Recent Advances in Biological Control of Pest Insects by Using Viruses in China^*

To date, eight nucleopolyhedroviruses (NPVs), three granuloviruses (GVs) and one cypovirus (CPV) have been registered as commercial insecticides in China (Table 1). HearNPV is the first commercial insecticide authorized by the Ministry of Agriculture of the P.R. China (17). This product is the most produced virus insecticides in China estimated at about 1600 tons of formulated HearNPV in 2005 (Investment and consulting report on biological agrochemicals, 2006-2007). Spodoptera litura NPV and S. exigua NPV have also been applied at a large scale to control pests on vegetables (1, 8).

Table 1.  Viruses authorized as commercial insecticide in China

Recently, efforts to improve the control efficacy of viral insecticides by selecting enhancers have been effective. For example, combination of a chitin synthase inhibitor, chlorfluazuron, with HearNPV formulationat a concentration of 4.2 ppm reduced the median lethal time in 3^rd instar H. armigera larvae from 4.90 days to 2.24 days (18). Chitinase, expressed by Serratia marcesscens, was also used to enhance the infectivity of HearNPV (15). An enhancing protein and its truncated fragment from a granulovirus have been expressed and used as synergists of HearNPV and AcMNPV in pest control (2, 3, 4, 6, 16).

Techniques have also been developed to increase the yield of viruses in host larvae. For example, incorporation of a juvenile hormone analogue, methoprene, with S. litura NPV might increase virus yield by 227% (5). Similarly, the juvenile hormone analogue ZR515 had been used to increase the yield of HearNPV (Chinese paten: ZL98121687.0).

A major limitation to wide use of baculoviruses for insect control is their slow speed of action. To enhance the efficacy of HearNPV, it was genetically modified either by deletion of the ecdysteroid UDP-glucosyltransferase (egt) gene from the genome (the recombinant HearNPV-Δegt) or by insertion of an insectspecific scorpion toxin (AaIT) gene at the egt gene locus (the recombinant HearNPV-AaIT) (13). To develop the recombinant HearNPVs as a commercial insecticide, the efficacy and biosafety of the recombinant viruses have been investigated.

Application of HearNPV-AaIT on cotton plantations protected the fruit from damage by the bollworm better than HearNPV or HearNPV-Δegt over entire cotton seasons in 2000 and 2001. Yield of cotton lint from HearNPV-AaIT-treated plants was about 22% higher than from plantations treated with wild-type virus alone (13). At an optimal regime to produce the recombinant HearNPV in vivo, the yield of Hear NPV-AaIT was about 44% of the wild-type virus (10). When all conditions are the same, the cost to spray of formulated HearNPV-AaIT was about 50% higher than that of wild-type virus. By all accounts, the increased value of cotton lint after sprays of HearNPV-AaIT formulation is much higher than sprays of the wildtype virus (Table 2). Thus the potential for comercialization of the recombinant HearNPV is promising.

Table 2.  Comparison of economic effects of application of wild-type and recombinant HearNPVs in cotton

To assess the risk of releasing recombinant HearNPV into the environment, the effects on nontarget species, possibility of AaIT gene flow to other organisms, and their environment fitness were studied.

Effects on non-target species: The median lethal dose of HearNPV-AaIT was above 2 000 mg/kg against female and male rats administered by either intra dermal or per os. This recombinant was also found to be non toxic for bobwhite quail, zebra fish, silkworm and bees. It is not an allergen to a guinea pig skin. Furthermore, it there was no pathological response when rats were inoculated with the recombinant virus.

