转基因金龟子绿僵菌获得的口服毒力评价与自噬基因ATG5对球孢白僵菌生防潜能的贡献
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摘要
金龟子绿僵菌(Metarhizium anisopliae)和球孢白僵菌(Beauveria bassiana)等昆虫病原真菌被广泛用于农林害虫防治,因而称为害虫虫生真菌。它们的杀虫机理无一例外地都是通过昆虫体壁侵入血腔而诱发昆虫病害和死亡,而不能通过口器摄入孢子的途径从消化道侵入寄主。虫生真菌的生防潜能既取决于侵染体对靶标害虫的侵染力或毒力,也取决于侵染体对夏季高温、阳光紫外辐射等胁迫因素的耐受力。本研究的目标,一是解决表达异源昆虫肠毒蛋白转基因工程菌的绝对口服毒力评价的技术难题,在本实验室前期工作基础之上进一步夯实虫生真菌通过遗传改良获得胃毒杀虫活性而研制新一代双途径侵染的安全高效广谱真菌杀虫剂的科学基础；二是解析球孢白僵菌自噬相关基因ATG5(BbATG5)的功能,首次探索细胞自噬可能对虫生真菌生长发育及生防潜能的影响,为虫生真菌细胞自噬的深入研究积累经验。主要结果总结如下。
表达昆虫肠毒蛋白的金龟子绿僵菌工程菌构建及其口服毒力测定以高效侵染蚜虫、飞虱、叶螨等刺吸式口器害虫但不能经体壁有效侵染斜纹夜蛾(Spodoptera litura)低龄幼虫的金龟子绿僵菌野生菌株Ma456为受体菌,运用根癌农杆菌(Agrobacterium tumefaciens)介导的遗传转化技术,将苏云金芽孢杆菌(Bacillus thuringiensis)营养生长期微量分泌的非晶体昆虫肠毒蛋白Vip3Aa1基因导入受体菌株中组成性表达,筛选获得一株生长产孢性状良好且稳定表达目标基因的工程菌株MaVipB01.将野生菌株与工程菌株的分生孢子配制成1×106、1×107和1×108个孢子/mL的悬液,在自动喷塔中将各菌株低、中、高浓度的孢子悬液依次等量(1mL)喷雾到直径约为13cm的甘蓝叶圆片上,空气干燥数分钟后供斜纹夜蛾一至四龄幼虫取食,每天按需更换相同喷菌处理的新鲜叶片。如此供食8天,每天观察记录试虫死亡率、存活幼虫的平均叶片取食量(cm2/头)及体重(mg/头),并根据不同喷菌处理叶片上的孢子沉降量(孢子数/mm2)估计各处理存活幼虫的孢子累计摄入量(孢子数／头),每浓度处理重复三次,每重复试虫28～40头,以综合评价表达Vip3Aa1毒蛋白的工程菌株对试虫的胃毒杀虫活性和口服毒力。
结果显示,一至四龄幼虫通过取食野生株高浓度喷菌叶片的摄入量分别达到2.28×105、1.88×106、5.22x106和8.11×106个孢子／头,而无论虫龄高低其死亡率始终与对照无异(5%左右)。所获少量虫尸经保温培养,也未见真菌病害致死的典型症状,而且同一虫龄的各处理间在幼虫的叶片取食量和体重方面均无显著差异。显然,野生株无法通过大量摄入孢子诱发试虫的实质性感染,与喷菌叶片接触也未能诱发孢子经体壁附着的实质性感染。与此形成鲜明对比的是,取食工程菌株喷菌叶片的各龄幼虫,其累计死亡率、叶片摄入量、体重及摄孢子入量,均随供食叶片的喷菌浓度而变化。其中,一龄幼虫取食高浓度喷菌叶片第2-4天的死亡率分别为44%、87%和97%,叶片取食量分别比对照降低69%、76%和83%,期间存活幼虫体重分别下降47%、57%和80%,相对应的孢子摄入量仅为2.8、4.2和5.2x103个／头。随着个体和虫龄的增大,带菌叶片取食量或孢子摄入量更大才能诱发与较小个体或较低虫龄相同的死亡率。取食高浓度喷菌叶片的四龄幼虫第6-8天摄入7.9、9.9和12.1×105个孢子后才分别死亡45%、62%和69%,期间存活幼虫的叶片取食量和体重分别下降～85%和84～91%。取食工程菌分生孢子而死亡的幼虫,无论虫龄高低,均表现典型的中毒症状,如个体显著变小、食量减少及行动迟缓。
工程菌株对各龄幼虫的生测数据经时间—30量一死亡率模型的拟合分析,获得随取食带菌叶片时间而变化的半致死浓度LC5o和随供食叶片喷菌浓度而变化的半致死时间LT50,即为口服毒力的定量指标。据此,叶片上工程菌孢子达到1293个/mm2,初孵一龄幼虫取食第2天即可死亡50%,取食第3-5天死亡一半所需的理论喷菌浓度分别降为103、28和8个孢子/mm2,事实上该龄幼虫取食高浓度处理叶片的第5天即全部死亡。二龄幼虫需要取食1500个/mm2的带菌叶片4天才死亡一半,第5-8天死亡一半的叶片带菌量分别降为537、152、83和45个孢子/mm2。杀三龄幼虫50%所需的喷菌浓度与二龄幼虫的接近,或许因为其增加的体重刚好抵销摄食量提高的影响。进入暴食期的四龄幼虫个体更大,取食带菌叶片第6-8天死亡一半所需的喷菌浓度分别为1760、730和410个孢子/cm2。
自噬因子BbATG5对球孢白僵菌生长发育及毒力的影响从球孢白僵菌野生株Bb2860中克隆获得自噬相关基因BbATG5的全长ORF(905bp)及其2382、1129bp的上下游侧翼序列。其ORF带一个95bp的内含子,编码由269个氨基酸组成的蛋白质,以单拷贝的形式存在于基因组中。将BbATG5的部分片段(49bp)用带bar标记基因的表达盒(942bp)进行替代而同源重组,构建靶基因敲除突变株ABbATG5。随后在敲除株中转入由BbATG5全长编码序列及其启动子(上游2.6kb)和磺酰脲抗性基因sur组成的表达盒进行靶标基因的拯救而构建回补株ΔBbATG5::BbATG5。
将野生株、敲除株和回补株的液培菌丝在含50mM LiCl的除氮察氏培养液Cz-N(氮饥饿胁迫)中诱导培养3h后,收集菌丝并用单丹磺酰戊二胺MDC染色。荧光显微镜观察显示,野生株和回补株的菌丝细胞中均有大量自噬体和溶酶体出现,而敲除株细胞中虽可见较明显的溶酶体,但自噬体稀有。透射电镜观察进一步证实诱导后的野生株和回补株菌丝细胞的液泡中存在大量自噬体,而同样诱导的敲除株细胞中少有自噬体。这些结果证实球孢白僵菌在氮饥饿条件下会发生细胞自噬现象,而BbATG5的缺失可阻碍自噬发生。
用流式细胞仪进行各菌株分生孢子的前向散射(FSc)和侧向散射(SSc)检测,发现敲除株分生孢子增大约20%,且伴随明显的形态畸变。在察氏培养基(CzA)上,野生株和回补株的孢子萌发率基本保持100%,而敲除株的却降低～20%。在无氮源的CzA-N平板和无营养的吐温-80水琼脂平板上,野生株和回补株的孢子萌发率分别降低60%和43%左右,而敲除株的分别仅有～20%和不足5%。将各菌株在萨氏培养基SDA、燕麦培养基OMA、CzA和CzA-N等不同营养的平板上培养7天,发现敲除株在CzA上的营养生长显著慢于野生株和回补株。而其在CzA-N上的营养生长则减慢50%左右,且敲除株菌落中的菌丝变得稀疏。在富营养的SDA和OMA上,敲除株的营养生长虽与野生株和回补株无明显差异,但其产孢量却大幅度下降80%左右。在富营养的培养液SDB中接种106个分生孢子/mL进行36h摇床培养,敲除株仅产0.52×107个芽生孢子/mL,比野生株和回补株少产芽生孢子94～97%。用斜纹夜蛾二龄幼虫对各菌株进行同一浓度(107个孢子/mL)等量孢子悬液的标准化喷菌生物测定,野生株、敲除株和回补株对试虫的LT50分别为5天、7天和5天,即敲除株的毒力降低40%。
结语以上结果表明,组成性表达异源肠毒蛋白的金龟子绿僵菌工程菌株,通过获得胃毒杀虫活性,不仅拓宽了真菌的寄主范围,而且使原本对斜纹夜蛾无侵染力的菌株的口服毒力达到可应用的水平。这充分说明利用现代生物技术将虫生真菌与苏云金芽孢杆菌丰富多样的Vip3A家族蛋白结合起来的科学意义和应用前景。另一方面,通过解析自噬相关基因BbATG5的生物学功能,证明BbATG5不仅是球孢白僵菌细胞正常自噬所必须的,而且参与调节营养吸收、孢子萌发、营养生长、产孢及毒力等一系列重要表型而影响真菌的生防潜能。这为深化虫生真菌细胞自噬现象及其意义的科学认识奠定了基础。
Entomopathogenic fungi, such as Beauveria bassianaandMetarhizium anisopliae, have been widely used as biocontrol agents against agricultural and forestry insect pests. Their killing action unexceptionally stems from the capability of their infecting target pests by integument penetration into host hemocoel for proliferation but rarely from the ingestion of infective cells by chewing insects. The fungal biocontrol potential depends not only on cell infectivity to target pests but also on cell tolerance to outdoor stresses, such as high temperature and solar UV irradiation during the summer of pest infestation. This study aimed at:(1) solving technical problems with the assessment of absolute oral virulence in transgenic fungiwith per os infectivity being acquired byexpressing a heterogeneous insect midgut-specific toxin; and (2) characterizing the function of the autophage-relating geneBbATG5in B. bassiana and probing possible effect of cell autophage on the fungal growth, development, and biocontrol potential. The results are summarized below.
Construction of a transgenic M. anisopliae strain expressing a midgut toxin and assessment of its oral virulence.As an insect midgut-specific toxin gene from Bacillus thuringiensis, the vegetative insecticidal proteigeneVip3Aa1was integrated into Ma456, a wild-type M. anisopliae strain that can kill sucking pests, such as aphids, planthoppers and spider mites, but is unable to infect Spodoptera litura neonatesby cuticlar infection, via Agrobacterium-mediated transformation. A transformant (MaVipB01) selected from several putative mutant colonies was confirmed expressing constitutively the target protein. To assess per osvirulenceacquired by the transformant, conidia preparations of parental Ma456and transgenic MaVipB01were suspended in0.02%Tween80respectively and stardardized to the low, median and high concentraions of1×106,1×107and1×108conidia per milliliter. For each strain, equal-volume (1ml) aliquots of the conidial suspensions (treatments) and aqueous0.02%Tween80(control) were separately sprayed onto cabbage leaf discs (～13cm diameter) in an automatic spray tower at the uniform working pressure of0.7kg/cm2. Air-dried for minutes, sprayed leaf discs were supplied with the larvaeof S. lituraInstars IV in large Petri dishes (15cm diameter) for their feeding. During8-day rearing, leaf disc residues were daily changed with fresh leaf discs sprayed as above in advance. Daily observations and meaurements in each dish included larval mortality, mean leaf consumption (cm2) and mean body weight (mg) per survivor, and the number of conidia ingested per larva in terms of consumed leaf area and conidial deposit (no. conidia per mm2) on the leaf disc sprayed. The bioassay of each strain against each instar was repeated three times and each replicate (dish) of the treatments including2840larvae.
Consequently, all the larvae showed consistently null responses to the wild-type strain althoughInstars ⅡⅤ ingested up to2.28×105,1.88×106,5.22x106and8.11×106conidia per capita from theleaf discs with high conidial deposits respectively. Larval mortalities fell in a range of-5%observed in the controls irrespective of different larval instars or fungal sprays. Notypical mycosis syndrome was observed from a very few cadavers in each treatment after incubation for several days under saturated humidity. Nor were significant differences in both leaf consumption and body weight of a given instar found between the fungal sprays. Thus, the wild-type strain failed to cause substantial infection by either conidial ingestion or cuticle attachment during the ingestion. In contrast, the larvae of each instar feeding on theleaf discs under differentsprays of transgenic conidia showed significant differences in mortality, leaf consumption and body weight. The first-instar larvae ingesting2.8,4.2and5.2x103conidia per capita suffered from the mortalities of44%,87%and97%after feeding the high-spray leaf discs for42days respectively, accompanied with the reductions of69%,76%and83%in leaf comsuption and of47%,57%and80%in survivor body weight. Similar mortalities of larger and older larvae required longer leaf feeding or more conidial ingestion. For Instar IV, for instance, cumulative ingestions of7.9,9.9and12.1×105conidia per capita on days68resulted in the mortalities of45%,62%and69%respectively and the reductions of-85%in leaf consumption and8491%in survivor body weight during the period. Interestingly, all the larvae died of the ingestion of transgenic conidia exhibited typical poisoning syndrome, such asbody shrunkage, much less leaf consumption and drastic body weight decrease.
Time-concentration-mortality modeling analysis of the transgenic strain against Instars IIVgenerated the trends of median lethal concentration (LC50) over thefeeding time of sprayed leaf discs and of median lethal time (LT50) over the conidial deposits on the leaf discs. Based on the trends for the oral virulence of the transgenic strain, ingestion of leaves with1293conidia/mm2could cause50%neonate mortality on day2and the sprays decreased drastically to103,28and8conidia/mm2for the same mortality if the feeding time was allowed for3to5days. For the second-instar larvae, LC50required the leaf spray of1500conidia/mm2for4-day feeding and decreased to537,152,83and45conidia/mm2on days58respectively. The oral virulence trend for Instar Ⅲ was close to that for Instar Ⅱ perhaps due to the deducted effects of theirlarger bodiesand more leaf consumption. The50%mortality of the fourth-instar larvae on days68required the leaf sprays of1760,730and410conidia/cm2for their feeding respectively.
Function of autophage-relating gene BbATG5and its contributions to the growth, asexual development and virulence of B. bassiana.Autophagy is a highly conserved process, representing the major eukaryotic degradative pathwayof cellular components. Autophagy-mediated recycling of cellular materials contributes to celldifferentiation, tissue remodelling and proper development. In fungi, autophagy is required fornormal growth and cell differentiation. The filamentous entomopathogen Beauveria bassiana and itsinvertebrate targets represent a unique model system with which to examine host-pathogeninteractions. The ATG5gene is one of17involved in autophagosome formation, and the B.bassiana homologue (BbATG5) with the coding sequence of905bp and the upstream and downstream flanking regions of2382and1129bp was identified. The role of autophagy in B. bassiana growth andvirulence was investigated via construction of a targeted gene knockout of BbATG5. The mutant ΔBbATG5displayed increased sensitivity to nutrient limitation, with decreased germination and growthas compared with the wild-type parent. Conidiation became severely defective and conidiaderived from the ΔBbATG5mutant were altered in morphology. Cell differentiation intoblastospores was also reduced by a great deal. Apart from the significant defects in growth and development,insect bioassays using the oriental leafworm moth, Spodoptera litura, indicated～40%decrease in virulence in the ΔBbATG5strain. All the phenotypic defects of the ΔBbATG5straincould be restored by integration of an intact copy of BbATG5into the delta mutant. These data suggest that unlikeseveral plant and animal pathogenic fungi, where ATG5is required for infection, in B. bassiana itis dispensable for pathogenesis despite its significant influence on the fungal virulence.
