Abstract: A survey for natural occurrence of entomopathogenic fungi of two spotted spider mite Tetranychus urticae Koch was made in Coimbatore District of Tamil Nadu, India during 2009. Occurrence of two entomopathogenic fungi viz., Cladospoirum cladosporioides (Fresenius) de Vries to the tune of 75.25, 87.00 and 96.75% and Beauveria bassiana (Bals.-Criv.) Vuill to the tune of 7.50, 12.00 and 5.25% were recorded on T. urticae infesting cowpea, red gram and okra, respectively. Both the fungal isolates were assessed for their pathogenicity against the spider mite, T. urticae and the papaya mealybug, Paracoccus marginatus Williams and Granara de Willink commonly occurring on okra and cotton, using leaf disc bioassay. The results revealed that the fungus, C. cladosporioides was more effective followed by the B. bassiana against two spotted spider mite and for mealybug, B. bassiana was found to be more effective. C. cladosporioides and B. bassiana recorded the LC50 values of 4.30x106 and 5.27x106 conidia mL-1 with LT50 values of 63.80 and 110.30 h, respectively against T. urticae. Against P. marginatus, C. cladosporioides and B. bassiana recorded the LC50 values of 5.20x107 and 3.60x107 conidia mL-1 with LT50 values of 191.04 and 176.64 h, respectively. This is the first record on natural infection of T. urticae by C. cladosporioides and B. bassiana in Tamil Nadu, India.
INTRODUCTION
The two spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae) is responsible for significant yield losses in many horticultural, ornamental and agricultural crops worldwide (Zhang, 2003). T. urticae is of major concern in vegetables causing heavy damage leading to 7-48% yield loss (Srinivasa and Sugeetha, 1999). One of the major problems in the control of T. urticae is its ability to rapidly develop resistance to many important acaricides after only a few applications (Nauen et al., 2001). To circumvent the problems of acaricide resistance and also to enable farmers and growers to respond to consumer concerns about pesticide residues, there is a need for an effective method of phytophagous mite control that does not involve chemicals. This is most likely to be achieved with a suite of natural enemies that complement one another’s activities at different times during crop and pest development. Several insect pathogens viz., Hirsutella thompsonii Fisher, (Aghajanzadeh et al., 2006), Beauveria bassiana (Bals.-Criv.) Vuill (Irigaray et al., 2003) and Metarhizium anisopliae (Metschn.) Sorokin (Chandler et al., 2005) are found promising both under laboratory and field conditions against the phytophagous mites. Entomopathogenic fungi are more advantageous as they are capable of infecting them directly through the integument and are amenable for easy culturing. In recent years, more attention has been given to fungal pathogens of insects, but less attention has been given to the exploitation of fungal pathogens for the control of acarine pests. Hence, there is a scope for the use of fungal pathogens against plant mites. Fungal isolates from different geographical locations will have varying virulence and adaptability to environmental conditions like temperature and humidity. Identification of such type of isolates will pave way for the development of biopesticide with high virulence and temperature adaptability. Moreover, not much work has been done on these aspects in India, against mites. With this view, the present investigation was undertaken to isolate and identify strains of entomopathogenic fungi infecting T. urticae in nature and test the pathogenicity of these fungi under the laboratory conditions
MATERIALS AND METHODS
Survey for natural occurrence of spider mite fungal pathogens: Survey was undertaken during 2009 to isolate and identify the fungi associated with T. urticae on cowpea, redgram and okra in Coimbatore district of Tamil Nadu. Ten plants in each crop viz., cowpea, red gram and okra were selected at random and the number of live and mycosed mites per square cm area from top, middle and bottom leaves were counted and the percentage mortality due to fungi was worked out. Mite cadavers showing natural external growth of fungi were collected and transferred to the laboratory for isolation and identification. Sabouraud Dextrose Agar with yeast extract (SDAY) medium was used for isolation of the fungi and the slants were incubated in BOD incubator at 25±2°C and 80±10% RH until sporulation. After sporulation, T. urticae adults were inoculated with the fungi and reisolated in pure form from the cadavers showing typical mycosis as per the procedure outlined by Goettel and Inglis (1997). The fungal species were identified with the experts at Indian Agricultural Research Institute, New Delhi and USDA, ARS, USA. The isolated cultures were maintained at 25±2°C in an incubator on SDAY. The pure stock cultures were sub-cultured at 15 days intervals in Petri plates (10 cm diameter). Pure stocks in slants were held under refrigerated condition until further use.
