Review on safety of the entomopathogenic fungi Beauveria bassiana and Beauveria brongniartii

The commercial use of entomopathogenic fungi and their products as mycoinsecticides necessitates their registration. Worldwide, several registration guidelines are available, however, most of them focus on similar or even the same safety issues. With respect to the two entomopathogenic fungi, Beauveria bassiana (Bals.-Criv.) Vuill. and Beauveria brongniartii (Sacc.) Petch, many commercial products have been developed, and numerous papers on different biological, environmental, toxicological and other safety aspects have been published during the past 30-40 years. The aim of the present review is to summarise these data. The following safety issues are presented: (1) identity of Beauveria spp.; (2) biological properties of Beauveria spp. (history, natural occurrence and geographical distribution, host range, mode of action, production of metabolites/toxins, effect of environmental factors); (3) analytical methods to determine and quantify residues; (4) fate and behaviour in the environment (mobility and persistence in air, water and soil); (5) effects on non-target organisms (non-target microorganisms, plants, soil organisms, aquatic organisms, predators, parasitoids, honey bees, earth worms and nontarget arthropods); (6) effects on vertebrates (fish, amphibia, reptiles and birds); and (7) effects on mammals and human health. Based on the present knowledge it is concluded that both Beauveria species are considered to be safe.     

Transformation of the entomopathogenic fungus Metarhizium anisopliae to benomyl resistance

Abstract Protoplasts of the entomopathogenic fungus Metarhizium anisopliae were transformed to benomyl resistance using cosmid pSV50 which harbours a β-tubulin gene cloned from a Neurospora crassa benomyl-resistant mutant. Transformant colonies, which appeared at a frequency of 4 per 50 μg DNA, grew and sporulated on 10 μg/ml benomyl, whereas the wild type was inhibited by 3 μg/ml. Southern blot hybridization of DNA from transformants showed that, in each case, tandem repeats of the cosmid had integrated at several chromosomal loci. The transformants were mitotically stable when subcultured on non-selective agar and retained the ability to infect and kill larvae of Manduca sexta . Two transformants were less virulent than the wild type and one of them showed slower in vitro spore germination. The benomyl-resistant phenotype persisted in reisolates from insect cadavers.     

Investigations on the destruxin production of the entomopathogenic fungus Metarhizium anisopliae

The dynamics of cyclic peptide destruxins (dtxs) produced by Metarhizium anisopliae strains V245 and V275 were monitored both on solid and in liquid media. The results showed that both strains did not produce dtxs in large-scale fermenter cultures or solid Czapek Dox (CD) agar. Production of the major dtxs A and B could be determined in both strains when grown on rice for up to 10-30 days. The main dtxs A, B, E, and E diol were detected in CD liquid culture filtrate from both strains after three days post-inoculation on. Parallel decrease of dtx E and increase of E diol in the culture medium were found, indicating that the latter is the hydrolytic product from the former. Production of dtxs A and B was significantly positively correlated. A negative correlation was observed between the production of the metabolites and pH value of the medium. The influence of different nutrient sources on dtx production was evaluated by using media with different carbon and nitrogen ratios as well as with different insect homogenates. The findings showed that the amount of dtxs A, B, and E increased with the increasing content of peptone in the medium. When insect homogenate was used as single nutrient source or added to CD medium, no toxins were detected in the culture filtrate. The potential risk posed by the toxic metabolites during mass production is discussed.     

Development of a simple and rapid Agrobacterium tumefaciens-mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var. acridum

AIMS: To examine the ability of Agrobacterium to attach to Metarhizium anisopliae var. acridum strain CG423 under co-cultivation and to develop an Agrobacterium-mediated method of gene delivery into strain CG423, a promising agent for biological control of grasshoppers. METHODS AND RESULTS: The co-cultivation of Agrobacterium tumefaciens and M. anisopliae var. acridum was analysed under scanning electron microscopy. We observed that Agrobacterium attached to and formed aggregates around Metarhizium conidia and germ tubes. We also observed the occurrence of fibril-like structures connecting neighbouring bacterial-fungal cells. The Agrobacterium-mediated transformation was applied using two binary vectors carrying a benomyl resistance gene as a selection marker. The efficiency of transformation was up to 53 transformants per 10(5) target conidia. High mitotic stability of the transformants (89-97%) was demonstrated after five successive transfers on non-selective media. Molecular analysis revealed the occurrence of high frequency of gene conversion. CONCLUSIONS: In our study, we report that A. tumefaciens strain AGL-1 attaches to and genetically transforms the entomopathogenic fungus Metarhizium anisopliae var. acridum. SIGNIFICANCE AND IMPACT OF THE STUDY: We report for the first time, the attachment of Agrobacterium to fungal cells opening new avenues for the study of this essential step of the T-DNA transfer process. Considering the efficiency of the transformation protocol herein described, this is a useful tool for gene disruption in M. anisopliae var. acridum.     

