Role of beneficial fungi in managing diseases and insect pests of tea plantation

The effectiveness of Trichoderma atroviride, T. asperellum, T. harzianum, against targeted tea disease causing pathogen, Fusarium solani (dieback), Beauveria bassiana against tea mosquito (Helopeltis theivora), and Metarhizium anisopliae against the red spider mite, Oligonychus coffeae Nietner, and the live wood eating termite (Microcerotermes beesoni Snyder), respectively were evaluated under laboratory and field conditions. The Trichoderma isolates showed effective control of F. solani (64.6 to 71.7%) under laboratory conditions, while the wettable powder (WP) formulation could reduce dieback disease incidence (57.1 to 77.7%) over control, under field conditions. The post prune application of Trichoderma asperellum on light pruned (LP) and application of T. asperellum as well as T. harzianum on deep skiffed (DS) tea fields showed a significant growth promotional response. The treated bushes could produce an average number of 421.2 and 398.4 shoots when it was applied 7.5 and 5.0% (w/v), respectively, which was higher than the number of shoots recorded in the control plots (259.4). The antagonist also induced comparatively higher shoot length (6.85–7.99 cm) than the untreated control plots (5.13–6.38 cm). The B. bassiana isolates (2 × 10 conidia/ml) exhibited 71.5 to 93.0% control of 2nd instar nymphs of H. theivora under in vitro conditions. However, addition of different adjuvants resulted in enhanced mortality. Under field conditions, the highest reduction of tea mosquito population of 52.3% was noted when B. bassiana (1 × 10cfu/ml) was sprayed along with Tween 20 plus and crude sugar. However, the wettable powder formulation of this EPF along with jaggery reduced the insect incidence. The M. anisopliae effectively controlled the red spider mite to the tune of 46.3 to 63.8% under laboratory conditions, while the wettable powder formulation of this isolate was more effective against the termite, resulting in the reduction of infestation to the tune of 84.2% under field conditions.


Background
Tea (Camellia sp.) is one of economically important plantation crops of India. It is perennial in nature and hence provides favorable micro as well as macroclimate for thriving of different insect pests and fungal diseases, which together cause enormous crop loss in terms of quantity and quality. Synthetic agrochemicals have been used for the management of these pests and diseases.
Certainly, they provide efficient control for longer period; however, their frequent and injudicious application had definitely invited numerous other associated problems such as environmental pollution, development of pesticide resistance in insects, and unwanted pesticide residues in made tea (Roy et al. 2011).
The biological control agents (BCAs) like the species of Trichoderma, Beauveria, and Metarhizium are reported to be safer and promising components of integrated pest and disease management (IPDM) strategies, which have been adopted in various crops including tea (Hall and Papierok 1982). Although research works have been carried out in this direction (Debnath 1996;Kumhar 2013, 2014), however very little information is available on the commercialization of such beneficial fungi as potential microbial pesticides.
The present study aimed to isolate Trichoderma spp., B. bassiana, and M. anisopliae from the tea soils and development of suitable formulations for in vitro as well as in vivo evaluation against targeted diseases and insect pests.

Isolation and identification of fungal species
The species of Trichoderma, Beauveria, and Metarhizium were isolated from soil through standard technique (Askew and Laing 1993;Ghanbary et al. 2009 andQazzaz 2012) during 2014-2015. Purification of isolates was done through hyphal tip culture technique and stored in refrigerator. Then, pure cultures were grown on potato dextrose agar (PDA) plates, followed by incubation at 25 ± 2°C for 48-96 h. The pure cultures were identified based on their colony characters like colony color, growth pattern and formation of conidial rings, and color of conidia. The shapes of conidiophores and phialides were observed using microscope. Later on, the identity of isolates was re-confirmed from Indian Type Culture Collection, Division of Mycology and Plant Pathology, Indian Agricultural Research Institute, New Delhi, India.
For isolation of pathogen, i.e., Fusarium solani, dieback diseased tender tea shoots were collected from tea bushes during 2014-15. The diseased shoots were cut in to small pieces, followed by surface sterilization with mercuric chloride (0.1%) and subsequent 2 washing with distilled water. Then, these pieces were inoculated in to PDA plates, and plates were incubated at 26 ± 2°C for 1 week. Colonies developed in plates were purified, using PDA plate. After colony development, they were identified on the basis of morphological characteristics.
In vitro bioefficacy of fungi Trichoderma spp.
Bioefficacy of Trichoderma spp. was assessed by dual culture technique (Stack et al. 1986). Five-millimeter discs of both fungi (Trichoderma spp. and F. solani) were transferred in to plates at equidistance. In control, only F. solani was inoculated for comparison. Each treatment was replicated 5 times. Plates were incubated at 25 ± 2°C for 1 week. Pathogen's mycelial growth was measured and its inhibition was worked out, using the following formula: Mycelial growth inhibition % ð Þ ¼ Colony dia:in control À Colony dia:in treatment Colony dia:in control Â 100

