biomass and produced blastospores more rapidly (1.2×10⁹ blastospores mL^?1 in 2 days) than the other culture volumes tested. Dissolved oxygen was depleted in culture volumes of 100, 150, and 200 mL after 20.5, 16.8, and 13.5 h, respectively. The DO in 150 and 200 mL cultures remained exhausted (<3%) throughout the growth period resulting in significantly lower blastospore yields and increased hyphal growth. These results were used to establish oxygen levels (>20% DO) for I. fumosorosea growth in 100-L bioreactors resulting in blastospore production (1.1×10⁹ blastospores mL^?1 in 2 days) comparable to highly aerated, low volume shake flask cultures. In addition, maintaining higher DO levels resulted in increased blastospore production by cultures of I. fumosorosea grown on low-cost nitrogen sources (cottonseed meal and soy flour) that previously elicited excessive hyphal growth. These studies showed that oxygen availability is essential for significant yeast-like growth by I. fumosorosea cultures and that continuous monitoring of oxygen concentrations in shake flask cultures can be used to establish aeration conditions for bioreactors.     

碳源对玫烟色虫草菌丝生长、芽生孢子产生及其耐热性的影响

提高虫生真菌孢子应对热胁迫的能力是生防菌应用研究的关键,为研究菌丝培养阶段碳源对玫烟色虫草Cordyceps fumosorosea IF-1106耐热性的影响,选择了麦芽糖、可溶性淀粉、蔗糖、葡萄糖、果糖、海藻糖为碳源的培养基对玫烟色虫草IF-1106进行液体培养,评估了不同碳源条件下菌丝的生长、产孢及所产芽生孢子的耐热性。结果表明,在菌株培养阶段,培养基中碳源的种类及浓度对菌丝产量、产孢量及所产芽生孢子的耐热性有显著影响,其中蔗糖为碳源时,所产芽生孢子的耐热性强,45 ℃热胁迫条件下LT₅₀为1.65 h;蔗糖浓度为40 g/L时,可产生大量耐热芽生孢子,液体培养3 d后产孢量可达3.43×10⁷个孢子/mL。为探索不同培养条件下所产芽生孢子耐热性不同的原因,提取了孢子内的海藻糖并采用离子色谱法对其进行了定量分析,发现耐热性高的芽生孢子胞内海藻糖含量普遍较低,可见海藻糖是与芽生孢子耐热性密切相关的内源物质。综上所述,选择适宜的培养基是调控孢子耐热性的有效途径,本研究为生产高耐热的玫烟色虫草生防制剂提供了有益的指导。     

Impact of carbon and nitrogen nutrition on the quality, yield and composition of blastospores of the bioinsecticidal fungus Paecilomyces fumosoroseus

The impact of growing cultures of Paecilomyces fumosoroseus in liquid media containing four combinations of glucose and casamino acids (8 g l^–1 or 80 g l^–1 glucose, 1.32 g l^–1 or 13.2 g l^–1 casamino acids) was evaluated, based on blastospore production, germination rate, viability after freeze-drying and short-term storage stability. When blastospores were produced using a high casamino acid concentration, blastospore yields and germination rates were significantly higher (13.2–18.5×10⁷ blastospores ml^–1, 50–60% germination after 4 h), compared to cultures grown in media containing lower casamino acid concentrations (0.4–2.3×10⁷ blastospores ml^–1, 10–20% germination after 4 h). Chemical analyses of blastospore composition showed that accelerated blastospore germination may be related to increased proteinaceous reserves rather than to glycogen or lipid accumulation. Tolerance to freeze-drying by blastospores suspended in spent medium was enhanced by a high initial casamino acid concentration in the culture medium (75% survival) and by the residual glucose concentrations in the spent medium. Under the conditions of this study, the storage stability of blastospores of P. fumosoroseus was unaffected by the nutritional condition in which they were produced.     

Influence of formulation additives on the desiccation tolerance and storage stability of blastospores of the entomopathogenic fungus Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes)

Blastospores of the entomopathogenic fungus Paecilomyces fumosoroseus were formulated with 10% lactose/1% bovine serum albumin (BSA) or various compositions of Fantesk™, a starch-oil composite prepared by jet-cooking an aqueous dispersion of starch and oil. Storage stability studies with wet blastospore formulations showed that maximum blastospore survival was achieved during low-temperature storage at -20°C with lactose/BSA formulations or starch-oil formulations supplemented with sucrose, zein protein, and whole milk. Under conditions of wet storage at -20°C, the addition of whole milk to starch-oil formulations significantly improved blastospore stability while the addition of sucrose or zein protein had no effect. In freeze-drying studies, no significant differences were seen in blastospore desiccation tolerance or in stability during storage at either 4 or -20°C when blastospores of P. fumosoroseus were formulated with lactose/BSA or starch-oil formulations with sucrose, zein protein, and whole milk. Freeze-dried blastospore formulations stored at 4°C showed no loss in blastospore viability after 3 months storage and blastospore formulations stored at -20°C showed no loss in viability during the entire 12-month study. For freeze-dried, starch-oil formulations, sucrose was shown to improve blastospore survival during the freeze-drying process. The addition of whole milk to starch-oil formulations significantly improved the stability of freeze-dried blastospores stored at 4°C. Compared to unformulated blastospore suspensions that showed blastospore settling after 30 min, suspensions of blastospores formulated with lactose/BSA or starch-oil composites remained stable for up to 2 h after mixing.     

Biodegradation of entomopathogenic hyphomycetes: Influence of clay coating on Beauveria bassiana blastospore survival in soil

The survival of naked and clay-coated Beauveria blastospores in soil was investigated with an experimental biodegradation method using a trap technique. At various times of incubation, traps were collected to study changes in biomass, inoculum potential, and infection potential of degraded blastospores, and to investigate the colonization of blastospore biomass by antagonistic soil-borne microorganisms. Infection potential was tested on larvae of Plutella maculipennis and Leptinotarsa decemlineata. Naked blastospores were inactivated after 3 weeks incubation, while clay-coated blastospores were still active after 2 months incubation in soil at 20°C. All investigations demonstrated the protective role of clay coating against biodegradation of fungal propagules by soil bacteria and protozoa. Consequently, clay coating of blastospores is proposed for the formulation of entomopathogenic hyphomycetes propagules. Moreover, antagonists implicated in lysis of blastospores, must be considered as an important part of the environmental response to a massive introduction of a fungus used for insect control.