71. The Use of SlNPV as A Biological Agent to Control Cutworm on Soybean

Arifin, M. 1999. The use of SlNPV as a biological agent to control cutworm on soybean. Seminar on Pest Surveillance and Forcasting. Direktorat Bina Perlindungan Tanaman Pangan. Bogor, 31 Januari 1999. 11 p.

Muhammad Arifin

Research Institute for Food Crops Biotechnology, Bogor

INTRODUCTION

Spodoptera litura nuclear-polyhedrosis virus (SlNPV) (Borrelinavirus litura) is one of the insect pathogens infecting cutworm (Spodoptera litura) on soybean. This virus was first discovered in cutworm larvae in Central Lampung (Arifin and Waskito, 1986). Since 1985, experiments on the use of SlNPV to control cutworm were carried out at the now defunct, Bogor Research Institute for Food Crops.

Numerous experiments reported that SlNPV have high biotic potency (Arifin, 1993). As a biological agent, SlNPV is compatible with the integrated pest management (IPM) concept because: (a) its host-specificity only to the soybean cutworm and some other noctuids species, (b) it does not affect predators and parasitoids, and does not upset non-target host, human body, and environment, (c) it may alleviate insecticide resistant problem, and (d) it is compatible with most other control methods (Maddox, 1975; Starnes et al., 1993). There are several major reasons why SlNPV is suitable for a biological agent to control cutworm: (a) cutworms is a major pest attacking various kinds of vegetable and food crops, (b) considerable amount of broadspectrum, toxic, synthetic insecticides are used against cutworm, and (c) many cutworms are resistant to most major insecticides group. Efforts to develop SlNPV as a biological control agent can be conducted in three steps: (a) production of SlNPV, (b) solve the constraints affecting the effectiveness of SlNPV, and (c) optimizing application techniques of SlNPV.

The purpose of this paper is to provide information on biological properties, production, and application techniques of SlNPV as a guidance in the control of cutworm on soybean.

BIOLOGICAL PROPERTIES OF SlNPV

A. Description

SlNPV is a typical virus which produces irregular, crystal-like, proteinaceous polyhedra inclusion bodies (PIBs) in the nuclei of infected cells. The polyhedra which measured 0.5-15 µm, contain isometric virus particles (virions) with a surrounding envelope. The virions are rod-shaped, 40-70 nm X 250-400 nm, contain double-stranded, circular deoxy ribonucleic acid (DNA) molecules (Ignoffo and Couch, 1981; Tanada and Kaya, 1993). The morphological features of polyhedra inclusion bodies by light and electron microscopy is shown in diagrammatic representations as in Fig. 1.

B. Mode of Action

SlNPV is normally transmitted from one insect to another by oral ingestion of polyhedra. When ingested by a larva, the polyhedra dissolved and release virions into midgut. Liberated virions passed from midgut to haemocoel and subsequently infected nuclei of susceptible cells, e.g., fat body, hypodermis, tracheal matrix, epithelial, and blood. Infected larvae appeared shiny, discolored and sometimes pink-white in color. Feeding is greatly reduced so larvae grow slowly. Larvae may gather at the top of plants and hang in a typical inverted position. Cell lysis and disintegration of larval tissue began shortly after polyhedra formation. Younger larvae died in 2 days, but older larvae died in 4 to 9 days after virus ingestion. Shortly after death, larvae become flaccid and integument ruptures, releasing billions of inclusion bodies (Ignoffo and Couch, 1981; Tanada and Kaya, 1993).

C. Pathogenicity

Bioassays were used to determine the pathogenicity of SlNPV. Several models of virus administering were tried, the easiest large-scale, analitycal method, however, was to incorporate polyhedra into a diet and feed it to neonatal or 24 h-old larvae. The concentration-mortality relationship for SlNPV is usually expressed as a median lethal concentration (LC₅₀). This represents the concentration polyhedra that will cause death in half the test subjects. The LC₅₀ for third instar larvae was 5.4 X 10³ polyhedra inclusion bodies (PIBs)/ml and the LC₉₀ was 4,7 X 10⁵ PIBs/ml. Susceptibility of larvae decreased with age. First instar larvae was 100 times more susceptible to the virus than fifth instar larvae. Three days were required for initial mortality regardless of concentration (Arifin and Waskito, 1986).

