Research Article
Practical Utilization of Botanical Extracts and Microbial in Controlling Dieback Disease of Tea [Camellia sinensis (L) O. Kuntze] Caused by Fusarium solani (Mart.) Sacc. 
,
Prodip Kumar Baruah2 
,
Suresh Chandra Das2 2 School of Biological Sciences, University of Science & Technology, Meghalaya, India
Author Correspondence author
Journal of Tea Science Research, 2017, Vol. 7, No. 2 doi: 10.5376/jtsr.2017.07.0002
Received: 09 Feb., 2017 Accepted: 01 Mar., 2017 Published: 03 Mar., 2017
Sarmah S.R., Baruah P.K., and Das S.C., 2017, Practical utilization of botanical extracts and microbial in controlling dieback disease of tea [Camellia sinensis (L) O. Kuntze] caused by Fusarium solani (Mart.) Sacc., Journal of Tea Science Research, 7(2): 11-19 (doi: 10.5376/jtsr.2017.07.0002)
In Northeast Indian tea plantations infection of Fusaruim solani, resulting dieback of tea plant is increasing which causes considerable crop loss during the recent times. In this investigation, native plant extracts, i.e. Acorus calamus L., Azadirachta indica A. Juss., Clerodendrum viscosum Vent., and Xanthium strumarium L. and microbials i.e. Bacillus subtilis and Trichoderma viride Pers. were utilized to evaluate the efficacy in controlling dieback disease of tea. These extracts inhibited the growth of Fusarium solani by 60-90%. In field application of C. viscosum and X. strumarium extracts reduced the disease up to 89.3% and 81% respectively. More than 70% disease reduction was observed when aqueous extracts of A. calamus and A. indica were used separately. Maximum disease reduction was achieved up to 86.9% due to application of T. viride. The results, thus, suggested the potential use of herbal extracts and microbial strains as an effective component of integrated disease management (IDM) schedule in the organic tea farming. The methods and rate of application are also discussed.
Introduction
Tea plant is susceptible to infection of several pathogenic microbes which results outbreak of serious diseases. Recently, Fusarium solani (Mart.) Sacc. is causing dieback to tea branches in certain planting materials of tea plantation in North-East India. Due to attack of F. solani, wilting occurs in the tender shoots and cause dieback of the tissues. Such infected shoots die off and fail to regenerate new shoots. Considerable crop loss occurs due to the damage caused by the pathogen in the harvestable part of the tea plant (Sarmah et al., 2006). The pathogen is reported as predominant in the humid and temperate climate of north east India. Increasing use of pesticide in plant protection sector is a serious concern. It builds up the risk of chemical residue in tea, degradation of soil health and environmental hazards. The guideline of the regulatory bodies to monitor the maximum residue limit (MRL) of pesticide in the consumed product is also another important issue of the tea growers for their sustainability in world market. Tea industry provides employment to more than 1.1 million Indian workers and almost half them are women. The indiscriminate and continuous use of fungicides results serious threat to human health also. Hence the implementation of IDM strategy by the use of plant extracts and microbial biocides is gaining importance and becoming popular in the agriculture sector for controlling different diseases. This will minimize the pesticide application as well as the residual effect in consumed tea.
Trichoderma isolates inhibit the mycelia growth of Fusarium moniliformae (upto 60.78%) the causal organism of sugarcane wilt disease which proves its biocontrol potentiality (Gawade et al., 2012). Sundaramoorthy and Balabaskar (2013) evaluated the efficacy of the native isolates of Trichoderma species in promoting growth and yield parameters of tomato and management of Fusarium wilt disease caused by Fusarium oxysporum f. sp. lycopersici (FOL) under in vitro and in vivo conditions and found to inhibit the radial growth of mycelia of the pathogen (by 53%). The effects of Trichoderma species on the wilt disease complex of tomato (Solanum lycopersicum) caused by F. oxysporum f. sp. ciceri and Rhizoctonia solani were investigated under greenhouse conditions. Song et al., (2014) developed a biocontrol system with Bacillus sp. against ginseng root rot caused by Fusarium cf. incarnatum. Gurjar et al., (2012) reported the efficacy of aromatic secondary metabolites obtained from plant extracts in plant disease management. It is established that plants have ability to synthesize aromatic secondary metabolites, which are highly active against the pathogen. These groups of compounds show antimicrobial effect and serves as plant defense mechanisms against pathogenic microorganisms (Das et al., 2010).
