Production of blastospores by three strains of metarhizium anisopliae (metch.) sorokin in submerged culture

Studies on blastospore production in different liquid media were conducted with three strains of Metarhizium anisopliae var. anisopliae (M. a.) derived from various countries (M. a. 43: Austria, M. a. 57: Brazil, M. a. 97: Philippines). Variation of six fermentation parameters (cornsteep products, carbohydrates, pH values, temperature, Tween 80, and polyethyleneglycol (PEG) 200) disclosed that the three strains of M. anisopliae differed in their growth pattern and physiology. In standard medium and in all tests, M. a. 57 produced the highest number of blastospores invariably amounting to > 10⁸ per ml, while mycelial pellets were never formed. The preferred carbohydrates were glucose and fructose. Blastospore production of M. a. 43 was increased by growth at 30°C, at pH 6.5 or by addition of 5% PEG 200. However, it was impaired by different concentrations of Tween 80 or higher concentrations of PEG 200 (10–15%). M. a. 97 produced most blastospores at 30°C, and the strain preferred basic (pH 8.0) as well as acid (pH 4.5) media. Blastospore production was increased by the addition of 5% PEG 200 or 0.4–1.2% Tween 80. Moreover, PEG 200 suppressed pellet formation effectively. Altogether, our results showed that for optimal blastospore production of Metarhizium anisopliae, suitable strain‐specific parameters have to be evaluated.     

Evaluation of Isaria fumosorosea (Hypocreales: Cordycipitaceae) for control of the Asian citrus psyllid,Diaphorina citri (Hemiptera: Psyllidae)

A laboratory bioassay was developed to evaluate strains of Isaria fumosorosea Wize, against Diaphorina citri. Up to 100% of adult psyllids were killed at concentrations between 10⁶ and 10⁷ blastospores/ml after 12 days, with derived LC₅₀ values (at 7 days post treatment) between 1.4 × 10⁵ and 2.0 × 10⁶ blastospores/ml for strains ARSEF 3581, FE 9901 and Apopka-97. A significantly higher value (1.5 × 10⁷) was obtained with a conidial formulation of Apopka-97. Average survival times were dosage dependent, i.e. between 10.2 days at 10³ blastospores/ml and 3.5 days at 10⁸ blastospores/ml. Rates of mycosis were also dosage dependent, with up to 100% sporulation on cadavers at 10⁸ blastospores/ml but declining at lower concentrations. The Apopka-97 strain (commercially available as PFR-97) was tested against established D. citri infestations in potted citrus in greenhouse cages. Treatments at label rates reduced psyllid populations by approximately 50% over 3 weeks. The combination of PFR-97 with emulsifiable oils (0.25% v/v) did not increase psyllid mortality compared with either agent alone. Imidacloprid applied as a drench killed 100% of psyllids within 3 weeks. Subsequent greenhouse tests during humid conditions were hampered by natural dissemination of I. fumosorosea to untreated psyllids, suggesting that this fungus is spread by air movement and may be highly effective under very humid conditions. In later tests, a Cladosporium sp. rapidly colonised psyllid cadavers and leaf surfaces, but was not pathogenic in laboratory tests. Our studies confirm the potential of I. fumosorosea to be used in IPM strategies for D. citri that rely on other tactics, such as insecticidal oils and native or introduced biological control agents.     

Compatibility of OMRI-certified surfactants with three entomopathogenic fungi

The Organic Materials Review Institute (OMRI) is a non-profit organisation providing an independent review of products intended for use in organic production systems to certify compliance with US national organic standards. Since all adjuvants to be used in organic agriculture production are required to meet these standards, OMRI's certified list of products is a convenient starting point when developing organic pest control formulations. In the current study, six OMRI-certified surfactants are tested for their compatibility with three common entomopathogens: Beauveria bassiana, Metarhizium brunneum and Isaria fumosorosea. The fungi were evaluated in two common propagule forms, solid-state produced conidia and liquid-media produced blastospores. The results show that most of the surfactants are compatible with the fungi at a high surfactant concentration (2% w/v). In general, the conidia showed a higher susceptibility (greater reduction in spore germination) to the surfactants than the blastospores under these conditions. In addition, the surface tension and foaming properties of the surfactants were determined.     