Test insect cultures: The red spider mites, T. urticae were collected from okra fields, mass reared and maintained in the okra plants at glass house of Insectary, Department of Agricultural Entomology, Tamil Nadu Agricultural University and Coimbatore by following the method developed by Krishnamoorthy (1988). The mealybugs, P. marginatus were collected from the naturally infested okra and cotton plants, mass reared and maintained on potato sprouts. The mites and the mealybugs from the culture were used for the fungal bioassays.
Bioassays: Two fungi viz., C. cladosporioides and B. bassiana isolated from the T. urticae were assayed in the laboratory against T. urticae and P. marginatus for its pathogenicity. Pure cultures of the fungi were subcultured and the plates showing luxuriant fungal growth (11 to 15 days after inoculation) were selected for harvesting conidia and flooded with 20 mL of sterile distilled water containing 0.02% surfactant, Tween 80 (Feng et al., 1994). The conidia were liberated by gentle agitation and collected in sterile 250 mL Erlenmeyer flask. The final volume was made upto 100 mL with sterile distilled water. Conidia count was determined using a double ruled Neubauer haemocytometer using phase contrast microscope (Goettel and Inglis, 1997).
Adult mites and mealybugs were used for the bioassays. For bioassay against T. urticae, okra leaf discs were placed in a petri dish (10 cm diameter) lined with moist cotton wool and sprayed individually with conidial suspensions of C. cladosporium and B. bassiana at different doses viz., 5x105, 1x106, 5x106, 1x107, 5x107 and 1x108 conidia mL-1 using hand atomizer and allowed to dry for 20 min in a laminar flow. Fifty mites were then transferred to the leaves using a camel hair brush. The control leaves were treated with sterile distilled water containing a 0.02% of Tween 80. All dishes were incubated at 25±2°C. Five replications were maintained for each treatment. Observations on the mortality of mites were taken at 24 h interval upto 8 days after treatment. All dead mites removed from the petridishes were kept in an incubator and the cadavers showing mycosis were considered to be dead as a result of infection by the fungus (Hall, 1984).
Similar procedures were adopted for bioassay against mealybugs using cotton leaf discs as substratum.
Statistical analysis: The concentration and time mortality responses were subjected to probit analysis (Finney, 1971) using a Statistical Package for Social Sciences (SPSS), Ver. 10.00 SPSS Inc., USA.
RESULTS
Survey for natural occurrence of spider mite fungal pathogens: Survey for the natural infection of entomopathogenic fungi on T. urticae revealed the occurrence of two fungi viz., Cladosporium cladosporioides (Fresenius) de Vries (ITCC No. 7641; TNAU 2 - ARSEF 9616) with dirty green color and a white colored fungus, Beauveria bassiana (Bals.-Criv.) Vuill (ITCC No.7654). Occurrence of C. cladosporioides to the tune of 75.25, 87.00 and 96.75% and 7.50, 12.00 and 5.25% by B. bassiana were recorded for the first time on the two spotted spider mite, T. urticae infesting cowpea, red gram and okra, respectively at Coimbatore, Tamil Nadu, India (Table 1).
Bioassays: Fungal pathogens viz., C. cladosporioides and B. bassiana isolated from the spider mite was assayed for its efficacy against the red spider mite, T. uticae, an important pest of okra and against the papaya mealybug, P. marginatus which is a growing concern over most of the crops including okra and cotton. Results of the bioassays revealed significant differences in the concentration and time mortality responses. C. cladosporioides was more effective with lowest LC50 and shortest LT50 values followed by B. bassiana against two spotted spider mite and for mealybug B. bassiana was found to be more effective. C. cladosporioides and B. bassiana recorded the LC50 values of 4.30x106 and 5.27 x106 conidia mL-1 with LT50 values of 63.80 and 110.30 h, respectively against T. urticae (Table 2, 3). Against P. marginatus, C. cladosporioides and B. bassiana recorded the LC50 values of 5.20x107 and 3.60x107 conidia mL-1 with LT50 values of 191.04 and 176.64 h, respectively (Table 4, 5).
