Cloning of a cuticle-degrading protease from the entomopathogenic fungus, Beauveria bassiana

Abstract A Beauveria bassiana extracellular subtilisin-like serine endoprotease is a potential virulence factor by virtue of its activity against insect cuticles. A cDNA clone of the protease was isolated from mycelia of B. bassiana grown on cuticle/chitin cultures. The amino acid sequence of this gene was compared to that of Metarhizium anisopliae Pr1, the only pathogenicity determinant so far described from an entomopathogenic fungus, and proteinase K, isolated from Tritirachium album , a saprophytic fungus. The cDNA sequence revealed that B. bassiana Prl is synthesized as a large precursor ( M _r 37 460) containing a signal peptide, a propeptide and the mature protein predicted to have an M _r of 26 832.     

Light stimulates conidiation of the entomopathogenic fungus Beauveria bassiana

Beauveria bassiana is a commercially important entomopathogenic fungus. Like other insect fungal pathogens, B. bassiana usually produces asexual reproductive bodies, conidia, for dispersal, transmission and infection of insects. Adequate mass-production of high quality conidia is crucial to development of an efficient B. bassiana insecticide. However, little is known about details of conidiation in this fungus in response to environmental signals, which limits understanding of the mechanism of conidiation and improvement in conidia production. Here, morphologenetic changes of B. bassiana under different light conditions are reported. When cultured in total darkness, B. bassiana hyphae can grow continuously with few reproductive structures differentiated, while illumination with white light resulted in prolific formation of conidiophores bearing abundant conidia, indicating that light could stimulate conidiation of B. bassiana. Among the single colour lights tested, blue light was the most effective to stimulating sporulation. Colonies became adapted for blue light stimulus only after hyphae had grown in total darkness for at least 96 h, whereas the photoadaptation obviously declined after 144 h. For the exposure time, 3 min of blue light pulse was enough to stimulate conidiation in the photoadapted mycelia, while prolonged light exposure over 3 min resulted in a decrease in conidia yield. Our results provided useful clues for understanding the mechanism of conidiation mediated by light in B. bassiana.     

Biochemical and molecular characteristics of indigenous strains of the entomopathogenic fungus Beauveria bassiana of Central India

Forty-eight isolates of indigenous strains of Beauveria bassiana from various insect hosts collected from Central India were characterised by employing protease zymography and RAPD analysis. Results of protease zymographic profiles were reproducible and significant enough to contribute to the biochemical diversity of this species. RAPD analysis revealed the presence of high level of genetic diversity and indicated that 0-66% significant differences has evolved between these isolates. The sets of amplified bands showing identical pattern to others were grouped at 100% similarity level. A wide range in the value (0.25-1.00) of Jaccard similarity coefficient was observed among all the isolates. The grouping of the indigenous strains, obtained from numerical analysis of these data, appears to be related to the host specificity in B. bassiana. Clear groups were seen for strains isolated from Lepidopteran and Coleopteran insect hosts.     

High-throughput insertion mutagenesis and functional screening in the entomopathogenic fungus Beauveria bassiana

The entomopathogenic fungus Beauveria bassiana displays a broad insect host range and serves as a model for examining host-pathogen interactions. Rapid construction and screening of random-insertion mutants of B. bassiana provides a powerful tool to dissect the molecular mechanisms of fungal virulence. LiAc/DMSO treated B. bassiana blastospores were found to be highly competent to transformation using linear DNA and a polyethylene glycol-based method. Selection on cellophane-layered Czapek-Dox agar at a lowered pH (from 7.5 to 6.3) greatly decreased background growth of non-transformed cells and improved screening of transformants. Optimization of the protocol using integration of the bar phosphinothricin resistance gene resulted in high transformation rates (200-250 transformants/μg DNA/10⁸ cells). A collection of ∼4000 insertion mutants was examined via high-throughput screens for hydrocarbon utilization. One mutant was isolated that grew poorly on both n-hexadecane and tributyrin. The random insertion site was mapped to a gene that displayed homology to vitamin H (biotin)/tartrate transporters. Insect bioassays using Galleria mellonella as the target host revealed decreased virulence in the mutant. This system provides a simple and rapid method for the generation and screening of insertion mutants and should expand our ability to genetically analyze the B. bassiana lifestyle.     

