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; Babu and 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.

Materials and methods

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 and Qazzaz 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:

$$ \mathrm{Mycelial}\ \mathrm{growth}\ \mathrm{inhibition}\ \left(\%\right)=\frac{\mathrm{Colony}\ \mathrm{dia}.\mathrm{in}\ \mathrm{control}-\mathrm{Colony}\ \mathrm{dia}.\mathrm{in}\ \mathrm{treatment}}{\mathrm{Colony}\ \mathrm{dia}.\mathrm{in}\ \mathrm{control}}\times 100 $$

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 × 109) 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).

$$ \mathrm{Corrected}\ \mathrm{mortality}\ \left(\%\right)=\frac{\mathrm{No}.\mathrm{of}\ \mathrm{live}\ \mathrm{in}\mathrm{sect}\ \mathrm{in}\ \mathrm{control}-\mathrm{No}.\mathrm{of}\ \mathrm{live}\ \mathrm{in}\mathrm{sect}\ \mathrm{in}\ \mathrm{treatment}}{\mathrm{No}.\mathrm{of}\ \mathrm{live}\ \mathrm{in}\mathrm{sect}\ \mathrm{in}\ \mathrm{control}}\times 100 $$

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 × 108 to 2 × 108) 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.

$$ \mathrm{Insect}\ \mathrm{mortality}\ \left(\%\right)=\frac{\mathrm{Number}\ \mathrm{of}\ \mathrm{dead}\ \mathrm{insect}}{\mathrm{Total}\ \mathrm{number}\ \mathrm{of}\ \mathrm{insect}\ \mathrm{used}}\times 100 $$
Plate 1
figure 1

Leaf disc technique for in vitro bioassay of Metarhizium anisopliae

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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 × 108 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.

$$ \mathrm{Disease}\ \mathrm{reduction}\ \left(\%\right)=\frac{\mathrm{No}.\mathrm{of}\ \mathrm{disease}\ \mathrm{shoots}\ \mathrm{in}\ \mathrm{control}-\mathrm{No}.\mathrm{of}\ \mathrm{disease}\ \mathrm{shoots}\ \mathrm{in}\ \mathrm{treatment}}{\mathrm{Number}\ \mathrm{of}\ \mathrm{disease}\ \mathrm{shoots}\ \mathrm{in}\ \mathrm{control}}\times 100 $$

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.

Statistical analysis of data

Statistical analysis of the data was carried out with the help of online statistical package OPSTAT of Chaudhary Charan Singh Haryana Agricultural University, Hisar, Haryana, India.

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. Later on, Dr. T. Prameela Devi, Principal Scientist, Indian Type Culture Collection, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi, identified the isolates as Trichoderma atroviride, T. harzianum, T. asperellum, B. bassiana, and M. anisopliae. Two strains, i.e., Trichoderma harzianum (ITCC 7765) and T. asperellum (ITCC 7764), got accession number from ITCC, New Delhi.

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 (Table 1). Vidhya Pallavi et al. (2010) found a very good control of gray blight (Pestalotiopsis sp.) and wood rot (Hypoxylon sp.) pathogens of tea through Trichoderma spp. Kumhar and Babu (2015a, b) noted control of F. solani (26.03–59.05%) by Trichoderma spp. Similarly, Naglot et al. (2015) reported efficacy of Trichoderma spp. against this pathogen.

Table 1 In vitro bioefficacy of Trichoderma spp. against Fusarium solani
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B. bassiana isolate I caused the highest mortality (93.0%) of H. theivora, followed by isolate III; however, the performance of all isolates was statistically at par with each other (Table 2). Different strain of B. bassiana was reported to be pathogenic to shot hole borer beetle (Euwallacea fornicatus) of tea, H. antonii of guava, and H. theivora of tea (Selvasundaram and Muraleedharan 2000, Visalakshy and Mani 2011 and Babu and Kumhar 2014). Shophiya et al. (2014) noted that B. bassiana as an effective biological control agent of different larval stages of castor hairy caterpillar, Pericallia ricini Fab.

Table 2 In vitro bioassay of Beauveria bassiana isolates against tea mosquito (Helopeltis theivora)
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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 × 106 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.

Table 3 In vitro bioefficacy of Beauveria bassiana against Helopeltis theivora in combination with different adjuvants
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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 × 108 and the lowest at 1 × 108 conidia/ml (Fig. 1) under laboratory conditions. Roobak Kumar et al. (2011) 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 × 105 to 1 × 109 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.

Fig. 1
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Pathogenicity of Metarhizium anisopliae against red spider mite

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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).

Fig. 2
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View of Trichoderma applied fields. 1 Foliar spray and 2 Post prune spray

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Table 4 In vivo bioefficacy of Trichoderma spp. against dieback disease
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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.

Fig. 3
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Comparison of application of concentration of Trichoderma asperellum for light pruned (LP) tea

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Fig. 4
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Vegetative growth promotional effect of Trichoderma spp. on DS tea (2 MAS)

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In vivo testing of B. bassiana

The highest mortality of 52.3% was noted when B. bassiana (1 × 108 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) established that B. bassiana in combination with 2 adjuvants like Triton AE and Teepol enhancing the mortality of shot hole borer beetles (Euwallacea fornicatus) of tea plants under field conditions. The formulated wettable formulation of B. bassiana exhibited a better control of tea mosquito (56.4–58.4%) at both locations, i.e., Kurti TE and North Tukvar TE (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.

Table 5 In vivo evaluation of Beauveria bassiana in different combination of adjuvants
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Fig. 5
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In vivo bioefficacy of Beauveria bassiana against Helopeltis theivora

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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.

Fig. 6
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In vivo bioefficacy of Metarhizium anisopliae against termite

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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.