真菌病毒Beauveria bassiana chrysovirus 2(BbCV2)外壳蛋白多克隆抗体制备及应用

球孢白僵菌Beauveriabassiana是一种在害虫生物防治中应用广泛的虫生真菌,真菌病毒能够影响球孢白僵菌的致病力和生物学性状,但真菌病毒相关蛋白的抗体制备和利用其进行病毒检测和定位鲜有报道。本研究原核表达了一株普遍流行并且造成寄主球孢白僵菌毒力下降的双链RNA(double stranded RNA,dsRNA)病毒Beauveria bassiana chrysovirus 2(Bb CV2)的外壳蛋白(Coat protein,CP),并制备了多克隆抗体,进行了病毒在寄主球孢白僵菌胞内和胞外的检测,以及利用间接免疫荧光进行病毒在寄主内的定位。结果表明,表达的BbCV2-CP蛋白主要以包涵体的形式存在,相对分子质量约为85.7kD;经间接ELISA方法测定,制备的BbCV2-CP兔源多抗效价达到1:8192000;Westernblot结果显示,制备的BbCV2兔源多抗能与抗原蛋白进行特异性结合,且能检测出感染寄主球孢白僵菌的BbCV2;ELISA测定结果表明病毒BbCV2可在寄主真菌体内复制,并能够游离到寄主体外;经间接免疫荧光观察,发现BbCV2病毒定位于真菌细胞核中。本研究建立了球孢白僵菌真菌病毒的血清学检测体系,为球孢白僵菌-真菌病毒互作机制研究提供材料。

Relationship between Biological Characteristics of Beauveria bassiana (Bals.) Vuill and Pathogenicity to Bombyx mori L.

[Objective] This study was to investigate the relationship between biological characteristics of Beauveria bassiana （Bals.） Vuill and pathogenicity to Bombyx rnori L, with the aim to provide scientific basis for the control of white muscardine in Bombyx mori L. [Method] The strains isolated and purified from the 6 Beauveria bassiana biocontrol agents from all over the country and the 3 white muscardine silkworms collected from Guangxi provincial silkworm rearing areas were identified by the morphological observation and molecular biology technology. The pathogenicity of B. bassaina to silkworms was determined, and the biological characteristics such as growth diameter, sporulation and the extracellular protease activity of the different B. bassiana strains were compared. [Result] The isolated 9 strains were all B. bassaina （Bals.） Vuillemin, and all strains had high pathogenicity to silkworm, but with different pathogenicities. The growth diameter, sporulation and extracellular protease activity of different B. bassiana strains were also different, and showed correlation with the patheogenicity to silkworms. [Conclusion] B. bassiana spores production amount and exocellular protease activity had significant positive correlation with their pathogenicity to silkworm.

A Mass and Rapid Method for DNA Extraction of Beauveria bassiana

[Objective] The aim was to optimize the mass and rapid method for DNA extraction of Beauvena bassiana. [Method] Boiling water DNA extraction method was improved, DNA extraction liquid was heated by PCR instrument and the extraction process was finished rapidly. [ Resuit] The quality of DNA obtained through mass and rapid extraction of fungal genomic DNA could meet the requirement of RAPD amplification analysis. The clear bands were amplified from 22 tested strains, the number of clear bands were different in the range of 2 -6 and the size of band were mainly concentrated in 450 -800 bp. The DNA extracted by this method also could completely meet the requirement of SCAR amplification. The amplified specific DNA bands used to mark the strain F263 were very clear. [Conclusion] This research provided relatively perfect method for mass and rapid extraction of fungal clenomic DNA.

