Introduction

Locusts and grasshoppers are common pests of agriculture throughout the world. In China, there are a number of locusts and grasshoppers that can cause substantial damage; one of the most important is the oriental migratory locust Locusta migratoria manilensis (Meyen), a species that is found in much of eastern China. To limit damage to crops, treatment is required virtually every year, usually with a variety of chemical insecticides. The migratory locust is common near lakes and rivers, thus during the past decade a biological agent, Nosema locustae (Microsporida: Nosematidae), has been used near water to limit the use of chemicals there (Zhang et al. 2001). Another promising biological agent is the fungus Metarhizium anisopliae var. acridum (formerly Metarhizium flavoviride) (Deuteromycotina: Hyphomycetes) (Driver et al. 2000). High mortality of locusts and grasshoppers under laboratory and field conditions has been demonstrated by more than 10-years' research by the LUBILOSA program (Lutte Biologique contre les Locustes et les Sauteriaux) in Africa and by CSIRO (Commonwealth Scientific and Industrial Research Organisation) in Australia (Lomer et al. 1993; Hooper et al. 1995; Douro-Kpindou et al. 1997; Langewald et al. 1997; Hunter et al. 1999, 2001). An African isolate has been field tested against grasshoppers in China (Li et al. 2000), but the high mortality of the Australian migratory locust, Locusta migratoria (L.), treated with the Australian isolate (Hunter et al. 1999), led to laboratory and field trials to demonstrate the efficacy of the Australian isolate against the oriental migratory locust in China.

Materials and Methods

The conidia of M. anisopliae var. acridum (strain FI-985) were produced in Australia at the Becker Underwood factory. The spores were stored as dry conidial powder at 4°C until required. The dry conidial powder was formulated in corn oil at a concentration of 300 g of spores (1.2 × 10¹³ conidia)/L of oil to form the Green Guard^® product, which was shipped to China by air. Within China, the formulated Green Guard^® was stored in a cool cellar (10°C in winter, 20 to 25°C in summer) until used.

In a laboratory test to assess whether L. m. manilensis was affected by Green Guard^®, laboratory-reared 4^th instar oriental migratory locusts were treated with Green Guard^® concentrate (1.2 × 10¹³ conidia/L) diluted 50×, 1000×, 3000×, in soybean oil. Each concentration was sprayed onto a sample of 20 nymphs, using a hand-held sprayer. Nymphs treated with various dilutions of Green Guard^® or with soybean oil as a control, were then reared at 28°C in 40×10-cm chambers, and the dead were counted at regular intervals thereafter.

The field trials were conducted against infestations of mid-to late-instar nymphs that local authorities had found near waterways. The trial during 2002 at Tianjin was an area of fallow interspersed with areas of short and tall grass near the Dagang reservoir, while the trial during 2003 in Henan province was adjacent to the Yellow River in a 0.5-m soya bean crop, interspersed with strips of grass. Both were localized infestations that allowed treatment of most of the infestation to prevent the invasion of untreated locusts from the outside. The Green Guard^® used in the treatments was transported to the field where it was mixed with soybean oil and applied by local authorities at a 10-m track spacing, using backpack ULV sprayers. During 2002, the 2 treatments (50 g of spores in 1500 mL oil, 75 g of spores in 1500 mL oil) were in adjacent 20-ha blocks. On one side of the treated blocks, a 10-ha area was treated with oil alone (oil-treated) while an adjacent 20-ha area was left untreated. During 2003, 2 adjacent 80 ha blocks were treated (50 g of spores in 1125 mL of oil, 125 g of spores in 1125 mL of oil).

Decline in locust density after treatment was the main method of assessing efficacy. Locust density in each block was assessed about the time of treatment and a second assessment made when local authorities reported a noticeable decline in numbers, usually 8 to 11 d later. Density estimates were made in the central part of each block, where at each of 20 to 30 points, the number of locusts/m² was estimated. When a locust band was encountered, the density of the band and its size (length × width) was measured. After each assessment, a quick walk was undertaken diagonally across the whole of the treated area to confirm that locust densities throughout the area were similar to densities in the intensively assessed center. During 2003, the number of locust natural enemies seen was recorded at each point where locust density was estimated.

Ground temperature where the locusts were marching was measured at intervals throughout the day. Thermometers were placed flat on the soil surface in the sun, and in the shade, such that the underside of the thermometer was just touching the soil surface.

Results

In laboratory tests against the oriental migratory locust, there was high mortality at all dilutions tested, but little mortality in the controls (Table 1). These promising results led to field trials at Tianjin during 2002 and in Henan province during 2003. In the initial trial at Tianjin, 3^rd to 5^th instar nymphal bands were treated and within 8 d, there was substantial mortality at both the 50 and 75g/ha doses (Table 2A). There was little change in locust density in a block treated with soybean oil or in a block left untreated (Table 2A). The Green Guard^® was stored for a year in a cellar (10°C in winter, 20 to 25°C in summer) and used during the second year of trials. During August 2003, Green Guard^® was applied at sites in Henan province at a dose of 50g and 125 g of spores in 1125mL oil/ha to an infestation of to 4^th instar nymphs: there was a 76% and 65% decline, respectively, after 11 d (Table 2B). At the 2 treatment sites, the most common natural enemies were the generalist robber flies (Diptera: Asilidae) (Ommatius spp., Promachus spp) and Bombyliid flies (Anastoechus chinensis), that attack a variety of insects, including locusts. There was no significant change (Student t-test, p>0.05) in the number of these natural enemies: at the 50 g/ha site there were 0.85 ± 0.15 natural ememies/m² at treatment, and 0.80 ± 0.23/M² 11 d later. At the 125g/ha site, there were 0.84 ± 0.16 natural ememies/M² at treatment, and 1.00 ± 0.19/m², 11 d later.