Regulation of production of a trypsin-like protease by the insect pathogenic fungus Metarhizium anisopliae

Abstract The entomopathogenic fungus metarhizium anisopliae produces several cuticle-degrading proteases which may play a role in pathogenesis. The regulation of one of these, a trypsin-like protease PR2, has been investigated using depressed mycelia. Three insoluble protein sources, insect cuticle, elastin and collagen, as well as two soluble proteins, BSA and gelatin, induced PR2. The polymeric carbon sources cellulose and xylan resulted in depressed basal levels but not induced production of PR2. An approximately 15-fold increase in PR2 activity per mg dry weight of mycelium was observed when the fungus was grown in the presence of bovine serum albumin (BSA), as compared with conditions of depression alone. This indicates that PR2 is induced by BSA, and probably by other proteins. Basal levels of PR2 were detected after 8 h when mycelium was starved for both carbon and nitrogen but only after 16 h when starved for either nitrogen or carbon. In the presence of a protein source, nitrogen strongly repressed PR2 whereas carbon had little effect. There was no effect of sulphur on PR2 production.     

Metarhizium anisopliae var. Anisopliae Pr1A基因的克隆表达及抗血清研究

采用RT-PCR方法从本实验室分离筛选到的金龟子绿僵小孢变种Metarhizium anisopliae vat．anisopliae中,扩增得到PrlA基因全长,此基因全长为1242bp,经Blastn分析此基因序列与M．anopliae的PrlA基因(M73795)同源率为98%。以pET- 22b( )为基础载体,构建pET-PrlA重组表达载体,在大肠杆菌(Escherichia．coli)BL 21(DE3)中进行表达。经SDS—PAGE分析,获得了约42kDa大小的重组目的蛋白,目的蛋白占表达总蛋白含量的63．2%。将表达的PrlA蛋白切胶回收后制备成抗原,免疫家兔4次后,采血收集抗血清,用ELISA测定效价为1/10000。结果表明,获得的抗体可用于更进一步的研究,将有利于我们进一步了解M．anisopliaeis的侵染机理,弄清楚各Pr蛋白酶的作用方式和对寄主的选择优势,提高生防控制的有效性。     

Toxicity testing of destruxins and crude extracts from the insect-pathogenic fungus Metarhizium anisopliae

Increasing sensitivity towards secondary metabolites from fungal biological control agents (BCAs) has prompted the toxicological risk assessment of metabolites produced by the insect pathogenic fungus Metarhizium anisopliae. Viability studies on one human and one insect cell line were used to compare the two approaches of testing individual metabolites (destruxins A, B and E) or the complete crude extract from liquid cultures. Furthermore, crude extract was separated into fractions, which did not contain the main destruxins A, B and E. Evaluation of the cytotoxic activity of these different compounds suggested that a wide range of metabolites with synergistic or adverse effects are present in the crude extract. The results indicate that identification and toxicological assessment of each individual metabolite produced by a BCA is not only time and cost-intensive, but also does not convey the whole picture. Testing of the crude extract offers an alternative approach and is recommended when assessing the risks of metabolites for registration purposes.     