Isolation and characterization of microsatellite loci from the entomopathogenic fungus Beauveria bassiana (Ascomycota: Hypocreales)

Beauveria bassiana, an entomogenous fungus used for the biological control of pest insects, comprises a globally‐distributed species complex of regionally endemic lineages. In order to study the population genetics of B. bassiana, detail species boundaries, conduct ecological studies of natural populations and track fates of experimentally‐released strains, sensitive genetic markers are required. We describe the isolation and characterization of eight microsatellite loci that amplify successfully from strains representative of the phylogenetic diversity in the B. bassiana complex.     

Effect of nutrition on growth and virulence of the entomopathogenic fungus Beauveria bassiana

Three isolates of the entomopathogen Beauveria bassiana along with one strain of Metarhizium anisopliae were cultured on seven media with different carbon/nitrogen (C/N) ratios. The effect of nutrition on virulence of the isolates was evaluated via measurement of colony growth, spore yield, germination speed, conidial C/N ratio and Pr1 (a serine protease) activity. 'Osmotic stress' medium produced the lowest colony growth with low numbers of conidia in all isolates. However, these conidia showed a high germination rate and virulence. However, conidial Pr1 activity was low in some isolates. In most but not in all cases conidia from 1% yeast extract, 2% peptone and low (10 : 1) C/N medium had higher Pr1 activity compared with conidia from other media. However, in some instances we could not conclude that there was a relationship among germination rate, conidial Pr1 activity and virulence. C/N ratio of conidia was statistically different among various media and fungal isolates. Conidia with lower C/N ratio generally produced lower LT(50) (lowest median lethal time) values (more virulent). Insect-passaged conidia from different media had lower C/N ratio compared with similar conidia from artificial cultures. Therefore, they should be more virulent than in vitro produced conidia. As germination rate, conidial Pr1 activity and C/N ratio are independent of host, it seems that host-related determinants such as insect cuticle and physiology and environmental conditions may influence host susceptibility and therefore fungal isolate virulence towards host insects.     

桃蚜脱皮对球孢白僵菌毒力的影响

室内测定了球孢白僵菌Beauveria bassiana BBSG8702菌株对桃蚜Myzus persicae 若蚜的毒力,并分析了若蚜脱皮与白僵菌有效侵染之间的关系。在21℃、1×10⁶/mL孢子浓度下, 1～4龄若蚜感菌后的累积死亡率分别为10.1%、2.1%、3.1%和40.2%,明显低于同样条件下成蚜的感菌死亡率(98.4%)。在若蚜各龄感菌死亡的个体中,1～4龄中分别有8.1%、50.0%、44.4%和98.0%的若蚜在死亡前发育到成蚜,且都能产下正常发育的后代。若蚜的感菌死亡率与接菌后到第一次脱皮的时间密切相关,脱皮早则感菌死亡率低,反之亦然。但试验中也发现有部分若蚜特别是低龄若蚜感菌后未完成第一次脱皮便死亡。观察了桃蚜在1、4龄早期感染球孢白僵菌后的发育、存活和生殖情况,结果表明：1、4龄早期若蚜接菌后第一次脱皮的时间早于未接菌若蚜,但提前幅度少于5%,在羽化为成蚜之前被致死的概率分别只有4.1%和0,在羽化为成蚜后8天内的生殖率与未接菌蚜虫的无明显差异。这些结果表明,若蚜的脱皮,尤其当蚜虫在低龄若蚜时受感染后的多次脱皮,可有效地摆脱球孢白僵菌分生孢子的侵染,降低该菌的毒力。     

感染球孢白僵菌的烟粉虱若虫免疫应答转录组分析

【目的】筛选烟粉虱Bemisia tabaci(Gennadius)若虫应对白僵菌Beauveria bassiana侵染的应答基因,以进一步研究烟粉虱免疫反应的分子机制。【方法】采用新一代高通量测序技术对感染和非感染白僵菌的烟粉虱4龄若虫进行了测序分析,并筛选了差异表达基因;利用生物信息学工具对转录组测序得到的基因进行了功能注释、分类以及参与的信号通路展示。【结果】组装得到非冗余Unigene 232 554个,其N50和N90分别为1 153 bp和260 bp,平均长度为67 424 bp。对所有的基因进行差异表达分析发现:以P<0.05为标准筛选得到了1 166个差异表达基因,其中上调表达基因有474个,下调表达基因有692个,其中,与免疫相关的基因有405个;GO富集分析发现:有416个GO term有富集现象,包括156个生物学过程(66 402个Unigenes),89个细胞组分(27 645个Unigenes)和154个分子功能(73 417个Unigenes);KEGG代谢通路分析将烟粉虱转录组1 145个DEG匹配到309个通路上,其中76个通路得到了富集。【结论】对405个可能参与到烟粉虱若虫对白僵菌侵染的免疫识别和防御的基因进行了测序。研究结果为从分子水平上开展应用虫生真菌防治烟粉虱的研究奠定信息学基础。     