D. Stability

Short-wave UV ray (215-260 nm) is the major environmental factor inactivating polyhedra (Ignoffo and Couch, 1981). Nearly 50% of the polyhedra applied on the upper surface of leaves was inactivated within 3 hours. Although these polyhedra was completely inactivated after 15 hours exposure to the sunlight, the polyhedra existed on the under surface of leaves still maintain 50% of its original infectio-capability after 20 hours. Application of polyhedra to the under surface of leaves was considered advantageous under direct sunlight condition.

There had been concerted efforts to minimize sunlight inactivation of polyhedra. Particulate sun-shields e.g., carbon, carbon-based dyes, alumunium oxide, titanium dioxide, clays, flour, and flourescent materials (poly flavonoids, whitening agents, and baits) were all increased the persistence of polyhedra. Ignoffo et al. (1977) suggested that sunlight inactivation of polyhedra probably is caused by peroxide or peroxide radicals produced by the UV irradiation of amino acids. Sunlight inactivation was inhibited but not completely stopped by addition of peroxidase.

Temperature likely to be encountered in the field are not so harmful to insect pathogens as does direct sunlight. Although field temperature (<45⁰ C) had no effect on the stability of polyhedra, viral replication was inhibited at 40⁰ C. Relative humidity plays a minor role in the persistence of polyhedra. Acids or alkalis disrupt polyhedra, thus, presumably destroy viral activity. Viral activity was not affected at pH 7.0, but was reduced slightly (<15%) at pH 4.0 and 9.0 and reduced significantly (>95%) at pH 1.2 and 12.4. Most insecticides were compatible with SlNPV. In a sensitive bioassay, only methyl parathion inactivated polyhedra. Formalin and sodium hypochlorite destroy viruses and thus are commonly used as disinfectants for SlNPV (Ignoffo and Couch, 1981).

The role of SlNPV as a mortality factor is greatly influenced by its ability to persist in a particular areas under natural conditions. Pathogens that do not persist must be applied repeatedly as microbial insecticides for temporary control of an insect pest (Maddox, 1975).

E. Efficacy

A preliminary test of unformulated SlNPV was conducted in 1992 to determine the effective dosages of polyhedra. Results indicated that polyhedra suspension of 1.5 X 10¹² PIBs/ha was effective in the greenhouse (73% mortality), but not effective in the field (33% mortality). The data revealed that sunlight was probably the most destructive environmental factor affecting activity of SlNPV. To increase its effectiveness, SlNPV must be formulated into wettable powder concentrate (Arifin et al., 1993).

The next semi-field trials attempted to determine the effectiveness of SlNPV at different formulation materials was conducted in 1993. Results indicated that SlNPV formulated with lactosum when applied at a rate of 9 X 10¹² PIBs/ha was effective against the cutworm. The mortality level of the larvae was 81%, initial mortality of the larvae occurred at 4 days after application, and larval mortality level of 80% occurred at 9 days after application. It was concluded that wettable powder using lactosum as inner carrier contributed to SlNPV efficacy (Arifin and Villayanti, 1994).

The effectiveness of SlNPV was compared with a standard insecticide (monocrotophos) and an untreated check in a field experiment in 1994. Results indicated that SlNPV at a rate of 1.5 X 10¹¹ PIBs/ha (applied twice each at 7.5 x 10¹⁰ PIBs/ha with an interval of a week) gave better control than single application. At that rate, the cutworm population was reduced by 88%, whereas insecticide treatment was less 27% than untreated check. Leaf damaged level was less 22% than insecticide treatment and less 26% than untreated check. Soybean yield on SlNPV plots was higher 14% (161 kg/ha) than monocrotophos treated plots and 31% higher (301 kg/ha) than untreated check. The net benefit was more 25% than insecticide treatment and more 35% than untreated check (Arifin et al., 1995).

