Entomopathogenic Fungal Screening against Two Spotted Spider Mites, Tetranychus urticae Koch in Tomato and Broad Mite, Polyphagotarsonemus latus (Banks) in Chilli

The 21^st century came with great challenges and difficulties for humanity. The main need for crop production is to protect plants including the management of arthropod pests. The widespread use of chemical pesticides, the resulting pollution, the adverse impact on human health, other organisms and the need for chemical reduction in agriculture have spurred the development of other pest control measures in recent times. Entomopathogenic fungi are parasitic microorganisms with an ability to infect and kill arthropods. They are mainly used as biopesticides in ecologic farming as a safe alternative to toxic chemical insecticides (Jaihan et al., 2016; Ríos-Moreno et al., 2016; Lovett and Leger, 2017).
       
According to Mustafa and Kaur (2008), the benefits of EPF over chemical pesticides are their high specificity of capture and the inability of pests to develop resistance as EPF simultaneously uses several methods of action. The distinctions associated with evacuation in different locations will have different intensity and adaptability to environmental conditions such as temperature and humidity. The application of the traditional pressure of entomopathogens with greater virulence and persistence in the environment requires an hour. Identification and detection of that type of isolation will enhance the synthesis of biopesticides with high virulence and temperature. In addition, there is not much work done on these factors in India, especially in Tamil Nadu against the constraints and selection of hazardous areas for pest management is still in the early stages of our country. To keep this in mind, the present investigation is being conducted on the classification of indigenous fungal species and their relative stability in Tetranychus urticae Koch and Polyphagotarsonemus latus (Banks) under laboratory and field conditions.

Extensive studies have been carried out in various agro- climatic regions of Tamil Nadu to document the natural occurrence of entomofungal pathogens on insects and insects from August 2012 to December 2013.
 
Isolation, maintenance and growth conditions of entomopathogenic fungi
 
Insect / mite cadavers that appear to be infected by fungi were collected during the survey and brought to the laboratory for isolation in specific media. To isolate the entomopathogenic fungi, mycoses / insects collected from the fields were surface sterilized with 4% sodium hypochloride and then washed several times with sterile water. In a sterile petri dish, diseased samples were placed and a small portion of the infected area was transferred to a culture plate containing Sabouraud’s maltose agar, enriched with one percent yeast extract (SMAY) media and incubated at 25±2°C. After two days of incubation, the organisms were subcultured for purification (Butt and Goettel, 2000). Pathogenesis of isolated fungi was established by spraying seed suspensions in healthy insect colonies. Fungi were again isolated from dead insects and compared with the original culture. Pure cultures of fungal isolates were maintained in the laboratory for further study. Isolated new fungal pathogens were identified at the Plant Pathology Division of Indian Agricultural Research Institute, New Delhi, India.
 
Source of entomopathogenic fungal isolates
 
Pure cultures of the entomopathogenic fungal strains, Beauveria bassiana (Balsamo) Vuillemin, Metarhizium anisopliae (Metchinkoff) Sorokin and Lecanicillium lecanii (Zimm.) Zare and Gams were obtained from the Department of Agricultural Entomology and Plant Pathology, Tamil Nadu Agricultural University (TNAU), National Bureau of Agricultural Insect Resources (NBAIR), Bangalore and Sugarcane Breeding Institute (SBI), Coimbatore. Local isolates viz. B. bassiana (Bb 111) and Metarhizium flavoviride Gams and Rozsypal var. minus (Mf) were obtained during the survey. The details of fungal isolates are as follows:


 
Mass culturing of broad mite, Polyphagotarsonemus latus and two-spotted spider mite, T. urticae
 
Chilli K2 seedlings were grown in pots (size: 30 cm dia.) and used to maintain broad mite cultures in the glass house. Heavily infested leaves from the plants in field were collected and released onto 45 days old chilli plants raised in pots. The mites were maintained and replaced with fresh ones within 45 days. For culturing T. urticae, potted tomato plants (PKM1) were maintained in the glasshouse at a temperature of 28 ± 1°C with 70 ± 5% RH. The leaves of the host plant, which were severely affected by T. urticae, were collected and placed in potted plants to begin the basic culture of the test pests.
 
