In vitro compatibility of Beauveria bassiana strain ATCC 74040 with copper fungicides

Copper fungicides and mycoinsecticides based on entomopathogenic fungi Beauveria spp. are the most common pesticides used in organic crop production systems. The in vitro effects of the copper fungicides copper oxide, copper hydroxide, copper oxychloride, copper sulphate, dicopper chloride trihydroxide and tribasic copper sulphate were investigated for mycelial growth, sporulation and conidial germination of the ATCC 74040 commercial strain of Beauveria bassiana. Mycelial growth was evaluated on potato dextrose agar plates with 100%, 75%, 50%, 25%, 12.5%, 6.25% and 0% of the recommended application rates of each fungicide at 15 and 25°C. Sporulation and conidial germination were determined at the recommended field doses of each fungicide at 25°C. All copper fungicides had fungistatic or fungicidal effects on B. bassiana that varied according to the dose. Only in two cases, copper oxide at 15°C and copper hydroxide at 25°C, at the lowest concentration of 6.5%, was mycelial growth not statistically significantly inhibited. Inhibition of mycelial growth depended both on the fungicide and its concentration, and partly on temperature. Both sporulation and conidial germination of B. bassiana were significantly inhibited by all fungicides. All fungicides inhibited the sporulation in a similar way (99.8%–100%). With the exception of copper oxychloride (inhibition, 13.6%), the other fungicides showed high detrimental effects on conidial germination (inhibition, 91.7%–100%). The fungus was strongly affected by some fungicides even at the lowest doses. The biological index used for the B. bassiana with copper fungicides ranged from 0.6 (copper sulphate) to 18.1 (copper oxychloride). Therefore, the tested fungicides were classified into the upper half of the highly toxic (T) category and are considered incompatible with the entomopathogenic fungus B. bassiana strain ATCC 74040 under in vivo experimental conditions. These results need to be further verified in vitro under both greenhouse and open-field conditions.     

Effect of combination treatment with entomopathogenic fungi Beauveria bassiana and Nomuraea rileyi (Hypocreales) on Spodoptera litura (Lepidoptera: Noctuidaeae)

In biocontrol of insect pests, efficacy of treatment with multiple pathogens has not been frequently investigated but may have potential for effective management. The possible advantage of a combination treatment with two entomopathogenic fungi - Beauveria bassiana and Nomuraea rileyi - was assessed in laboratory bioassays on second instar Spodoptera litura. From among the fungal isolates of an epizootic population, two isolates of each fungus differing in virulence to S. litura were chosen, one highly virulent and the other with low virulence. The bioassays were carried out at either a continuous temperature of 25±1°C or at a temperature cycle of 32±2°C 8 h/21±2°C 16 h to mimic the field temperatures during the epizootic. Treatments with the two fungi were done both simultaneously and sequentially. In combination treatments at 25±1°C, in all isolate combinations, a majority of the larvae showed N. rileyi induced mycosis; the percentage mortality and speed of kill of insects in these treatments was similar to the N. rileyi isolate used in the combination treatments. At the temperature cycle of 32±2°C 8 h/21±2°C 16 h, in all combination treatments, all the dead insects exhibited B. bassiana mycosis; the mortality pattern was similar to the B. bassiana isolate used in the combination treatments. The adverse effect of high temperature on virulence of N. rileyi was however, not evident in in vitro growth assays. Combination treatment with both fungi did not have a synergistic effect on insect mortality.     

球孢白僵菌对四种十字花科蔬菜害虫的兼控潜力评价

研究了分离自小猿叶甲的一株球孢白僵菌(Beauveria bassiana(SCAU-BB01D))对十字花科蔬菜4种非目标害虫烟粉虱Bemisia tabaci、小菜蛾Plutella xylostella、黄曲条跳甲Phyllotreta striolata和菜缢管蚜Lipaphis erysimi的致病能力。结果表明,在实验室条件下,该菌株对以上4种害虫均有不同程度的侵染力。1×108孢子/mL的白僵菌悬浮液对1,2,3,4龄烟粉虱若虫的侵染率分别为84.88,86.81,55.94和38.78%;对小菜蛾2,3和4龄幼虫的侵染率为67,59和44%;对黄曲条跳甲幼虫和成虫分别为63%和60%;对菜缢管蚜成蚜和若蚜的侵染率分别为44%和35%。通过机率值分析,得出该菌株对烟粉虱1~4龄若虫的LT50分别为4.14,3.78,6.24和7.59 d;对小菜蛾2~4龄幼虫的LT50分别为7.29,8.16和9.82d;对黄曲条跳甲成虫和幼虫的LT50分别为11.22和8.11 d;对菜缢管蚜成蚜和若蚜的LT50分别为11.01和12.15 d。说明该球孢白僵菌菌株对烟粉虱、小菜蛾和黄曲条跳甲3种非目标害虫具有一定的兼控作用,在蔬菜害虫的生物防治中具一定的应用潜力。     