The infection of F. solani in tea is increasing in an alarming way. It may be due to the change in climatic conditions or increasing pathogen virulence. In the present investigation the herbal extracts and microbial biocides were evaluated for the first time to control the said disease in tea cultivation of North East India to minimise the deleterious effects of chemical fungicide. We have evaluated in vitro potentiality of A. calamus, A. indica, C. viscosum, and X. strumarium extracts (both aqueous and solvent) at 2, 4, 6, 8 and 10% concentrations and established their field efficacy. Microbial strains, like T. viride and B. subtilis were also tried through field trials for their efficacy in reducing the dieback disease in tea. The present study thus, aimed at exploration and popularization of the native plant extract and microbial biocides in controlling dieback disease in tea under field evaluations. The importance of the study is to develop an alternative disease control strategy against dieback pathogen as well as to reduce the dependence on chemical fungicides.
1 Materials and Methods
1.1 Collection and preparation of the plant extracts
Four test plants were used in the present investigation viz, A. calamus L, var. calamus, A. indica A. Juss var. indica, C. viscosum Vent, and X. strumarium L var. strumarium respectively. These were collected from their natural habitats, in and around the Tocklai Tea Research Institute (TTRI), Tea Research Association (TRA), Jorhat (26°45′0″N, latitude and 94°13′12″E longitude and Elevation: 116 m), Assam, India, Roy et al. (2010). During the collection of plant species utmost care was being concentrated to collect the mature leaves (one year age group of plants except A. indica) along with the juvenile stem. Collected plants were sopped and dried under shade for 15-20 days. It was powdered by using an electric grinder and passed through 20 mesh sieve and kept in polypropylene bag. The required amount of the dried powder (200 g, 500 g, 400 g, 600 g, 800 g and 1000 g representing 2, 4, 5, 6, 8 and 10% w/v respectively) were extracted separately in distilled water and solvent (hexane) by soaking and shaking in a mechanical shaker for 24 hour. The emulsion thus obtained was filtered using a double-folded muslin cloth and vacuum filter through whatman filter paper and make up the volume to obtain 2, 4, 5, 6, 8 and 10% concentration. The aqueous extract was directly used and the solvent extract was evaporated in a rotary evaporator at 60°C. The volume was made up to 10 ml by adding emulsifier and thus a homogeneous suspension was prepared. From this suspension, required concentration was made, labeled and make it ready to use in vitro as well as field experimentation.
1.2 Preparation of microbial bioagents
Strains of Trichoderma sp. was isolated from rhizosphere tea soils collected from nearby tea gardens of TTRI, by using serial dilution plate technique on PDA medium. Trichoderma was purified by hyphal tip technique. Initial identification was done on the basis of cultural and morphological characters and finally at International Mycological Institute (IMI) as T. viride (IMI No. 356099). B. subtilis was isolated from the phylloplane region of tea plants by leaf washing method on PDA medium. Cultural and morphological characters and identification at IMI (IMI No. 356098) confirmed as B. subtilis and these cultures were preserved. Thus, the stock cultures T. viride and B. subtilis were obtained from host research institute (Kumar et al., 2014). From the stock culture microbial inoculants were prepared by sub culturing in PDA plates. These plates were incubated at 25± 2°C for 5 days and at 28± 2°C for 2 days in case of T. viride and B. subtilis respectively.
Potato broth (10%) supplemented with 0.2% glucose was used for mass culture of Trichoderma and Bacillus cultures. 500 ml of liquid culture medium was prepared in 1000 ml Erlenmeyer flasks and sterilized by autoclaving at 15 lb/inch2 for 20 minutes. After sterilization, the bioagents were aseptically inoculated in the medium. Agar discs of 5 mm diameter of Trichoderma and one loopful inocula of Bacillus was added in each flask of 500 ml medium. The growth of the inoculated microbes was measured by analyzing the cfu count 5×106 cfu /ml and 6×107 cfu /ml for Trichoderma and Bacillus respectively. The cultures were incubated at 25± 2°C for 10 days for Trichoderma and 2 days for Bacillus at 28± 2°C. These cultures were macerated in a mixer grinder to obtain a homogenize mixture. From this mixture 0.2, 0.5 and 1 liter was mixed with 9.8, 9.5 and 9 liters of water respectively to get 2, 5 and 10% dilutions and applied in field immediately.