Laboratory bioassay
 
Bioassay was conducted with the isolates of entomopathogenic fungi to test their pathogenicity against broad mite and two-spotted spider mites under laboratory conditions. For the bioassays, sub-cultures were prepared in SMAY Plus 1% yeast extract and incubated for 10-14 days at a temperature of 25 ± 2°C. Conidia were removed from culture media using sterile spatula, suspended in 10 ml sterile distilled water containing 0.05% Tween 80 and filtered through moist filter papers. Conidial suspensions were rotated for 15 minutes to create a homogeneous suspension and conidia were counted using a Neubauer hemocytometer under a phase contrast microscope (Leica TM1000). The suspensions were prepared at concentrations of 1 × 10⁴, 1 × 10⁵, 1 × 10⁶, 1 × 10⁷ and 1 × 10⁸ conidia ml^-1. The seed suspensions used were less than four hours and stored in the refrigerator (Sangeetha, 2013).
       
Insects from infected chilli and tomato seedlings were used for bioassay studies. The chilli and tomato leaf affected with mites was placed underside up in a Petri dish (9 cm diameter) lined with a surgical cotton wad (8 cm diameter) above which a water-soaked 2-layer filter papers were kept to ensure high relative humidity. The leaf petiole was inserted into a wet cotton swab and kept for a long time to maintain the freshness of the leaves. Thirty adult mites were selected from the chilli leaves placed on the petri dish and the remaining nymphs and adults were removed using a fine brush. Bioassays were performed with conidial concentration ranging from 1 × 10⁴ to 1 × 10⁸ conidial ml^-1 in 0.05% aqueous Tween 80 with three replications each. Mites sprayed with 0.05% Tween80® solution served as control. The percentage of affected adults was recorded for up to seven days of treatment. Cadavers with fungal growth were considered as a successful infection.

Relative susceptibility of T. urticae and P. latus adults to fungal pathogens
 
Bioassays were performed with fourteen fungal isolates viz., eight isolates of  B. bassiana (Bb 111, Bb 112, Bb 113, Bb 114, Bb 101, B2. Bb NBAIR and Bb SBI), three isolates of Metarhizium anisopliae (Metchinkoff) Sorokin (M2, Ma NBAIR and Ma SBI), one isolate of M. flavoviride var. minus (Mf) and two isolates of Lecanicillium lecanii (Zimm.) Zare and Gams (Ll NBAIR and L2) against T. urticae and P. latus. The bioassay results showed that T. urticae and P. latus were susceptible to all the fungal isolates tested. Investigation of smears of mite cadavers confirmed mycosis as the cause of death in variants treated with conidial suspensions of the fungal isolates.
       
The pathogenicity of entomopathogenic fungi differed between each other. The red spider mite infected by fungi were mummified, hard to touch and mycelial growth develops after 24 to 48 h of death. Initially, the growth of the fungus was uneven in the inter membrane of the abdomen and eventually the entire cadaver was covered by the growth of the fungus. Two days after the treatment. The broad mite entered the state of moribund two days after treatment and then quickly covered the entire body followed by sporulation.
 