Beauveria bassiana
The in vitro bioassay of B. bassiana against H. theivora was carried out by employing the methodology of Amarsena et al. (2011) with slight modifications. To prepare spray suspension, 10 ml of distilled sterilized water was added in to 2 weeks old B. bassiana culture grown on potato dextrose agar (PDA) slant, and biomass was harvested, followed by filtration twice through muslin cloth. The conidial concentration (2 × 10 9 ) was determined, using hemocytometer and sprayed on the nymphs of H. theivora with the help of an atomizer. Each treatment was replicated 3 times. Insect mortality was recorded till 192 h, and percent mortality was corrected using Abbott's formula (Abbott 1925 Different adjuvants, namely, Tween 20 (2 ml/l of water), glycerol (5 ml/l of water), and crude sugar/molasses (5 g/l of water), were in vitro studied to find out their role in enhancing the efficacy of B. bassiana against 2nd instar tea mosquito. B. bassiana conidial suspension in combination with these adjuvants separately was sprayed on tea shoots, and known numbers of nymphs were released on to these shoots. Each treatment was replicated thrice times, and observations on insect mortality were recorded and 24-h interval for a period of 1 week.

Metarhizium anisopliae
The pathogenicity of M. anisopliae was carried out against the red spider mite (O. coffeae) employing leaf disc technique (Plate 1). The fungal culture was mass multiplied, using potato dextrose broth (PDB). The conidial concentration (1 × 10 8 to 2 × 10 8 ) was determined, using hemocytometer and sprayed on the red spider mite with atomizer. Twenty insects per treatment were taken, and each treatment was replicated 4 times. The insect mortality was calculated by the following formula.
The dead insects were collected and re-inoculated in the PDA (potato dextrose agar) plates followed by incubation at 26 ± 2°C for 1 week to confirm insect mortality.

In vivo testing of fungi
The liquid fermentation technique was adopted with minor modifications for the mass production of Trichoderma atroviride, T. asperellum, T. harzianum, and B. bassiana using 25-l capacity fermenter (Bhat et al. 2009). The potato dextrose broth (Hi-media) was used as basal medium. The biomass was harvested after 11 days, and wettable powder formulations were prepared. M. anisopliae was mass multiplied on PDB medium. After 2 weeks, the medium and biomass was homogenized and used for field application. It was sprayed on tea stem and drenched uniformly in collar region during 2015-16.

Trichoderma spp.
Developed wettable powder (WP) formulation (2 × 10 8 cfu/g) of Trichoderma isolates and commercial formulation was tested against dieback disease in field during 2015-16. Two sprays at weekly interval were given immediately after plucking the shoots. Sixty bushes were taken per treatment, and each treatment was repeated four times. Pre-and post-spray observations on number of dieback shoots were recorded. One hundred shoots from plucking basket were taken, and infected shoots were counted. The disease reduction over control was calculated by the following formula. The formulation was tested during cold weather in the first week of December on light pruned (LP) and deep skiffed (DS) teas at experimental plot of Tea Research  Association North Bengal Regional Research and Development Centre. It was sprayed immediately after pruning operation. After 2 months, the number of bud break (five bushes) and shoot length (5 shoots of 5 bushes) was recorded from each treatment.

Beauveria bassiana
The B. bassiana formulation was evaluated during 2015-2016 at TRA NBRRDC plot in combination with different adjuvants, namely, Tween 20 (2 ml/l of water) and crude sugar (5 g/l of water). Randomized block design (RBD) with 3 replications was followed.

Metarhizium anisopliae
The in vivo bioefficacy of M. anisopliae was also assessed for the control of termite during 2015-2016.
The broth culture at 5% v/w sprayed on the bush stem and drenched in the collar region properly two times at an interval of 3 months. Thiamethoxam was used as standard check for comparison. Pre-and post-treatment observations on number of bushes showed presence of earth runs, or live termite was calculated.

Results and discussion
Isolation and identification of fungal species On the basis of colony and morphological characters, the isolated antagonistic and EPFs were identified by the first author. Pathogen was identified as Fusarium solani, which produced cream-colored colonies with pinkish pigmentation on PDA. It produced sickle-shaped macro-conidia  (mostly mono, bi, tri, rarely tetra septate) and aseptate micro-conidia.