Conclusive remarks.The aquirement of per os infectivity by the transgenic strain MaVipB01expressing constitutively the insect midgut-specific toxin Vip3Aal broadens a spectrum of M. anisopliae against insect pests.The high oral virulence of the transformant to S. litura larvae which cannot be killed by the parental wild-type stain through normal cuticle infection indicates a potential for its commercial development and application. This highlights prospects for developing a newgeneration of green mycoinsecticidesby the engineering of various entomopathogenic fungi with diverse midgut-specific toxins in the Vip3A family fromB. thuringiensis. On the other hand, the functional characterization of BbATG5demostrates for the first time that BbATG5is not only essential for the normal autophage of B. bassiana but also involved in regulating the fungal nutrition uptake, conidial germination, vegetative growth, conidiation and virulence, thereby affecting the biocontrol potential of B. bassiana against insect pests.
表达昆虫肠毒蛋白的金龟子绿僵菌工程菌构建及其口服毒力测定以高效侵染蚜虫、飞虱、叶螨等刺吸式口器害虫但不能经体壁有效侵染斜纹夜蛾(Spodoptera litura)低龄幼虫的金龟子绿僵菌野生菌株Ma456为受体菌,运用根癌农杆菌(Agrobacterium tumefaciens)介导的遗传转化技术,将苏云金芽孢杆菌(Bacillus thuringiensis)营养生长期微量分泌的非晶体昆虫肠毒蛋白Vip3Aa1基因导入受体菌株中组成性表达,筛选获得一株生长产孢性状良好且稳定表达目标基因的工程菌株MaVipB01.将野生菌株与工程菌株的分生孢子配制成1×106、1×107和1×108个孢子/mL的悬液,在自动喷塔中将各菌株低、中、高浓度的孢子悬液依次等量(1mL)喷雾到直径约为13cm的甘蓝叶圆片上,空气干燥数分钟后供斜纹夜蛾一至四龄幼虫取食,每天按需更换相同喷菌处理的新鲜叶片。如此供食8天,每天观察记录试虫死亡率、存活幼虫的平均叶片取食量(cm2/头)及体重(mg/头),并根据不同喷菌处理叶片上的孢子沉降量(孢子数/mm2)估计各处理存活幼虫的孢子累计摄入量(孢子数／头),每浓度处理重复三次,每重复试虫28～40头,以综合评价表达Vip3Aa1毒蛋白的工程菌株对试虫的胃毒杀虫活性和口服毒力。
结果显示,一至四龄幼虫通过取食野生株高浓度喷菌叶片的摄入量分别达到2.28×105、1.88×106、5.22x106和8.11×106个孢子／头,而无论虫龄高低其死亡率始终与对照无异(5%左右)。所获少量虫尸经保温培养,也未见真菌病害致死的典型症状,而且同一虫龄的各处理间在幼虫的叶片取食量和体重方面均无显著差异。显然,野生株无法通过大量摄入孢子诱发试虫的实质性感染,与喷菌叶片接触也未能诱发孢子经体壁附着的实质性感染。与此形成鲜明对比的是,取食工程菌株喷菌叶片的各龄幼虫,其累计死亡率、叶片摄入量、体重及摄孢子入量,均随供食叶片的喷菌浓度而变化。其中,一龄幼虫取食高浓度喷菌叶片第2-4天的死亡率分别为44%、87%和97%,叶片取食量分别比对照降低69%、76%和83%,期间存活幼虫体重分别下降47%、57%和80%,相对应的孢子摄入量仅为2.8、4.2和5.2x103个／头。随着个体和虫龄的增大,带菌叶片取食量或孢子摄入量更大才能诱发与较小个体或较低虫龄相同的死亡率。取食高浓度喷菌叶片的四龄幼虫第6-8天摄入7.9、9.9和12.1×105个孢子后才分别死亡45%、62%和69%,期间存活幼虫的叶片取食量和体重分别下降～85%和84～91%。取食工程菌分生孢子而死亡的幼虫,无论虫龄高低,均表现典型的中毒症状,如个体显著变小、食量减少及行动迟缓。
工程菌株对各龄幼虫的生测数据经时间—30量一死亡率模型的拟合分析,获得随取食带菌叶片时间而变化的半致死浓度LC5o和随供食叶片喷菌浓度而变化的半致死时间LT50,即为口服毒力的定量指标。据此,叶片上工程菌孢子达到1293个/mm2,初孵一龄幼虫取食第2天即可死亡50%,取食第3-5天死亡一半所需的理论喷菌浓度分别降为103、28和8个孢子/mm2,事实上该龄幼虫取食高浓度处理叶片的第5天即全部死亡。二龄幼虫需要取食1500个/mm2的带菌叶片4天才死亡一半,第5-8天死亡一半的叶片带菌量分别降为537、152、83和45个孢子/mm2。杀三龄幼虫50%所需的喷菌浓度与二龄幼虫的接近,或许因为其增加的体重刚好抵销摄食量提高的影响。进入暴食期的四龄幼虫个体更大,取食带菌叶片第6-8天死亡一半所需的喷菌浓度分别为1760、730和410个孢子/cm2。
自噬因子BbATG5对球孢白僵菌生长发育及毒力的影响从球孢白僵菌野生株Bb2860中克隆获得自噬相关基因BbATG5的全长ORF(905bp)及其2382、1129bp的上下游侧翼序列。其ORF带一个95bp的内含子,编码由269个氨基酸组成的蛋白质,以单拷贝的形式存在于基因组中。将BbATG5的部分片段(49bp)用带bar标记基因的表达盒(942bp)进行替代而同源重组,构建靶基因敲除突变株ABbATG5。随后在敲除株中转入由BbATG5全长编码序列及其启动子(上游2.6kb)和磺酰脲抗性基因sur组成的表达盒进行靶标基因的拯救而构建回补株ΔBbATG5::BbATG5。
将野生株、敲除株和回补株的液培菌丝在含50mM LiCl的除氮察氏培养液Cz-N(氮饥饿胁迫)中诱导培养3h后,收集菌丝并用单丹磺酰戊二胺MDC染色。荧光显微镜观察显示,野生株和回补株的菌丝细胞中均有大量自噬体和溶酶体出现,而敲除株细胞中虽可见较明显的溶酶体,但自噬体稀有。透射电镜观察进一步证实诱导后的野生株和回补株菌丝细胞的液泡中存在大量自噬体,而同样诱导的敲除株细胞中少有自噬体。这些结果证实球孢白僵菌在氮饥饿条件下会发生细胞自噬现象,而BbATG5的缺失可阻碍自噬发生。
用流式细胞仪进行各菌株分生孢子的前向散射(FSc)和侧向散射(SSc)检测,发现敲除株分生孢子增大约20%,且伴随明显的形态畸变。在察氏培养基(CzA)上,野生株和回补株的孢子萌发率基本保持100%,而敲除株的却降低～20%。在无氮源的CzA-N平板和无营养的吐温-80水琼脂平板上,野生株和回补株的孢子萌发率分别降低60%和43%左右,而敲除株的分别仅有～20%和不足5%。将各菌株在萨氏培养基SDA、燕麦培养基OMA、CzA和CzA-N等不同营养的平板上培养7天,发现敲除株在CzA上的营养生长显著慢于野生株和回补株。而其在CzA-N上的营养生长则减慢50%左右,且敲除株菌落中的菌丝变得稀疏。在富营养的SDA和OMA上,敲除株的营养生长虽与野生株和回补株无明显差异,但其产孢量却大幅度下降80%左右。在富营养的培养液SDB中接种106个分生孢子/mL进行36h摇床培养,敲除株仅产0.52×107个芽生孢子/mL,比野生株和回补株少产芽生孢子94～97%。用斜纹夜蛾二龄幼虫对各菌株进行同一浓度(107个孢子/mL)等量孢子悬液的标准化喷菌生物测定,野生株、敲除株和回补株对试虫的LT50分别为5天、7天和5天,即敲除株的毒力降低40%。
结语以上结果表明,组成性表达异源肠毒蛋白的金龟子绿僵菌工程菌株,通过获得胃毒杀虫活性,不仅拓宽了真菌的寄主范围,而且使原本对斜纹夜蛾无侵染力的菌株的口服毒力达到可应用的水平。这充分说明利用现代生物技术将虫生真菌与苏云金芽孢杆菌丰富多样的Vip3A家族蛋白结合起来的科学意义和应用前景。另一方面,通过解析自噬相关基因BbATG5的生物学功能,证明BbATG5不仅是球孢白僵菌细胞正常自噬所必须的,而且参与调节营养吸收、孢子萌发、营养生长、产孢及毒力等一系列重要表型而影响真菌的生防潜能。这为深化虫生真菌细胞自噬现象及其意义的科学认识奠定了基础。
Entomopathogenic fungi, such as Beauveria bassianaandMetarhizium anisopliae, have been widely used as biocontrol agents against agricultural and forestry insect pests. Their killing action unexceptionally stems from the capability of their infecting target pests by integument penetration into host hemocoel for proliferation but rarely from the ingestion of infective cells by chewing insects. The fungal biocontrol potential depends not only on cell infectivity to target pests but also on cell tolerance to outdoor stresses, such as high temperature and solar UV irradiation during the summer of pest infestation. This study aimed at:(1) solving technical problems with the assessment of absolute oral virulence in transgenic fungiwith per os infectivity being acquired byexpressing a heterogeneous insect midgut-specific toxin; and (2) characterizing the function of the autophage-relating geneBbATG5in B. bassiana and probing possible effect of cell autophage on the fungal growth, development, and biocontrol potential. The results are summarized below.
Construction of a transgenic M. anisopliae strain expressing a midgut toxin and assessment of its oral virulence.As an insect midgut-specific toxin gene from Bacillus thuringiensis, the vegetative insecticidal proteigeneVip3Aa1was integrated into Ma456, a wild-type M. anisopliae strain that can kill sucking pests, such as aphids, planthoppers and spider mites, but is unable to infect Spodoptera litura neonatesby cuticlar infection, via Agrobacterium-mediated transformation. A transformant (MaVipB01) selected from several putative mutant colonies was confirmed expressing constitutively the target protein. To assess per osvirulenceacquired by the transformant, conidia preparations of parental Ma456and transgenic MaVipB01were suspended in0.02%Tween80respectively and stardardized to the low, median and high concentraions of1×106,1×107and1×108conidia per milliliter. For each strain, equal-volume (1ml) aliquots of the conidial suspensions (treatments) and aqueous0.02%Tween80(control) were separately sprayed onto cabbage leaf discs (～13cm diameter) in an automatic spray tower at the uniform working pressure of0.7kg/cm2. Air-dried for minutes, sprayed leaf discs were supplied with the larvaeof S. lituraInstars IV in large Petri dishes (15cm diameter) for their feeding. During8-day rearing, leaf disc residues were daily changed with fresh leaf discs sprayed as above in advance. Daily observations and meaurements in each dish included larval mortality, mean leaf consumption (cm2) and mean body weight (mg) per survivor, and the number of conidia ingested per larva in terms of consumed leaf area and conidial deposit (no. conidia per mm2) on the leaf disc sprayed. The bioassay of each strain against each instar was repeated three times and each replicate (dish) of the treatments including2840larvae.
Consequently, all the larvae showed consistently null responses to the wild-type strain althoughInstars ⅡⅤ ingested up to2.28×105,1.88×106,5.22x106and8.11×106conidia per capita from theleaf discs with high conidial deposits respectively. Larval mortalities fell in a range of-5%observed in the controls irrespective of different larval instars or fungal sprays. Notypical mycosis syndrome was observed from a very few cadavers in each treatment after incubation for several days under saturated humidity. Nor were significant differences in both leaf consumption and body weight of a given instar found between the fungal sprays. Thus, the wild-type strain failed to cause substantial infection by either conidial ingestion or cuticle attachment during the ingestion. In contrast, the larvae of each instar feeding on theleaf discs under differentsprays of transgenic conidia showed significant differences in mortality, leaf consumption and body weight. The first-instar larvae ingesting2.8,4.2and5.2x103conidia per capita suffered from the mortalities of44%,87%and97%after feeding the high-spray leaf discs for42days respectively, accompanied with the reductions of69%,76%and83%in leaf comsuption and of47%,57%and80%in survivor body weight. Similar mortalities of larger and older larvae required longer leaf feeding or more conidial ingestion. For Instar IV, for instance, cumulative ingestions of7.9,9.9and12.1×105conidia per capita on days68resulted in the mortalities of45%,62%and69%respectively and the reductions of-85%in leaf consumption and8491%in survivor body weight during the period. Interestingly, all the larvae died of the ingestion of transgenic conidia exhibited typical poisoning syndrome, such asbody shrunkage, much less leaf consumption and drastic body weight decrease.
Time-concentration-mortality modeling analysis of the transgenic strain against Instars IIVgenerated the trends of median lethal concentration (LC50) over thefeeding time of sprayed leaf discs and of median lethal time (LT50) over the conidial deposits on the leaf discs. Based on the trends for the oral virulence of the transgenic strain, ingestion of leaves with1293conidia/mm2could cause50%neonate mortality on day2and the sprays decreased drastically to103,28and8conidia/mm2for the same mortality if the feeding time was allowed for3to5days. For the second-instar larvae, LC50required the leaf spray of1500conidia/mm2for4-day feeding and decreased to537,152,83and45conidia/mm2on days58respectively. The oral virulence trend for Instar Ⅲ was close to that for Instar Ⅱ perhaps due to the deducted effects of theirlarger bodiesand more leaf consumption. The50%mortality of the fourth-instar larvae on days68required the leaf sprays of1760,730and410conidia/cm2for their feeding respectively.
