- Top of page
- 1 INTRODUCTION
- 2 MATERIALS AND METHODS
- 3 RESULTS
- 4 DISCUSSION
Entomopathogenic fungi play a crucial role in controlling insect populations.[1, 2] To date, 13 species or subspecies of insect-pathogenic fungi, such as Metarhizium spp. and Beauveria spp., have been formulated and registered as mycoinsecticides. Compared with bacteria and viruses, the entomopathogenic fungi are particularly well suited to being developed as biological pesticides for use in dry grasslands and deserts. Metarhizium acridum, previously named M. anisopliae var. acridum or M. flavoviride var. minus, is a locust-specific entomopathogenic fungus. M. acridum biological agents have been widely used to control locusts or grasshoppers in Australia, West Africa and China. However, their actions against target pests are quite slow compared with chemical insecticides. Poor efficacy is a primary obstacle to their commercialisation and large-scale application against agriculturally important insect pests, especially those species that exhibit sudden outbreaks.[8, 9]
A certain deficiency in efficacy is probably inbuilt in pathogens because an evolutionary balance will have developed, involving their hosts, so that a quick kill, even at high doses, is not possible for the entomopathogenic fungus. Thus, efforts have been focused on enhancing fungal virulence by genetic manipulation. Entomopathogenic fungi infect insects by direct penetration of the cuticle and then propagate within the insect haemocoel following penetration. Several endogenous genes encoding the cuticle-degrading protease Pr1 and chitinase have been transferred into entomopathogenic fungi to enhance their efficacy against pest Insects.[8, 12, 13] Another approach to improving fungal virulence is through the expression of some insect molecules, such as the diuretic hormone and the trypsin-modulating oostatic factor. The insecticidal toxins from some scorpion and spider venoms are also a major resource for genetically modified biopesticidal agents. The remarkable extent to which virulence can be increased was shown by expressing a scorpion toxin (AaIT) in an M. anisopliae strain against tobacco hornworm (Manduca. sexta), mosquitoes (Aedes aegypti) and coffee berry borer beetle (Hypothenemus hampei).[16, 17] Similarly, Beauveria bassiana expressing AaIT demonstrated a significant increase in insecticidal activity against Masson's pine caterpillar (Dendrolimus punctatus) and the wax moth (Galleria mellonella). These studies have shown the feasibility of enhancing fungal biocontrol potential by gene transformation.
BjαIT, an insect-selective, 64-amino-acid neurotoxin, was isolated from the venom of the Judean black scorpion (Buthotus judaicus). However, its potential to improve microbial insecticides has not been explored. The insect-specific neurotoxin gene of BjαIT was synthesised on the basis of its amino acid sequence and successfully expressed in P. pastoris. The expressed product, when injected, exhibited insecticidal activity against locusts and cockroaches. This shows that BjαIT is a potential candidate for increasing the efficacy of entomopathogenic fungi against pest insects by genetic engineering.
As a commercial bioinsecticide strain, M. acridum CQMa102 has been used to control Locusta migratoria manilensis in a wide variety of locust habitats in China. Now this strain has become a model pathogen for exploring the relationship between an entomopathogenic fungus and its host. In the present study, M. acridum was genetically modified with the BjαIT gene. The virulence of the genetically engineered strain BjαIT-102 against L. migratoria manilensis was then tested in comparison with the wild type (WT) in order to determine whether BjαIT is suitable for use in entomopathogenic fungi as a candidate virulence factor.
- Top of page
- 1 INTRODUCTION
- 2 MATERIALS AND METHODS
- 3 RESULTS
- 4 DISCUSSION
As a key regulator of insect populations, entomopathogenic fungi may have significant applications as biocontrol agents. In recent years, enhancement of fungal virulence by genetic manipulation has become a research hotspot owing to the reduced effects and higher costs of fungal pesticides compared with chemical insecticides. The subtilisin-like protease (Pr1) and chitinases influence the virulence of entomopathogenic fungi, as they are important cuticle-degrading enzymes.[26, 27] Constitutive expression of Pr1 or hybrid chitinase gene caused a 23–25% reduction in time to the death of the host compared with WT.[8, 28] An insect-specific neurotoxin (AaIT) from the scorpion Androctonus australis has become a candidate for improving the virulence of entomopathogenic fungi. The LC50 of M. anisopliae expressing AaIT was reduced 22-, nine- and 16-fold against M. sexta, A. aegypti and H. hampei respectively.[16, 17] Similarly, B. bassiana expressing AaIT showed a 15-fold increase in insecticidal activity against D. punctatus. In the present study, the median lethal dose (LC50) for BjαIT-102 was 18.2-fold lower. The median lethal times (LT50) for BjαIT-102 were reduced by 28.1 and 30.4% after topical inoculation and injection respectively, suggesting that the BjαIT gene has the potential to improve the virulence of entomopathogenic fungi.