球孢白僵菌(Beauveria bassiana)培养特征多样性与寄主及地理来源的相关性分析

球孢白僵菌不同菌株微观萌发特征及宏观培养特性表现出高度的多样性，2个性状集团经主成分分析后，得到微观萌发特征的主因子为单芽管萌发孢子比例、芽管总平均长度及芽管整齐度；宏观培养性状的主因子分别为察氏培养基上的菌落颜色、菌落基质颜色、菌落生长速率及液体培养液的pH值。由主因子指标进行典型相关分析，两性状集团总体相关不显著（p＝0. 1378），但菌落生长速率与单芽管萌发孢子比例及芽管总平均长度表现出显著的相关性。由主因子指标进行的聚类及Shannon－Wiener多样性指数统计表明，相同地理来源菌株间的相似性程度高于相同分离寄主间的菌株相似性。

玉米根际土球孢白僵菌(Beauveria bassiana)的分离与鉴定

为明确虫生真菌在玉米根际的分布情况,通过选择性培养基对采自安徽省涡阳、萧县、蒙城3地的玉米根际土壤中的虫生真菌进行了分离,并结合形态特征及r DNA ITS序列分析对分离的菌株进行了鉴定。结果表明,从玉米根际土壤中共分离得到39个白僵菌菌株,经鉴定均为球孢白僵菌(Beauveria bassiana),且不同菌株间的菌落生长速度和分生孢子产生量存在显著差异。本研究对丰富玉米地下害虫及土传病害的生物防治资源具有重要的意义。

球孢白僵菌Beauveria bassiana B6菌株对桔小实蝇的控制作用

室内研究了球孢白僵菌Beauveria bassianaB6菌株对桔小实蝇Bactrocera dorsalis的控制作用。结果表明:采用地面撒施固体菌剂对地面桔小实蝇老熟幼虫和土壤中蛹的致死作用较弱,但对成虫致死作用明显,在0.5×108~2.0×108孢子/g内,成虫死亡率可达82.89%~94.04%。在蛹期、幼虫期和蛹期施2.25×108孢子/g固体菌剂,2个处理对成虫致死效果很好,成虫死亡率高达91.29%~100%,与对照差异极显著(P【0.01)。沙土表面喷施1.0×108孢子/ml的孢子悬浮液,桔小实蝇总死亡率为75.18%,防效为71.93%;养虫笼内壁喷施1×107~1×108孢子/ml孢子悬浮液,成虫死亡率为81.00%~100%,防效为78.94%~97.94%。因此可以明确该白僵菌固体菌剂对桔小实蝇成虫有一定防效,有望成为桔小实蝇防控的一项有效措施。

球孢白僵菌(Beauveria bassiana)NO6的分离、鉴定与除草活性

旨在研究土壤真菌NO6发酵液中对杂草野燕麦具有抑制作用的次生代谢产物。从坎布拉土壤中分离纯化微生物菌株,利用培养皿分析法测定其次级代谢产物除草活性,利用菌落形态和ITS序列对除草活性最高的菌株进行鉴定,进而利用正相硅胶柱色谱、TLC和半制备HPLC方法对其代谢产物中的除草活性物质进行分离纯化。结果表明,共分离获得9株微生物菌株,其中菌株NO6对野燕麦的除草活性最高,将其鉴定为球孢白僵菌(Beauveria bassiana)。发酵液经乙酸乙酯萃取、硅胶柱层析和半制备HPLC分离纯化得到保留时间为66.868min的除草活性物质3。

Compatibility of Beauveria bassiana with Neoseiulus barkeri for Control of Frankliniella occidentalis

The entomopathogenic fungus Beauveria bassiana （Balsamo） Vuillemin and predatory mite Neoseiulus barked Hughes are effective biological control agents of Frankliniella occidentalis Pergande （Thysanoptera： Thripidae）, one of the most important pests of ornamentals and vegetables world-wide. Combined application of both may enhance control efficiency. The functional response for N. barked on the first instar larvae of western flower thrips which were infected by B. bassiana for 12 and 24 h in the laboratory （（25＋1）℃, （70＋5）% RH, L：D=16 h：8 h） was determined. The virulence of B. bassiana against the second instar and pupae of the thrips attacked by N. barked were also tested. The results showed that N. barked exhibited a Holling type II functional response on treated thrips. After having been treated with the fungus for 12 h and then offered to the predator, thrips were more vulnerable to be killed by N. barked. The second instar larvae and pupae of the thrips which had been attacked by predatory mites were markedly more susceptible to B. bassiana infection than normal thrips; the cumulative corrected mortality of B. bassiana of the second instar and pupae which were attacked by N. barked were 57 and 94%, respectively, compared to 35 and 80% in controls on the day 8. These findings highlight the potential use of B. bassiana in combination with N. barkeri to control F. occidentalis.