Production,formulation and application of the entomopathogenic fungus Beauveria bassiana for insect control: current status

This review summarizes the progress and achievements made in the last decade in mass production formulation and application technology of the entomopathogenic fungus Beauveria bassiana. Reports published on relevant research from Belgium, Canada, China, Cuba, Czechoslovakia (former), France, Germany, Great Britain, Philippines, Poland, Switzerland, USA and USSR (former) regarding this topic have been covered. Much of the non‐English language literature, particularly that from Eastern European and Chinese sources, has not been translated and is inaccessible to most English or other western language readers. We have done this translation and through this review provide technological details about mass production of B. bassiana in China. Various aspects of B. bassiana growth, substrate use, production of mycelia, conidiospore and blastospores, process technologies associated with separation, drying and milling, formulation, storage and ‘shelf‐life’, and field efficacy are reviewed. Data are presented on: a modified diphasic production technology developed in China during the 1980s; comparisons between submerged fermentation, which usually produces blastospores, and those producing conidia; the use of mycelial preparations pelletized with alginate or gelatinized with cornstarch or cornstarch‐oil; and data on low or ultra‐low volume sprays of emulsifiable or oil conidial suspensions and dust formulations. B. bassiana has proved to be competitive with chemical insecticides for the annual protection of 0.8–1.3 million hectares against forest and farm insect pests in China. It is hoped that this review will help to bridge the language gap between eastern and western scientists in microbial control using B. bassiana.     

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.     

Isolation and characterization of nucleotide excision repair deficient mutants of the entomopathogenic fungus, Beauveria bassiana

To better understand DNA repair in the entomopathogenic fungus Beauveria bassiana, three ultraviolet (UV) light sensitive mutants were isolated and characterized to be deficient in nucleotide excision repair (NER). The UV sensitive mutants were scored by comparison to survival of the parental isolate, GK2016, after 36 J/m² UV-C irradiation. At this dose, conidial survival of GK2016 was 98% and the mutants LC75, LC194, and LC85 had survival values of 63%, 45%, and 31%, respectively. An immunological method which measured the removal of pyrimidine-(6-4)-pyrimidone photoproducts during repair confirmed the decreased ability of LC75, LC194, and LC85 to remove these UV-induced dimers by NER. The mutants were also found to be deficient in NER at swollen/ germinating conidia and blastospore life cycle stages. The germination of the moderately UV sensitive mutant, LC75, was similar to that of the parental isolate, GK2016, after UV irradiation and incubation to enhance NER. The more sensitive mutants, LC194 and LC85 were 2.1- or 2.7-fold, respectively, less likely to germinate after UV irradiation based on their ability to carry out NER. These NER deficient mutants, the first to be derived from B. bassiana, reveal the importance of NER in spore survival post-UV irradiation.     

油松毛虫受球孢白僵菌感染的组织病理学变化

为了研究昆虫病原真菌对松毛虫的致病机理, 提供北方地区松毛虫生物防治的科学依据, 本研究采用球孢白僵菌Beauveria bassiana (Bals.) Vuillemin 菌株1573感染油松毛虫Dendrolimus tabulaeformis Tsai et Liu, 通过扫描电镜和石蜡切片光学显微镜观察技术, 研究了菌株的感染过程和虫体的组织病理学变化。结果表明, 该病原真菌通过穿透表皮入侵油松毛虫, 染菌后24 h, 观察到分生孢子附着于头部的颅顶, 单眼、触角和口器的基部, 在胸、腹部附着于毛簇、毛瘤、棘状突和节间褶。染菌后36 h, 孢子萌发长出菌丝, 在入侵部位, 菌丝的端部特化成附着胞和侵入钉。染菌后48 h, 菌丝依靠机械力和胞外酶的作用穿透表皮, 虫体表皮出现了裂痕和黑化。染菌后72 h, 菌丝已进入体腔, 感染血淋巴、脂肪体、肌肉、消化道、丝腺和神经组织, 并利用血液和内部组织器官作为营养大量繁殖, 此时, 虫体发涨, 表皮变暗。染菌后96 h, 菌丝占据了松毛虫的血腔, 内部的组织结构被完全瓦解, 松毛虫死亡。最后, 菌丝冲破体壁, 在尸体表面长出新的分生孢子。这些结果说明, 球孢白僵菌B. bassiana菌株1573是一种油松毛虫的高致病性菌株, 可以引起虫体的一系列组织病理变化而致其死亡。     

