Trichoderma Asperellum as A Promising Mycofungicide for Managing The Dieback Disease of Tea (Camellia Sinensis)

Background: The dieback disease of tea caused by Fusariumsolani adversely affects its production and quality. Genus Trichoderma is a promising biocontrol agent to control it without any residual effect and most suitable for inclusion into an integrated disease management approach. Isolation of Trichoderma from rhizosphere soil of the Dooars zone was done. It was identied as T. asperellum based on its cultural characters and DNA ngerprinting. A liquid formulation (2% Aqueous Suspension) was prepared from it and bio-ecacy was evaluated under eld conditions for the control of dieback disease and other parameters for two seasons in three zones. Its different concentrations, T. harzianum, and Hexaconazole were included in the study. Results: The fungal isolate (KBN-29), identied as T. asperellum, was found nearest to the isolate TV-3 (Genebank-KX538814.1) with 99% similarity. Plots treated with T. asperellum 2% AS at 1000 and 1200ml/ha concentration gave better disease control as the yield of green leaves as compared to Hexaconazole 5% EC. The formulation was safe to non-target benecial organisms’ in all three zones without any phytotoxicity to tea leaves at 4, 8, and 16ml/L concentrations. Conclusions: The present study conrms that the developed liquid formulation of T. asperellum 2% AS was found signicantly superior for the management of dieback disease of tea plantations in Darjeeling, Dooars, and Assam zones when used at concentrations of 1200 and 1000 ml followed by 800 and 600 ml/ha during both seasons. The maximum made tea yield was recorded in plots treated with T. asperellum at 1000 and 1200ml/ha followed by Hexaconazole 5% EC. The T. asperellum 2% AS was observed to be safe for non-target benecial organisms viz., C. carnea, O. javanus, and S. gilvifrons. The developed T. asperellum 2% AS formulation was not phytotoxic to tea leaves at 4, 8, and 16ml/L concentrations. medium (HiMedia) plates in triplicates. Plates were then properly sealed with paralm and incubated at 26±2 0 C for 72-96 hours. Appeared fungal colonies were observed and antagonist’s colonies were identied based on its mycelia color and fast-growing character. Such colonies were transferred into another potato dextrose agar (PDA) plates for pure culture and re-incubated at 26±2 0 C for 96 hours in the BOD incubator.

The local fungal antagonist was isolated from the tea rhizosphere of Dooars zone, District Jalpaiguri, West Bengal, India, and was designated as KBN-29. Based on cultural and morphological characters it was identi ed as T. asperellum( Fig. 2A). Further, its identity was established as T. asperellum by ITCC, Division of Mycology and Plant Pathology, IARI, Pusa, New Delhi -110012, and it has assigned an accession number as ITCC-7764. The DNA ngerprinting (16s ITS) outsourced from the National Bureau of Agriculturally Important Microorganisms, Indian Council of Agricultural Research, Kushmaur, Mau, Uttar Pradesh, India, revealed that its nucleotide sequence ( Fig. 1) was the nearest similar to T. asperellum isolate TV-3 (Genebank-KX538814.1) possessed both query coverage and similarity 99% when followed NCBI blast. The NBAIM provided its accession number as NAIMCC-SF-0041. The liquid formulation from T. asperellum was prepared in the form of 2% Aqueous Suspension (AS) using a liquid fermentation technique to carry out different bioe cacy studies.
Multilocation eld Bio-e cacy of T. asperellum 2% AS on dieback disease during the season I and II In the Darjeeling zone, the dieback infection (Fig. 2 B) before imposing treatment (pre-assessment) ranged from 15.78-18.11 shoots. The rst spray could reduce dieback infection in all the treated plots as compared to untreated ones. Among the treatments, T. asperellum 2% AS at 1000 ml and 1200ml/ha concentrations were found more effective in controlling the disease with two rounds of spraying ( Fig. 2 C and D) which gave superior control of disease as compared to lower concentrations (T. asperellum 2% AS at 600ml and 800ml/ha) and market sample (T. harzianum 1% WP) @2500g/ha. However, hexaconazole 5% EC exhibited the maximum disease reduction in both seasons (Table  1).
In Dooars and Assam zones, pre-assessment infestation ranged from 19.89 to 22.67 and 20.67 to 23.33, respectively in the rst season. All doses of T. asperellum formulation yielded better over untreated control and T. harzianum 1% WP (market sample); however, at higher concentrations viz., 1000 ml and 1200 ml were found more effective. In the second season also a similar trend of disease control was recorded in both zones, ( Table 2 and 3).

