The microbial control of horticultural pest insects using entomopathogenic fungi, Beauveria bassiana and Verticillium lecanii

The diamondback moth, Plutella xylostella, whitefly, Trialeurodes vaporariorum, and the aphids, Aphis gossypit and Myzus persicae are major horticultural pests because of their resistance to chemical insecticides. This study developed microbial controls for the diamondback moth and whitefly and aphids using entomopathogenic fungus Beauveria bassiana and VerticUIium lecanii, respectively. 1)The pathogenicity of five isolates of B. bassiana, and an isolate of Metarhizium anisopliae in P. xylostella were investigated. Treatment of 4th-instar larvae, with each isolate, resulted in 0-100% mortality 5days after inoculation. Detailed studies were made of the most virulent B. bassiana isolate, MG-Bb-1, obtained from P. xylostella, to determine its efficacy at the different developmental stages of P. xylostella, and at different doses and humidities. MG-Bb-1 was highly pathogenic in larvae of all instars, but only slightly pathogenic in eggs, and not pathogenic in pupae. Humidity influenced its virulence in 4th-instar larvae; MG-Bb-1 caused mortality exceeding 80% at an RH of at least 76%, but only 30% mortality at 52% RH. The mortality at various temperatures, and the conidial invasion time after inoculation, were investigated. When larvae were inoculated with a suspension of MG-Bb-1( 1 X 10 7conidia/ml), the mortality exceeded 95%, regardless of the incubation temperature. However, the longevity of infected larvae was prolonged as temperature decreased. The mortality in larvae exposed to cycles of 25 deg C (12h) and 15 deg C(12h) was very similar to that in larvae reared at a constant temperature of 20 deg C. These results indicate that the speed of killing depends on the thermal constant. Based on the linear relationship between the temperature (t) and time to death ( D), V=1/D=0.023t- 0.1285 (r**2=0.972), 5,5 deg C and 43.2 days-degrees were estimated as the lower threshold for lethality and the thermal constant, respectively. Conidial invasion of the host began approximately 6h post-inoculation, and was completed in 15 h at 25 t. In laboratory bioassays, a high rate of infection of P. xylostella larvae by B. bassiana required a suitable temperature (20-30 deg C) and nigh humidity (75-100%), and these conditions had to be maintained for 10-15 hours after conidial adhesion. 2) Conidial suspensions (10 7conidia/ml) of MG-Bb-1 were sprayed on cabbage seedlings planted in plastic pots, and on a cabbage field, to control P. xylostella. Fungus treatment was effective against P. xylostella larvae on cabbage seedlings in plastic pots. In the field experiment, the application controlled P. xylostella as well as flufenoxuron did. The efficacy of the pilot formulation of MG-Bb-1 at controlling P. xylostella was evaluated in six field trials, conducted between June and October. Applications of the pilot formulation at a dose of 2-5 X 10**6conidia/ml significantly affected P. xylostella larvae. However, this dose was ineffective against larvae of Pieris(Artogenia) rapae crudvora. When the pilot formulation of MG-Bb-1 was applied once or twice in the field trials, both applications had similar effects on P. xylostella. One application was sufficient to control P. xylostella, given favorable environmental conditions. However, MG-Bb-1 had almost no pathogenicity in eggs or pupae, so two applications were necessary to treat P. xylostella outbreaks. Temperature and humidify were measured at the lower, middle, and upper leaves of the cabbage plants. No temperature differences were found, while the humidity was greater at the lower and middle, leaves than at the upper leaves. P. xylostella larvae are usually found on the lower and middle leaves of cabbage, where conditions favor the control of P. xylostella byerrtomopathogenic fungi. 3) MG-Bb-1 was. pathogenic in 1st-instar larvae of Mamestra- brassicae and 3rd-instar larvae of P. (A.) rapae curicivora. However, their susceptibilities to MG-Bb-1 were lower than that of P. xylostella; 1 X 10**8conidia/ml were needed to produce mortality over 90%. It might be possible to control these insects with an increased dosage, but it is advisable to use other control methods, such as physical control, in order to mitigate production costs as well as unwanted effects on non-target insects. 4) The pathogenicity of MG-Bb-1 against key arthropod pests in greenhouse cultures was investigated. MG-Bb-1 was pathogenic in the aphid Myzus persicae,, the thrip Furankliniella occidentalis, and the spider mite tetranychus urtica. MG-Bb-1 caused high mortality in M. persicae at concentrations above 10**6conidia/ml. Conversely, F. occidentalis. and T. urtica were, less susceptible to MG-Bb-1 than was M. persicae. Nevertheless, F. occidentalis and T.urtica are major greenhouse pests because of their resistance to chemical insecticides, so MG-Bb-1 should be used to control F. occidentalis and T. urtica. 