BbRho5对球孢白僵菌生长速率的作用研究

为阐明BbRho5对球孢白僵菌生防潜能的作用,构建了Bbrho5单基因敲除菌株ΔBbrho5,以野生型菌株WT作为对照,在不同培养基上测定菌落生长速率,并测定了菌株对多菌灵胁迫耐受性及对大蜡螟幼虫体壁侵染能力。进一步获取和分析了ΔBbrho5和WT细胞内基因转录组数据。结果表明,BbRho5蛋白功能缺陷显著抑制球孢白僵菌菌丝生长速率,同时微弱影响其多菌灵胁迫抗逆性及生防能力。相较于WT,ΔBbrho5中具有770个差异表达基因(DEGs),其中上调基因395个,下调基因375个。GO分析显示,ΔBbrho5 VS WT中DEGs主要富集于氧化还原酶活力(oxidoreductase activity)和单加氧酶活力(monooxygenase activity)功能。KEGG通路富集结果显示,DEGs主要富集于氮代谢及多种氨基酸代谢通路。在氮代谢通路中富集到7个功能基因,其中有5个上调,2个下调,说明敲除菌株可能采用增强氮源利用及谷氨酸合成以应对Bbrho5缺陷引起的生长迟缓。以上研究结果揭示了球孢白僵菌中小GTP酶BbRho5对球孢白僵菌生长速率具有重要影响,且氮代谢和氨基酸代谢可能为其重要的响应代谢通路。     

BbRho5对球孢白僵菌生长速率的作用研究

为阐明BbRho5对球孢白僵菌生防潜能的作用,构建了Bbrho5单基因敲除菌株ΔBbrho5,以野生型菌株WT作为对照,在不同培养基上测定菌落生长速率,并测定了菌株对多菌灵胁迫耐受性及对大蜡螟幼虫体壁侵染能力。进一步获取和分析了ΔBbrho5和WT细胞内基因转录组数据。结果表明,BbRho5蛋白功能缺陷显著抑制球孢白僵菌菌丝生长速率,同时微弱影响其多菌灵胁迫抗逆性及生防能力。相较于WT,ΔBbrho5中具有770个差异表达基因(DEGs),其中上调基因395个,下调基因375个。GO分析显示,ΔBbrho5 VS WT中DEGs主要富集于氧化还原酶活力(oxidoreductase activity)和单加氧酶活力(monooxygenase activity)功能。KEGG通路富集结果显示,DEGs主要富集于氮代谢及多种氨基酸代谢通路。在氮代谢通路中富集到7个功能基因,其中有5个上调,2个下调,说明敲除菌株可能采用增强氮源利用及谷氨酸合成以应对Bbrho5缺陷引起的生长迟缓。以上研究结果揭示了球孢白僵菌中小GTP酶BbRho5对球孢白僵菌生长速率具有重要影响,且氮代谢和氨基酸代谢可能为其重要的响应代谢通路。     

Growth and sporulation of Beauveria bassiana and Metarrhizium anisopliae on media containing various amino acids

Natural isolates of two entomogenous fungi, Beauveria bassiana and Metarrhizium anisopliae, were cultured in liquid culture media containing 24 amino acids and KNO₃ to determine their effect on growth and sporulation. In addition, the growth and medium pH changes for each isolate grown on an asparagine-containing medium were compared. Tryptophan and alanine were most effective for growth and sporulation of B. bassiana, although glutamine and KNO₃ also produced large numbers of regularly shaped spores. Tryptophan, glutamic acid, and histidine were all well utilized for both growth and sporulation of M. anisopliae. Nitrogen sources containing sulfur were poorly utilized for sporulation by M. anisopliae. In general, B. bassiana produces greater mycelial mass and much larger numbers of spores than M. anisopliae. Both fungi attained nearly the same growth maximum on asparagine medium though B. bassiana exhibited an initially more rapid growth rate. In both fungi this rapid growth phase was accompanied by a decline in medium pH followed by a rise in pH during the decline phase of growth.     

