CONTROL OF COTTON APHID AND GREENHOUSE WHITEFLY WITH A FUNGAL PATHOGEN Jeong Jun Kim, Min Ho Lee, Cheol-Sil Yoon 1, Hong-sun Kim, Jai-Ki Yoo, and Kyu-Chin Kim 2 Division of Entomology, National Institute of Agricultural Science and Technology, (NIAST), RDA, Suwon, 441-707 Korea 1 Research Institute of Engineering and Technology, Korea University, Seoul 136-701, Korea 2 Department of Agrobiology, Chonnam National University, Kwangju, 500-757, Korea ABSTRACT This Bulletin reports on the control of aphid and whitefly using a domestic strain of the fungal pathogen V. lecanii in Korea. Of the six isolates collected in Korea, V. lecanii CS-625 caused the highest mortality of cotton aphid (Aphis gossypii). Strain CS-626 was selected as a biological control agent for whitefly, based on bioassay and field tests. The pesticides dimethomorph and procymidone did not affect spore germination and mycelial growth of V. lecanii CS-625, provided they were used at the recommended concentration. INTRODUCTION Aphid and whitefly are serious insect pests in greenhouses all over the world. They damage a range of crops, especially cucumber, pepper, and tomato. They rapidly increase in numbers, and they transmit plant viruses. To control them, farmers apply high doses of pesticides. The overuse of pesticides has resulted in insect resistance to insecticides. Environmental residues are also a concern, because consumers demand pesticide-free food. Thus, many countries are trying to reduce their use of pesticides. Biological control is an alternative control method. This includes entomopathogenic fungi (i.e. fungal pathogens which affect insects) (Latge and Papierok 1988, Hajek and St. Leger 1994). They are found in the field under natural conditions. There is also interest in the commercial development of selected strains of such fungi (Milner 1997). Fungi which cause disease in insect pests include Erynia neoaphidis and Zoophthora radicans. They are characterized by rapid sporulation and germination, high virulence, and active discharge of conidia, all attractive traits in a fungal pathogen. However, mass production and storage of these fungi are difficult. In contrast, hypho-mycete fungi such as Verticillium lecanii and Beauveria bassiana require several days of high humidity for sporulation and infection. They also depend on the movement of aphids to come into contact with their conidia, and a much greater number of conidia are needed to transmit infection. Nevertheless, hyphomycete fungi are cheap to mass produce, easy to store, and effective over a wide range of temperatures and humidity levels. They also provide a rapid kill at economical doses. Two isolates of V. lecanii were introduced by Koppert into the Netherlands in 1981 and 1982. One controls aphids on chrysanthemum, while another controls whitefly on cucumber and tomato (Milner 1997). In Korea, eight species of fungi which infest insects, including Pandora neoaphid and Zoophthora radicans, were found on several Keywords: biological control, cotton aphid, fungal pathogen, Korea, V. lecanii, whitefly, 7
species of aphid (Yoon et al. 2000). This Bulletin reports on laboratory virulence tests against cotton aphid, using indigenous strains of hyphomycete fungi, and field tests of selected isolates of V. lecanii CS-626 to control whitefly. CONTROL EFFICACY OF THE FUNGAL PATHOGEN AGAINST COTTON APHID Testing for virulence Cotton aphid (Aphis gossypii) is one of the most important insect pests of vegetable crops in Korea. We tested the potential for biological control of several species of hyphomycete fungi native to Korea. These included two isolates of Beauveria bassiana, one isolate of Verticillium lecanii and three isolates of Paecilomyces spp.. The time intervals required for a mortality rate of 50% (LT 50 ) among the A. gossypii varied with different fungal strains (Table 2). V. lecanii CS-625 had the highest virulence against A. gossypii, which was found in both the conidia and the blastospores. The time taken for V. lecanii CS-625 conidia and blastospores to inflict LT 50 of A. gossypii was shorter than for the other isolates, at 2.74 and 3.31 days, respectively. These findings suggest that V. lecanii CS-625 is ideal for further development as a microbial pesticide to control aphids. Effect of temperature on virulence Tests to study the effect of temperature on virulence were carried out. A suspension of conidia was sprayed