1.3 In vitro evaluation
The antagonistic effect of B. subtilis and T. viride on growth of F. solani was tested in the laboratory. PDA medium were poured in 9 cm. petriplates and both the cultures i.e. bioagent and F. solani were inoculated by following the dual culture technique of (Fokkema, 1978). The petriplates were incubated in room temperature for 15 days to see the effect of the tested bioagents.
The in vitro efficacies of the herbs were tested following food poisoned technique (Groover and Moore, 1962). The required amounts of extracts were mixed with freshly prepared sterilized 100 ml. of potato dextrose agar (PDA) medium to obtain different concentrations. These were poured in 9 cm. petriplates under aseptic condition. To maintain inoculum uniformity, agar discs of 5 mm diameter were punched out aseptically by cork borer from the seven days (incubated at 25± 2°C) culture of F. solani. The discs were then transferred aseptically with their mycelial surface downward in the petriplates. This was done separately for aqueous and solvent extract against each herb and incubated under normal laboratory environment for their optimal growth. The growth of the pathogen in terms of colony diameter was measured in respect to different treatments. Control set was kept without any herbal extract. The data were recorded and the percent growth inhibition over control was estimated to evaluate the efficacy of the treatment in restricting the growth of the test pathogen.
1.4 Field evaluation
F. solani disease infected tea area situated (26.67°N 94.33°E and 26°43'29"N 94°2'47"E) nearby Tocklai Tea Research Institute were selected to conduct the trials. Plants of 10-15 years of age were considered. In this study the severity of the disease infection was considered during the selection of the experimental areas. The climatic condition of the study area is tropical monsoon.
Bioassay trials were laid out in Completely Randomized Block Design (CRD) area to assess the disease incidence and severity. All the treatments were replicated five times in field trials (herbal and microbial) having fifty numbers of plants in each treatment in each location. Herbal extracts and microbial biocides were mixed with water to obtain the required dilutions and the plants were not watered separately during spraying. Each treatment was separated by a buffer row of non treated tea as guard row. Two rounds of treatment were imposed at 15 days interval by taking special care to drench the infected portions with hand operated calibrated backpack sprayer (hollow cone NMD 60/450 nozzle, droplet diameter 1.6 mm, droplet size 140 lm, discharge 450 ml/min at 40 psi pressure and distance between nozzle and target 30–45 cm) at 400 l/ha. Post treatment observation was assessed after one year in the next cropping season in 0-4 (0= no infection, 1=25%, 2=50%, 3=75%, 4=100% of infection) scale of severity (McKinney, 1923) by observing each treated individual plant. Thus the percent disease reduction was calculated to evaluate the efficacy of the treatment in reducing the disease incidence.
1.5 Data analysis
The data were subjected to analysis of variance (ANOVA) following RBD and critical difference (CD; P=0.01 and 0.05) and critical variance (CV) in accordance with Snedecor and Cochran (1989).
2 Results and Discussion
2.1 In-vitro experimentation
The in-vitro efficacy of the plant extracts at different concentrations was evaluated in terms of percent growth inhibition over control as shown in Table 1. The presented data are the mean of seven replications for each treatment. Tested plant extracts inhibited the growth of F. solani. Aqueous extract of X. strumarium was found maximum with 88.4 to 99.8% inhibition of F. solani. A. calamus, A. indica and C. viscosum were also resulted more than 90% inhibition at 8 and 10% concentration. The efficacies of solvent extracts were found to be slightly superior to their respective aqueous extracts. Growth inhibition data indicates the significance of treatments (p<0.05) in terms of percent control. In the present investigation, more than 90% growth inhibition of the pathogen was achieved with the tested herbal extract and it can be considered as important information. Based on which biocontrol agents (BCAs) were evaluated in field to control dieback disease of tea caused by F. solani.