Use of chitin to improve a Beauveria bassiana alginate-pellet formulation

A study was undertaken to evaluate optimum concentrations of chitin in sodium alginate pellet formulations to enhance conidia production. Chitin concentrations tested were 0, 0.5, 1, 2, 3 and 4% (w/v), with (2%, w/v) or without wheat bran. The different chitin-wheat bran pellet combinations were prepared with Beauveria bassiana isolate Qu-B306 at 10⁸ conidia mL^-1. After 21 days of incubation in a humid chamber at 28°C, conidia production was assessed. Improvements up to three times the initial conidia number were achieved using 2% chitin and 2% wheat bran. Higher levels of chitin decreased the number of conidia per pellet. For all chitin concentrations, conidia number increased with the addition of wheat bran (P≤0.05). Contamination by saprophytic fungi was reduced by the incorporation of chitin in the pellet formulation.     

Successive subculturing alters spore-bound Pr1 activity,germination and virulence of the entomopathogenic fungus,Beauveria bassiana

One of the hurdles in the development of entomopathogenic fungi such as Beauveria bassiana is loss of virulence when successively maintained in vitro. This may result in products of inferior quality in mass production programs. Also, there are many contradicting data and unclear points in this case. Three isolates of B. bassiana were subcultured successively 15 times. Spore-bound Pr1 activity, germination rate, and virulence of conidia against mealworm (Tenebrio molitor L.) larvae were studied. Results showed that isolates normally retained their virulence during 10 subculturings. However, they clearly offered decreased virulence (elevated LT₅₀ values and lower percent mortality). The activity of Pr1 bound to conidia declined as subculturing continued; the lowest spore-bound activity and germination potential of conidia was recorded for the 15th subculture. Virulence data were in agreement with Pr1 activity and germination rate as there was a positive correlation between germination rate and spore-bound Pr1 activity with fungal virulence. This explains that at least a part of attenuation in fungal virulence can be explored in enzymatic activity, especially in the important cuticle-degrading protease, Pr1.     

A study of host specificity in the entomopathogenic fungus Beauveria bassiana (Hypocreales,Clavicipitaceae)

Beauveria bassiana (Balsamo – Crivelli) Vuillemin based mycoinsecticides are used against agricultural, veterinary and medical insect pests. The fungus has a very diverse and extensive host range. Variation in virulence among isolates of B. bassiana to different insect species has been abundantly documented. Given the effect of multiple factors on virulence, it is not certain whether the observed difference in virulence can be labelled as host specificity. Environmental conditions and susceptibility of the insect population are two main factors that affect successful fungal infection. Keeping the environmental factors constant, if virulence of an isolate to different insect species and different populations within an insect species is compared, the scale of difference between the two responses can be estimated. If differences in virulence of an isolate to different insect species are greater than the difference in virulence to different insect populations within an insect species, then, the isolate can be considered as exhibiting specific preference to those insect species towards which it exhibits high virulence. To examine this feature, a worldwide sample of B. bassiana was bioassayed on nine insect species and two different populations within two insect species. Laboratory bioassays were done on: Bombyx mori (Lepidoptera), Spodoptera litura (Lepidoptera), Chilo partellus (Lepidoptera), Helicoverpa armigera (Lepidoptera), Epilachna vigintioctopunctata (Coleoptera), Mylabris pustulata (Coleoptera), Aphis craccivora (Homoptera), Maconellicoccus hirsutus (Hemiptera) and Oecophylla smaragdina (Hymenoptera). The range of variation in virulence of a B. bassiana isolate to different insect species was not more than that observed with different populations within a single insect species. B. bassiana is thus a generalist with no strict host preference. B. bassiana based biopesticide can be used as a broad spectrum insecticide against a myriad of insect pests.     