Function of autophage-relating gene BbATG5and its contributions to the growth, asexual development and virulence of B. bassiana.Autophagy is a highly conserved process, representing the major eukaryotic degradative pathwayof cellular components. Autophagy-mediated recycling of cellular materials contributes to celldifferentiation, tissue remodelling and proper development. In fungi, autophagy is required fornormal growth and cell differentiation. The filamentous entomopathogen Beauveria bassiana and itsinvertebrate targets represent a unique model system with which to examine host-pathogeninteractions. The ATG5gene is one of17involved in autophagosome formation, and the B.bassiana homologue (BbATG5) with the coding sequence of905bp and the upstream and downstream flanking regions of2382and1129bp was identified. The role of autophagy in B. bassiana growth andvirulence was investigated via construction of a targeted gene knockout of BbATG5. The mutant ΔBbATG5displayed increased sensitivity to nutrient limitation, with decreased germination and growthas compared with the wild-type parent. Conidiation became severely defective and conidiaderived from the ΔBbATG5mutant were altered in morphology. Cell differentiation intoblastospores was also reduced by a great deal. Apart from the significant defects in growth and development,insect bioassays using the oriental leafworm moth, Spodoptera litura, indicated～40%decrease in virulence in the ΔBbATG5strain. All the phenotypic defects of the ΔBbATG5straincould be restored by integration of an intact copy of BbATG5into the delta mutant. These data suggest that unlikeseveral plant and animal pathogenic fungi, where ATG5is required for infection, in B. bassiana itis dispensable for pathogenesis despite its significant influence on the fungal virulence.
Conclusive remarks.The aquirement of per os infectivity by the transgenic strain MaVipB01expressing constitutively the insect midgut-specific toxin Vip3Aal broadens a spectrum of M. anisopliae against insect pests.The high oral virulence of the transformant to S. litura larvae which cannot be killed by the parental wild-type stain through normal cuticle infection indicates a potential for its commercial development and application. This highlights prospects for developing a newgeneration of green mycoinsecticidesby the engineering of various entomopathogenic fungi with diverse midgut-specific toxins in the Vip3A family fromB. thuringiensis. On the other hand, the functional characterization of BbATG5demostrates for the first time that BbATG5is not only essential for the normal autophage of B. bassiana but also involved in regulating the fungal nutrition uptake, conidial germination, vegetative growth, conidiation and virulence, thereby affecting the biocontrol potential of B. bassiana against insect pests.
引文
陈建武,唐丽霞,汤慕瑾(2002)苏云金芽孢杆菌vip3A基因的克隆、表达及活性分析.生物工程学报18：686-692.
陈守文,喻子牛(2003)微生物农药及产业化发展.化学与生物工程5：12-15.
樊美珍,李增智(1994)营养物和培养条件对虫生真菌附着胞形成的影响.安徽农业大学学报21：13-130.
方卫国,张永军,肖月华,马金成,杨星勇,裴炎(2003)金龟子绿僵菌羧基转运蛋白MaJEN1及其启动子的克隆与分析.遗传学报30：283-288.
方卫国(2003)昆虫病原真菌降解寄主体壁酶基因的克隆及球孢白僵菌高毒力重组菌株的获得.西南农业大学博士学位论文.
冯明光(1997)迁徙蚱蜢经口器摄入感染球孢白僵菌的时间与剂量效应.浙江农业大学学报23(5)：491-498.
冯明光(1998)生物杀虫剂与新的农业科技革命.植物保护21世纪展望暨第三届全国青年植物保护科技工作者学术研讨会论文集,北京：中国科学技术出版社,37-40.
郭慧芳,方继朝,罗伟杰,张海(2003)不同昆虫病毒对斜纹夜蛾和甜菜夜蛾的联合增效作用.中国生物防治19：23-26.
黄大防(2003)农业微生物基因工程研究与展望.农业生物技术学报11：111-114.
黄亚丽,潘玮,蒋细良,郭平,田云龙,李记鹏,朱昌雄(2007)根癌农杆菌介导丝状真菌遗传转化的研究进展.生物技术通报3：111-114.
蒋冬花,邓日强,庞义,龙綮新(1997)对斜纹夜蛾高毒力的Bt新分离株及其特性的研究.微生物学通报24：262-265.
李文华,王中康,殷幼平,夏玉先,彭国雄(2004)理化因子对白僵菌附着孢形成的影响.重庆大学学报(自然科学版)27：102-106.
李增智,韩宝瑜,樊美珍,汤坚(1998)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.应用生态学报9：503-510.
李增智(1999)菌物在害虫、植病和杂草治理中的现状和未来.中国生物防治15：35-40.
李增智,樊美珍(2000)真菌生物技术与真菌杀虫剂的发展.微生物农药及其产业化.北京：科学出版社,115-121.
李增智,樊美珍(2002)真菌生物技术与真菌杀虫剂的发展.微生物农药及其产业化.喻子牛主编,北京：科学出版社,469-487.
吕善运,王惠杰,阎建平,罗绍彬(1997)苏云金杆菌DL-5789的杀虫谱测定.中国生物防治13：72-75.
蒲蛰龙,李增智(1996)昆虫真菌学.合肥：安徽科技出版社,301-446.
僧明华,应盛华,冯明光(2009)模拟昆虫中肠环境对球孢白僵菌分生孢子的影响.浙江大学学报(农业与生命科学版)35(3)：261-265.
沈志成(2006a)一种杀虫基因及其用途.中华人民共和国专利200610049610.6.
石志琦,范永坚,王裕中(2002)天然化合物在农药中的应用研究.江苏农业学报18：241-245.
唐启义,冯明光(2007)实用统计分析及其DPS数据处理系统.北京：科学出版社.
喻子牛,柯云,刘子铎,孙明(2000)微生物农药在病虫害可持续控制中的应用及发展策略.喻子牛主编,微生物农药及其产业化.北京：科学出版社,1-12.
张爱文,邓春生,农向群(1991)虫生真菌育种方法的进展.微生物学杂志11：89-92.
张永军,彭国雄,方卫国,杨星勇,裴炎,王中康(2000)球孢白僵菌胞外蛋白酶及类枯草杆菌蛋白酶的诱导.应用与环境生物学报6：182-186.
Abellovich H (2004) Regulation of autophagy by the Target of Rapamycin(Tor) proteins, p.60-69 Jn Klionsky DJ (ed.), Autophagy. Landes.Bioscience, Georgeand town, TX.
Ashford TP, Porter KR (1962) Cytoplasmic components in hepatic cell lysosomes. J Cell Biol 12: 198-202.
Bampton ET, Goemans CG, Niranjan D, Mizushima N, Tolkocsky AM (2005) The dynamics of autophagy visualized in live cells:from autophagosome formation to fusion withendo/lysosomesAutophagy 1:23-26.
Bartoszewska M, Kiel JAKW, Bovenberg RAL, Veenhuis M, van der Klei IJ (2011) Autophagy deficiency promotes (3-lactam production in Penicillium chrysogenum. ApplEnviron Microb77: 1413-1422.
Beard CE, Court L, Boets A, Mourant R (2008) Unusually high frequency of genes encoding vegetative insecticidal proteins in an Australian Bacillus thuringiensiscollection.Curr Microbiol57:195-199.
Behie SW, Zelisko PM, Bidochka MJ (2012) Endophytic insect-parasitic fungi translocate nitrogen directly from insects to plants. Science 336:1576-1577.
Biederbick A, Kern HF, Elsasser HP (1995) Monodansylcadaverine(MDC)is a specific in vivo marker for autophagic va.cuoles.Eur J Cell Biol 66:3-14.
Bogo MR, Vainstein MH, Aragao FJL, Rech E, Schrank A (1996) High frequency gene conversion among benomyl resistance transformants in the entomopathogenic fungus Metarhizium anisopliae.FEMS Microbiol Lett 142:123-127.
Bogo MR, Rota CA, Pinto HJr, Ocampos M, Correa CT, Vainstein MH, Schrank A (1998) A chitinase encoding gene (chid gene) from the entomopathogen Metarhizium anisopliae:isolation and characterization of genomic and full length cDNA.Curr Microbiol 37:221-225.
Boucias DG, Pendland JC, Latge JP (1988) Nonspecificfactors involved in attachment of entonomopathogenic deuterromycetes to host insect cutice.ApplEnviron Microb54:1795-1805.
BradfischCA, Harmer SL (1990)ω-conotoxin giva and nifedipine inhibit the depolarizing action of the fungal metabolite, Desruxin B on muscle from the tobacco budworm(Heliothis virescens).Toxicon 28:1249-1254.
Brake J, Faust M, Stein J (2005) Evaluation of transgenic hybrid corn(VIP3A)in broiler chickens.Poultry Sci 84:503-512.
Brown JS, Aufauvre-Brown A, Holden DW (1998) Insertional mutagenesis of Aspergillus fumigatus.Mol Gen Genet 259:327-335.
Bundock P, Dulk-Ras A, Beijersbergen AGM, Hooykaas PJJ (1995) Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to saccharomyces cerevisiae.EMBOJ 14:3206-3214.
Bundock P, Hooykaas PJJ (1996) Integration of Agrobacterium tumefaciens T-DNA in the saccharomyces cerevisiae genome by illegitimate recombination. Proc Natl Acad Sci USA93:15272-15275.
Bundock P, Mroczek K, Winkler A, Steensma HY, Hooykaas PJJ (1999) T-DNA from Agrobacterium tumefaciens as efficient tool for gene targeting in Kluyveromyces lactis. Mol Gen Genet261: 115-121.
Campoy S, Perez F, Martin JF (2003) Stable transformants of the azaphilong pigment-producing Monascus purpureus obtained by protoplast transformation and Agrobacterium-media.ted DNA transfer.Curr Genet 43:447-452. Cantone FA, Vandenberg JD (1999) Genetic transformation and mutagenesis of the entomopathogenic
fungus Paecilomyces fumosoroseus.JInvertebr Pathol74:281-288. CharnleyAK, St Leger RJ (1991) The role of cuticle-degrading enzymes in fungal pathogenesis in
insects, p.267-287.InCole E and Hoch HC (eds.), Fungal spore disease initiation in plants and animals. Plenum Press, New York. CharnleyAK(1997) Entomopathogenic fungi and their role in the pest control.InWicklow DT,
Soderstrom B (eds.), The MycotaⅣ Environmental and Microbial Relationships, pp. 185-201.Springer-Verlag, New York. Chamley AK, Cobb B, Clarkson JM (1997) Towards the improvement of fungal insecticides. In Evans
HF (Ed.), Microbial insecticides:novelty or necessity?No.68 BCPC Symposium Proceedings, pp. 115-126. Chiang HL, Terlecky SR, Plant CP,Dice JF (1989) A role for a 70-kilodalton heat shock protein in
lysosomal degradation of intracellular proteins.Science 246:382-385. Cho EM, Liu L, Farmerie W, Keyhani NO (2006)EST analysis of cDNA libraries from the
entomopathogenic fungus Beauveria (Cordyceps) bassiana.I.Evidence for stage-specific gene expression in aerial conidia, in vitro blastospores, and submerged conidia. Microbiol-SGM 152: 2843-2855. Clarkson J, CharnleyAK(1996) New insights into the mechanisms of fungal pathogenesis in insects.
Trends microbiol 4:197-203. Codogno P, Meijer AJ (2004) Signalin pathways in mammalian autophagy.In Klionsky DJ
(ed.),Autophagy, pp.26-47. Landes Bioscience, Georgeand town, TX.
Combier JP, Melayah D, Raffier C (2003) Agrobacterium tumefaciens-mediated transformation as a too, for insertional mutagenesis in the symbiotic ectomycorrhizal fungus Hebeloma cylindrosporum. FEMSMicrobiol Lett 220:141-148.
Crotzer VL,Blum JS (2005) Autophagy and intracellular surveillance:Modulating MHC class II antigen presentation with stress.Proc Natl Acad Sci USA 102:7779-7780.
Daboussi MJ, Djeballi A, Gerlinger C, Blaiseau PL, Bouvier I,Cassan M, Lebrun MH, Parisot D,Brygoo Y (1989) Transformation of seven species of filamentous fungi using the nitrate reductase gene of Aspergillus nidulans.Curr Genet15:453-456.
de Groot MJA, Bundock P, Hooykaas PJJ, Beijersbergen AGM (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi.Nat Biotechnol 16:839-842.
Dong B, Liu XH, Lu JP, Zhang FS, Gao HM, Wang HK, Lin FC (2009) MgAtg9 trafficking in Magnaporthe oryzae. Autophagy 5:946-953.
Duarte RTD, Staats CC, Fungaro MHP, Schrank A,Vainsten MH, Furlaneto-Maia L, Nakamura CV, de Souza W, Furlaneto MC (2007) Development of simple and rapid Agrobacterium tumefaciens-mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var. acridum. Lett ApplMicrobiol 44:248-254.
Drif L, Brehelin M (1994) Purification and characterization of an agglutinin from the hemolymph of Locusta migratoria(Otrhoptera). Insect Biochem Molec 24:283-289.
Eskelinen EL (2004) Macroautophagy in mammalian cells.InPSaftig (ed.), Lysosomes.Landes Bioscience, Georgeand town, TX.
Estruch JJ, Warren GW (1996) Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects.P Natl Acad Sci USA93:5389-5394.
Fan Y, Fang W, Guo S, Pei X, Zhang Y, Xiao Y, Li D, Jin K, Bidochka MJ, Pei Y (2007) Increased insect virulence in Beauveria bassiana strains over-expressing an engineered chitinase. Appl Environ Microbiol 73:295-302.
Fang W,Zhang Y, Yang X, Zhang X, Duan H, Li Y, Pei Y (2004) Agrobacterium tumefaciem-mediated transformation of Beauveria bassiana using an herbicide resistance gene as a selection maker.J Invertebr Pathol85:18-24.
Fang W, Leng B, Xiao Y, Jin K, Ma J, Fan Y, Yang X, Zhang Y, Pei Y (2005) Cloning of Beauveria bassiana chitinase gene Bbchitl and its application to improve fungal strain virulence. Appl Environ Microb71:363-370.
Fang W, Pei Y, Bidochka MJ (2006) Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens. Can J Microbiol 52:623-626.
Fang WG, Feng J, Fan, YH, Zhang YJ, Bidochka MJ, St. Leger RJ, Pei Y (2009) Expressing a fusion protein with protease and chitinases activities increases the virulence of the insect pathogen Beauveria bassiana. J Invertebr Pathol 102:155-159.
Fargues RS, Maurette MT, Oliveros E, Riviere E, Latters A (1984) Chemical and photochemical reactivity in micelar media and microemulsions VII-Effect of the interface on the reactivity of excited states.Tetrahedron 40:2381-2384.
Feng MG, Poprawski TJ, Khachatourians GG (1994) Production, formulation and application of the entomopathogenic fungus Beauveria bassiana for insect control:current status. Biocontrol Sci Technol4:3-34.
Feng MG, Liu CL, Xu JH, Xu Q (1998) Modeling and biological implication of time-dose-mortality data for the entomophthoralean fungus, Zoophthora anhuiensis, on the green peach aphid Myzus persicae. J Invertebr Pathol72:246-251.
Feng MG, Chen B, Ying SH (2004) Trials of Beauveria bassiana, Paecilomyces fumosoroseus and imidacloprid for management of Trialeurodes vaporariorum (Homoptera:Aleyrodidae) on greenhouse grown lettuce. Biocontrol Sci Techn 14:531-544.
FinchamJRS (1989) Transformation in fungi.Microbiol Rev 53:148-170.
Fungaro MHP, Rech E, Mohlen GS, Vainstein MH, Pascon RC, de Queiroz MV, Pizzirani-Kleiner AA, de Azevedo JL (1995) Transformation of Aspergillus nidulans by microprojectile bombardment on intact conidia. FEMS Microbiol Lett 125:293-298.
