| 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. |
PDF Full Text Request