绿僵菌对小麦纹枯病菌的抑制作用研究

在实验室条件下,研究了金龟子绿僵菌（Metarhizium anisopliae）对小麦纹枯病菌（Rhizoctonia cerealis）的拮抗作用及其机理。结果表明,金龟子绿僵菌与小麦纹枯病菌对峙培养以及在培养基中加入金龟子绿僵菌孢子悬浮液,对小麦纹枯病菌菌丝生长均有较好的抑制作用。测定了培养不同天数的金龟子绿僵菌Ma55发酵液对小麦纹枯病菌菌丝生长、菌核产生量及菌核萌发率的影响。结果表明,液体振荡培养25 d的金龟子绿僵菌Ma55发酵液对小麦纹枯病菌的菌丝生长、菌核产生量及菌核萌发具有显著的抑制作用,且Ma55发酵液中的抑菌活性物质具有较好的热稳定性。在光学显微镜下,未观察到Ma55对小麦纹枯病菌的重寄生现象,但发现金龟子绿僵菌与小麦纹枯病菌对峙培养处小麦纹枯病菌营养菌丝的细胞质变稀薄、菌丝部分消解或断裂。上述结果显示,金龟子绿僵菌对小麦纹枯病菌的拮抗机制主要是营养竞争、空间竞争及抗生作用等。     

绿僵菌Ma83几丁质酶的发酵研究

本实验从虫生真菌中筛选出金龟子绿僵菌M a83菌株,它的几丁质酶合成能力最强。其产酶的适宜条件是,碳源为胶体几丁质加葡萄糖,氮源为NaNO3,培养温度为28℃,培养基起始pH 6.0;接种量为5 mL液态种,最适装液量为5 mL,添加维生素C可以提高酶活;正交实验表明培养因子的最佳组合是:NaNO31 g/L,胶体几丁质0.6 g/L,酵母膏0.05 g/L,葡萄糖0.10 g/L。根据液态培养产酶过程结果可知,当M a83菌培养6天时,几丁质酶活力达到8.1 U/mL。     

Culture conditions affect virulence and production of insect toxic proteins in the entomopathogenic fungus Metarhizium anisopliae

Conidial spores are often used as the infectious agent during insect biocontrol applications of entomopathogenic fungi. Here we show differential virulence of conidia derived from Metarhizium anisopliae strain EAMa 01/58-Su depending upon the solid substrata used for cultivation, where LC₅₀ values differed by up to ~10-fold (5.3×10⁶?4.5×10⁵ conidia/ml) and LT₅₀ values by ~40% (9.8?7.1 d). This fungal strain is also known to secrete proteins that are toxic towards adult Mediterranean fruit flies, Ceratitis capitata, and the Greater wax moth, Galleria mellonella, larvae. In vitro production and intrahemoceol injection using G. mellonella as the host was used to test fractions during purification of the protein toxins, demonstrating that they elicited defence-related responses including melanisation and tissue necrosis. Production of these proteins/peptides along with a number of potential cuticle degrading enzymes was confirmed both in vitro and during the infection process (in vivo). Two-dimensional gel electrophoresis, followed by gel elution and bioassay, was used to identify at least three proteins or peptides (molecular mass=11, 15 and 15 kDa) as mediating the observed insect toxicity. These data demonstrate that in vitro screening for insect toxins can mimic in vivo (i.e. during the infection process) secretion and applies the use of proteomics to invertebrate pathology.     

绿僵菌侵染对椰心叶甲酚氧化酶活性的影响

对椰心叶甲Brontispa longissima(Gestro)成虫血淋巴中酚氧化酶的特性进行分析,并研究绿僵菌(Metarhizium anisopliae)侵染对血浆甲酚氧化酶活性的影响。结果显示,椰心叶甲成虫的血浆及血细胞裂解液中均检测到酚氧化酶活性,且昆布多糖及胰蛋白酶可显著提高其活性。绿僵菌MA-4侵染组在侵染后第1至第5d的血浆酚氧化酶活性高于未侵染组(P<0.05),但是椰心叶甲成虫体内注射10μg昆布多糖后,侵染组的酚氧化酶活性显著低于未侵染组(P<0.05),表明绿僵菌一方面对可激活椰心叶甲的酚氧化酶原激活系统,另一方面又可抑制昆布多糖对椰心叶甲酚氧化酶原激活系统的诱导作用。     