In eastern China, it is very common for the sun to be hidden by haze. During June 2002, in the Beijing-Tianjin area, there were clear sunny skies on only 4 of 16 d. During observations made in July–August 2003, there were clear sunny skies on only 6 of 22 d in Beijing and 3 of 11 d in Shandong/Henan. On clear hot days, when it was hot from mid morning to late afternoon (shade temperature: 27 to 33°C), locust nymphs were commonly seen marching across the soil surface. The temperature on the soil surface was 10 to 15°C higher than that in the shade and was above 40°C from late morning to late afternoon (Fig. 1). But most days were cloudy or hazy and then the temperature on the soil surface was only 4 to 6°C higher than temperatures in the shade, remaining between 30 to 35°C for most of the day (Fig. 1).

Discussion

There was high mortality of L. migratoria manilensis at a dose of 50g (2 × 10¹² conidia) of M. anisopliae per hectare. The dose of 50 g/ha (2 × 10¹² spores/ha) is lower than the dose of 5 × 10¹² spores/ha of the African isolate applied to grasshoppers in northwest China (Li et al. 2000) and is lower than the 3 to 4 × 10¹² spores/ha dose that gave high mortality to L. migratoria in Australia (Hunter et al. 1999). An even lower dose (17 to 25 g/ha) was found to cause high mortality of the Australian plague locust, Chortoicetes terminifera (Walker) both during field trials and during control operations where nearly 40,000 ha were treated (Hunter et al. 2001, unpubl. data). However, the Australian plague locust is found in habitats with short grass and sparse to moderate vegetation cover, where lower doses are likely to be sufficient according to the modeling of Scanlan et al. (2001). Migratory locusts are often found in thick vegetation, which protects most locusts from direct impact at the time of spraying, so infection of many of the locusts is by secondary pickup from the vegetation. In thick vegetation, there are less spores per unit area of vegetation and models (Scanlan et al. 2001) indicate that a moderately high dose is required to ensure locusts pick up a lethal infection. Further work at a large number of sites, as is possible under operational conditions, will determine whether or not doses below 50 g/ha are sufficient for control of the oriental migratory locust in China.

Development of M. anisopliae is most rapid at 25 to 35°C with mortality of Australian migratory locusts reaching 50% in 5 to 6 d at constant laboratory temperatures (Milner 1997). But mortality is very temperature dependent: mortality took nearly twice as long (11 d) at while at there was almost no mortality at all (Milner 1997). In the field on sunny days, grasshoppers and locusts can increase their body temperatures by basking in the sun (Inglis et al. 1996, 1997; Blanford 1998, 2000). During spring in mid-temperate Australia, it is generally too cool for Metarhizium development at night, but locusts can increase their body temperatures to the 25 to 35°C favored by M. anisopliae, for much of the daylight hours, and mortality in the field takes about 2 weeks (Hunter et al. 2001). During the hot days of summer, locust body temperatures are high for most of the daylight hours (Blanford et al. 1998, 2000), being at or above the 37 to 40°C where development of M. anisopliae is nearly completely inhibited (Milner 1997). However, temperatures are more moderate at night, being in the 25 to 35°C range preferred by Metarhizium, for much of the night and in the early morning, so that mortality is more rapid (1.5 to 2 w) (Kooyman & Godonou 1997; Langewald et al. 1997; Hunter et al. 1999, 2001). In eastern China most control of L. migratoria manilensis occurs during summer when night temperatures are in the 25 to 35°C range preferred by Metarhizium. However, there is often a thick haze that hides the sun, so that locusts are also at 25 to 35°C where M. anisopliae development is most rapid (Milner 1997, Scanlan et al. 2001). Both the warm nights and hazy days are favorable for M. anisopliae, leading to rapid mortality in the field (8 to 11 d) and making this locust a particularly good candidate for control by M. anisopliae.

M. anisopliae is ideally suited to the preventive control programs conducted against the oriental migratory locust. M. anisopliae provides a way of killing a high proportion of locusts in their favored habitats: in vegetation around lakes and in river floodplains and avoids the need to apply chemicals there. At a dose of 50 g/ha, it is slightly more expensive than the most recent chemical insecticides, but does not have the high level of risk associated with the use of chemicals near water. Mortality after treatment is slower than that for chemicals, so treatments should begin when locusts are middle instars, a practice that is already common in current preventive programs in China. Locusts treated as 2^nd to 4^thinstars would die in the 2^ndweek after treatment, before reaching the adult stage. Even treatment of last-instar nymphs is possible if the nymphs are near lakes or rivers and not near crops: locusts will die as young adults, before they can fly well and reach more distant crops.

Increasing constraints on insecticide use mean that biological agents like M. anisopliae may contribute to the continued success of preventive control programs against the oriental migratory locust in China. Product efficacy is maintained after storage for over a year and results in moderately high mortality in 10 d or so. The preliminary results here show that Green Guard^®does not reduce the number of natural enemies. When mixed with vegetable oils (corn oil, soybean oil), it provides a natural product that will not leave chemical residues, allowing it to be used near water and on products destined for export into markets where chemical residues are not wanted. Large-scale field trials are planned in support of moves towards registration and use of Metarhizium as a significant part of a program of integrated pest management of locusts and grasshoppers in China.