onto cucumber plants, which were then kept at different temperatures (20, 25, 30 and 35 C). The mortality rate among the cotton aphids was recorded each day for five days. The mortality rates varied greatly according to the temperature. They were lower at 20 C than at 25 and 30 C (Fig. 1). However, because V. lecanii CS-625 showed the highest germination rate at temperatures of 20-25 C (Yoon 2000), temperatures of around 20 C are considered optimal for the use of Table 1.Original hosts and geographical location of isolates examined Table 2.Corrected mortality and LT 50 of A. gossypii 5 days following treatment, using native strains of entomogenous fungi (mean ± SD) 8
this biocontrol agent. Effect of concentration of the inoculum Tests were made to identify the most effective concentration of conidia in the inoculum. Application of inoculum at a concentration of 10 4-10 7 conidia/ml gave relatively low mortality. A higher concentration of 10 8 conidia/ml gave nearly 100% mortality after five days (Fig. 2). This higher concentration should therefore be used. The type of formulation must be studied in future to improve efficacy in the field. Influence of fungicides on V. lecanii CS-625 Each crop suffers from many pests and diseases, which are controlled by chemical as well as biological methods. For this reason, it is necessary Fig. 1. Effect of temperature on virulence of conidia of Verticillium lecanii CS-625 on nymphs of Aphis gossypii Fig. 2. Virulence of Verticillium lecanii CS-625 on Aphis gossypii on cucumber at different conidial concentrations 9
to assess the effect of pesticides on biological agents. The fungicides tested were wettable powders. To study the influence of pesticides on mycelial growth, a 6 mm plug of V. lecanii was transferred onto a plate containing a pesticide, and incubated at 25±1 C for seven days. Colony diameters were measured after 7 days. Significant differences were found in the effect of various fungicides on the spore germination and mycelial growth of V. lecanii CS-625 (Table 3). Two fungicides, Fenbuconazole + thira and propineb, strongly inhibited both spore germination and mycelial growth. Two others, azoxystrobin and chlorothalonil, strongly inhibited spore germination but did not seriously impair mycelial growth. Two fungicides, dimethomorph and procymidone, did not affect either spore germination or mycelial growth. Tests carried out by Hall (1981) included a different range of fungicides. He found that benomyl inhibited mycelial growth, but only slightly reduced spore germination. Wilding (1972) also found that benomyl inhibited the mycelial growth of V. lecanii on agar. As in our study, Hall (ibid) found that clorothalonil, dimethomorph, and thiram were toxic to V. lecanii. Hall found that carbendazim + kasugamycin and fenarimol strongly affected mycelial growth but did not impair spore germination. CONTROL EFFICACY OF THE FUNGAL PATHOGEN AGAINST WHITEFLY Two species which infest field crops, Bemisia tabaci (sweetpotato or silverleaf whitefly) and Trialeurodes vaporariorum (greenhouse whitefly) may cause severe damage in any country with a suitable climate. The two species are both present in South Korea, but T. vaporariorum is the more important pest of vegetable crops. In recent years, many of the conventional insecticides used to control whitefly have come under scrutiny, because of resistance problems and/or undesirable effects on natural enemies and the environment. New approaches are being tried, including the use of entomopathogenic fungi such as Verticillium lecanii. For this purpose, five strains of V. lecanii from Korea and other countries were tested and compared. The strain CS-626 was selected as being highly virulent against greenhouse whitefly at various temperatures (Table 4). Mortality of the CS-626 strain was nearly 100% at 25 C, and 80% at 15 and 35 C on the 7th day. This result indicates that the CS-626 strain should be effective in Korean greenhouses, in which the temperature during spring and fall normally ranges from 20 C to 35 C (Yoon et al. 1999). Since the virulence of the strain changes Table 3.Inhibition by fungicide of spore germination and mycelial growth of Verticillium lecanii 10