Efficacy of Metarhizium anisopliae microsclerotial granules

The entomopathogenic fungus Metarhizium anisopliae has very recently been shown to produce microsclerotia (MS) – compact, heavily melanised, hyphal aggregates – in liquid media. Soil incorporation bioassays of dried MS preparations of three isolates of M. anisopliae were conducted using third instar Tetanops myopaeformis (sugarbeet root maggot) in clay and/or clay loam field soils as a model system to demonstrate efficacy. At rates as low as 23 mg MS granules/100 g dry soil, the biocontrol efficacy of MS granules of M. anisopliae Strain F52 produced in liquid media with a high carbon concentration (36 g/L) and high C:N ratios (30:1, 50:1) were superior to MS preparations produced in low carbon (8 g carbon/L) media and a high carbon medium with a 10:1 C:N ratio. Bioassays using MS formulations of M. anisopliae strains MA1200 and TM109 produced in high carbon and high C:N ratio media were superior in efficacy to the other MS production media tested. MS preparations of M. anisopliae F52 showed superior efficacy against the sugarbeet root maggot in comparison with more conventional, conidia-covered nutritive (corn grit) granules in a clay and clay soil. The MS granules were also highly efficacious against the sugarbeet root maggot at soil moisture levels as low as 0.983 A _w (?2.33 MPa). Granular preparations incorporating Metarhizium MS can serve as a viable formulation for the use of this fungus against soil insects.     

A technique for accelerating and synchronising germination of conidia of the entomopathogenic fungus Metarhizium anisopliae

The entomopathogenic fungus Metarhizium anisopliae (strain ME1) failed to swell or form germ-tubes in distilled water. However, a period of soaking in distilled water (10–44 h) accelerated the process of germination when a suitable nutrient source was provided. The implications of this novel observation are discussed in terms of mechanisms of germination and the use of parasitic fungi for insect pest control.     

金龟子绿僵菌原生质体的制备和再生及其羟化酶活性研究

对金龟子绿僵菌(Metarhizium anisopliae)原生质体的制备和再生的影响因素进行实验,并在此基础上考察了甾体底物对原生质体羟化酶的诱导作用。结果表明,原生质体制备的合适条件是:42 h的菌丝体用纤维素酶(10 mg/mL)和蜗牛酶(5 mg/mL)的混合酶在含有0.8 mol/L甘露醇的pH 5.8磷酸缓冲液中,28℃震荡(80 r/min)酶解3 h,原生质体产量可达到6.12×10~7/mL,在含有0.6 mol/L KCl的双层马铃薯培养基上再生率达到7.79%。经过6 h底物诱导的菌丝体制备的原生质体细胞色素P450的表达量比没经过诱导的菌丝体制备的原生质体高约40%,证明该菌羟化酶系统的可诱导性。由于没有细胞壁的阻碍经过底物诱导的原生质体能够高效的将底物转化为产物,且副产物相对较少。     

Studies on the regeneration-restore and karyotype of protoplast in Metarhizium anisopliae var. majus

Abstract:  The characteristics and regeneration-restore of protoplasts and its karyotype of an insect pathological fungus, Metarhizium anisopliae var. majus were studied. Among the protoplasts, 25.3% were without a nucleus, and 74.7% contained a nucleus. Among the nucleus protoplasts, 53.6% contained a single nucleus. The regeneration-restore of protoplasts was of three distinct shapes. Considering the frequency of regeneration and the growing speed of the colony, 0.7 mol/l glucose was the optimum as osmotic stabilizer of culture medium in the regeneration-restore of the protoplasts. The chromosomal DNA molecules of M. anisopliae var. majus have been separated into seven bands by pulsed-field gel electrophoreses. Using the Schizosaccharomyces pombe chromosomes as size standard, the size of chromosomal DNA was estimated to be 1.1–6.5 Mb and its karyotype exhibited polytypism among strains.     

Studies on adaptations of Metarhizium anisopliae to life in the soil

Metarhizium anisopliae is an important fungal model for elucidating the basis of entomopathogenicity. A field trial conducted in 2000 with a strain expressing the gfp gene as a marker unexpectedly identified the rhizosphere (the root-soil interface) as the site where insects and pathogen most likely interact. However, the implications of rhizosphere events in maintaining large populations of M. anisopliae remain unclear. The extent to which plant ecology is impacted by the rhizosphere competence of M. anisopliae is also uncertain, but it could be considerable with implicit co-evolutionary implications. Rhizosphere competence may need to be considered as a feature for selecting fungal strains for biocontrol and this raises the possibility of managing the rhizosphere microflora to achieve insect control. Rhizosphere competence also raises some concerns, particularly if it resulted in introduced or hypervirulent transgenic strains persisting in the environment. It is hoped that field trials and other studies currently underway to extend our knowledge of host-pathogen-plant interactions will help identify containment strategies as well as lead to new and important ways of exploiting insect pathogenic fungi for crop protection.     