Effects of Tenebrio molitor L.Powder on Growth of Beauveria bassiana

[ Objective] The study aimed to reseamh the effect of Tenebrio molitor powder on the growth of Beauveria bassiana. [ Method ] The larvae of T. molitorwas used as the additive to add in the PDA medium, preparing 7 concentrations including 4, 2, 1,0.5, 0.25, 0.125 and 0.062 5 g/100 ml, the medium without adding any T. molitorlarva was set as control. The indexes such as the growth rate, spore germination rate, mycelium diameter, sporulaUon time, sporulatian quantity and POD activity of B. bassiana were determined. [ Result] Adding T. molitor larva to the PDA medium could increase the growth rate, spore germination rate, sporulation time, sporulatJon quantity and POD actJvity of B. bassiana, while had relatively small effect on spore size and mycelium diameter. The comprehensive evaluation screening showed that the medium with 4 g additive was the best for the growth of B. bassiana, different indexes all had obvious predominance. [ Conclusion] The study provided the basis for using T. molitor to culture B. baasianaa

Joint Toxicity of Beauveria bassiana and Two Insect Growth Regulators on Beet Armyworm,Spodoptera exigua(Hübner)

Joint toxicity of Beauveria bassiana and two insect growth regulators, methoxyfenozide and hexaflumuron, on beet annywonn larvae, Spodoptera exigua （ Htthner）, were investigated. An obvious synergism effect of the two insect growth regulators with B. beauveria was observed. When the insect growth regulators at LC50 concentrations （25 × 10^-6 g/L for methoxyfenozide and 1 000 × 10^ -6 g/L for hexaflumuren） were mixed with B. bassiana at the volume ratio of 4： 1, the mixtures achieved the best efficacy. The co-toxicity co-efficient （CTC） of methexyfenozide and B. bass/ana was 285, and that of hexaflumuron and B. bass/ana was 208. Scanning electron microscope observation of the longitudinal section of S. exigua revealed obviously different patterns of integument in different treatment groups.

苏云金杆菌(Bacillus thuringiensis)与球孢白僵菌(Beauveria bassiana)复配对小菜蛾幼虫(Plutella xylostella)的影响研究

研究不同浓度苏云金杆菌与球孢白僵菌复配混合剂对佳木斯地区小菜蛾幼虫死亡率的影响。用PBS缓冲液将苏云金杆菌与球孢白僵菌稀释成不同浓度梯度的菌悬液,采用正交分析法对苏云金杆菌及球孢白僵菌复配成7种不同浓度梯度的混合剂,并测定不同浓度混合剂对小菜蛾幼虫的死亡率,从而筛选出较适幼虫死亡的LD_(50)的比例及其浓度。经观察分析,在96 h内苏云金杆菌与球孢白僵菌复配为3:1时,小菜蛾幼虫的死亡率达到72%,其中在1×10^(-1)~1×10^(-6) g/mL浓度下幼虫死亡率较高。各混合液对小菜蛾幼虫致死效果不一,其中当苏云金杆菌与球孢白僵菌复配比例为3:1且浓度在1×10^(-1)~1×10^(-6) g/mL时,对昆虫幼虫致死效果明显。

白僵菌(Beauveria bassiana)的致病性及其在地下害虫防治中的应用

白僵菌是一种具有潜力的微生物杀虫剂 ,其杀虫作用受到环境因子、菌株和害虫自身因素等的影响。在土壤环境中应用白僵菌防治害虫 ,应从白僵菌侵染害虫的途径和方式入手 ,用特定害虫对白僵菌进行专化培养 ,选育专一性强的菌株 ,并与其它试剂混合使用 。

Toward the efficient use of Beauveria bassiana in integrated cotton insect pest management