Gao Q, Jin K, Ying SH, Zhang YJ, Xiao GH, Shang YF, Duan ZB, Hu X, Xie XQ Zhou G, Peng GX, Luo ZB, Huang W, Wang B, Fang WG, Wang SB, Zhong Y, Ma LJ, St Leger RJ, Zhao GP, Pei Y, Feng MG, Xia YX, Wang CS (2011) Genome sequencing and comparative transcriptomics of the model entomopathogenic fungi Metarhizium anisopliae and M. acridum. PLoS Genet 7:e1001264.
Godio RP, Fouces R, Gudina EJ (2004)Agrobacterium tumefaciens-mediated transformation of the antitumor clavaric acid-producing basidiomycete Hypholoma sublateritium.Curr Genet 46:287-294.
Gottar M, Gobert V, Matskevich A, Reichhart J, Wang C, Butt T, Belvin M, Hoffmann J, Ferrandon D (2006) Dual detection of fungal infections in Drosophila via recognition of glucans andsensing of virulence factors.Cell 127:1425-1437.
Gouka RJ, Gerk C, Hooykaas PJJ, Wouter VCT, de Groot MJA (1999) Transformation of Aspergillus awamori by Agrobacterium tumefaciens-mediated homologous recombination. Nat Biotechnol 17: 598-601.
Gurulingappa P, SwordGA, Murdoch G, Mcgee PA (2010) Colonization of crop plants by fungal entomopathogens and their effects on two insect pests when in planta. Biol Control 55:34-41.
Hajek AE, St.Leger RJ (1994) Interactions between fungal pathogenesis and insect host. Annu Rev Entomol 39:293-322.
Hanada T, Noda NN, Satomi Y, Ichimura Y, Fujioka Y, Takao T, Inagaki F, Ohsumi Y (2007) The Atgl2-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy. J Biol Chem 282:37298-37302.
Hanif M, Pardo AG, Gorfer M (2002) T-DNA transfer and integration in the ectomycorrhizal fungus Suillus bovinus using hygromycin B as a selectable marker.Curr Genet 41:183-188.
Heinemann JA, Spragnw JR, George F (1989) Bacterial conjugative plasmid mobilize DNA transfer between bacteria and yeast.Nature 340:205-209.
Holder DH, Keyhani NO (2005) Adhesion of the entomopathogenic fungus Beauveria (Cordyceps) bassiana to substrata. Appl Environ Microbiol71:5260-5266.
Hosmer DW, Lemeshow S (1989) Applied logistic regression. New York:Wiley.
Ichimura Y, Kirisako T, Satomi Y, Shimonishi Y, Ishihara N, Mizushima N, Tanida I, Kominami E,Ohsuni M, Noda T, Ohsumi Y (2000) A ubiquitin-like system mediates protein lipidation.Nature 408:488-492.
Inglis PW, Aragao FJL, Frazao H, Magalhaes BP, Valadares-Inglis MC (2000) Biolistic cotransformation of Metarhizium anisopliae var. acridum strain CG423 with green fuorescent protein and resistance to glufosinate ammonium. FEMS Microbiol Lett 191:249-254.
Jiang Q, YingSH, Feng MG (2007) Enhanced frequency of Beauveria bassiana blastospore transformation by restriction enzyme-mediated integration and electroporation.J Microbiol Meth 69: 512-517.
Jin K, Zhang Y, Fang W, Luo Z, Zhou Y, Pei Y (2010) Carboxylate transporter gene JEN1 from the entomopathogenic fubgus Beauveria bassiana is involved in conidiation and virulence. Appl Environ Microbiol 76:254-263.
Jin SF, Feng MG, Chen JQ (2008) Selection of global Metarhizium isolates for the control of the rice pest Nilaparvata lugens (Homoptera:Delphacidae). Pest Manag Sci 64:1008-1014.
Jin SF, Feng MG, Ying SH, Mu WJ, Chen JQ (2011) Evaluation of alternative rice planthopper control by the combined action of oil-formulated Metarhizium anisopliae and low-rate buprofezin.Pest Manag Sci67:36-43.
Kabeya Y, Kamada Y, Baba M, Takikawa H, Sasaki M, Ohsumi Y (2005) Atg17 functions in cooperation with Atgl and Atgl3 in yeast autophagy.MolBiolCell16:2544-2553.
Kamada Y, Funakoshi T, Shintani T, Nagano K, Ohsumi M, Ohsumi Y (2000) Tor-mediated induction of autophagy via an Apg1 protein kinase complex. J Cell Biol 150:1507-1513.
Kaur G, Padmaja V (2008) Evaluation of Beauveria bassiana isolates for virulence against Spodoptera litura (Fab.) (Lepidoptera:Noctuidae) and their characterization by RAPD-PCR. Afr J of Microbiol Res 2:299-307.
Kikuma T, Kitamoto K (2011) Analysis of autophagy in Aspergillus oryzae by disruption of Aoatgl3, Aoatg4, and Aoatgl5 genes. FEMS Microbiol Lett 316:61-69.
Kim J, Klionsky DJ (2000) Autophagy,cytoplasm-to-vacuole targeting pathway, and pexophagy in yeast and mammalian cells Annu Rev Biochem 69:303-342.
Kirkland BH, Westwood GS, Keyhani NO (2004) Pathogenicity of entomopathogenic fungi Beauveria bassiana and Metharhizium anisopliae to Ixodidaetick species Dermacentor variabilisRhipicephalus sanguineus, and Ixodes scapularis. JMed Entomol 41:705-711.
Klionsky DJ, Ohsumi Y (1999) Vacuolar import of proteins and organelles from the cytoplasm.Annu Rev Cell Dev Biol 15:1-32.
Klionsky DJ (2005) The molecular machinery of autophagy:unanswered questions. J Cell Sci 118:7-18.
Klionsky DJ (2007) Autophagy:from phenomenology to molecular understanding in less than a decade.Nat Rev Mol Cell Biol 8:931-937.
Klionsky DJ, Cuervo AM, Seglen PO (2007) Methods for monitoring autophagy from yeast to human. Autophagy 3:181-206.
Koukaki M, Giannoutsou E, Karagouni A, Diallinas G (2003) A novel improved method for Aspergillus nidulans transformation.J MicrobiolMeth 55:687-695.
Krappmann S (2006) Tools to study molecular mechanisms of Aspergillus pathogenicity. Trends Microbiol14:356-364.
Kruszewslca JS (1999) Heterologous expression of genes in filamentous fungiActaBiochimPol 46: 181-195.
Kuma A, Mizushima N, Ishihara N, Ohsumi Y (2002) Formation of the-350-kDa Apgl2-Apg5-Apg16 multimeric complex, mediated by Apg16 Oligomerization, is essential for autophagy in yeast. J Biol Chem 277:18619-18625.
Latge JP, Sampedro L, Brey P, Diaquin M (1987) Aggressiveness of Conidiobolus obscurus against the pea aphid-influence of cuticular extracts on ballistospore germination of aggressive and non-aggressive steains. J Gen Microbiol 138:1987-1997.
Lee MK, Walters FS, Hart H, Palekar N, Chen JS (2003) The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of CrylAb δ-endotoxin.Appl Environ Microbiol 69:4648-4657.
Lee MK, P Miles, J Chen (2006) Brush border membrane binding properties of Bacillus thuringiensis Vip3A toxin to Heliothis virescens and Helicoverpa zea midguts. Biochem Biophys Res Commun 339:1043-1047.
Leclerque A, Wan H, Abschutz A, Chen S, Mitina GV,Zimmermann G, Schairer HU (2004) Agrobacterium-mediated insertional mutagenesis (AIM) of the entomopathogenic fungus Beauveria bassiana.Curr Genet 45:111-119.
Levine B, Klionsky DJ (2004) Development by self-digestion:molecular mechanisms and biological functions of autophagy. Dev Cell 6:463-477.
Lewis MW, Robalino IV, Keyhani NO (2009) Uptake of the fluorescent probe FM4-64 by hyphae and hemolymph-derived in vivo hyphal bodies of the entomopathogenic fungus Beauveria bassiana. Microbiol-SGM155:3110-3120.
Li C, Xu N, Huang X, Wang W, Cheng J, Wu K, Shen Z (2007) Bacillus thuringiensis Vip3 mutant proteins:insecticidal activity and trypsin sensitivity. Biocontrol Sci Technol 17:699-708.
Liu Q, Ying SH, Feng MG (2011) Characterization of Beauveria bassiana neutral trehalase (BbNTH1) and recognition of crucial stress-responsive elements to control its expression in response to multiple stresses. Microbiol Res 166:282-293.
Liu TB, Liu XH, Lu JP, Zhang L, Min H, Lin FC (2010)The cysteine protease MoAtg4 interacts with MoAtg8 and is required for differentiation and pathogenesis in Magnaporthe oryzae. Autophagy6: 74-85.
Liu XH, Lu JP, Zhang L, Dong B, Min H, Lin FC (2007)Involvement of aMagnaporthe grisea serine/threonine kinase gene, MgATG1, in appressorium turgor andpathogenesis. Eukaryot Cell6: 997-1005.
Liu XH, Yang J, He RL, Lu JP, Zhang CL, Lu SL, Lin FC (2011)An autophagy gene, TrATG5, affects conidiospore differentiation in Trichoderma reesei. Res Microbiol162:756-763.
Llewellyn DJ, Mares CL, Fitt GP (2007) Field performance and seasonal changes in the efficacy against Helicoverpa armigera (Hubner) of transgenic cotton expressing the insecticidal protein vip3A. AgrForest Entomol 9:93-101.
Lu D, Pava-Ripoll M, Li Z,Wang C (2008) Insecticidal evaluation of Beauveria bassiana engineered to express a scorpin neurotoxin and a cuticle degrading protease.Appl Microbiol Biot 81:515-522.
Lu JP, Liu XH, Feng XX, Min H, Lin FC (2009)An autophagy gene, MgATG5, is required for cell differentiation and pathogenesis in Magnaporthe oryzae. Curr Genet55:461-473.
Luo XD, Keyhani NO, Yu XD, He ZJ, Luo ZB, Pei Y, Zhang YJ (2012) The MAP kinase Bbslt2 controls growth, conidiation, cell wall integrity, and virulence in the insect pathogenic fungus Beauveria bassiana. Fungal Genet Biol 49:544-555.
Manczinger L, Komonyi O, Antal Z and Rerenczy L (1997)A method for high-frequency transformation of Trichoderma viride.J Microbiol Meth 29:207-210.
Martinez-Vicente M, Cuervo AM (2007) Autophagy and neurodegeneration:when the cleaning crew goes on strike.Lancet Neurol 6:352-361.
Massey AC, Zhang C, Cuervo AM (2006) Chaperone-mediated autophagy in aging and disease. Curr Top Dev Biol 73:205-235.
Meyer V, Mueller D, Strowing T, Stahl U (2003) Comparison of different transformation methods for Aspergillus giganteus.Curr Genet 43:371-377.
McCoy CW, Samson RA, Boucias DG (1988)Entomogenous fungi. InIgnoffo CM, MandoreNB (eds.), CRC Handbook of Natural Pesticide, Vol V, Microbial Insecticides, Park A, pp.151-236.CRC press.Inc, Boca raton, Florida.
Michielse CB, Hooykaas PJJ, van den Hondel CAMJJ, Ram AFJ (2008)Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori. Nat Protoc3:1671-1678.
Milne R, Liu YH, Gauthier D, van Frankenhuyzen K (2008) Purification of Vip3Aal from Bacillus thuringiensis HD-1 and its contribution to toxicity of HD-1 to spruce budworm (Choristoneura fumiferana) and gypsy moth (Lymantria dispar) (Lepidoptera). J Invertebr Pathol 99:166-172.
MishraNC, Tatum EL (1973) Non-mendelian inheritance to DNA-induced inositol independence in Neurospora crassa.ProcNatlAcaSciUSA70:3875-3879.
Mizushima N, Yamamoto A, Hatano M, Kobayashi Y, Kabeya Y, Suzuki K, Tokuhisa T, Ohsumi Y, Yoshimori T (2001) Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells J CellBiol 152:657-667.
Mizushima N, Yamamoto A,Matsui M, Yoshimori T,Ohsumi Y (2004) In vivo analysis of antophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker.MolBiolCell 15:1101-1111.
Mizushima N (2007) Autophagy:process and function.Gene Dev 21:2861-2873.
Mullins ED, Chen X, Romaine P, Raina R, Geiser DM, Kang S (2001) Agrobacterium-mediated transformation of Fusarium oxysporum:an efficient tool for insertional mutagenesis and gene transfer.Phytopathology 91:173-180.
Murio M, Shiragami N, Takasurki A (1994) Destruxin B, a specific and readily reversible inhibitor if vacuolar-type H+-translocating ATPase. Biochem Biophys Res Commun 205:1358-1365.
Nadal M, Gold SE (2010) The autophagy genes ATG8 and ATG1 affect morphogenesis and pathogenicity in Ustilago maydis. Mol Plant Patho;11:463-478.
Noda T, Suzuki K, Ohsumi Y (2002) Yeast autophagosomes:de novo formation of a membrane structure.TrendsCellBiol 12:231-235.
Nowierski RM, Zeng Z, Jaronski S, Delgado F, Swearingen W (1996) Analysis and modeling of time-does-mortality of Melanoplus sanguinipes, Locusta migratoria migratorioides, and Schistocerca gregaria (Orthoptera:Acrididae) from Beauveria, Merarhizium, Paecilomyces isolates from Madagascar. J Invertebr Pathol 67:236-252.
Niemann A, Baltes J, Elsasser HP (2001) Fluorescence properties and staining behavior of monodansylpentane, a structural homologue of the lysosomotropic agent monodansylcadaverine. J Histochem Cytochem49:177-185.
Ohsumi Y (2001) Molecular dissection of autophagy:two ubiquitin-like systems.NatRevMolCellBiol 2:211-216.
Pal S, St Leger RJ, Wu LP (2007) Fungal peptide Destruxin A plays a specific role in suppressing the innate response in Drosophila melanogasterJBiol Chem 282:8969-8977.
Park Y, Kim W, Kim AY, Choi HJ, Choi JK, Park NS, Koh EK, Seo J, Koh YH (2011) Normal prion protein in Drosophila enhances the toxicity of pathogenic polyglutamine proteins and alters susceptibility to oxidative and autophagy signaling modulators. Biochem Biophys Res Commun404: 638-645.
Pava-Ripoll M, Posada F, Momen B, Wang C, St Leger RJ (2008) Increased pathogenicity against coffee berry borer, Hypothenemus hampei (Coleoptera:Curculionidae) by Metarhizium anisopliae expressing the scorpion toxin(AaIT)geneJ Invertebr Pathol 99:220-226.
Pendland JC, Heath M, Boucias DG (1988) Function of a galactose-binding lectin from Spodoptera exigua larval hemolymph:opsonization of blastospores from entomogeneous hyphomycetes. J Insect Physiol 34:533-540.