PRODUCTION OF SlNPV

Although several sophisticated procedures have been suggested, SlNPV can only be produced in cutworm larvae. Mass production of SlNPV began with production of a large quantity of selected isolate as inoculum. Production of SlNPV was conducted in four isolated facilities: (1) a diet-storage-preparation facility, (2) a facility with a unit for rearing stock culture of cutworm and another unit for mass rearing larvae for virus production, (3) a virus production facility, and (4) a product recovery and formulation facility. The production process of SlNPV is shown in diagrammatic representation in Fig. 2.

1. Mass rearing of cutworm

A large colony of the larvae was maintained on artificial diet (Table 1). During the first and second instars, larvae were supplied with a piece of diet (length 6 cm, width 3 cm and thickness 0.5 cm)/500 larvae in container A (diameter 9 cm, depth 2 cm). During the third and fourth instars, larvae were supplied with five pieces of diet (length 6 cm, width 1.5 cm and thickness 1.5 cm)/500 larvae in container B (length 20 cm, width 15 cm, depth 5 cm). Fifth instars larvae were supplied with five pieces of diet (length 6 cm, width 1.5 cm and thickness 1.5 cm) 100 larvae in container C (length 26 cm, width 20 cm, depth 6 cm). Sixth instars larvae were supplied with nine pieces of diet (length 6 cm, width 1.5 cm and thick 1.5 cm)/50 larvae in container C until pupation. Pairs of the adult moth were placed in an oviposition container and fed 10% honey solution. Eggs-masses were collected daily. A day before hatching, each egg-mass was placed in container A (Okada, 1977; Okada and Arifin, 1982).

2. Mass propagation of SlNPV

For maximum yield of polyhedra, the middle fifth instar larvae reared on the diet were infected by an addition of polyhedra suspension (5 X 10⁷ polyhedra/g of the diet) to the surface of the diet for 48 hours. After inoculation, larvae were transferred to individual rearing container with diet. After incubation period of about 10 days, dying and dead larvae are collected in glass distilled water, homogenized in a blender and passed through a layer of 100 mesh filter. Crude polyhedral suspension was centrifuged at 3500 rpm for 15 minutes and sedimented polyhedra were washed 4 times with water. The freeze-dried preparation of polyhedra was stored at 0⁰ C until use (Okada, 1977; Okada and Arifin, 1982).

3. Standardization and formulation of SlNPV

The preparation was suspended in water to make 10% (v/v) suspension. A serial five-fold dilution (10^-1 to 10^-5) were made. The concentration of 10^-5 suspension was determined using a haemacytometer. Washed polyhedra suspended in water was adjusted to 1 X 10⁹ PIBs/ml and the average number of polyhedra produced by a single sixth instar larva was 8 X 10⁹ polyhedra (Arifin, 1993).

Lactosum powder intensified the virulence of SlNPV and so it was used as main formulation component. Polyhedral suspension (1 X 10⁹ PIBs/ml) of 100 ml was mixed with 50 g of lactosum and the mixture was lyophilized. After lyophilization, both a sticker (0.01% Triton x-100) and an adjuvant (20% crude sugar) were added (to enhance the effectiveness of the polyhedra) and more lactosum powder was added to the mixture to make 100 g in total weight. Formulated preparation of SlNPV was adjusted to 1 X 10⁹ PIBs/g and then packed (Arifin, 1993; Okada, 1977).

Production cost of formulated SlNPV for 1 ha application was Rp 15,300,- per 150 g (1 US $= Rp 2,300,-). When it was compared with the price of monocrotophos insecticide for 1 hectare application (Rp 20,000,-), production cost of SlNPV was 1.3 times cheaper (Arifin et al., 1995).