不同温、湿度下白僵菌对棉铃虫幼虫的致病力

测试了不同温、湿度下白僵菌Beauveria bassiana对棉铃虫Helicoverpa armigera幼虫的致病力。实验设置了10、15、20、25、30℃ 5个温度水平,用高浓度（1×10<.sup8孢子/mL）、中浓度（1×10⁷孢子/mL）、低浓度（1×10⁶孢子/mL）的白僵菌孢子液分别感染1～4龄的棉铃虫。结果表明：三个浓度处理的棉铃虫在温度为25℃时的致死中时（LT₅₀）最短,死亡速度最快,死亡率最高;高于或低于此温度时,棉铃虫的LT₅₀延长,死亡速度减慢。相对湿度发生变化时,感病棉铃虫死亡速度和死亡率明显不同。相对湿度为95%左右时,棉铃虫死亡速度最快,死亡率最高;相对湿度低于70%时,棉铃虫死亡率显著降低。     

Variation in virulence of Beauveria bassiana isolates and its relatedness to some morphological characteristics

Ten Beauveria bassiana isolates were characterized using the following parameters: virulence, morphological measurements, germination in solid and liquid media, in vitro growth and physiological changes in liquid media. There were significant differences in mortality of second-instar larvae of the diamondback moth (DBM), Plutella xylostella (Lepidoptera: Plutellidae) and the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Coleoptera, Chrysomelidae) according to isolate used (DBM; 14.51-53.37%, CPB; 25.05-74.07%). The most virulent isolates to DBM and CPB were KCF107 (soil origin) and KCF106 (Chilo suppressalis origin), respectively. Several morphological and physiological characteristics of isolates were evaluated for a possible link to virulence against both insect species. Special mean diameter (2.45-3.58 µm) and area of conidia (5.72-10.25 µm²) were significantly different among isolates. Time for 50% of conidia to germinate (GT₅₀) varied from 11 to 21 h depending on isolate. There was no correlation to virulence of size of conidia, GT_50, red pigment production and mycelial growth. Acidity of 7-day-old broth cultures of isolates was positively correlated with virulence. Iranian isolates KCF106 and KCF107 were the most promising and virulent isolates but field testing is needed to further evaluate their potential for use in DBM and CPB management.     

应用白僵菌与溴氰菊酯混合防治马尾松毛虫研究

通过应用白僵菌粉与4种浓度溴氰菊酯粉混合后不同时间孢子萌发情况测定,以及用纯菌粉、药粉和菌药不同比例混合粉,对马尾松毛虫室内毒力测定和林间防治试验比较,筛选出菌粉与0．05％溴氰菊酯粉按4：1比例混合,防治效果最好,并经大面积推广防治示范,平均防治效果达95％以上,分别较纯菌粉和药粉效果提高14．9％和29．5％,具有明显的增效作用,马尾松毛虫死亡高峰期提前5－7天,减少松针损失量,能及时控制住高虫口林分害虫的严重危害、经济、社会和生态效益显著。     

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.     

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.     

Total soluble protein profiles of Beauveria bassiana and their relationship with virulence against Helicoverpa armigera

Intracellular total soluble proteins of Beauveria bassiana are believed to play an important role in virulence against insect hosts. Thirty B. bassiana isolates collected from different geographical regions and host ranges were characterised by total soluble proteins present in cells, using the SDS–PAGE technique to differentiate the isolates based on virulence and host insect origin. In vitro analysis of total soluble protein profiles of 30 isolates was studied to understand the relationship of isolates with their host of origin and virulence against Helicoverpa armigera. There was a positive relationship between virulence and host origin. All the non-virulent isolates are grouped together. Similarly, highly virulent isolates against H. armigera were grouped together. The relationship between total soluble proteins and pathogenicity was positively correlated. Thirty isolates shared only 22% similarity in their protein profiles.     

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.     

