Identification of group-I introns in the 28s rDNA of the entomopathogenic fungus Beauveria brongniartii

The length of the 28s ribosomal DNA differs significantly between two strains (Bt102 and Bt114) of the entomopathogenic fungus Beauveria brongniartii. RFLP analysis on PCR products revealed the presence of three insertional elements of 350–450 bp in strain Bt114. One of the insertions has been cloned and sequenced and shown to possess all the characteristic sequences and secondary structures of a group-IC intron. Its length is 428 bp and it is devoid of any long open reading frame. The distribution of this intron elsewhere in the genome of Bt114, as well as in the chromosomal ribosomal DNA, was studied. It seems to be present as seven copies in different genes not corresponding to the mitochondrial DNA. The presence of the intron in other strains of B. brongniartii was examined by the hybridization method. Some of them seemed to possess introns with a similar core although others presented no homology with the cloned fragment.     

Gene-disruption of the entomopathogenic fungus Beauveria bassiana incubated with dsRNA

The species of Beauveria bassiana is widely used for the management of agricultural insect pests. In this study, we integrated egfp-double-stranded RNA (dsRNA) to a previously generated egfp-expressing B. bassiana transformant (Bb-egfp#3) using a protoplast integration method. The Bb-egfp#3 protoplast was mixed with the dsRNA under PEG/CaCl₂ conditions and liquid-cultured in Sabouraud dextrose broth for 5 days. A control culture followed the same procedure without dsRNA. Bb-egfp#3/egfp-dsRNA cultures showed very low fungal growth (OD₆₃₀ = 0.2) compared to the control culture, Bb-egfp#3 only (OD₆₃₀ = 1.1). Screening of possible transformants on Sabouraud dextrose agar revealed a transformant T3, without egfp signal. T3 was confirmed as B. bassiana through sequencing of conserved genes and insect bioassays. Interestingly, the genomic egfp fragment of T3 was disrupted, and the egfp signal was not detected over four subcultures, which was also confirmed by RNA-seq of Bb-egfp#3 and T3. This study provides an interesting observation that protoplast integration with dsRNA could possibly generate significantly reduced gene expression in B. bassiana and it is stable across several generations.     

白僵菌转化的一个新蒽醌苷类化合物

目的：研究白僵菌对大黄酸葡萄糖甲基化的转化作用。方法：在白僵菌培养液中加入大黄酸底物，利用生长细胞对其进行生物转化。发酵液离心后用乙酸乙酯萃取，乙酸乙酯萃取部位通过各种色谱方法分离得到一个新化合物。运用氢谱碳谱以及二维核磁共振光谱鉴定结构。结果：转化产物为大黄酸糖基化衍生物，结构鉴定为7-O-（4’-甲氧基-β—D-葡萄糖）。结论：芦荟大黄素（1）是一个新化合物。     

Increased virulence using engineered protease-chitin binding domain hybrid expressed in the entomopathogenic fungus Beauveria bassiana

Insect cuticles consist mainly of interlinked networks of proteins and the highly insoluble polysaccharide, chitin. Entomopathogenic fungi, such as Beauveria bassiana, invade insects by direct penetration of host cuticles via the action of diverse hydrolases including proteases and chitinases coupled to mechanical pressure. In order to better target cuticle protein-chitin structures and accelerate penetration speed, a hybrid protease (CDEP-BmChBD) was constructed by fusion of a chitin binding domain BmChBD from Bombyx mori chitinase to the C-terminal of CDEP-1, a subtilisin-like protease from B. bassiana. Compared to the wild-type, the hybrid protease was able to bind chitin and released greater amounts of peptides/proteins from insect cuticles. The insecticidal activity of B. bassiana was enhanced by including proteases, CDEP-1 or CDEP:BmChBD produced in Pichia pastoris, as an additive, however, the augment effect of CDEP:BmChBD was significantly higher than that of CDEP-1. Expression of the hybrid protease in B. bassiana also significantly increased fungal virulence compared to wild-type and strains overexpressing the native protease. These results demonstrate that rational design virulence factor is a potential strategy for strain improvement by genetic engineering.     