Pendland J, Hung SY, Boucias DG (1993) Evasion of host defense by in vivo produced protoplast-like cells of the insect mycopathogen Beauveria bassiana. J Bacterial175:5962-5969.
Pendland JC, Boucias DG (1996) Phagocytosis of lectin-opsonized fungal cells and endocytosis of the ligand by insect Spodoptera esigua granular hemocytes:an ultra structural and immunocyto-chemical study.Cell Tissue Res 285:57-67.
Peng D, Chen S, Ruan L, Li L, Yu Z, Sun M (2006) Safety assessment of transgenic Bacillus thuringiensis with VIP insecticidal protein gene by feeding studies. Food Chem Toxicol 45: 1179-1185.
Petiot A, Pattingre S, Arico S, Meley D, Codogno P (2002) Diversity of signaling controls of macroautophagy in mammalian cells. Cell Struct Funct 27:431-441.
Pinan-Lucarre B, Balguerie A, Clave C (2005) Accelerated cell death in Podospora autophagy mutants. Eukaryot Cell4:1765-1774.
Pollack JK, Harris SD, Marten MR (2009) Autophagy in filamentous fungi. Fungal Genet Biol46:1-8.
Puttikamonkul S, Willger SD, Grahl N, Perfect JR, Movahed N, Bothner B, Park S, Paderu P, Perlin DS, Cramer RAJr (2010) Trehalose 6-phosphate phosphatase is required for cell wall integrity and fungal virulence but not trehalose biosynthesis in the human fungal pathogen Aspergillus fumigates. Mol Microbiol77:891-911.
Pu XY, Feng MG, Shi CH (2005) Impact of three application methods on the field efficacy of a Beauveria bassiana-based mycoinsecticide against the false-eye leafhopper, Empoasca vitis (Homoptera:Cicadellidae) in tea canopy. Crop Protect 24:167-175.
Qin Y, Ying SH, Chen Y, Shen ZC, Feng MG (2010) Integration of insecticidal protein Vip3Aal into Beauveria bassiana enhances fungal virulence to Spodoptera litura larvae by cuticle and per os infection. Appl EnvironMicrobiol 76:4611-4618.
Ramasamy R, Nadarajah VD (2008) A preliminary study of the bioactivity of vegetative proteins extracted from Malaysian Bacillus thuringiensis isolates-Trop Biomed 25:64-74.
Reumann S, Voitsekhovskaja O, Lillo C (2010) From signal transduction to autophagy of plant cell organelles:lessons from yeast and mammals and plant-specific features. Protoplasma 247: 233-256.
Roberts DW (1981) Toxins of entomopathogenic fungi.InBurges, HD (ed.), Microbial Control of Pest and Plant Diseases, pp.441-464. Acad Press,New York.
Roetzer A, Gratz N, Kovarik P, Schiiller C (2010) Autophagy supports Candida glabrata survival during phagocytosis. Cell Microbiol12:199-216.
Romeis J, Meissle M, Bigler F (2006) Transgenic crops expressing Bacillus thuringiensis toxins and biological control. Nat Biotechnol 24:63-71.
Samuels RI, Unkles SE, Rajak IC (1995) Cuticle degrading proteases from insect moulting fluid and culture filtrates of entomopathogenic fungi.Comp Biochem Phys B 110:661-669.
Sanchez O, Navarro RE, Aguirre J (1998) Increased transformation frequency and tagging of developmental genes in Aspergillus nidulans by restriction enzyme-mediated integration (REMI)Mol Gen Genet 258:89-94.
Sandhu SS, Unkles SE, Rajak RC, Kinghorn JR (2001) Generation of benomyl resistant Beauveria bassiana strains and their infectivity against Helicoverpa armigera.Biocontrol Sci Technol 11:250-254.
Selvapandiyan A, Arora N (2001) Toxicity analysis of N-and C-terminus-deleted vegetative insecticidal protein from Bacillus thuringiensisAppl Environ.Microbiol 67:5855-5858.
Schmelzle T, Hall MN (2000) TOR,a central controller of cell growth.Cell 103:253-262.
Schnepf E, Crickmore N, Rie JV (1998) Bacillus thuringiensis and its pesticidal crystal proteins.Microbiol MolBoil Rev 62:775-806.
Smith RJ, Grula EA (1982) Toxic components on the larval surface of the corn earworm(Heliothis zea)and their effects in germination and growth of Beauveria bassiana.J Invertebr Pathol39:15-22.
Shan LT, Feng MG (2010) Evaluation of the biocontrol potential of various Metarhizium isolates against green peach aphid Myzus persicae (Homoptera:Aphididae). Pest Manag Sci 66:669-675.
Shintani T (2006) Cytoplasm to vacuole targeting pathway in yeast .Tanpakushitsu Kakusan Koso 51:1480-1483.
Shi WB, Zhang LL, Feng MG (2008a) Field trials of four formulations of Beauveria bassiana and Metarhizium anisoplae for control of cotton spider mites (Acari:Tetranychidae) in the Tarim Basin of China. Biol Control 45:48-55.
Shi WB, Zhang L, Feng MG (2008b) Time-concentration-mortality responses of carmine spider mite (Acari:Tetranychidae) females to three hypocrealean fungi as biocontrol agents. Biol Control 46: 495-501.
Stafford H (2000) Crown gall disease and Agrobacterium tumefaciens:A study of the history,present knowledge,missiong information,and impact on molecular genetics.Bot Rev 66:99-118.
St Leger RJ, CharnleyAK, Cooper RM (1986) Cuticle-degrading enzymes of entomopathogenic fungi: mechanisms of interaction between pathogen enzymes and insect cuticle. J Invertebr Pathol 47:295-302.
St Leger RJ, CharnleyAK, Cooper RM (1987) Characterzation of cuticle degrading protease produced by the entomopathogen Metarhizium anisopliae.Arch Biochem Biophys253:221-232.
St Leger RJ, Laccetti LB, Staples RC, Robert DW (1990a) Protein kinase in the entomopathogenic fungus, Metarhizium anisopliae. J Gen Microbiol 136:1401-1411.
St Leger RJ, Joshi L, Bidochka MJ, Roberts DW (1996b) Construction of an improved mycoinsecticide overexpressing a toxic protease. PNatlAcaSciUSA 93:6349-6354.
St Leger RJ, Joshi L, Bidochka MJ, Rizzo NW, Roberts DW (1996c) Characterization and Ultrastructural Localization of Chitinases from Metarhizium anisopliae, M.flavoviride, and Beauveria bassiana during Fungal Invasion of Host(Manduca sexta)Cuticle.ApplEnvironMicrobiol162:907-912.
St Leger RJ, Screen SE (2001) Prospects for stain improvement of fungal pathogens of insects and weeds. In Butt TM, Jackson C, Magan N (eds), Fungi as biocontrol agent, pp.219-237. CABI International.
Suzuki K, Ohsumi Y (2007) Molecular machinery of autophagosome formation in yeast, Saccaromyces cerevisiae. FEBSLett581:2156-2161.
Talloczy Z, Jiang W, Virgin Iv HW (2002) Regulation of starvation-and virus-induced autophagy by the eIF2alpha kinase signaling pathway.ProcNatlAcaSciUSA 99:190.
Takahara H, Tsuji G,Kubo Y,Yamamoto M (2004) Agrobacterium tumefaciens-mediated transformation as a tool for random mutagenesis of Colletotrichum trifolii.JGen Plant Pathol 70:93-96.
Tanida I, Tanida-Miyake E, Komatsu M, Ueno T, Kominami E (2002) Human Apg3p/Autlp homologue is an authentic E2 enzyme for multiple substrates, GATE-16,GABARAP, and MAP-LC3,and facilitates the conjugation of hApgl2p to hApg5p.J BiolChem 277:13739-13744.
Tanida I, Sou Y, Ezaki J, Minematsu-Ikeguchi N,Ueno T, Kominami E (2004) HsAtg4B/HsApg4B/Autophagin-1 cleaves the carboxyl termini of three human Atg8 homologues and delipidates microtubule-associatedprotein light chain3-and GABAA Receptor-associatedprotein-phospholipid conjugates.J.Biol Chem 279:36268-36276.
Tsuji G, Fujii S, Fujihara N (2003) Agrobacterium tumefaciens-mediated transformation for random insertional mutagenesis in Colletotrichum lagenarium.J Gen Plant Pathol 69:230-239.
Tsukada M, Ohsumi Y (1993) Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBSLett333:169-174.
Veneault-Fourrey C, Barooah M, Egan M, Wakley G, Talbot NJ (2006) Autophagic fungal cell death is necessary for infection by the rice blast fungus. Science312:580-583.
Vey A, Quoit JM, Vago C (1985) Immunodepressive effect of fungal toxins-inhibition of the reaction of multicellular encapsulation by the destruxins. Cr Acad Scilll-Vie300:647-651.
Vilcinskas A, Matha V, G6tz P (1997a) Inhibition of phagocytic activity of plasmatocytes isolated from Galleria mellonella by entomogenous fungi and their secondary metabolites.J Insect Physiol43:1149-1159.
Vilcinskas A, Matha V, Gotz P (1997b) Effects of the entimopathogenic fungus Metahizium anisopliae and its secondary metabolites in morphology and cytoskeleton of plasmatocytes isolates from the greater wax moth Galleria mellonella. J Insect Physiol 43:1149-1159.
Wanchoo A, Lewis MW, Keyhani NO (2009) Lectin mapping reveals stage-specific display of surface carbohydrates in in vitro and hemolymph derived cells of the entomopathogenic fungus Beauveria bassiana. Microbiol-GSM155:3121-3133.
Wang B, Kang QL, Lu YZ, Bai LQ, Wang CS (2012) Unveiling the biosynthetic puzzle of destruxins in Metarhizium species.ProcNatlAcaSciUSA 109:1287-1292.
Wang C, St Leger RJ (2005)Developmental and transcriptional responses to host and non host cuticles by the specific locust pathogen Metarhizium anisopliae var. acridum.Eukaryot Cell 4:937-947.
Wang CS, St Leger RJ (2006) A collagenous protective coat enables Metarhizium amisopliae to evade insect immune responses.ProcNatlAcaSciUSA 103:6647-6652.
Wang CS, St Leger RJ (2007a)The MAD1 adhesin of Metarhizium anisopliae links adhesion with blastospore production and virulence to insects, and the MAD2 adhesin enables attachment to plants.Eukaryot cell 6:808-816.
Wang CS, St Leger RJ (2007b)The Metarhizium anisopliae perilipin homolog MPL1 regulates lipid metabolism, appressorial turgor pressure, and virulence. JBiolChem 282:21110-21115.
Wang CS, St Leger RJ (2007c)A scorpion neurotoxin increases the potency of a fungal insecticide. Nat Biotechnol 25:1455-1456.
Wang ZL, Lu JD,Feng MG (2012) Primary roles of two dehydrogenases in the mannitol metabolism and multi-stress tolerance of entomopathogenic fungus Beauveria bassiana. Environm Microbiol 14: 2139-2150.
Wang ZL, Ying SH, Feng MG (2013a)Recognition of a core fragment of Beauveria bassiana hydrophobin gene promoter (Phyd1) and its special use in improving fungal biocontrol potential.Microb Biotechnol 6:27-35.
Wang ZL, Zhang LB, Ying SH, Feng MG (2013b) Catalases play differentiated roles in the adaptation of a fungal entomopathogen to environmental stresses. Environ Microbiol 15:409-418.
Warren G.W (1997) Vegetative insecticidal proteins:novel proteins for control of corn pests. InCarozziNB and Koziel M (eds.), Advances in insect control:the role of transgenic plants, pp.109-121. Taylors& Francis Ltd, London.
Wheeler MB, Stuart GS, Hapner KD (1993) Agglutinin mediated opsonization of fungal blastospores in Melanoplus diferentialis (Insecta). J Insect Physiol 39:477-483.
Xia Y, Clarkson JM, CharnleyAK (2001) Acid phosphatases of Metarhizium anisopliae during infection of the tobacco hornworm Manduca sexta.Arch Microbiol 176:427-434.
Xiao GH, Ying SH, Zheng P, Wang ZL, Zhang SW, Xie XQ, Shang YF, Zheng HJ, Zhou Y, St Leger RJ, Zhao GP, Wang CS, Feng MG (2012) Genomic perspectives on the evolution of fungal entomopathogenicity in Beauveria bassiana. Sci Rep 2:483.
Xie XQ, Wang J, Ying SH, Feng MG (2010) A new manganese superoxide dismutase identified from Beauveria bassiana enhances virulence and stress tolerance when overexpressed in the fungal pathogenAppl Microbiol Biotechnol 86:1543-1553.
Xie XQ, Li F, Ying SH, Feng MG (2012) Additive contributions of two manganese-cored superoxide dismutases (MnSODs) to antioxidation, UV tolerance and virulence of Beauveria bassiana. PLoS ONE 7:e30298.
Xie XQ, Guan Y, Ying SH, Feng MG (2013) Differentiated functions of Rasl and Ras2 proteins in regulating the germination, growth, conidiation, multi-stress tolerance and virulence of Beauveria bassiana. Environ Microbiol 15:447-462.
Ye SD, Dun YH, Feng MG (2005) Time and concentration dependent interactions of Beauveria bassiana with sublethal rates of imidacloprid against the aphid pests Macrosiphoniella sanborni and Myzus persicae. Ann Appl Biol 146:459-468.
Ying SH, Feng MG (2006) Novel blastospore-bassd transformation system for integration of phosphinothricin resistance and green fluorescence protein genes into Beauveria bassiana.Appl Microbiol Biotechnol 72:206-210.
Ying SH, Feng MG (2011a) A conidial protein (CP15) of Beauveria bassiana contributes to the conidial tolerance of the entomopathogenic fungus to thermal and oxidative stresses. Appl Microbiol Biotechnol 90:1711-1720.
Ying SH, Feng MG (2011b)Integration of Escherichia coli thioredoxin (trxA) into Beauveria bassiana enhances the fungal tolerance to the stresses of oxidation, heat and UV-B irradiation. Biol Control 59:255-260.
Yorimitsu T, Klionsky DJ (2005) Autophagy:molecular machinery for self-eating.Cell Death Differ 12:1542-1552.
Yu CG, Mullins MA, Warren GW, Koziel MG, Estruch JJ (1997) The Bacillus thuringiensis vegetative insecticidal protein Vip3A lyses midgut epithelial cells of susceptible insects.ApplEnvironMicrobiol63:532-536.
Zhang S, Fan Y, Xia YX, Keyhani NO (2010) Sulfonylurea resistance as a new selectable marker for the entomopathogenic fungus Beauveria bassiana. Appl Microbiol Biotechnol87:1151-1156.
Zhang YJ, Feng MG, Fan YH, Luo ZB, Xing YY, Wu D, Pei Y (2008) A cuticle-degrading protease (CDEP-1) of Beauveria bassiana enhances virulence. Biocontrol Sci Technol18:551-563.