十种虫生真菌液体培养芽生孢子的形态发生

芽生孢子是许多真菌通过出芽方式产生的无性孢子,在液体发酵中主要以这种形式大量繁殖。真菌杀虫剂活性成分分生孢子的大量生产使用液体发酵得到的芽生孢子作为接种物,另外,它也是当今虫生真菌遗传转化的重要受体,因而研究虫生真菌芽生孢子的形态和发生特点具有重要意义。本研究分别对液体发酵中的球孢白僵菌、粉棒束孢、蝉棒束孢、环链棒束孢、玫烟色棒束孢、细脚棒束孢、斜链棒束孢、金龟子绿僵菌、蝗绿僵菌和蜡蚧霉等10种常见虫生真菌芽生孢子的形成过程进行显微观察,了解其分生孢子产生过程的异同。结果表明,芽生孢子的产生方式有两种类型：1）蝉棒束孢在其整个生活史中以菌丝生长为主,芽生孢子产生的数量很少。2）其他各种真菌芽生孢子产生方式相似,在菌丝体形成后就开始大量以菌丝出芽或缢缩产生芽生孢子,接着还可通过芽生孢子的出芽或缢缩断裂产生新的芽生孢子。芽生孢子的产生分为3个时期：初期先由菌丝形成芽生孢子;指数期芽生孢子大量增殖,菌丝和芽生孢子都可产生芽生孢子;后期以芽生孢子产新芽生孢子为主要方式。     

Evaluating a dual microbial agent biopesticide with Bacillus thuringiensis var. kurstaki and Beauveria bassiana blastospores

A biopesticide with a mixture of entomopathogenic microbial agents was studied for improvements in efficacy. Recently developed liquid fermentation techniques were used to produce blastospores of Beauveria bassiana (Balsamo) Vuillemin strain GHA (Bb), which were mixed with traditional fermentation of Bacillus thuringiensis Berliner kurstaki (Bt) spores and crystals. Based on a dosage response bioassay using Trichoplusia ni (Hübner) neonates exposed to treated leaf disks, the LC₅₀ values were determined to be 3.85?×?10⁷ spores mL^?1 for the unformulated Bt and 3.58?×?10⁷ blastospores mL^?1 for the unformulated Bb. Using the same bioassay conditions, mixtures of Bt:Bb at ratios of 1:0, 0.75:0.25. 0.5:0.5, 0.25:0.75, and 0:1 indicated that the greatest mortality was caused by the 0.5:0.5 mixture ratio of the microbial agents. Based on these results, the 0.5:0.5 mixture was formulated as a dry powder by spray drying and applied to field-grown cabbage for comparison with commercial Bt and Bb products. Overall, the mixed agent treatment caused equal or greater mortality of exposed insects when compared with commercial products. The insecticidal interaction was determined to be synergistic when combining these two microbial agents into a single product, providing greater mortality of target insects. Assuming similar fermentation costs, the combined treatment could provide improved efficacy without increasing production costs. These benefits support the concept of formulating mixed microbial agents as an environmentally friendly pest control tool.     

住院患者粪便标本中念珠菌检测分析

目的分析粪便中检出念珠菌孢子或假菌丝的临床意义。方法检测白求恩国际和平医院2008年4月住院患者送检的粪便标本,用科玛嘉显色培养基鉴定念珠菌菌种,并记录患者粪便常规及临床资料。结果其间共检测1011份粪便,有86份（8．51％）有真菌生长;其中念珠菌阳性76份（7．52％）。60岁以上老年人阳性者33份（占43．42％）。基础疾病以肿瘤,心、脑血管疾病三者为著（占31．58％）。有21例患者（27．63％）粪便性状有异常,以糊状便为主。结论粪便中检出芽生孢子和（或）假菌丝对诊断念珠菌性胃肠道感染有重要价值,尤其要关注抗生素应用患者。     

Dual selection of Beauveria bassiana strains and complex substrate media for the massive production of submerged propagules with activity against the eucalyptus bronze bug Thaumastocoris peregrinus