Background:For controlling the resistance to insects,in particular carpophagous and phyllophagous caterpillars,using chemical pesticides has led to contamination of cotton area in Benin.Facing this problem,alternative methods including the use of entomopathogenic fungi as biopesticide could be a sound measure to preserve the environment,biodiversity and ensure good quality of crops.Previous studies have revealed the insecticidal potential of the entomopathogenic Beauveria bassiana on some insect pest species.However,little is known about its effectiveness on cotton Lepidopteran pests.This review is done to learn more about B.bassina for its application in controlling cotton insect pests,especially Lepidopteran species.Main body:Different sections of the current review deal with the related description and action modes of B.bassiana against insects,multi-trophic interactions between B.bassiana and plants,arthropods,soil and other microbes,and biological control programs including B.bassiana during last decade.Advantages and constraints in applying B.bassiana and challenges in commercialization of B.bassiana-based biopesticide have been addressed.In this review,emphasis is put on the application methods and targeted insects in various studies with regard to their applicability in cotton.Conclusion:This review helps us to identify the knowledge gaps related to application of B.bassiana on cotton pest in general and especially in Lepidopteran species in Benin.This work should be supported by complementary laboratory bioassays,station and/or fields experiments for effective management of cotton Lepidopteran pests in Benin.

Cloning and expression in Saccharomyces cerevisiae of chit2 gene from Beauveria bassiana

To study recycled trashes from shrimps and crabs in the sea through chitinase secreted by microorganisms,the chitinase gene chit2 was cloned and sequenced from Beauveria bassiana by the polymerase chain reaction(PCR),and was ligated into the yeast expression vector pYES2.The expression vector plasmid was transformed into Saccharomyces cerevisiae H158.Gene expression took place upon induction with 2% galactose.The measurement of enzyme activity shows that the expression production can be expressed in active forms and secreted to the medium.The enzyme activity approaches the peak of 0.63 U/mL when the culture time is 36 h.

Assessment of Beauveria bassiana and Their Enzymatic Extracts against Metamasius spinolae and Cyclocephala lunulata in Laboratory

The application of enzymatic extracts and conidia of Beauveria bassiana in Metamasius spinolae and Cyclocephala lunulata was evaluated. The variables were mortality and time of death. In M. spinolae, mortality with extracts 29%, conidia 27% and the combination of both 31%, all had a time of death of four days. Although with different symptoms, used enzymatic extracts: contraction and softening of the joints;by conidia: mycelium in the joints;in the combination of conidia and enzymatic extracts: abundant aerial mycelium. In C. lunulata, 100% mortality in all treatments;Time of death: enzymatic extracts and extracts with conidia 1.2 days;conidia 2.8 days. Symptoms were different, enzymatic extracts: melanization and internal tissue lysis;enzymatic extract and conidia: mycelium emerged and melanization;conidia: mycelium emerged. Enzymatic extracts showed insecticidal activity in M. spinolae and C. lunulata. These results suggest the potential of enzymatic extracts as biocontrol agents to improve the use of entomopathogenic fungi.

Test of Culture and Biomass Production of the Entomopathogenic Fungus Beauveria bassiana （Bals.-Criv.） （Vu//., 1912） on Lactoserm, Margine and Olive Pomace

Entomopathogenic microorganisms occupy an important place among the alternative methods of fighting against pests insect. The fungus Beauveria bassiana is an agent naturally present in ecosystems. It has potential to control pest populations. In the context of biological control, the present work aims to the study of linear growth of Beauveria bassiana on different natural environments from the food industry. They are the raw whey, water and pomace, and followed the development of the fungus through a trial production of biomass on deproteinized whey.

Innovative Method to Control Dubas Bug, Ommatissus lybicus （Deberg） （Homoptera： Tropiduchidae） in Date Palm Orchards Using Endophytic Beauveria bassiana Isolates

The main objective of this study was to investigate the presence of natural endophytic Beauveria bassiana within date palm tissues using molecular technique and measure their field efficacies in controlling Dubas bug, Ornmatissus lybicus （Deberg）. Two entomopathogenic B. bassiana isolates （MARD 108 and 100） were isolated from date palm, Phoenix dactylifera L. leaves; in addition, one isolate （MARD 92） originally from soil was identified to have endophytic property. Concentration of 1 × 10^9 conidia/mL of each of three endophytic isolates was used in field experiments targeting Dubas bug nymphs via injection tree trunks. The results indicated that the high mortality rates reached 92%, 96% and 100% with infliction of the three endophytic isolates after 15 d from the treatment. The successful establishment of the fungal isolates in the date palm tissue was determined using B. bassiana species-specific primer for the first time via using conventional polymerase chain reaction （PCR） amplification technique before and after injection, and the positive gel band representation was the identification signs. The novel results depicted for the first time the presence of natural endophytic B. bassiana isolates within date palm tissues and their field efficacies in controlling Dubas bug, O. lybicus （Deberg） infestation.