Zhang YJ, Zhao JH, Fang WG, Zhang JQ, Luo ZB, Zhang M, Fan YH, Pei Y (2009) Mitogen-acticated protein kinase hogl in the entomopathogenic fungus Beauveria bassiana regulates environmental stress responses and virulence to insects. Appl Environ Microbiol 75:3787-3795.
Zhou G, Wang J, Qiu L, Feng MG (2012)A Group III histidine kinase (mhkl) upstream of high-osmolarity glycerol pathway regulates sporulation, multi-stress tolerance and virulence of Metarhizium robertsii, a fungal entomopathogen. Environm Microbiol 14:817-829.
Zwiers LH, De Waard MA (2001) Efficient Agrobacterium tumefaciens-mediated gene disruption in the phytopathogen Mycosphaerella graminicola.Curr Genet 39:388-393.
陈守文,喻子牛(2003)微生物农药及产业化发展.化学与生物工程5：12-15.
樊美珍,李增智(1994)营养物和培养条件对虫生真菌附着胞形成的影响.安徽农业大学学报21：13-130.
方卫国,张永军,肖月华,马金成,杨星勇,裴炎(2003)金龟子绿僵菌羧基转运蛋白MaJEN1及其启动子的克隆与分析.遗传学报30：283-288.
方卫国(2003)昆虫病原真菌降解寄主体壁酶基因的克隆及球孢白僵菌高毒力重组菌株的获得.西南农业大学博士学位论文.
冯明光(1997)迁徙蚱蜢经口器摄入感染球孢白僵菌的时间与剂量效应.浙江农业大学学报23(5)：491-498.
冯明光(1998)生物杀虫剂与新的农业科技革命.植物保护21世纪展望暨第三届全国青年植物保护科技工作者学术研讨会论文集,北京：中国科学技术出版社,37-40.
郭慧芳,方继朝,罗伟杰,张海(2003)不同昆虫病毒对斜纹夜蛾和甜菜夜蛾的联合增效作用.中国生物防治19：23-26.
黄大防(2003)农业微生物基因工程研究与展望.农业生物技术学报11：111-114.
黄亚丽,潘玮,蒋细良,郭平,田云龙,李记鹏,朱昌雄(2007)根癌农杆菌介导丝状真菌遗传转化的研究进展.生物技术通报3：111-114.
蒋冬花,邓日强,庞义,龙綮新(1997)对斜纹夜蛾高毒力的Bt新分离株及其特性的研究.微生物学通报24：262-265.
李文华,王中康,殷幼平,夏玉先,彭国雄(2004)理化因子对白僵菌附着孢形成的影响.重庆大学学报(自然科学版)27：102-106.
李增智,韩宝瑜,樊美珍,汤坚(1998)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.应用生态学报9：503-510.
李增智(1999)菌物在害虫、植病和杂草治理中的现状和未来.中国生物防治15：35-40.
李增智,樊美珍(2000)真菌生物技术与真菌杀虫剂的发展.微生物农药及其产业化.北京：科学出版社,115-121.
李增智,樊美珍(2002)真菌生物技术与真菌杀虫剂的发展.微生物农药及其产业化.喻子牛主编,北京：科学出版社,469-487.
吕善运,王惠杰,阎建平,罗绍彬(1997)苏云金杆菌DL-5789的杀虫谱测定.中国生物防治13：72-75.
蒲蛰龙,李增智(1996)昆虫真菌学.合肥：安徽科技出版社,301-446.
僧明华,应盛华,冯明光(2009)模拟昆虫中肠环境对球孢白僵菌分生孢子的影响.浙江大学学报(农业与生命科学版)35(3)：261-265.
沈志成(2006a)一种杀虫基因及其用途.中华人民共和国专利200610049610.6.
石志琦,范永坚,王裕中(2002)天然化合物在农药中的应用研究.江苏农业学报18：241-245.
唐启义,冯明光(2007)实用统计分析及其DPS数据处理系统.北京：科学出版社.
喻子牛,柯云,刘子铎,孙明(2000)微生物农药在病虫害可持续控制中的应用及发展策略.喻子牛主编,微生物农药及其产业化.北京：科学出版社,1-12.
张爱文,邓春生,农向群(1991)虫生真菌育种方法的进展.微生物学杂志11：89-92.
张永军,彭国雄,方卫国,杨星勇,裴炎,王中康(2000)球孢白僵菌胞外蛋白酶及类枯草杆菌蛋白酶的诱导.应用与环境生物学报6：182-186.
Abellovich H (2004) Regulation of autophagy by the Target of Rapamycin(Tor) proteins, p.60-69 Jn Klionsky DJ (ed.), Autophagy. Landes.Bioscience, Georgeand town, TX.
Ashford TP, Porter KR (1962) Cytoplasmic components in hepatic cell lysosomes. J Cell Biol 12: 198-202.
Bampton ET, Goemans CG, Niranjan D, Mizushima N, Tolkocsky AM (2005) The dynamics of autophagy visualized in live cells:from autophagosome formation to fusion withendo/lysosomesAutophagy 1:23-26.
Bartoszewska M, Kiel JAKW, Bovenberg RAL, Veenhuis M, van der Klei IJ (2011) Autophagy deficiency promotes (3-lactam production in Penicillium chrysogenum. ApplEnviron Microb77: 1413-1422.
Beard CE, Court L, Boets A, Mourant R (2008) Unusually high frequency of genes encoding vegetative insecticidal proteins in an Australian Bacillus thuringiensiscollection.Curr Microbiol57:195-199.
Behie SW, Zelisko PM, Bidochka MJ (2012) Endophytic insect-parasitic fungi translocate nitrogen directly from insects to plants. Science 336:1576-1577.
Biederbick A, Kern HF, Elsasser HP (1995) Monodansylcadaverine(MDC)is a specific in vivo marker for autophagic va.cuoles.Eur J Cell Biol 66:3-14.
Bogo MR, Vainstein MH, Aragao FJL, Rech E, Schrank A (1996) High frequency gene conversion among benomyl resistance transformants in the entomopathogenic fungus Metarhizium anisopliae.FEMS Microbiol Lett 142:123-127.
Bogo MR, Rota CA, Pinto HJr, Ocampos M, Correa CT, Vainstein MH, Schrank A (1998) A chitinase encoding gene (chid gene) from the entomopathogen Metarhizium anisopliae:isolation and characterization of genomic and full length cDNA.Curr Microbiol 37:221-225.
Boucias DG, Pendland JC, Latge JP (1988) Nonspecificfactors involved in attachment of entonomopathogenic deuterromycetes to host insect cutice.ApplEnviron Microb54:1795-1805.
BradfischCA, Harmer SL (1990)ω-conotoxin giva and nifedipine inhibit the depolarizing action of the fungal metabolite, Desruxin B on muscle from the tobacco budworm(Heliothis virescens).Toxicon 28:1249-1254.
Brake J, Faust M, Stein J (2005) Evaluation of transgenic hybrid corn(VIP3A)in broiler chickens.Poultry Sci 84:503-512.
Brown JS, Aufauvre-Brown A, Holden DW (1998) Insertional mutagenesis of Aspergillus fumigatus.Mol Gen Genet 259:327-335.
Bundock P, Dulk-Ras A, Beijersbergen AGM, Hooykaas PJJ (1995) Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to saccharomyces cerevisiae.EMBOJ 14:3206-3214.
Bundock P, Hooykaas PJJ (1996) Integration of Agrobacterium tumefaciens T-DNA in the saccharomyces cerevisiae genome by illegitimate recombination. Proc Natl Acad Sci USA93:15272-15275.
Bundock P, Mroczek K, Winkler A, Steensma HY, Hooykaas PJJ (1999) T-DNA from Agrobacterium tumefaciens as efficient tool for gene targeting in Kluyveromyces lactis. Mol Gen Genet261: 115-121.
Campoy S, Perez F, Martin JF (2003) Stable transformants of the azaphilong pigment-producing Monascus purpureus obtained by protoplast transformation and Agrobacterium-media.ted DNA transfer.Curr Genet 43:447-452. Cantone FA, Vandenberg JD (1999) Genetic transformation and mutagenesis of the entomopathogenic
fungus Paecilomyces fumosoroseus.JInvertebr Pathol74:281-288. CharnleyAK, St Leger RJ (1991) The role of cuticle-degrading enzymes in fungal pathogenesis in
insects, p.267-287.InCole E and Hoch HC (eds.), Fungal spore disease initiation in plants and animals. Plenum Press, New York. CharnleyAK(1997) Entomopathogenic fungi and their role in the pest control.InWicklow DT,
Soderstrom B (eds.), The MycotaⅣ Environmental and Microbial Relationships, pp. 185-201.Springer-Verlag, New York. Chamley AK, Cobb B, Clarkson JM (1997) Towards the improvement of fungal insecticides. In Evans
HF (Ed.), Microbial insecticides:novelty or necessity?No.68 BCPC Symposium Proceedings, pp. 115-126. Chiang HL, Terlecky SR, Plant CP,Dice JF (1989) A role for a 70-kilodalton heat shock protein in
lysosomal degradation of intracellular proteins.Science 246:382-385. Cho EM, Liu L, Farmerie W, Keyhani NO (2006)EST analysis of cDNA libraries from the
entomopathogenic fungus Beauveria (Cordyceps) bassiana.I.Evidence for stage-specific gene expression in aerial conidia, in vitro blastospores, and submerged conidia. Microbiol-SGM 152: 2843-2855. Clarkson J, CharnleyAK(1996) New insights into the mechanisms of fungal pathogenesis in insects.
Trends microbiol 4:197-203. Codogno P, Meijer AJ (2004) Signalin pathways in mammalian autophagy.In Klionsky DJ
(ed.),Autophagy, pp.26-47. Landes Bioscience, Georgeand town, TX.
Combier JP, Melayah D, Raffier C (2003) Agrobacterium tumefaciens-mediated transformation as a too, for insertional mutagenesis in the symbiotic ectomycorrhizal fungus Hebeloma cylindrosporum. FEMSMicrobiol Lett 220:141-148.
Crotzer VL,Blum JS (2005) Autophagy and intracellular surveillance:Modulating MHC class II antigen presentation with stress.Proc Natl Acad Sci USA 102:7779-7780.
Daboussi MJ, Djeballi A, Gerlinger C, Blaiseau PL, Bouvier I,Cassan M, Lebrun MH, Parisot D,Brygoo Y (1989) Transformation of seven species of filamentous fungi using the nitrate reductase gene of Aspergillus nidulans.Curr Genet15:453-456.
de Groot MJA, Bundock P, Hooykaas PJJ, Beijersbergen AGM (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi.Nat Biotechnol 16:839-842.
Dong B, Liu XH, Lu JP, Zhang FS, Gao HM, Wang HK, Lin FC (2009) MgAtg9 trafficking in Magnaporthe oryzae. Autophagy 5:946-953.
Duarte RTD, Staats CC, Fungaro MHP, Schrank A,Vainsten MH, Furlaneto-Maia L, Nakamura CV, de Souza W, Furlaneto MC (2007) Development of simple and rapid Agrobacterium tumefaciens-mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var. acridum. Lett ApplMicrobiol 44:248-254.
Drif L, Brehelin M (1994) Purification and characterization of an agglutinin from the hemolymph of Locusta migratoria(Otrhoptera). Insect Biochem Molec 24:283-289.
Eskelinen EL (2004) Macroautophagy in mammalian cells.InPSaftig (ed.), Lysosomes.Landes Bioscience, Georgeand town, TX.
Estruch JJ, Warren GW (1996) Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects.P Natl Acad Sci USA93:5389-5394.
Fan Y, Fang W, Guo S, Pei X, Zhang Y, Xiao Y, Li D, Jin K, Bidochka MJ, Pei Y (2007) Increased insect virulence in Beauveria bassiana strains over-expressing an engineered chitinase. Appl Environ Microbiol 73:295-302.
Fang W,Zhang Y, Yang X, Zhang X, Duan H, Li Y, Pei Y (2004) Agrobacterium tumefaciem-mediated transformation of Beauveria bassiana using an herbicide resistance gene as a selection maker.J Invertebr Pathol85:18-24.
Fang W, Leng B, Xiao Y, Jin K, Ma J, Fan Y, Yang X, Zhang Y, Pei Y (2005) Cloning of Beauveria bassiana chitinase gene Bbchitl and its application to improve fungal strain virulence. Appl Environ Microb71:363-370.
Fang W, Pei Y, Bidochka MJ (2006) Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens. Can J Microbiol 52:623-626.
Fang WG, Feng J, Fan, YH, Zhang YJ, Bidochka MJ, St. Leger RJ, Pei Y (2009) Expressing a fusion protein with protease and chitinases activities increases the virulence of the insect pathogen Beauveria bassiana. J Invertebr Pathol 102:155-159.
Fargues RS, Maurette MT, Oliveros E, Riviere E, Latters A (1984) Chemical and photochemical reactivity in micelar media and microemulsions VII-Effect of the interface on the reactivity of excited states.Tetrahedron 40:2381-2384.
Feng MG, Poprawski TJ, Khachatourians GG (1994) Production, formulation and application of the entomopathogenic fungus Beauveria bassiana for insect control:current status. Biocontrol Sci Technol4:3-34.
Feng MG, Liu CL, Xu JH, Xu Q (1998) Modeling and biological implication of time-dose-mortality data for the entomophthoralean fungus, Zoophthora anhuiensis, on the green peach aphid Myzus persicae. J Invertebr Pathol72:246-251.
Feng MG, Chen B, Ying SH (2004) Trials of Beauveria bassiana, Paecilomyces fumosoroseus and imidacloprid for management of Trialeurodes vaporariorum (Homoptera:Aleyrodidae) on greenhouse grown lettuce. Biocontrol Sci Techn 14:531-544.
FinchamJRS (1989) Transformation in fungi.Microbiol Rev 53:148-170.
Fungaro MHP, Rech E, Mohlen GS, Vainstein MH, Pascon RC, de Queiroz MV, Pizzirani-Kleiner AA, de Azevedo JL (1995) Transformation of Aspergillus nidulans by microprojectile bombardment on intact conidia. FEMS Microbiol Lett 125:293-298.
Gao Q, Jin K, Ying SH, Zhang YJ, Xiao GH, Shang YF, Duan ZB, Hu X, Xie XQ Zhou G, Peng GX, Luo ZB, Huang W, Wang B, Fang WG, Wang SB, Zhong Y, Ma LJ, St Leger RJ, Zhao GP, Pei Y, Feng MG, Xia YX, Wang CS (2011) Genome sequencing and comparative transcriptomics of the model entomopathogenic fungi Metarhizium anisopliae and M. acridum. PLoS Genet 7:e1001264.
Godio RP, Fouces R, Gudina EJ (2004)Agrobacterium tumefaciens-mediated transformation of the antitumor clavaric acid-producing basidiomycete Hypholoma sublateritium.Curr Genet 46:287-294.