Influence of fungicides and insecticides on the entomogenous fungus Metarhizium anisopliae a pathogen of the vine weevil,Otiorhynchus sulcatus

In the laboratory, the fungicides chlorothalonil and zineb prevented germination of Metarhizium anisopliae conidia when incorporated into Sabouraud dextrose agar (SDA) at the commercial concentration (based on the manufacturers’ recommended rates for horticultural crops). Twelve other fungicides and six insecticides had no effect on spore germination when applied at the same rate. Mycelial growth of M. anisopliae on SDA plates containing the recommended rate of all the pesticides (except propamocarb) was reduced compared with SDA alone. Two fungicides, benomyl and carbendazim, totally inhibited growth at 0.1 times the recommended rate. Growth was also completely prevented by the fungicides etridiazole, triforine and zineb, and the insecticides dichlorvos and hostathion, at 10 times the recommended rate. In a glasshouse experiment, a prophylactic drench of M. anisopliae conidia reduced vine weevil (Otiorhynchus sulcatus) populations on Impatiens plants by 88%. This level of control was not significantly reduced by subsequent application (7 days after egg infestation) of any of the pesticides at the recommended concentration. Larval control in pots treated with M. anisopliae plus any one of the 12 fungicides and four insecticides examined, ranged from 82% to 98%. The insecticide diazinon applied alone reduced larval numbers by 100%. Two other insecticides, dichlorvos and cypermethrin, and the fungicide pyrazaphos, also reduced weevil populations by over 50%. These experiments demonstrate the limitations of laboratory based in vitro screening programmes for assessing the chemical compatibility of M. anisopliae.     

Effects of Metarhizium anisopliae on Meccus pallidipennis (Hemiptera: Reduviidae) over different types of wall surfaces

The entomopathogenic fungus Metarhizium anisopliae is virulent for the insect triatomine Meccus pallidipennis. To evaluate the functionality of a fungal formulation (vegetable oil and emulsifiers) of this fungus, virulence was assayed by insect mortality on the pronotum of third instar nymphs (N3) M. pallidipennis in laboratory conditions and ST₅₀ was calculated. Mortality was evaluated directly: 100%, 97.33% and 98.66% mortalities were caused by formulation, emulsified formulation and fungal conidia, respectively, at day 8 of insect infection. Another bioassay was carried out in simulated external conditions (peridomicility) using red and gray brick walls, a stone fence and mountain soil (experimental units). These simulated conditions were infected with 10?ml of a 1?×?10⁹?conidia/ml emulsified formulation by means of a manual sprinkler prior to the placement of the nymphs. Ten N3 M. pallidipennis were deposited in each experimental unit and insect mortality was monitored every 12?h for 22 days. Each treatment was replicated four times. With the red brick wall, a mortality of 90% at day 22 and a ST₅₀ of 15 days were obtained on N3 M. pallidipennis; with the gray brick wall, 100% mortality and a ST₅₀ of 13 days; and with the stone fence, 88.33% mortality and a ST₅₀ of 21 days. The results obtained in this research work indicate that the formulation with conidia of the M. anisopliae strain EH-473/4 may be auxiliary in the development of strategies for the control of Chagas disease insect transmitters such as M. pallidipennis.     

Water content and water activity for the production of cyclodepsipeptides in solid-state fermentation by Metarhizium anisopliae

Metarhizium anisopliae was grown by solid-state fermentation on a series of compositions of rice, rice bran, or rice husk media for the production of cyclodepsipeptides, destruxins A and B. The best yield for destruxin A and destruxin B were 2.9 mg kg^–1 substrate and 227 mg kg^–1 substrate, respectively, after 14 days cultivation with rice/bran/husk medium at 71% water content together with water activity of 0.921.