APPLICATION TECHNIQUES OF SlNPV

To increase its effectiveness, SlNPV must be first formulated. Two of the most important goals in formulating a microbial insecticide are: (1) to increase the persistence of the pathogen for as long as possible, and (2) to place the pathogen in contact with its host in such a way to achieve maximum infection (Maddox, 1975).

SlNPV are presently applied with spraying equipment designed for the application of synthetic insecticides. Best results are usually attained if the SlNPV is applied during the early stages of cutworm development. Late larval instars are much more difficult to control with SlNPV than are earlier larval instars. This is generally true for synthetic insecticides as well, but this effect is much greater for SlNPV.

Direct sunlight is harmful to SlNPV. Application should be conducted in the late afternoon or early evening, allowing ingestion of the polyhedra before direct sunlight exposure and spraying equipment should be adjusted to give good coverage on the undersides of soybean leaves.

USE OF SlNPV IN IPM PROGRAMS

SlNPV can be used as a tool in iPM program, it conforms nicely with the objectives of IPM: (1) SlNPV is relatively host-specific and does not upset other biotic systems, (2) SlNPV is safe for humans and does not cause environmental contamination, and (3) SlNPV is compatible with most other control methods. Host specificity, however, may be a disadvantage in cases in which control of several insect pests is desired (Maddox, 1975; Starnes et al., 1993).

SlNPV often cause epizootic which drastically reduce large cutworm populations. Such epizootic can be used in IPM programs if two important factors are considered. First, the damage threshold of cutworm population is known. Second, entomologist are familiar with SlNPV symptom in the cutworm population (Maddox, 1975). If one is aware that cutworm is frequently controlled by natural occurrence of SlNPV, he or she may constantly monitored that population and avoid insecticides treatment except cutworm population exceeds damage threshold.

The economic threshold of cutworm density usually lower than the disease threshold level of SlNPV. For this reason, SlNPV can be considered as a microbial insecticide. This means that repeated applications of SlNPV are used similar to synthetic insecticides for temporary control of cutworm (Maddox, 1975; Starnes et al., 1993).

SlNPV may also be periodically introduced into cutworm populations in order to initiate epizootic at an earlier time than they would normally occur. Several factors should be considered when introducing SlNPV into cutworm population: (1) concentration of SlNPV must be sufficient to produce infection, (2) cutworm population in which the SlNPV is to be applied should have a relatively high density in order to assure propagation of the SlNPV and its survival from one generation to the next, and (3) SlNPV should be applied during a susceptible stage of the cutworm (Maddox, I975).

FUTURE ACTIVITIES OF SlNPV

1. Identification

We have several collections of SlNPV from different localities, unfortunately, not all of them have been identified. It can be expected to show different levels of virulence. Future activities should be focused to collect, characterize, and identify SlNPV isolates found throughout Indonesia by morphological, serological, and biochemical methods to select the most active strains for commercial production.

2. Large-scale production

SlNPV must be propagated in the living host. Although virus produces in cell lines, this technology is not practical from a commercial stand point. Production scale of SlNPV will be increased to on-farm research. For that purpose, the machinery equipments, especially the capacity of centrifugation should be increased and the working procedures should be improved.

3. Effectiveness

Even though SlNPV has been successfully used in cutworm-control programs, many problems are yet to be solved if SlNPV is expected to reach its fullest potential. Hence, SlNPV will play an increasingly important role in IPM program. For future activities, the potency and field effectiveness of SlNPV formulations are constantly improved. The development of new methods of formulation and application of SlNPV is needed. This will undoubtedly enhance the effectiveness of SlNPV.

CONCLUSIONS

SlNPV have high biotic potency and suitable to be used in IPM programs. To develop it as a biological control agent, main problems associated with the effectiveness of SlNPV should be solved. SlNPV must be formulated to prevent sunlight inactivation of polyhedra and applied repeatedly as a microbial insecticide for temporary control. Several activities should be conducted in the future, i.e., selection of more virulent strains of SlNPV, large-scale production of SlNPV for on-farm research, and the improvement of SlNPV effectiveness.

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