Median lethal concentration (LC₅₀)
 
Bioassay of entomofungal pathogens against T. urticae and P. latus showed variation in virulence. The results on LC₅₀ values against T. urticae revealed that the isolate, Bb 112 had highest virulence with the lowest LC₅₀ values of 0.6 × 10⁶ spores ml^-1 followed by Bb 101 (1.3 × 10⁶ spores ml^-1) with overlapping fiducial limits. This was followed by M. flavoviride var. minus which recorded LC₅₀ value of 1.4 × 10⁶ spores ml^-1 (Table 1). For other isolates viz., Bb 111, Bb 113, Bb 114, B2, Bb SBI, Ma NBAIR, Ma SBI, M2, Ll NBAIR and L2 the LC₅₀ values ranged from 1.8 × 10⁶ to 5.3 × 10⁶ spores ml^-1 and were not significantly different in their virulence as evidenced by the overlapping fiducial limits. Against P. latus, the LC₅₀ values ranged from 0.3 to 3.6 × 10⁶ spores ml^-1. Isolate Bb 112 and Bb 101 had higher virulence with lowest LC₅₀ values of 0.3 × 10⁶ and 0.8 × 10⁶ spores ml^-1 (Table 2). The other isolates, Bb111, Bb 113, Bb 114, Bb NBAIR, Bb SBI, Ma NBAIR, Ma SBI, M2, M. flavoviride var. minus, Ll NBAIR and L2 formed a group with overlapping fiducial limits.
 

 

 
Median lethal time (LT₅₀)
 
Considering the time mortality, variation in response of the T. urticae and P. latus adults to different fungal isolates was observed. Against T. urticae, the results indicated that Bb111 and Bb 112 killed the mites more quickly than other isolates. At the concentration of 10⁸ spores ml^-1, the LT₅₀ values of the Bb 112 and Bb 111 isolates were 92.60h and 95.37h, respectively followed by Ma SBI and M2 (98.44h and 104.45h) (Table 3). The time mortality of fungal pathogens against P. latus showed that the isolate Bb 112 had the lowest LT₅₀ value of 92.32h (Table 4). The isolates Ma SBI (95.24h), Mf (97.57h) and Ma NBAIR (97.84h) were next in the order of virulence with respect to time of kill and had overlapping fiducial limits with Bb112. The other isolates did not vary very much and had overlapping fiducial limits. The highest LT₅₀ value was recorded in Bb NBAIR (128.28h).
 

 

       
All the fungal pathogens tested indicated that the mortality of mites increased with increase in concentration. Muthukumar (2005) and Seiedy et al., (2010) also reported similar results with B. bassiana against T. urticae with the LC₅₀ values of 1.46 × 10⁵ spores ml^-1 and 3.7 × 10⁵ conidia ml^-1, respectively. Gatarahiya et al., (2012) also showed that B. bassiana strain PPRI 7315 had a median lethal concentration of 1.13 × 10⁶ conidia ml^-1 against T. urticae. Wekesa et al., (2005) reported the LC₅₀ values of 0.7 × 10⁷ and 2.5 × 10⁷ conidia ml^-1 for B. bassiana and M. anisopliae, respectively against T. urticae. The probit ­values obtained in the present study are also in accordance with Alves et al., (2002); Irigaray et al., (2003); Draganova and Simova (2010) and Seiedy et al., (2010) against T. urticae. Many isolates of Beauveria species have been found to produce toxic compounds after invasion of the host haemolymph (Roberts, 1981).
       
The findings of the present study against P. latus is in accordance with Nugroho and Ibrahim (2004) who reported the LC₅₀ value of B. bassiana was 2.74 × 10⁶ conidia ml^-1 and P. fumosoroseus was 3.23 × 10₆ conidia ml^-1 against P. latus on chilli with the LT₅₀ values of 3.5 and 2.4 days, respectively at the dosage of 1 × 10⁸ conidia ml^-1. The LC₅₀ against P. latus on bean plants was 1.16 × 10⁶ B. bassiana conidia ml^-1 (Pena et al., 1996). The effectiveness of M. anisopliae against P. latus in the present study was in agreement with the findings of Maketon et al., (2008) with the LC₅₀ of 1.3 × 10⁷ conidia ml^-1 and the corresponding LT₅₀ of 3.8 days.

The results of the present investigations on relative susceptibility are indicative that the fungi that were associated with the arthropod hosts showed pathogenicity to the adult mites with varying degree of virulence.