| In the present study, a seed-coating formulation was developed through compatibility test and optimization of verious additives, with Metarhizium anisopliae, an inportant entomogenous fungus as the active agent, and white chafers of corn as the target pests. GFP gene was used as a marker of Ma55, with which corn seeds were coated. The colonization of the fungus was checked regularly in the rhizosphere of the corn plants, and the control potential against the root pest was evaluated, which was confirmed by a field trial.By bioassay, 5 isolates of Beauveria brongnirtii and M. anisopliae were tested for their virulence on larvae of the brown mulberry chafer, Holotrichia parallela. Under a concentration of 1×107 conidia/ml of Bbr06,Bbr17,Ma09,Ma20 and Ma55, the grubs were infected by 13.33%,22.22%,42.22%,68.89% and 95.56%, respectively, with Ma55 resulting in the highest mortality. Its LT50 was at 10.93, 9.12 and 7.65d, respectively, for the concentrations of 1×107,1×108, and 1×109conidia/ml.With Ma55 selected as a candidate for the whole study, the optimal additives were determined in long-term tests of compatibility of the conidia to solvent oil, film agent, antioxidant, and plant growth regulators. Under the conidia-oil ratios at 0.2, 0.1 and 0.05g/ml, germination of M. anisopliae condia on the 90th day in solvent oil"B"was not only significantly higher than those in the other solvent oils (P<0.05), but also significantly higher than that in the control at ambient temperature and 25℃(P<0.05). As compared with the control at 4℃, there was no significantly difference of germination (P<0.05). The result suggested that solvent oil"B"was the optimum. Film agent JX was the best for all physical properties, and seeding ratio with it was not significantly different from that in the control. In addition, significantly more conidia germinated with JX as compared with the other two agents under the concentrations of 0.1%, 0.5%, 1% (P<0.05). Under the concentrations of 0.1mg/ml, 0.2mg/ml and 0.4mg/ml, TBHQ showed excellent free radical scavenger activity. In the germination test, the conidia in TBHQ germinated significantly more as compared with those in the other two scavengers under the concentrations of 0.1mg/ml (P<0.05). Germination of the conidia in NAA was significantly higher than in 6-BA under the concentrations of 1ppm, 2ppm and 3ppm (P<0.05). Finally, the composition of the formulation was determined to be seed-coating agent solvent oil B, film agent JX, antioxidant TBHQ, and growth regulator NAA. A quadratic universal rotary design was used for study of impact of film agent, antioxidant and plant regulator on conidial germination, resulting in a formulation as the follows: solvent oil"B", with conidia-oil ratios at 0.1 g/ml, film agent JX at 0.05%, antioxidant TBHQ at 0.1mg/ml, and plant growth regulator NAA at 2ppm.A plasmid, pGPE-GFP, carrying green fluorescent protein gene (gfp) and phosphinothricin resistance gene bar was constructed and transformed into Metarhizium anisopliae. The transformants grew normally and GFP fluorescence was detected. Corn seeds were coated with the seed-coating agent of M. anisopliae containing gfp and were sowed. At seedling stage, soil was regularly sampled from rhizosphere, non-rhizosphere and surface for detection and monitoring of the transformant. The result showed that the inoculum of the gfp-marked M. anisopliae was not only the highest in the rhizosphere soil, but also did not fluctuate significantly from the 20th to the 30th day. Comparatively, the inoculum in the non-rhizosphere soil was not only lower than in the rhizosphere soil, but also declined significantly with time. In surface soil, the inoculum was the lowest and also declined significantly. All the evidences suggest that the conidia of M. anisopliae from the seed-coating agent were very viable with good colonization effect in rhizosphere soil of corn, demonstrating that the seed-coating agent is of potential against root insect pests.A cornfield test was run, with corn seeds coated by seed-coating agent at the rate of 1:10, 1:25, 1:50. The seeds germination was estimated on the 10th day. At the dosage of 1:25 and 1:50, there was no significant difference of seeding rate between the treatments and the untreated control (P>0.05). On the 30th day, the efficacy of seedling protection from seedling pest infestation was estimated. With the coating rate at 1:10 and 1:25, the infestation rate of the seedlings was significantly lower than that of the untreated control (P>0.05), with 1:25 showing higher efficacy. The evidence suggested that the Metarhizium anisopliae seed-coating formulation has great potential for root pest control of corn.
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