The Impact of Beauveria bassiana, Bacillus thuringiensis （Bt） Spray, Trichogramma pretiosum and Spinosad on the Lepidoptera： Crambidae, European Corn Borer （Ostrinia nubilalis）

The research cxamined the effects of microbial and novel insecticidal control strategies and also Irichogramma pretiosum on the I,epidoptera： Crambidae, the European corn borer （ECB）, （Ostrinia nubilalis）. The treatments included ~ino^d, from bacteria the entomopathogenic fungus Beauveria bassiana, the egg parasitoid Trichogramma pretiosum, and foliar applied Bt spray, and t^vo combinations Bearveria bassiana plus Bt spray and Trichogramma pretiosum and spinosad. The infestations by the European corn borer larvae on stalks of corn were very＂ severe in all the control treatments both 2006 and 2007. The microbial treatments caused various levels of European corn borer mortality. In all the parameters there was a significant difference between the control and other treatments with a P 〈 0.00 l. In this research, treatment with spinosad emerged as the most effective biological agent in the control of the European corn borer. The treatments had no significant effects on the relative abundance and composition of non-target arthropods （P 〉 0.05）. This research is relevant in boosting underutilized control strategies and increasing stakeholder adoption of integrated pest management practices and thereby＇ reducing the use of conventional insecticides especially for orgamc thrmers. This is very important especially in certain countries where the commumities are skeptical about consuming transgenic crops.

Contribution of Different Agronomic Practices and the Fungus Beauveria bassiana on the Coffee Berry Borer

The research was carried out to study the effect of varying certain ecological conditions in the coffee through the implementation of different agronomic practices such as regulation of the shade, ground cover and rehabilitation of plantations on the populations of the coffee berry borer Hypothenemus hampei Ferrari （Coleoptera： Curculionidae）, it was also evaluated the influence of these conditions in the introduction and transfer of new variants of application of the fungus Beauveria bassiana （Balsamo） Vuillemin. The results showed that the pest damage was lower under a system of sustainable coffee, designed after the restoration of the population density of coffee, conducting systematic pruning and plant health, sanitation of the forest, shade management to double stratum with scattered light intensity of 60-70% and a combination of systems of denominated handling of weeds （Strip weeding - Ring weeding）, it was found that under these conditions the technical effectiveness of the fungus B. bassiana was higher compared with applications made in the trees less enlightened, being more advantageous to spray the foliage four times with a frequency of 40 days from 60 days to peak flowering with positive economic indicators even when used as single biological control strategy against the coffee berry borer.

球孢白僵菌Beauveria bassiana对茶蚜Toxoptera aurantii致病力筛选及毒力活性研究

茶蚜Toxoptera aurantii(Boyer de Fonscolombe, 1841)是茶园重要害虫之一,开展茶蚜的绿色防控是茶叶安全优质生产的重要保障。研究以无翅茶蚜为研究对象,通过喷雾塔喷雾接菌方法,对供试的9株球孢白僵菌菌株对无翅茶蚜的致病力活性进行筛选,获得了3株致死中时LT50在4.5 d以内,7 d校正死亡率在90%以上的潜在高致病菌株。进一步通过活性评价试验,优化出高致病球孢白僵菌Bb2032菌株,并对该菌株开展了侵染无翅茶蚜毒力学活性评价研究,其4 d、8 d致死中浓度LC50分别为2.881×107孢子/mL和2.102×104孢子/mL。最后通过显微技术观察高致病球孢白僵菌株Bb2032侵染茶蚜的症状,表明菌丝主要从无翅茶蚜的腹管和头部两个位置浸染。该研究将为白僵菌应用在茶园有害生物综合治理中提供理论基础,为茶叶的安全生产提供技术支撑,具有广阔的应用前景。