| Table 1: | Survey for the natural occurrence of fungal pathogens on two-spotted spider mite, T. urticae -in Coimbatore District (2009) |
| Table 2: | Concentration-mortality response of two-spotted spider mite, T. urticae to fungal pathogens |
| No. of mites used per treatment was 300 | |
| Table 3: | Time-mortality response of two-spotted spider mite, T. urticae to fungal pathogens |
| No. of mites used per treatment was 300 | |
| Table 4: | Concentration-mortality response of papaya mealybug, P. marginatus to fungal pathogens |
| No. of mealybugs used per treatment was 300 | |
| Table 5: | Time-mortality response of papaya mealybug, P. marginatus to fungal pathogens |
| No. of mealybugs used per treatment was 300 | |
DISCUSSION
Potential of entomopathogenic fungi for the management of phytophagous mites was reviewed by Van der Geest et al. (2000). Isolation and identification of indigenous fungal pathogens are the need of hour to manage the pests in an ecofriendly manner. In the present investigation, survey for the natural infection of entomopathogenic fungi on T. urticae revealed the occurrence of C. cladosporioides and B. bassiana. The entomogenous fungi, Cladosporium spp. have been isolated and reported from different insect (Abdel-Baky and Abdel-Salam, 2003; Perea et al., 2003) and mite (Pena et al., 1996; Van der Geest et al., 2000) hosts in nature in different regions of the world. Occurrence of C. cladosporioides on Bemisia sp., (Abdel-Baky et al., 1998), Cladosporium. uridenicola (Link ex Gray) on aphids and whiteflies (Abdel-Baky, 2000) and Cladosporium oxysporum (Berk. and Curt.) on Planococcus citri (Risso) (Samways and Grech, 1986) was also reported. Natural infection of T. urticae by C. cladosporioides was also reported by Eken and Hayat (2009) in Turkey. However, this is the first record of natural infection of fungal pathogens on T. urticae from Tamil Nadu, India.
Pathogenicity tests of the two fungal isolates revealed significant differences in the concentration and time mortality responses. C. cladosporioides was more effective with lowest LC50 and shortest LT50 values followed by B. bassiana against two spotted spider mite and for mealybug B. bassiana was found to be more effective. Similarly, Eken and Hayat (2009) reported that the C. cladosporioides isolates could cause 50.95 to 74.76% mortality of T. urticae with a LT50 values ranged from 2.34 to 3.90 days in Turkey. Tamai et al. (1999) reported that the B. bassiana could cause 50% mortality at concentrations ranging from 5x106 to 1x109 conidia mL-1. The isolate 447 (ATCC 20872) of B. bassiana applied at a concentration of 108 conidia mL-1 gave 74.4% mortality of T. urticae (Alves et al., 2002). Chandler et al. (2005) reported that B. bassiana 432.99 (cultured from 'Naturalis-L', Troy Biosciences, Phoenix, TX, USA) recorded 98% reduction in the numbers of T. urticae adults, nymphs and eggs under glass house condition. Efficacy of B. bassiana against P. marginatus was also reported by Banu et al. (2010).
CONCLUSION
Selection of successful and virulent isolates of fungal pathogens is the need of the hour for the biosuppression of the phytophagous mites. Present investigation demonstrates the effectiveness of the fungi viz., C. cladosporioides and B. bassiana against both T. urticae and P. marginatus under laboratory condition. Further, research on the field efficacy of these fungi could wide open their potential for the successful management of T. urticae and P. marginatus with less dependence on chemical control. Natural occurrence of other entomopathogenic fungal fauna against these pests needs to be explored.
ACKNOWLEDGMENTS
The authors thankfully acknowledges help rendered by staff, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi and Dr. Richard Humber, USDA-ARS-NAA-BioIPM, Insect Mycologist and Curator, ARSEF Collection of Entomopathogenic Fungal Cultures, USA for their identification services.