Temperature and moisture requirements for conidial germination of an isolate of Beauveria bassiana,pathogenic to Rhodnius prolixus

The effects of temperature, relative humidity and water activity on germination of conidia of an isolate of Beauveria bassiana (Bals.) Vuill. pathogenic to the triatomine vector of Chagas' disease, Rhodnius prolixus Stål., were investigated in vitro. Germination occurred at temperatures between 15 °C and 35 °C under saturated atmosphere and the optima ranged from 25 °C to 3O °C. At the extreme temperatures tested (15 °C and 35 °C) the germination process was delayed, but germination rates reached more than 95%. Germination of B. bassiana conidia was strongly affected by moisture conditions. The availability of water, in both atmospheric and liquid conditions, caused changes in germination times as well as in germination rates. For example, at 25 °C + O.5 °C, germination took place within 20 h at 95.5% RH, whereas it needed 72 h of incubation at 90% RH. Germination times increased as the water activity declined from 0.96 a_w to 0.92 a_w. Below 0.92 a_w, o germination was observed after a 72 h incubation time.     

Recovery of endophytic Beauveria bassiana on a culture medium based on cetyltrimethylammonium bromide

Selective media (potato dextrose agar plus yeast extract, different concentrations of cetyltrimethylammonium bromide (CTAB) and different antibacterials) were evaluated for the isolation of endophytic Beauveria cf. bassiana from dry bean (Phaseolus vulgaris) plants. CTAB amounts of 0.15 and 0.3?g/L plus either dihydrostreptomycin, oxytetracycline or doxycycline resulted in the lowest numbers of common saprophytic fungi and the highest proportional recovery of Beauveria colonies from internal plant tissues.     

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.     

Microsatellite markers to monitor a commercialized isolate of the entomopathogenic fungus Beauveria bassiana in different environments: Technical validation and first applications

Here, we report on the application of five previously developed microsatellite markers (simple sequence repeats, SSRs) to monitor an isolate of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. in different environments. Discriminatory power of these SSR markers was assessed in two commercialized B. bassiana isolates as well as in 16 B. bassiana isolates from a world-wide collection, and three of the five SSR markers were estimated to allow a confident discrimination among the given isolates. Sensitivity thresholds of 0.1 pg DNA were subsequently determined for all SSR markers in case pure genomic fungal B. bassiana DNA was used as a template for PCR assays, but threshold levels varied depending on the environment (soil, plant) of the PCR assay. Furthermore, presence of a commercialized B. bassiana isolate was monitored via these SSR markers in three different types of potting substrates over a period of 14 weeks. With two SSR markers, strain-specific products were detected up to 14 weeks after application of B. bassiana to the substrate. Infectivity of B. bassiana conidia in the respective soil samples was confirmed by the Galleria baiting technique. Together these results indicate that molecular markers like SSRs specific for commercialized strains of entomopathogenic fungi are important tools to monitor a particular fungal strain in complex environmental samples such as bulk soil or plant DNA.     

变温干燥固体发酵产物对球孢白僵菌分生孢子性能的影响

[目的]评价球孢白僵菌固体发酵产物的干燥温度对产后分生孢子性能的影响.[方法]采用28℃2和35℃组合的7种恒温或变温处理干燥发酵产物,分析收获的分生孢子质量.[结果]变温干燥可显著降低产后孢子粉的杂菌污染.干燥温度对活孢率和孢子萌发速度影响不一致.35℃恒温干燥5 h后活孢率与新鲜孢子无明显差异,但萌发中时缩短了9.3%.干燥处理提高了孢子对高温和紫外辐射的耐受性.适当的变温干燥比恒温干燥有利于增强孢子抗逆性.干燥温度影响分生孢子胞内海藻糖积累,但其含量与抗逆性无直接相关性.优化干燥温度可提高产后分生孢子毒力.在370～450孢子/mm2剂量下,经28℃ 24 h后升至35℃干燥2 h或35℃恒温干燥5 h的分生孢子对桃蚜的致死中时分别比新鲜孢子缩短了10.6 h和7.5 h.[结论]球孢白僵菌固体发酵产物的干燥温度是影响产后孢子粉杂菌污染、孢子活力、抗逆性和毒力的重要因素.