In vitro bioefficacy of fungi
Trichoderma isolates effectively controlled F. solani (64.6 to 71.7 %). Among tested isolates, the maximum growth inhibition was noted in case of T. asperellum (KBN-29), followed by T. atroviride (KBN-24) and T. harzianum (KBN-1/14) as indicated ( In this present investigation, it was observed that different adjuvant enhanced the efficacy of B. bassiana under laboratory conditions. The highest mortality (77.5%) was achieved when crude sugar was added with B. bassiana. Combination of surfactant plus humectant ranked 2nd when insect population was exposed to tea leaves, which were sprayed by the formulation 5 days prior to the initiation of the experiment. When insects were exposed to tea shoots sprayed 10 days prior to the experiment resulted in reduced insect mortality (17.5 to 30.0%). The sprayed shoots were supported with glass vial filled with water to keep the shoots without wilting (Table 3). The EPF Lecanicillium lecanii and NKAE in combination with jaggery resulted in to increase the insect mortality as high as 12.5% (Subramaniam Sankara Rama et al., 2011). Gatarayiha et al. (2010) observed 60.0 to 85.7% mortality rate of spotted spider mite due to application of B. bassiana (4.2 × 10 6 conidia per ml) in combination with Break-thru (polyether-polymethylsiloxane-copolymer, a silicone surfactant). In combination with oil emulsion, this fungus could cause 39.4 to 61.3% mortality of adult mites.
In the present investigation, the formulation of M. anisopliae could cause mortality of red spider mites to the tune of 46.3 to 63.8%. This mortality recorded was concentration dependent, and it was the highest at the concentration containing 2 × 10 8 and the lowest at 1 × 10 8 conidia/ml (Fig. 1) under laboratory conditions. Roobak   observed that Pseudomonas putida was capable of controlling of red spider mites under laboratory conditions. The mites (both nymphs and adults) when exposed to this bacterium, it resulted in reduced mobility, cessation of feeding, and leading to the ultimate death. Sileshi et al. (2013) assessed in vitro bioefficacy of 4 isolates of M. anisopliae and B. bassiana against termite by spraying of 1 × 10 5 to 1 × 10 9 conidia per milliliter concentrations, and they found that M. anisopliae and B. bassiana could cause 60-100 and 25-95% mortality, respectively, at different concentration.

In vivo testing of fungi
In vivo testing of Trichoderma spp.
The results of the present investigation revealed that Trichoderma isolates could reduce the dieback incidence by 57.1 to 68.6 and 66.7 to 77.7%, when sprayed at 2.5 and 5.0 g per liter, respectively, which was found to be superior to the treatment with a commercial formulation (Table 4) which is evident from lush green appearance both in foliar spray and post-prune spray treatments (Fig. 2). Panwar et al. (2014) assessed bioefficacy of T. harzianum and T. viride against Fusarium head blight pathogen of wheat (Fusarium graminearum) under greenhouse conditions and found that foliar application of T. harzianum and T. viride alone and in combination significantly reduced the disease severity than the control. T. harzianum at 5 g/l resulted in better control of root disease in Sri Lanka (Balasuriya 2005). Species of Trichoderma were found to be potential in controlling the blister blight of tea under field conditions in North East India (Sarmah et al. 2005). At the experimental plot of Tea Research Association, the applied formulation had promoted vegetative growth of tea bushes in terms of increased number of bud break, shoot length, and shoot weight than the untreated control. The average number of bud break was 398.4 and 421.2 at 5 and 7.5% concentration, respectively, which was higher than that recorded in untreated control (259.4) as shown in Fig. 3. Similar trend of promotion of vegetative growth was observed on deep skiffed tea plantation also (Fig. 4). In both cases, health of bushes was found better than control. Kumhar and Babu (2015a, b) noted that foliar spray of Trichoderma formulation could promote the vegetative growth of tea plantation in terms of increased number of shoots and their length.

In vivo testing of B. bassiana
The highest mortality of 52.3% was noted when B. bassiana (1 × 10 8 cfu/ml) was sprayed by Tween 20 plus crude sugar (Table 5) against tea mosquito bug. The infected insects were observed in almost all the treatments with varying numbers except in T5, T6, and T7. Selvasundaram and Muraleedharan (2000) (Fig. 5), than commercial formulation (38.0-40.5%). Ghatak and Reza (2007) observed that B. bassiana at different doses was found effective against tea pest under field conditions, and its efficacy was comparable with that of synthetic chemical insecticides. Annamalai et al. (2016) reported that B. bassiana and L. lecanii were effective for the control of thrips, T. tabaci on onion.

In vivo testing of M. anisopliae
Application of M. anisopliae formulation under field conditions resulted in significant reduction of termite population as indicated in Fig. 6. The field bioassay of B. bassiana and M. anisopliae was carried out (Singha et al. 2011) and was noticed that the application of these entomopathogens could reduce the number of termites per tea plant until the 5th week after treatment as compared to control.

Conclusion
It is concluded that, although Trichoderma atroviride, T. asperellum, and T. harzianum effectively controlled the dieback disease and promoted vegetative growth of tea plantation, the effectiveness was more pronounced by T.  asperellum than the other 2 species. The EPFs B. bassiana and M. anisopliae were found effective in controlling the tea mosquito and red spider mite, respectively. Hence, application of these fungal species may be an alternate measure to minimize the hazardous effect of synthetic agrochemicals for the management of selected tea pests and disease. Commercialization and utilization of these microbial formulations may be helpful in solving the problems associated with tea cultivation including the pesticide residues in made teas to a great extent.