In experiments of multiple application of HearNPV-AaIT on cotton in 2000, 2001 and 2002, biodiversity and the dynamics of the natural enemies were also monitored under field conditions. No significant difference in biodiversity and in the number of natural enemies was observed when plots were treated with either the recombinant or the wild-type virus (Sun et al., unpublished data). Furthermore, the survival time and fecundity of ladybeetles did not differ significantly when they were fed H. armigera larvae infected with either HearNPV-AaIT or wildtype virus (Sun et al., unpublished data). The toxin AaIT expressed by yeast was biologically active against Spodoptera exigua and Argyrogramma agnata larvae when injected into the haemocoel. Whereas, it was not toxic to the two insect species when the toxin was administered per os (14). These data indicate that AaIT was active when it was injected into or expressed in the neurosystem of the insects and it was not active when it was given per os.

Possibility of transferring of AaIT gene from recombinant virus to its surrounding organisms: PCR and dot-blot hybridization were used to test the possiblity AaIT gene flow from HearNPV-AaIT to other organisms after field release. AsIT sequences were not detected in the genome of a pathogen of cotton, Verticillium dahliae Lleb., which was cocultivated with HearNPV-AaIT DNA or virions for up to 90 days. Similarly, AsIT sequences were not detected in the genomic DNA of ladybeetles collected from fields where the recombinant virus was applied several times (9). It is concluded that possibility of the AaIT gene of the recombinant HearNPV transferred to surrounding organisms was very low.

Fitness of the recombinant HearNPV in the environment: Virus yield per larva, inoculated with HearNPV-AaIT in the 1st, 2nd, 3rd, 4th or 5th larval stage, was 23%, 32%, 41%, 44% and 47% of the yield of HearNPV-wt, respectively (14). These data indicate that production of recombinant virus in host larvae was lower than that of wild-type virus.

In the field, there was no significant difference among the inactivation rates of the two recombinant HearNPVs and the wild-type virus on cotton plants (11). Furthermore, the persistence of recombinant viruses in soil was not significantly different from that of wild-type virus (12).

In the field, HearNPV-AaIT exhibited a significantly lower rate of transmission than the wild-type virus. The vertical transmission of HearNPV-AaIT from infected females to offspring was also significantly lower than that of wild-type HearNPV (19).

Based upon the results mentioned above, there is no evidence that the recombinant baculovirus provides an increased hazard to non target organisms including humans or has a deleterious effect on environmental health in comparison to wild-type viruses. In order to commercialize the genetically modified HearNPV containing AaIT gene, a data package on the safety the virus has been submitted to the Ministry of Agriculture, P.R. China.

A new method for delivering viruses to target insects by using Trichogramma spp. as vector has been developed in China (7). By taking into account the biology and the ecology of T. dendrolimi, a special box was designed for releasing the wasps and accompanying viruses. When T. dendrolimi was released with Dendrolimus punctatus cypovirus to control the masson pine moth (D. punctatus), it was observed that 20%-40% of eggs had been parasitized and 40%-47% of D. punctatus larvae had been infected with the virus (7). In the case of release T. dendrolimi combined with HearNPV to control the cotton bollworm, it was observed that 33%-37% of eggs had been parasitized and 61%-70% of H. armigera larvae had been infected (20).

This technique has also been used for control of H. armigera, S. litura, Plutella xylostella, Ectropis oblique, Pieris rapae, etc. on more than 20 000 hectares since 1991 in China.

The abuse of chemical insecticides has led to pest resistance, resurgence and environmental problems in many developing countries. Society now demands safer pesticides and less-toxic chemical residues in food and in water. Effective management of pests requires diverse and flexible tools. Among classical biocontrol agents, insect viruses are attractive tools that appear supplement or and, in many cases, replace synthetic chemical pesticides in IPM schemes. Despite their high potential and documented cases of success, the market for viral insecticides is still limited in China. The problem is mainly derived from their relative slow speed of action, relatively low virulence for the older instars and a narrow host range. However, many of these perceived disadvantages can be addressed by modern molecular technologies or in combination with other biocontrol strategies. Genetically modified baculoviruses have already been eluded to and appear to be effective in insect pest control. Furthermore, they can be produced in large amounts by village industries, indicating that they have the potential to deliver sustainable pest control, especially in developing countries.