Gottar M, Gobert V, Matskevich A, Reichhart J, Wang C, Butt T, Belvin M, Hoffmann J, Ferrandon D (2006) Dual detection of fungal infections in Drosophila via recognition of glucans andsensing of virulence factors.Cell 127:1425-1437.
Gouka RJ, Gerk C, Hooykaas PJJ, Wouter VCT, de Groot MJA (1999) Transformation of Aspergillus awamori by Agrobacterium tumefaciens-mediated homologous recombination. Nat Biotechnol 17: 598-601.
Gurulingappa P, SwordGA, Murdoch G, Mcgee PA (2010) Colonization of crop plants by fungal entomopathogens and their effects on two insect pests when in planta. Biol Control 55:34-41.
Hajek AE, St.Leger RJ (1994) Interactions between fungal pathogenesis and insect host. Annu Rev Entomol 39:293-322.
Hanada T, Noda NN, Satomi Y, Ichimura Y, Fujioka Y, Takao T, Inagaki F, Ohsumi Y (2007) The Atgl2-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy. J Biol Chem 282:37298-37302.
Hanif M, Pardo AG, Gorfer M (2002) T-DNA transfer and integration in the ectomycorrhizal fungus Suillus bovinus using hygromycin B as a selectable marker.Curr Genet 41:183-188.
Heinemann JA, Spragnw JR, George F (1989) Bacterial conjugative plasmid mobilize DNA transfer between bacteria and yeast.Nature 340:205-209.
Holder DH, Keyhani NO (2005) Adhesion of the entomopathogenic fungus Beauveria (Cordyceps) bassiana to substrata. Appl Environ Microbiol71:5260-5266.
Hosmer DW, Lemeshow S (1989) Applied logistic regression. New York:Wiley.
Ichimura Y, Kirisako T, Satomi Y, Shimonishi Y, Ishihara N, Mizushima N, Tanida I, Kominami E,Ohsuni M, Noda T, Ohsumi Y (2000) A ubiquitin-like system mediates protein lipidation.Nature 408:488-492.
Inglis PW, Aragao FJL, Frazao H, Magalhaes BP, Valadares-Inglis MC (2000) Biolistic cotransformation of Metarhizium anisopliae var. acridum strain CG423 with green fuorescent protein and resistance to glufosinate ammonium. FEMS Microbiol Lett 191:249-254.
Jiang Q, YingSH, Feng MG (2007) Enhanced frequency of Beauveria bassiana blastospore transformation by restriction enzyme-mediated integration and electroporation.J Microbiol Meth 69: 512-517.
Jin K, Zhang Y, Fang W, Luo Z, Zhou Y, Pei Y (2010) Carboxylate transporter gene JEN1 from the entomopathogenic fubgus Beauveria bassiana is involved in conidiation and virulence. Appl Environ Microbiol 76:254-263.
Jin SF, Feng MG, Chen JQ (2008) Selection of global Metarhizium isolates for the control of the rice pest Nilaparvata lugens (Homoptera:Delphacidae). Pest Manag Sci 64:1008-1014.
Jin SF, Feng MG, Ying SH, Mu WJ, Chen JQ (2011) Evaluation of alternative rice planthopper control by the combined action of oil-formulated Metarhizium anisopliae and low-rate buprofezin.Pest Manag Sci67:36-43.
Kabeya Y, Kamada Y, Baba M, Takikawa H, Sasaki M, Ohsumi Y (2005) Atg17 functions in cooperation with Atgl and Atgl3 in yeast autophagy.MolBiolCell16:2544-2553.
Kamada Y, Funakoshi T, Shintani T, Nagano K, Ohsumi M, Ohsumi Y (2000) Tor-mediated induction of autophagy via an Apg1 protein kinase complex. J Cell Biol 150:1507-1513.
Kaur G, Padmaja V (2008) Evaluation of Beauveria bassiana isolates for virulence against Spodoptera litura (Fab.) (Lepidoptera:Noctuidae) and their characterization by RAPD-PCR. Afr J of Microbiol Res 2:299-307.
Kikuma T, Kitamoto K (2011) Analysis of autophagy in Aspergillus oryzae by disruption of Aoatgl3, Aoatg4, and Aoatgl5 genes. FEMS Microbiol Lett 316:61-69.
Kim J, Klionsky DJ (2000) Autophagy,cytoplasm-to-vacuole targeting pathway, and pexophagy in yeast and mammalian cells Annu Rev Biochem 69:303-342.
Kirkland BH, Westwood GS, Keyhani NO (2004) Pathogenicity of entomopathogenic fungi Beauveria bassiana and Metharhizium anisopliae to Ixodidaetick species Dermacentor variabilisRhipicephalus sanguineus, and Ixodes scapularis. JMed Entomol 41:705-711.
Klionsky DJ, Ohsumi Y (1999) Vacuolar import of proteins and organelles from the cytoplasm.Annu Rev Cell Dev Biol 15:1-32.
Klionsky DJ (2005) The molecular machinery of autophagy:unanswered questions. J Cell Sci 118:7-18.
Klionsky DJ (2007) Autophagy:from phenomenology to molecular understanding in less than a decade.Nat Rev Mol Cell Biol 8:931-937.
Klionsky DJ, Cuervo AM, Seglen PO (2007) Methods for monitoring autophagy from yeast to human. Autophagy 3:181-206.
Koukaki M, Giannoutsou E, Karagouni A, Diallinas G (2003) A novel improved method for Aspergillus nidulans transformation.J MicrobiolMeth 55:687-695.
Krappmann S (2006) Tools to study molecular mechanisms of Aspergillus pathogenicity. Trends Microbiol14:356-364.
Kruszewslca JS (1999) Heterologous expression of genes in filamentous fungiActaBiochimPol 46: 181-195.
Kuma A, Mizushima N, Ishihara N, Ohsumi Y (2002) Formation of the-350-kDa Apgl2-Apg5-Apg16 multimeric complex, mediated by Apg16 Oligomerization, is essential for autophagy in yeast. J Biol Chem 277:18619-18625.
Latge JP, Sampedro L, Brey P, Diaquin M (1987) Aggressiveness of Conidiobolus obscurus against the pea aphid-influence of cuticular extracts on ballistospore germination of aggressive and non-aggressive steains. J Gen Microbiol 138:1987-1997.
Lee MK, Walters FS, Hart H, Palekar N, Chen JS (2003) The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of CrylAb δ-endotoxin.Appl Environ Microbiol 69:4648-4657.
Lee MK, P Miles, J Chen (2006) Brush border membrane binding properties of Bacillus thuringiensis Vip3A toxin to Heliothis virescens and Helicoverpa zea midguts. Biochem Biophys Res Commun 339:1043-1047.
Leclerque A, Wan H, Abschutz A, Chen S, Mitina GV,Zimmermann G, Schairer HU (2004) Agrobacterium-mediated insertional mutagenesis (AIM) of the entomopathogenic fungus Beauveria bassiana.Curr Genet 45:111-119.
Levine B, Klionsky DJ (2004) Development by self-digestion:molecular mechanisms and biological functions of autophagy. Dev Cell 6:463-477.
Lewis MW, Robalino IV, Keyhani NO (2009) Uptake of the fluorescent probe FM4-64 by hyphae and hemolymph-derived in vivo hyphal bodies of the entomopathogenic fungus Beauveria bassiana. Microbiol-SGM155:3110-3120.
Li C, Xu N, Huang X, Wang W, Cheng J, Wu K, Shen Z (2007) Bacillus thuringiensis Vip3 mutant proteins:insecticidal activity and trypsin sensitivity. Biocontrol Sci Technol 17:699-708.
Liu Q, Ying SH, Feng MG (2011) Characterization of Beauveria bassiana neutral trehalase (BbNTH1) and recognition of crucial stress-responsive elements to control its expression in response to multiple stresses. Microbiol Res 166:282-293.
Liu TB, Liu XH, Lu JP, Zhang L, Min H, Lin FC (2010)The cysteine protease MoAtg4 interacts with MoAtg8 and is required for differentiation and pathogenesis in Magnaporthe oryzae. Autophagy6: 74-85.
Liu XH, Lu JP, Zhang L, Dong B, Min H, Lin FC (2007)Involvement of aMagnaporthe grisea serine/threonine kinase gene, MgATG1, in appressorium turgor andpathogenesis. Eukaryot Cell6: 997-1005.
Liu XH, Yang J, He RL, Lu JP, Zhang CL, Lu SL, Lin FC (2011)An autophagy gene, TrATG5, affects conidiospore differentiation in Trichoderma reesei. Res Microbiol162:756-763.
Llewellyn DJ, Mares CL, Fitt GP (2007) Field performance and seasonal changes in the efficacy against Helicoverpa armigera (Hubner) of transgenic cotton expressing the insecticidal protein vip3A. AgrForest Entomol 9:93-101.
Lu D, Pava-Ripoll M, Li Z,Wang C (2008) Insecticidal evaluation of Beauveria bassiana engineered to express a scorpin neurotoxin and a cuticle degrading protease.Appl Microbiol Biot 81:515-522.
Lu JP, Liu XH, Feng XX, Min H, Lin FC (2009)An autophagy gene, MgATG5, is required for cell differentiation and pathogenesis in Magnaporthe oryzae. Curr Genet55:461-473.
Luo XD, Keyhani NO, Yu XD, He ZJ, Luo ZB, Pei Y, Zhang YJ (2012) The MAP kinase Bbslt2 controls growth, conidiation, cell wall integrity, and virulence in the insect pathogenic fungus Beauveria bassiana. Fungal Genet Biol 49:544-555.
Manczinger L, Komonyi O, Antal Z and Rerenczy L (1997)A method for high-frequency transformation of Trichoderma viride.J Microbiol Meth 29:207-210.
Martinez-Vicente M, Cuervo AM (2007) Autophagy and neurodegeneration:when the cleaning crew goes on strike.Lancet Neurol 6:352-361.
Massey AC, Zhang C, Cuervo AM (2006) Chaperone-mediated autophagy in aging and disease. Curr Top Dev Biol 73:205-235.
Meyer V, Mueller D, Strowing T, Stahl U (2003) Comparison of different transformation methods for Aspergillus giganteus.Curr Genet 43:371-377.
McCoy CW, Samson RA, Boucias DG (1988)Entomogenous fungi. InIgnoffo CM, MandoreNB (eds.), CRC Handbook of Natural Pesticide, Vol V, Microbial Insecticides, Park A, pp.151-236.CRC press.Inc, Boca raton, Florida.
Michielse CB, Hooykaas PJJ, van den Hondel CAMJJ, Ram AFJ (2008)Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori. Nat Protoc3:1671-1678.
Milne R, Liu YH, Gauthier D, van Frankenhuyzen K (2008) Purification of Vip3Aal from Bacillus thuringiensis HD-1 and its contribution to toxicity of HD-1 to spruce budworm (Choristoneura fumiferana) and gypsy moth (Lymantria dispar) (Lepidoptera). J Invertebr Pathol 99:166-172.
MishraNC, Tatum EL (1973) Non-mendelian inheritance to DNA-induced inositol independence in Neurospora crassa.ProcNatlAcaSciUSA70:3875-3879.
Mizushima N, Yamamoto A, Hatano M, Kobayashi Y, Kabeya Y, Suzuki K, Tokuhisa T, Ohsumi Y, Yoshimori T (2001) Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells J CellBiol 152:657-667.
Mizushima N, Yamamoto A,Matsui M, Yoshimori T,Ohsumi Y (2004) In vivo analysis of antophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker.MolBiolCell 15:1101-1111.
Mizushima N (2007) Autophagy:process and function.Gene Dev 21:2861-2873.
Mullins ED, Chen X, Romaine P, Raina R, Geiser DM, Kang S (2001) Agrobacterium-mediated transformation of Fusarium oxysporum:an efficient tool for insertional mutagenesis and gene transfer.Phytopathology 91:173-180.
Murio M, Shiragami N, Takasurki A (1994) Destruxin B, a specific and readily reversible inhibitor if vacuolar-type H+-translocating ATPase. Biochem Biophys Res Commun 205:1358-1365.
Nadal M, Gold SE (2010) The autophagy genes ATG8 and ATG1 affect morphogenesis and pathogenicity in Ustilago maydis. Mol Plant Patho;11:463-478.
Noda T, Suzuki K, Ohsumi Y (2002) Yeast autophagosomes:de novo formation of a membrane structure.TrendsCellBiol 12:231-235.
Nowierski RM, Zeng Z, Jaronski S, Delgado F, Swearingen W (1996) Analysis and modeling of time-does-mortality of Melanoplus sanguinipes, Locusta migratoria migratorioides, and Schistocerca gregaria (Orthoptera:Acrididae) from Beauveria, Merarhizium, Paecilomyces isolates from Madagascar. J Invertebr Pathol 67:236-252.
Niemann A, Baltes J, Elsasser HP (2001) Fluorescence properties and staining behavior of monodansylpentane, a structural homologue of the lysosomotropic agent monodansylcadaverine. J Histochem Cytochem49:177-185.
Ohsumi Y (2001) Molecular dissection of autophagy:two ubiquitin-like systems.NatRevMolCellBiol 2:211-216.
Pal S, St Leger RJ, Wu LP (2007) Fungal peptide Destruxin A plays a specific role in suppressing the innate response in Drosophila melanogasterJBiol Chem 282:8969-8977.
Park Y, Kim W, Kim AY, Choi HJ, Choi JK, Park NS, Koh EK, Seo J, Koh YH (2011) Normal prion protein in Drosophila enhances the toxicity of pathogenic polyglutamine proteins and alters susceptibility to oxidative and autophagy signaling modulators. Biochem Biophys Res Commun404: 638-645.
Pava-Ripoll M, Posada F, Momen B, Wang C, St Leger RJ (2008) Increased pathogenicity against coffee berry borer, Hypothenemus hampei (Coleoptera:Curculionidae) by Metarhizium anisopliae expressing the scorpion toxin(AaIT)geneJ Invertebr Pathol 99:220-226.
Pendland JC, Heath M, Boucias DG (1988) Function of a galactose-binding lectin from Spodoptera exigua larval hemolymph:opsonization of blastospores from entomogeneous hyphomycetes. J Insect Physiol 34:533-540.
Pendland J, Hung SY, Boucias DG (1993) Evasion of host defense by in vivo produced protoplast-like cells of the insect mycopathogen Beauveria bassiana. J Bacterial175:5962-5969.
Pendland JC, Boucias DG (1996) Phagocytosis of lectin-opsonized fungal cells and endocytosis of the ligand by insect Spodoptera esigua granular hemocytes:an ultra structural and immunocyto-chemical study.Cell Tissue Res 285:57-67.
Peng D, Chen S, Ruan L, Li L, Yu Z, Sun M (2006) Safety assessment of transgenic Bacillus thuringiensis with VIP insecticidal protein gene by feeding studies. Food Chem Toxicol 45: 1179-1185.