Effect of T. asperellum formulation on the yield of green leaves
The average green leaf yield of rst six rounds harvesting at the weekly interval in T. asperellum sprayed plots with 1000 ml and 1200 ml/ha was found to be signi cantly superior over the Hexaconazole 5% EC and T. harzianum 1%WP at 2500g/ha. T. asperellum 2% AS at 1000 ml and 1200 ml/ha showed an increased average made tea yield of 422 and 425 kg/ha, respectively during the rst season and 395 and 399 kg/ha, respectively during the second season as compared to untreated control where it was 377 and 354 kg /ha in the rst and second season, in Darjeeling zone. However, in Dooars zone, the yield of made tea was 1694 and 1703 Kg at concentrations of 1000 ml and 1200 ml/ha as compared to 1519 kg in control in the rst year whereas it was 1724 and 1729 kg at both higher doses as compared to control (1543) in the second season. The same yield trend was observed in the Assam zone, where yield in the rst year was higher (2315 and 2324 kg) at both higher concentrations as compared to control (2071 kg). In the second season, it was 2216 and 2229 kg at higher doses against 1983 kg in control during the second year (Table 4). Two higher doses of T. asperellum had increased the green leaf yield over control during both seasons in Darjeeling (11.93 -12.73%), Dooars (11.73 -12.11%) and Assam (11.78 -12.40%).

Effect of the formulation on non-target bene cial organisms
The population level of important insect predators viz., C. carnea, O. javanus, and S. gilvifrons was recorded at 0 days, 7 th day of the rst spray, and 7 th , 14 th and 21 st day of the second spray during season 1 and 2 ( Fig. 3-5). The experimental results indicated that T. asperellum 2% AS did not show any adverse effect on the population-level of these non-target bene cial organisms.
Testing for phytotoxicity, tainting and organoleptic attributes Tea leaves on the bushes were observed for phytotoxic effects after the spray of T. asperellum 2% AS. The test result indicated that there were no phytotoxic effects in form of wilting of leaves, vein clearing, necrosis, epinasty, and hyponasty on the tea leaves at the concentrations used in the study (Table 5).

Discussion
In the present study, different concentrations of T. asperellum liquid formulation (2% AS) successfully controlled the dieback disease at all locations, and hence, it could be an alternate approach of disease management under organic crop production system of Darjeeling and other areas. Foliar spray of Trichoderma formulation resulted in an increased number of shoots and their length [14]. It was found that Trichoderma WP formulation controlled the dieback disease of tea to a great extent when sprayed at 2.5 and 5.0 g/liter concentration and performed better than the commercial formulation of the antagonist. Light pruned (LP) and deep skipped (DS) tea bushes showed enhanced vegetative growth as compared to control [15]. Foliar spray of T. harzianum and T. viride on wheat crop managed head blight disease caused by Fusarium graminearum under greenhouse conditions better than control [16]. Trichoderma spp was found to be promising under eld conditions for the management of blister blight of tea in North East India [17]. The e cacy of T. atroviride strains has proven for the protection of pruning wounds in the grapevine [18]. T. asperellum was successful in managing a broad array of fungal phytopathogens [19,20] such as F. oxysporum and Curvulariaaeria [21,22].
Our study showed increased production of healthy green tea shoots due to the application of T. asperellum liquid formulation as compared to chemical fungicide, the market sample of T. harzianum, and control. Hence there are chances to get bountiful production of tea crop through by applying as a foliar spray besides controlling the disease. T. asperellum showed synergistic activity with Bacillus amyloliquefaciensand combined application of both microbes signi cantly enhanced the growth of wheat as well as protected the crop against plant pathogens [23].
The liquid formulation of the local antagonist (T. asperellum 2% AS) did not adversely affect the bene cial insects in the tea ecosystem and con rmed the earlier ndings that the biopesticides are less toxic, decomposes quickly, free from pollution, and residue problems. They generally affect the targeted and closely related organisms in the same environment [24,25]. The spray of sh emulsion increased the yield of tomatoes and peppers with no observable phytotoxic effect on crop foliage under the eld conditions [26]. The developed liquid formulation was found to be non-phytotoxic without showing any kind of toxicity symptoms on tea leaves.