according to both the concentration of conidia and environmental conditions, we need to determine the optimal concentration (Fargues 1972, Hall and Papierok 1982). Table 5 shows the effect of different concentrations of the CS-626 conidial suspension on the mortality of adult whitefly. The LC 50 was estimated at 2.3 x 10 6 conidia/ml. Low concentrations of 1x10 3-10 7 conidia/ml showed relatively low mortality. Higher concentrations of 1x10 9 and 10 8 conidia/ml gave almost 100% mortality after 7 and 9 days, respectively. Since the difference at LT 50 between the two concentrations was only one day (Table 5), spraying a conidial suspension at a concentration of 10 8 conidia/ml seems to be an economical and effective way of controlling whitefly. However, these data may not be directly applicable to the prediction of mortality in greenhouses, which needs further research. Experiments in whitefly control Pot and greenhouse tests were carried out to test the effectiveness of V. lecanii strain CS-626 in controlling whitefly. Tomato plants infested with more than 75 second instar larvae of whitefly (T. vaporariorum) per leaf were sprayed with inoculum containing various concentrations of conidia (10 6-10 9 conidia/ml). All the conidia were the CS-626 strain. The same strain was used on tomato plants in two greenhouse tests. The number of adult whitefly decreased by 60.9% in plants grown on higher shelves and 72% in plants grown on lower shelves, compared to the control (Fig. 3). Fig. 4 and Fig. 5 show the control efficacy of the same CS-626 strain against whitefly in a greenhouse. The conidial solution was effective for Table 4.Mortality of whitefly (T. vaporariorum) inoculated with conidia of different strains of V. lecanii, and maintained at different temperatures Table 5.LT 50 for V. lecanii strain CS-626 tested against T. vaporariorum scales and the number of conidia adhering to a single scale at various concentrations of the conidial suspension (mean ± SD) 11
only a short period of time. Repeated applications were needed to further decrease whitefly population densities. Some kind of forecasting system is needed to determine the optimum times of application. CONCLUSION The CS-626 strain of the fungal entomopathogen V. lecanii was an effective biological control agent against T. vaporariorum in South Korean greenhouses. It gave good control of both cotton aphid (Aphis gossypii) and whitefly (Trialeurodes vaporariorum) infesting vegetable crops grown in greenhouses. For both pests, a suitable concentration of conidia in the suspension applied as inoculum was 10 8 /ml. The optimum temperature range for maximum virulence was 20-25 C. Since all crops suffer from a range of pests and diseases, not just one, an important aspect of biological control is the tolerance of the agent to chemical pesticides. We tested the tolerance of V. lecanii CS-625 to a range of pesticides. The pesticides dimethomorph and procymidone did not affect spore germination or mycelial growth, provided they were applied according to the recommended dosage. More research is needed on formulations which will improve the effectiveness of V. lecanii in controlling aphids and whitefly. It is important to prolong the period of its effectiveness. At present, the conidial solution is capable of infecting target insect pests only for a short time after it is applied to crops. REFERENCES Chandler, D., J.B. Heale, and A.T. Gillespie. 1993. Germination of entomopathogenic fungus Verticillium lecanii on scales of the glasshouse whitefly Trialeurodes vaporatiorum. Biological Science and Technology 3: 161-164. Fargues, J. 1972. Étude des conditions d infections des larves de doryphore Leptinotarsa decemlineata Say. par Beauveria bassiana (Bals.) Vuill. (Fungi imperfecti). Entomophaga 17: 319-337. Hajek, A.E. and St Leger, R.J. 1994. Interactions between fungal pathogens and insect hosts. Annual Review of Entomology 39: 293-322. Hall, R.A. 1976. A bioassay of the pathogenicity of Verticillium lecanii conidiospores on the aphid, Macrosiphoniella sanborni. Journal of Fig. 3. Mortality of whitefly (T. vaporariorum) after treatment with fungal pathogen V. lecanii, strain CS-626, in a greenhouse. Temperature was 28.3±6.7 C, and relative humidity was 72.9±33.6% during the experiment. 12
Fig. 4. Mean density of adult whitefly (T. vaporariorum) in higher shelves of cherry tomato plants in a greenhouse. Temperatures are 17.0±7.8 C, and relative humidity was 66.5 ± 22.9% at the first application. Fig. 5. Mean density of adult whitefly (T. vaporariorum) per sticky trap in greenhouse. 13
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