The bronze bug Thaumastocoris peregrinus is an invasive pest, affecting Eucalyptus plantations worldwide. Although its natural enemy Cleruchoides noackae has been tested for the biological control of this pest, other strategies like the use of native entomopathogenic fungi are needed. For this, native virulent fungal isolates should be selected, massively multiplied in an efficient way, and prepared to obtain a stable product. Isolates of native Beauveria bassiana obtained from T. peregrinus and from different collections were screened for their virulence towards this insect and for their amenability to be massively produced in a low-input liquid submerged fermentation and prepared as a dry powder. Three out of six virulent strains were suitable for their massive production in a 2% corn flour suspension, achieving 10⁹ submerged propagules/g of dehydrated preparation. The LC50 achieved by the dry submerged propagules did not differ from the LC50 of fresh aerial conidia. The proposed dual selection of strain and a complex substrate, and the procedures leading to the production of a dry preparation, allowed for high viability and virulence of the fungal spores of three strains.     

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.     

Infection sites of the entomopathogenic fungus Beauveria bassiana in the larvae of the mosquito Aedes aegypti

A series of experiments were conducted to determine the infection sites of the entomopathogenic fungus Beauveria bassiana after ingestion by the larvae of Aedes aegypti. The timing of the host death in relation to larval molting, the principle sites of fungal infection and development in host tissues were studied. Fungal conidiospores (CS) and blastospores (BS) were used separately for treatment of mosquito larvae. Although in most instances CS germinated and developed within the host, in others there was a premature abortion of the fungal development cycle. On the other hand, BS ingested by the larvae showed differences in the fungal development stages in the larval tissues. While the two primary infection sites were the head and the anal region, the most preferred site for fungal development was the larval gut. No more than two cycles of fungal development can occur in the host. Although both CS and BS are effective as larvicides, BS is far more pathogenic.

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Résumé Une série d'expériences a été menée afin de déterminer les emplacements d'infection du fongus entomopathogénique Beauveria bassiana (souche GK 2016) lequel avait été ingesté des larves d'Aedes aegypti. On a étudié le moment de la mort des hôtes en relation des mues des larves, les emplacements principaux où se conduit l'infection fongale et le développement du fongus dans des tissus divers d'hôte. On a utilisé les conidiospores (CS) et les blastospores (BS) séparément pour traitement des larves des moustiques. Bien que dans la plupart des cas CS ait germiné et se soit développé chez l'hôte, en d'autres cas, CS a germiné mais n'avait pas produit du BS évidemment grâce à l'avortement prématuré du cycle de développement des fongus chez l'hôte. Le BS qui avait été ingesté dans les larves présentait des stages différents de développement du fongus dans les tissus des larves. Quoique les deux emplacements placements principaux étaient la tête et la région anale, l'emplacement préféré pour le développement du fongus était l'intestin des larves. Nous montrons qu'il n'y a plus de deux cycles de développement du fongus chez l'hôte. Bien que CS et BS soient efficaces comme larvicides, BS est de loin plus pathogène.

     

Effect of photoperiod and host distribution on the horizontal transmission of Isaria fumosorosea (Hypocreales: Cordycipitaceae) in greenhouse whitefly assessed using a novel model bioassay

A model bioassay was used to evaluate the epizootic potential and determine the horizontal transmission efficiency of Isaria fumosorosea Trinidadian strains against Trialeurodes vaporariorum pharate adults under optimum conditions (25±0.5°C, ~100% RH) at two different photoperiods. Untreated pharate adults were arranged on laminated graph paper at different distributions to simulate varying infestation levels on a leaf surface. Four potential hosts were located 7, 14 and 21 mm away from a central sporulating cadaver simulating high, medium and low infestation levels, respectively. Percent hosts colonized were recorded 7, 12, 14 and 21 days post-treatment during a 16- and 24-h photophase. After 21 days, mean percent hosts colonized at the highest, middle and lowest infestation levels were 93 and 100%, 22 and 58%, 25 and 39% under a 16- and 24-h photophase, respectively. From the results, it was concluded that the longer the photophase, the greater the percentage of hosts colonized, and as host distance increased from the central sporulating cadaver, colonization decreased. The use of this novel model bioassay technique is the first attempt to evaluate the epizootic potential and determine the horizontal transmission efficiency of I. fumosorosea Trinidadian strains under optimal environmental conditions at different photoperiods. This bioassay can be used to assess horizontal transmission efficiency for the selection of fungi being considered for commercial biopesticide development.