Methods
Isolation, identi cation, and the accession of Trichoderma sp Isolation of the antagonist was carried out following standard technique with slight modi cation [27]. Soil samples were collected from tea rhizosphere in sterilized polyethylene bags, brought to mycology laboratory, stored in the refrigerator at 4±1 0 C. For isolation of antagonistic fungi, soil samples were homogenized processed following multiple serial dilution plate technique (MSDP). From 6 th and 8 th dilutions, 0.5 mL was drawn and uniformly distributed in solidi ed Trichoderma speci c medium (HiMedia) plates in triplicates. Plates were then properly sealed with para lm and incubated at 26±2 0 C for 72-96 hours. Appeared fungal colonies were observed and antagonist's colonies were identi ed based on its mycelia color and fast-growing character. Such colonies were transferred into another potato dextrose agar (PDA) plates for pure culture and re-incubated at 26±2 0 C for 96 hours in the BOD incubator.
Identi cation: The antagonistic isolate was identi ed based on its cultural and morphological characters by the rst author. Later, its identity was recon rmed from Indian Type Culture Collection, Division of Mycology and Plant Pathology, Indian Agricultural Research Institute, Pusa, New Delhi, India and it has assigned an accession number. To establish its perfect identity, DNA ngerprinting (16s ITS) was get done from ICAR National Bureau of Agriculturally Important Microorganisms, Indian Council of Agricultural Research, Kushmaur, Mau, Uttar Pradesh, India.
The obtained nucleotide sequence blasted at the NCBI website to match the nearest similar.

Development of formulation: T. asperellum 2% Aqueous Suspension
The liquid formulation of T. asperellum was manufactured using liquid-state fermentation techniques by M/s Varsha Bioscience and Technology India Pvt Ltd, Hyderabad -500059, Telangana, India using the slightly modi ed method [28]. The mother culture of the antagonist was sub-cultured on to plates containing Trichoderma Speci c Media (TSM) followed by incubation at 28±0.5°C for 5 days.
Then 10 liters of seed inocula was prepared by inoculating 120 hr old mother culture into 1000 ml conical ask lled with 250 ml autoclaved potato dextrose agar and incubated in an orbital shaker at 28±1°C and 180 rpm for seven days. From seed inocula, submerged large scale fermentation (maintained temp. 28±2 0 C, 45 RPM) was done to scale up its quantity using another autoclaved medium containing 30g sugarcane molasses and 5g yeast extract per liter of water. One week old seed inocula (10%) was inoculated in the fermenter incubated for 7 days and the cultural biomass was separated by centrifuging the culture broth through the on-line centrifugation system and both the conidia, as well as mycelia were collected. Active ingredient (2x10 8 CFU/ ml) was determined by the MSDP technique in nal formulation by adding required distilled sterilized water.
Multilocation eld Bio-e cacy of T. asperellum 2% AS on dieback disease during the season I and II The T. asperellum 2% AS formulation was tested under eld conditions at Darjeeling, Dooars, and Assam zones against dieback disease caused by F. solani from 2016 to 2018. The plot size was kept 84 m 2 for each treatment with 100 bushes. Experiments were laid out in randomized block design (RBD) with seven treatments in three replications. Common tea cultivars/clones of particular zone namely TRA-AV-2, TV-25, TV-26, and TV-1 of 28 to 54 years old were chosen for the study.
The plots having dieback disease incidence above 5% ETL were selected for eld bio-e cacy study. The disease incidence was recorded by placing a 1 x 1-foot quadrate at 3 randomly selected spots per treatment then healthy and infected tea shoots were plucked. Then the rst spray with hand operated knapsack sprayer tted with NMD 450 nozzle was done immediately after the plucking (0-day). Observations on disease incidence were recorded on the 7 th day of the rst spray using the same quadrate. Subsequently, both healthy and infected shoots were plucked on the same day and weight (Kg) of the fresh healthy shoots was recorded. The second spray was given on the 7 th day after the 1 st spray and disease incidence was recorded on the 7 th , 14 th, and 21 st days of the second spray.

Effect of T. asperellum formulation on the yield of green leaves
Green leaf yield (kg/plot) was recorded from the rst six rounds of plucking and at every plucking round it was converted into made tea per hectare using the formula [29]. The collected data of bioe cacy trials and yield were statistically analyzed to nd out the critical difference among treatment at a 5% level of signi cance (p =0.05) through the online statistical package "OPSTAT" of Chaudhary Charan Singh Haryana Agricultural University, Hisar (www.hau.ac.in). The study was conducted using different tea plant species those are abundant in the ecosystem hence do not require ethical approval.

Consent for publication
The authors agree to publish this paper. The data has not been published partially or completely in any other journal.

Availability of data and materials
The datasets generated and analyzed during the current study are not publicly available due to privacy reasons but are available from the corresponding author on reasonable request.

Competing interests
The authors declare that there is no con ict of interest regarding the publication of this paper. It is declared that the authors have no competing interests.

Funding
At present we do not have any nancial support from any funding agency for this research work to get it published. However, the waivers (BMC Plant Biology Journal) considered it for the review process without processing charges.