球孢白僵菌不同分离株的生物学及对小猿叶甲成虫致病性测定

球孢白僵菌Beauveriabassiana是一种寄主范围广、致病性强、适应性广的昆虫病原真菌。对采自广东阳山小猿叶甲Phaedonbrassicae的球孢白僵菌 6个分离株 (A ,B ,C ,D ,E ,F)的菌落生长速率、产孢量、分生孢子萌发率进行了实验 ,测定和比较了这 6个分离株对小猿叶甲成虫的致病力。结果表明 :各分离株在菌落生长速率上没有显著差异 ,但各分离株的孢子萌发率有显著差异 ,其中C ,D ,E分离株产孢量明显高A ,F分离株。用 5× 1 0 7分生孢子 mL的悬浮液处理小猿叶甲成虫 ,分离株间的致病力有显著的差异 ,以D分离株对小猿叶甲成虫的感染死亡率最高 ,为 86 67%。综合比较 ,产孢量和致病性较高的D分离株可作为进一步研究的材料。     

应用球孢白僵菌防治马尾松毛虫的策略及其生物多样性基础的研究

对３种不同方式应用球孢白僵菌防治马尾松毛虫的结果表明,３个林场的虫口在连续４年内均被抑制在低水平,但松毛虫种群动态不同：以淹没式施放白僵菌为主,但有时也淹没式施放化学杀虫剂的麻姑山林场平均虫口密度为３．１６±４．９４条·株－１,波动最大;长期接种式应用白僵菌的戴公山林场平均虫口密度只有０．０９±０．１４条·株－１,一直处于低密度状态;以化学农药处理小面积虫源地结合不频繁地淹没式施放白僵菌的金寺山林场平均虫口密度为１．４５±２．２９条·株－１,偶可达防治指标．对群落组成的调查结果表明,从麻姑山、金寺山到戴公山,动物和虫生真菌群落各物种的总个体数递减,而总物种数、总物种数与总个体数的比值,天敌与害虫种数和个体数的比值以及群落多样性指数皆递增,表明群落多样性渐趋丰富,食物链网渐趋复杂,群落稳定性和对害虫的自然控制能力逐渐增强．对群落动态的主成分分析进一步表明,从麻姑山、金寺山到戴公山,动物和虫生真菌群落的演化时序分明,趋于稳定,自我调节力强．     

Genetic diversity among Brazilian isolates of Beauveria bassiana: comparisons with non-Brazilian isolates and other Beauveria species

Aims:  The genetic diversity of Beauveria bassiana was investigated by comparing isolates of this species to each other (49 from different geographical regions of Brazil and 4 from USA) and to other Beauveria spp.
Methods and Results:  The isolates were examined by multilocus enzyme electrophoresis (MLEE), amplified fragment length polymorphism (AFLP), and rDNA sequencing. MLEE and AFLP revealed considerable genetic variability among B. bassiana isolates. Several isolates from South and Southeast Brazil had high similarity coefficients, providing evidence of at least one population with clonal structure. There were clear genomic differences between most Brazilian and USA B. bassiana isolates. A Mantel test using data generated by AFLP provided evidence that greater geographical distances were associated with higher genetic distances. AFLP and rDNA sequencing demonstrated notable genotypic variation between B. bassiana and other Beauveria spp.
Conclusion:  Geographical distance between populations apparently is an important factor influencing genotypic variability among B. bassiana populations in Brazil.
Significance and Impact of the Study:  This study characterized many B. bassiana isolates. The results indicate that certain Brazilian isolates are considerably different from others and possibly should be regarded as separate species from B. bassiana sensu latu . The information on genetic variation among the Brazilian isolates, therefore, will be important to comprehending the population structure of B. bassiana in Brazil.     

Gene structure and expression of the gene from Beauveria bassiana encoding bassiasin I, an insect cuticle-degrading serine protease

Genomic and cDNA encoding Beauveria bassiana bassiasin I, a potential cuticle-degrading serine protease, were isolated and analysed. Bassiasin I gene is comprised of 1137 bp (379 aa) and 3 introns which are 69, 62 and 68 bp long. The comparison of a deduced amino acid sequence with Metarhizium anisopliae Pr1, B. bassiana Pr1, and proteinase K showed high homology. When the cDNA including the intact signal peptide was expressed in E. coli, a clear proteolytic-degraded zone on LB-skimmed milk plates was observed.