Petiot A, Pattingre S, Arico S, Meley D, Codogno P (2002) Diversity of signaling controls of macroautophagy in mammalian cells. Cell Struct Funct 27:431-441.
Pinan-Lucarre B, Balguerie A, Clave C (2005) Accelerated cell death in Podospora autophagy mutants. Eukaryot Cell4:1765-1774.
Pollack JK, Harris SD, Marten MR (2009) Autophagy in filamentous fungi. Fungal Genet Biol46:1-8.
Puttikamonkul S, Willger SD, Grahl N, Perfect JR, Movahed N, Bothner B, Park S, Paderu P, Perlin DS, Cramer RAJr (2010) Trehalose 6-phosphate phosphatase is required for cell wall integrity and fungal virulence but not trehalose biosynthesis in the human fungal pathogen Aspergillus fumigates. Mol Microbiol77:891-911.
Pu XY, Feng MG, Shi CH (2005) Impact of three application methods on the field efficacy of a Beauveria bassiana-based mycoinsecticide against the false-eye leafhopper, Empoasca vitis (Homoptera:Cicadellidae) in tea canopy. Crop Protect 24:167-175.
Qin Y, Ying SH, Chen Y, Shen ZC, Feng MG (2010) Integration of insecticidal protein Vip3Aal into Beauveria bassiana enhances fungal virulence to Spodoptera litura larvae by cuticle and per os infection. Appl EnvironMicrobiol 76:4611-4618.
Ramasamy R, Nadarajah VD (2008) A preliminary study of the bioactivity of vegetative proteins extracted from Malaysian Bacillus thuringiensis isolates-Trop Biomed 25:64-74.
Reumann S, Voitsekhovskaja O, Lillo C (2010) From signal transduction to autophagy of plant cell organelles:lessons from yeast and mammals and plant-specific features. Protoplasma 247: 233-256.
Roberts DW (1981) Toxins of entomopathogenic fungi.InBurges, HD (ed.), Microbial Control of Pest and Plant Diseases, pp.441-464. Acad Press,New York.
Roetzer A, Gratz N, Kovarik P, Schiiller C (2010) Autophagy supports Candida glabrata survival during phagocytosis. Cell Microbiol12:199-216.
Romeis J, Meissle M, Bigler F (2006) Transgenic crops expressing Bacillus thuringiensis toxins and biological control. Nat Biotechnol 24:63-71.
Samuels RI, Unkles SE, Rajak IC (1995) Cuticle degrading proteases from insect moulting fluid and culture filtrates of entomopathogenic fungi.Comp Biochem Phys B 110:661-669.
Sanchez O, Navarro RE, Aguirre J (1998) Increased transformation frequency and tagging of developmental genes in Aspergillus nidulans by restriction enzyme-mediated integration (REMI)Mol Gen Genet 258:89-94.
Sandhu SS, Unkles SE, Rajak RC, Kinghorn JR (2001) Generation of benomyl resistant Beauveria bassiana strains and their infectivity against Helicoverpa armigera.Biocontrol Sci Technol 11:250-254.
Selvapandiyan A, Arora N (2001) Toxicity analysis of N-and C-terminus-deleted vegetative insecticidal protein from Bacillus thuringiensisAppl Environ.Microbiol 67:5855-5858.
Schmelzle T, Hall MN (2000) TOR,a central controller of cell growth.Cell 103:253-262.
Schnepf E, Crickmore N, Rie JV (1998) Bacillus thuringiensis and its pesticidal crystal proteins.Microbiol MolBoil Rev 62:775-806.
Smith RJ, Grula EA (1982) Toxic components on the larval surface of the corn earworm(Heliothis zea)and their effects in germination and growth of Beauveria bassiana.J Invertebr Pathol39:15-22.
Shan LT, Feng MG (2010) Evaluation of the biocontrol potential of various Metarhizium isolates against green peach aphid Myzus persicae (Homoptera:Aphididae). Pest Manag Sci 66:669-675.
Shintani T (2006) Cytoplasm to vacuole targeting pathway in yeast .Tanpakushitsu Kakusan Koso 51:1480-1483.
Shi WB, Zhang LL, Feng MG (2008a) Field trials of four formulations of Beauveria bassiana and Metarhizium anisoplae for control of cotton spider mites (Acari:Tetranychidae) in the Tarim Basin of China. Biol Control 45:48-55.
Shi WB, Zhang L, Feng MG (2008b) Time-concentration-mortality responses of carmine spider mite (Acari:Tetranychidae) females to three hypocrealean fungi as biocontrol agents. Biol Control 46: 495-501.
Stafford H (2000) Crown gall disease and Agrobacterium tumefaciens:A study of the history,present knowledge,missiong information,and impact on molecular genetics.Bot Rev 66:99-118.
St Leger RJ, CharnleyAK, Cooper RM (1986) Cuticle-degrading enzymes of entomopathogenic fungi: mechanisms of interaction between pathogen enzymes and insect cuticle. J Invertebr Pathol 47:295-302.
St Leger RJ, CharnleyAK, Cooper RM (1987) Characterzation of cuticle degrading protease produced by the entomopathogen Metarhizium anisopliae.Arch Biochem Biophys253:221-232.
St Leger RJ, Laccetti LB, Staples RC, Robert DW (1990a) Protein kinase in the entomopathogenic fungus, Metarhizium anisopliae. J Gen Microbiol 136:1401-1411.
St Leger RJ, Joshi L, Bidochka MJ, Roberts DW (1996b) Construction of an improved mycoinsecticide overexpressing a toxic protease. PNatlAcaSciUSA 93:6349-6354.
St Leger RJ, Joshi L, Bidochka MJ, Rizzo NW, Roberts DW (1996c) Characterization and Ultrastructural Localization of Chitinases from Metarhizium anisopliae, M.flavoviride, and Beauveria bassiana during Fungal Invasion of Host(Manduca sexta)Cuticle.ApplEnvironMicrobiol162:907-912.
St Leger RJ, Screen SE (2001) Prospects for stain improvement of fungal pathogens of insects and weeds. In Butt TM, Jackson C, Magan N (eds), Fungi as biocontrol agent, pp.219-237. CABI International.
Suzuki K, Ohsumi Y (2007) Molecular machinery of autophagosome formation in yeast, Saccaromyces cerevisiae. FEBSLett581:2156-2161.
Talloczy Z, Jiang W, Virgin Iv HW (2002) Regulation of starvation-and virus-induced autophagy by the eIF2alpha kinase signaling pathway.ProcNatlAcaSciUSA 99:190.
Takahara H, Tsuji G,Kubo Y,Yamamoto M (2004) Agrobacterium tumefaciens-mediated transformation as a tool for random mutagenesis of Colletotrichum trifolii.JGen Plant Pathol 70:93-96.
Tanida I, Tanida-Miyake E, Komatsu M, Ueno T, Kominami E (2002) Human Apg3p/Autlp homologue is an authentic E2 enzyme for multiple substrates, GATE-16,GABARAP, and MAP-LC3,and facilitates the conjugation of hApgl2p to hApg5p.J BiolChem 277:13739-13744.
Tanida I, Sou Y, Ezaki J, Minematsu-Ikeguchi N,Ueno T, Kominami E (2004) HsAtg4B/HsApg4B/Autophagin-1 cleaves the carboxyl termini of three human Atg8 homologues and delipidates microtubule-associatedprotein light chain3-and GABAA Receptor-associatedprotein-phospholipid conjugates.J.Biol Chem 279:36268-36276.
Tsuji G, Fujii S, Fujihara N (2003) Agrobacterium tumefaciens-mediated transformation for random insertional mutagenesis in Colletotrichum lagenarium.J Gen Plant Pathol 69:230-239.
Tsukada M, Ohsumi Y (1993) Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBSLett333:169-174.
Veneault-Fourrey C, Barooah M, Egan M, Wakley G, Talbot NJ (2006) Autophagic fungal cell death is necessary for infection by the rice blast fungus. Science312:580-583.
Vey A, Quoit JM, Vago C (1985) Immunodepressive effect of fungal toxins-inhibition of the reaction of multicellular encapsulation by the destruxins. Cr Acad Scilll-Vie300:647-651.
Vilcinskas A, Matha V, G6tz P (1997a) Inhibition of phagocytic activity of plasmatocytes isolated from Galleria mellonella by entomogenous fungi and their secondary metabolites.J Insect Physiol43:1149-1159.
Vilcinskas A, Matha V, Gotz P (1997b) Effects of the entimopathogenic fungus Metahizium anisopliae and its secondary metabolites in morphology and cytoskeleton of plasmatocytes isolates from the greater wax moth Galleria mellonella. J Insect Physiol 43:1149-1159.
Wanchoo A, Lewis MW, Keyhani NO (2009) Lectin mapping reveals stage-specific display of surface carbohydrates in in vitro and hemolymph derived cells of the entomopathogenic fungus Beauveria bassiana. Microbiol-GSM155:3121-3133.
Wang B, Kang QL, Lu YZ, Bai LQ, Wang CS (2012) Unveiling the biosynthetic puzzle of destruxins in Metarhizium species.ProcNatlAcaSciUSA 109:1287-1292.
Wang C, St Leger RJ (2005)Developmental and transcriptional responses to host and non host cuticles by the specific locust pathogen Metarhizium anisopliae var. acridum.Eukaryot Cell 4:937-947.
Wang CS, St Leger RJ (2006) A collagenous protective coat enables Metarhizium amisopliae to evade insect immune responses.ProcNatlAcaSciUSA 103:6647-6652.
Wang CS, St Leger RJ (2007a)The MAD1 adhesin of Metarhizium anisopliae links adhesion with blastospore production and virulence to insects, and the MAD2 adhesin enables attachment to plants.Eukaryot cell 6:808-816.
Wang CS, St Leger RJ (2007b)The Metarhizium anisopliae perilipin homolog MPL1 regulates lipid metabolism, appressorial turgor pressure, and virulence. JBiolChem 282:21110-21115.
Wang CS, St Leger RJ (2007c)A scorpion neurotoxin increases the potency of a fungal insecticide. Nat Biotechnol 25:1455-1456.
Wang ZL, Lu JD,Feng MG (2012) Primary roles of two dehydrogenases in the mannitol metabolism and multi-stress tolerance of entomopathogenic fungus Beauveria bassiana. Environm Microbiol 14: 2139-2150.
Wang ZL, Ying SH, Feng MG (2013a)Recognition of a core fragment of Beauveria bassiana hydrophobin gene promoter (Phyd1) and its special use in improving fungal biocontrol potential.Microb Biotechnol 6:27-35.
Wang ZL, Zhang LB, Ying SH, Feng MG (2013b) Catalases play differentiated roles in the adaptation of a fungal entomopathogen to environmental stresses. Environ Microbiol 15:409-418.
Warren G.W (1997) Vegetative insecticidal proteins:novel proteins for control of corn pests. InCarozziNB and Koziel M (eds.), Advances in insect control:the role of transgenic plants, pp.109-121. Taylors& Francis Ltd, London.
Wheeler MB, Stuart GS, Hapner KD (1993) Agglutinin mediated opsonization of fungal blastospores in Melanoplus diferentialis (Insecta). J Insect Physiol 39:477-483.
Xia Y, Clarkson JM, CharnleyAK (2001) Acid phosphatases of Metarhizium anisopliae during infection of the tobacco hornworm Manduca sexta.Arch Microbiol 176:427-434.
Xiao GH, Ying SH, Zheng P, Wang ZL, Zhang SW, Xie XQ, Shang YF, Zheng HJ, Zhou Y, St Leger RJ, Zhao GP, Wang CS, Feng MG (2012) Genomic perspectives on the evolution of fungal entomopathogenicity in Beauveria bassiana. Sci Rep 2:483.
Xie XQ, Wang J, Ying SH, Feng MG (2010) A new manganese superoxide dismutase identified from Beauveria bassiana enhances virulence and stress tolerance when overexpressed in the fungal pathogenAppl Microbiol Biotechnol 86:1543-1553.
Xie XQ, Li F, Ying SH, Feng MG (2012) Additive contributions of two manganese-cored superoxide dismutases (MnSODs) to antioxidation, UV tolerance and virulence of Beauveria bassiana. PLoS ONE 7:e30298.
Xie XQ, Guan Y, Ying SH, Feng MG (2013) Differentiated functions of Rasl and Ras2 proteins in regulating the germination, growth, conidiation, multi-stress tolerance and virulence of Beauveria bassiana. Environ Microbiol 15:447-462.
Ye SD, Dun YH, Feng MG (2005) Time and concentration dependent interactions of Beauveria bassiana with sublethal rates of imidacloprid against the aphid pests Macrosiphoniella sanborni and Myzus persicae. Ann Appl Biol 146:459-468.
Ying SH, Feng MG (2006) Novel blastospore-bassd transformation system for integration of phosphinothricin resistance and green fluorescence protein genes into Beauveria bassiana.Appl Microbiol Biotechnol 72:206-210.
Ying SH, Feng MG (2011a) A conidial protein (CP15) of Beauveria bassiana contributes to the conidial tolerance of the entomopathogenic fungus to thermal and oxidative stresses. Appl Microbiol Biotechnol 90:1711-1720.
Ying SH, Feng MG (2011b)Integration of Escherichia coli thioredoxin (trxA) into Beauveria bassiana enhances the fungal tolerance to the stresses of oxidation, heat and UV-B irradiation. Biol Control 59:255-260.
Yorimitsu T, Klionsky DJ (2005) Autophagy:molecular machinery for self-eating.Cell Death Differ 12:1542-1552.
Yu CG, Mullins MA, Warren GW, Koziel MG, Estruch JJ (1997) The Bacillus thuringiensis vegetative insecticidal protein Vip3A lyses midgut epithelial cells of susceptible insects.ApplEnvironMicrobiol63:532-536.
Zhang S, Fan Y, Xia YX, Keyhani NO (2010) Sulfonylurea resistance as a new selectable marker for the entomopathogenic fungus Beauveria bassiana. Appl Microbiol Biotechnol87:1151-1156.
Zhang YJ, Feng MG, Fan YH, Luo ZB, Xing YY, Wu D, Pei Y (2008) A cuticle-degrading protease (CDEP-1) of Beauveria bassiana enhances virulence. Biocontrol Sci Technol18:551-563.
Zhang YJ, Zhao JH, Fang WG, Zhang JQ, Luo ZB, Zhang M, Fan YH, Pei Y (2009) Mitogen-acticated protein kinase hogl in the entomopathogenic fungus Beauveria bassiana regulates environmental stress responses and virulence to insects. Appl Environ Microbiol 75:3787-3795.
Zhou G, Wang J, Qiu L, Feng MG (2012)A Group III histidine kinase (mhkl) upstream of high-osmolarity glycerol pathway regulates sporulation, multi-stress tolerance and virulence of Metarhizium robertsii, a fungal entomopathogen. Environm Microbiol 14:817-829.
Zwiers LH, De Waard MA (2001) Efficient Agrobacterium tumefaciens-mediated gene disruption in the phytopathogen Mycosphaerella graminicola.Curr Genet 39:388-393.