一种白僵菌代谢产物提取物对利血平拮抗实验的影响

目的研究一种白僵菌代谢产物提取物 (BCEF)对利血平拮抗实验的影响。方法采用预防性一次灌胃给药、预防性连续灌胃给药、治疗性连续灌胃给药观察BCEF对利血平拮抗实验的影响。结果皮下注射 (sc)利血平后动物呈现利血平化 :出现眼睑下垂、体温下降及活动抑制。小鼠、大鼠BCEF2 5、5 0、10 0mg·kg-1预防性一次灌胃对利血平 (sc ,2、2 .5mg·kg-1,qd× 1d)、大鼠BCEF2 5、5 0、10 0mg·kg-1预防性连续灌胃 14d ,对利血平 (sc ,2 .5mg·kg-1,qd× 1d)致上睑下垂及活动抑制均无明显改变 ;小鼠BCEF5 0、10 0mg·kg-1治疗性连续灌胃 14天 ,可明显改善利血平 (sc ,0 .2mg·kg-1,qd× 14d)化小鼠的眼睑下垂及活动抑制。结论BCEF对利血平拮抗实验具有一定的改善作用。提示BCEF具有部分增强单胺递质系统的作用。     

Beauveria brongniartii subjected to spray-drying in a composite carrier matrix system

The negative aspects of chemical pesticides are of growing concern to the public. Thus, there is a strong effort to exploit environmentally friendly possibilities for pest management. One strategy is the application of biocontrol agents such as the fungus Beauveria brongniartii. In this context, the central objective of the research presently described is to investigate spray drying as a preservation method for fungal conidia to obtain a practical formulation for spray application. An aqueous binary mixture composed of skim milk (SM) and polyvinylpyrrolidone (PVP K90) was examined as encapsulation matrix. The influence of different inlet/outlet temperature adjustments, the composition of the carrier system and the conidia concentration were examined with respect to their influence on spore viability. Results indicate that air outlet temperatures up to 53 ± 2°C resulted in a slight reduction of conidial viability (≈3%). Microencapsulated conidia have been subjected to storage tests with and without the addition of silica gel capsules at various temperatures. Results show that survival is inversely related to storage temperatures and residual moisture levels of the spray dried powders. The highest survival rates were observed at moisture contents of 3% and a temperature of 10°C. Moreover, production characteristics like entrapment efficacy, shape and size were investigated. Furthermore, the composition of the carrier matrix was optimised to result in production yields of 25%. Results show that spray drying is a useful, economic encapsulation technology for aerial conidia of Beauveria brongniartii resulting in highly concentrated, spray dried powders of 92% viability.     

A Distinctive Pattern of Beauveria bassiana‐biotransformed Ginsenoside Products Triggers Mitochondria/FasL‐mediated Apoptosis in Colon Cancer Cells

Ginseng is one of the most commonly used adaptogens. Transformation into the minor ginsenosides produces compounds with more effective action. Beauveria bassiana, a teleomorph of Cordyceps bassiana, is a highly efficient producer of mammalian steroids and produces large amounts of sugar‐utilizing enzymes. However, the fermentation of steroid glycosides in ginseng with B. bassiana has never been studied. Thus, we evaluated the bioconversion of the major ginsenosides in white ginseng by B. bassiana. Interestingly, B. bassiana increased the total amount of protopanaxadiols and hydrolyzed Rb1 into minor ginsenosides, exhibiting high levels of Rd and Rg3, as well as moderate levels of Rb2 and Rc analyzed by high‐performance liquid chromatography coupled with evaporative light‐scattering detection. The β‐glucosidase activity was highly increased, which led to the selective elimination of sugar moiety at the 20‐C position of Rb1 to Rd, followed by Rg3. Rb2 and Rc accumulated because of the minimal activities of α‐L‐arabinopyranosidase and α‐L‐arabinofuranosidase, respectively. The fermentation product exerted dose‐dependent cytotoxicity in HCT‐15 cells, which are resistant to ginseng. The product, but not white ginseng, exhibited apoptotic effects via the Fas ligand and caspase 8/9. This study demonstrates for the first time that the B. bassiana‐fermented metabolites have potent apoptotic activity in colon cancer cells, linking to a therapeutic use. Copyright © 2015 John Wiley & Sons, Ltd.     

Endophytic colonization of entomopathogenic fungi increases plant disease resistance by changing the endophytic bacterial community

Various mechanisms are involved in plant disease resistance mediated by entomopathogenic fungi; however, the role of plant endophytic microbes in disease resistance is unknown. In the present study, we showed that the disease incidence of northern corn leaf blight caused by Exserohilum turcicum (Et) on maize was reduced significantly by soil inoculation with Beauveria bassiana (Bb). Meanwhile, B. bassiana colonization and E. turcicum infection increased the diversity and abundance and diversity of endophytic bacteria and fungi, respectively, while the abundance of endophytic bacterial of the Bb + Et treatment decreased significantly compared with that of Et treatment alone. However, Bb + Et treatment increased the relative abundance of plant beneficial bacteria significantly, for example, Burkholderia and Pseudomonas. Network analyses showed that the microbiome complexity increased after soil inoculation with B. bassiana. Taken together, these results revealed the potential mechanism by which entomopathogenic fungi exert biological control of maize leaf spot disease.