Entomopathogenic fungi infect the insect by direct penetration of the cuticle, and the fungus then kills the host via proliferation and secreting toxins in the insect haemocoel. It was suggested that the production of toxins in M. acridum is limited, or that none is produced in the insect, which was confirmed by analysing the genome of M. acridum and testing the effect of the toxin on the fever of locusts. It was suggested that M. acridum virulence against insects chiefly depends on abundant proliferation in the haemocoel at the post-penetration infection stage. The results of in vivo growth of M. acridum confirmed the speculations above. The virulence of engineered strains expressing AaIT was astonishingly enhanced, but the effects of AaIT on the penetration and in vivo growth are not clear. For example, it is not possible to assess the effects of AaIT on the germination and appressoria of entomopathogenic fungi because the expression of AaIT is driven by Mcl1 promoter, which works in the insect haemolymph. In the present study, the germination and appressorium formation rates of BjαIT-102 and its growth in locusts were investigated. There were no differences in germination or appressorium formation between WT and the transformants, and differences in early growth (2 days after inoculation) in the locust haemolymph were not significant. These results demonstrated that the effects of BjαIT on virulence are not related to cuticle penetration. However, the expression of BjαIT accelerated the growth of M. acridum in the locust haemolymph 3 and 4 days after inoculation. This may be due to a physiological derangement of the insect, caused by BjαIT, which can affect the voltage-gated sodium channels of the host Insect.
The safety issue associated with genetic manipulation of entomopathogenic fungi has become a focus of attention. Engineering the overexpression of the Pr1 gene prevents spore production on the carcasses, thus blocking the fungus from reproducing in the field and decreasing the threat to non-target Insects. The threat of AaIT-expressing strains to the environment could be reduced by applying tissue-specific promoters. However, it would be more straightforward to engineer virulence genes (e.g. AaIT) in entomopathogenic fungi that have a narrow host range. M. acridum specifically targets acridids such as locusts and grasshoppers. Furthermore, BjαIT enhanced the insecticidal efficacy during post-penetration, as did AaIT, and did not alter the host range of the strain because its specificity is chiefly controlled by events on the cuticle. This was further demonstrated by the fact that BjαIT-102 did not infect a number of non-target insects, such as Galleria mellonella, Pyrausta nubilalis and Blattella germanica (data not shown). Thus, it should not compromise environmental safety to engineer BjαIT to increase the virulence of M. acridum. The safety of M. acridum expressing BjαIT should be further enhanced by using tissue-specific promoters, such as Mcl1 promoter.
In the natural environment, entomopathogenic fungi infect host insects and cause epidemics, chiefly through the conidia on host carcasses, which play an important role in the sustainable control of target pests in the field.[26, 32] BjαIT-102 produced normal conidia on dead locusts, although the conidial yield was reduced. Similarly, expressing AaIT in M. anisopliea or B. bassiana produced a significant decrease in the production of conidia on the carcasses.[17, 18] However, the long-term control exerted by BjαIT-102 against locusts would be enhanced if the virulence of BjαIT-102 were to be taken into consideration. The 50% lethal concentration (LC50) of BjαIT-102 against the locusts was reduced 18.2-fold compared with the values for WT; in other words, the equivalent control could be achieved with far fewer conidia.
In conclusion, the results showed that expressing BjαIT in M. acridum significantly increased its virulence against locusts but did not affect the pre-penetration of the cuticle and conidium formation on the carcasses. Moreover, they demonstrated that engineering BjαIT offers great potential as a means by which specific entomopathogenic fungi can increase their virulence in a relatively safe and cost-effective way. However, practical application will require the accumulation of more data on the characteristics of the genetically modified fungi, e.g. the effects of these modifications on the mass production and storage of conidia and the dependence of their viability on the conditions of heat, solar radiation and low humidity. Furthermore, BjαIT could be a candidate for improving the virulence of other entomopathogens as microbial insecticides.