5) The pathogenicity of MG-Bb-1 in non-target insects was. investigated. MG-Bb-1 was pathogenic in Bombyx mori larvae and a predatory mite, Phytoseiulus persimilis, but not in two predators of P. xylostella: Pheropsophus jessoensis and Dolichus halensis. 6)The pilot formulation of MG-Bb-1(5 X 10 6conidia/ml) was sprayed on. greenhouse strawberry to control the two-spotted spider mite, T. urtica, and to confirm its effect on the mite predator, P. persimilis. The population density of T, urtica was suppressed as a result of the interaction between the effects of the pilot formulation and predation pressure, due to P, persimilis. Nymphs and adult P. persimilis infected with MG-Bb-1 were not observed in this greenhouse trial at that dosage. 7) The pathogenicity of two V. lecanii isolates (MG-VI-18 isolated from whitefly, T. vaporariorum, and MG-V1-45 from cotton aphid, A. gossypii) was investigated. MG-VI-18 was highly pathogenic in larvae and adults, with intermediate effects in pupae, and no pathogenicity in eggs of T. vaporariorum. The Silver-leaf whitefly, Bemisia argeritifolii, was more susceptible to.MG-Vl-18 than T. vaporariorum. MG-Vl-45 was highly pathogenic in the aphids, A, gossypii and M. persicae. These isolates had almost no pathogenicity in B. mori. The sizes of conidia and blastospores of MG-VI-18 and MG-VI-45 differed; MG-V1-45 produced larger conidia and blastospores than did MG-VI-18. Conversely, the mycelial growth of MG-VI-18 was faster than that of MG-Vl-45, and the growth temperature range of NG-V1-18 was greater than that of MG-VI-45. Both MG-VI-18 and MG-Vl-45 were pathogenic in T-. vaporariorum, A. gossypii and M..persicae, but host specificity was observed. MG-VI-18 was more pathogenic in larvae and adults of T. vaporariorum than was MG-VI-45. The difference was especially marked in the larval stage. MG-VI-45 was more pathogenic than MG-VI-18 in A: go.ssypii (apterous. adults) and M. persicae (apterous and alate adults). . MG-VI-45 was inoculated using two methods: dipping insects( A. gossypii and M. persicae), and dipping food plants (detached cucumber and cabbage leaf), in conidial suspensions. Dipping food plants resulted in higher aphid mortality with MG-Vl-45, as compared to the result from dipping insects. MG-VI-45 frequently, invades the leg segments of aphids, mainly the tarsus. These results indicate that MG-Vl-45 does not need the systemic adhesion of conidia to kill aphids, so MG-Vl-45 has a residual effectiveness and may be used for preventive application. The optimum temperature of MG-Vl-45 was 25 deg C for mycelial growth, and for the inoculation tests of aphids. Fungus infection occurred at temperatures as low as 10 deg C, but the longevity of infected aphids was prolonged as temperature decreased. Temperatures as high as 35 deg C inhibit mycelial growth of MG-V1-45. MG-V1-45. as a microbial control agent must be used at temperatures between 15 deg C and 30 deg C. The optimum humidity of MG-V1-45 was 100% RH, but high mortalities were obtained above 85% RH. Conidial invasion of the host begins approximately 14-16h post-inoculation and is complete in 20h, at 20 deg C and 100% RH. 8)Conidial suspensions (10 7conidia/ml) of MG-V1-18 were sprayed on tomatoes planted in flowerpots and on greenhouse cucumber plants to control T. vaporariorum. In both trials, infected larvae and adults of T. vaporariorum increased up to 10days after application, and then the population density of T. vaporariorum was suppressed for a period that exceeded 20days. MG-V1-18 was effective against T. vaporariorum, but was slow-acting. Conidial suspensions (10 7conidia/ml) of MG-V1-45 were sprayed on greenhouse cucumber plants to control A. gossypii. In the first greenhouse experiment, with a low density of A. gossypii, one application of MG-V1-45 resulted in lower numbers of A. gossypii, as compared to controls; it controlled A, gossypii as well as Ethyophencarb, In the second greenhouse experiment, with a high density of A. gossypii, one application of MG-V1-45 reduced the numbers of A. gossypii, but the effect of MG-V1-45 was similar to that of MG-V1-18. In these experiments, no aphid predators infected with MG-V1-45 were observed. The two isolates of V. lecanii take 5-10 days, after application, to kill their host insects, which include whiteflies and aphids. Subsequently, many infected hosts are found, and the population density of hosts is suppressed for a prolonged period. These results indicate that the secondary infection of healthy individuals from insect cadavers, covered with newly produced conidia, constitutes an important factor for the success of V. lecanii. V. lecanii must be used when the density of target insects is low. 9)B. bassiana and V. lecanii show great potential for the control of horticultural pest insects. The microbial control agents MG-Bb-1 for P. xylostella, MG-VI-18 for T. vaporariorum, and MG-VI-45 for A. gossypii and M. persicae constitute particularly valuable components of integrated pest management (IPM) systems.