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Table1 In vitro effect of plant extracts (both aqueous and solvent) in growth inhibition of F. solani (in cm.) Note: CD = Critical difference; CV = Critical variance |
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2.2 Antagonistic effect of microbes
The results shown in Figure 1 (a-b) indicates the antagonistic / hyperparasitic action of the tested microbes. T. viride and B. subtilis are showing the antagonistic effect on growth of F. solani. In the control petriplates full growth of F. solani was observed (Figure 1c). This proves the biocontrol potential of T.viride and B.subtilis in controlling F. solani, the causal agent of dieback disease of tea.
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Figure 1 (a-b) Antagonistic effect of T.viride against F. solani (a) and B. subtilis against F. solani (b); Pure F. solani culture(c) |
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Scientific investigation and bioefficacy evaluation of microbial biocides and botanicals to incorporate in the integrated disease management (IDM) schedule in tea are the prime area of research in the present investigation. The effect of BCAs particularly the Trichoderma in reduction of dieback disease in tea caused by F. solani was found quite encouraging which can be popularized as an alternative of chemical fungicides. Gupta et al. (1999) established the effect of Trichoderma pseudokoningii as compatible to chemical fungicides and established as an effective biocontrol component in IDM schedule which reduces stem canker (Botryodiplodia theobromae) and cutting rot (F. solani) of mulberry (Morus sp.).
Trichoderma is an opportunistic plant symbiont with the ability to serve as antagonist, plant growth promoter, inducer of plant defense mechanism, rhizosphere colonizer and neutralizer of pathogen’s activity. Trichoderma may directly kill the pathogen by mycoparasitism, antibiosis or competition (Junaid et al., 2013). Trichoderma is also known to produce hydrolytic enzymes, steroids etc. that may inhibit the growth of pathogenic fungi. Indirectly, it may also contribute effective plant growth in environments of biotic and abiotic stresses through altering the nutritional status of the plant. The exploitation of plant growth promoting activities of Trichoderma in tea by foliar application is also another promising area for future study. T. viride at 10% concentration reported 62.2% reduction of poria branch canker disease of tea caused by Poria hypobrunnea (Sarmah et al., 2005).
Beneficial bacterial strains efficiently colonize leaf surfaces and root systems and their surrounding soil layer and thereby protect the plants from pathogenic infection through competition for ecological niche, production of inhibitory allelochemicals and induction of systemic resistance in host plants. Biocontrol potential of Bacillus was reported against leaf spotting bacterial and fungal pathogens, root –Knot nematodes, stem blight, damping-off, blue mold, and late blight diseases in both in vitro assays and greenhouse conditions (Choudhary and Johri, 2009). Production of antimicrobial agents, biofilm formation, and triggering of host systemic resistances are the activities contributes by B. subtilis (Chen et al., 2013). Synthesis of a wide variety of metabolites and antibiotics with antifungal activity are seemed to be associated with this biocontrol mechanism (Jetiyanon and Kloepper, 2002). According to Podile and Parkash (1996), B. subtilis may adhere to the pathogen directly and later multiply there and colonize the surface which ultimately results significant damage to the fungal cell walls. The aspects thus appeared as essential in association with the antifungal properties of Bacillus sp. used in the biological control of plant diseases. Disease reduction in tea was achieved possibly due to diverse action of B. subtilis as biocontrol agent. Both Trichoderma and Bacillus have shown their biocontrol potential in reducing dieback disease of tea in field evaluations, however, Trichoderma was found as superior than Bacillus during the study.
2.3 Field experimentation
2.3.1 Field experiment during 1st year with aqueous plant extract
The result indicated the efficacy of the tested plant extracts in reducing dieback disease of tea. Among them C. viscosum and X. strumarium reduces the disease severity up to 76.8% and 74.2% at 5% concentration and 78.2% reduction at 10% concentration which was observed as slightly higher in comparison to other treatments. More than 70% disease was also reduced by application of A. calamus and A. indica at 5 and 10% concentration.
2.3.2 Field experiment during 2nd year using plant extract
The trial was repeated for the 2nd year and the result indicates the efficacy of the tested plant extracts. At 10% concentrations 89.3% and 81% disease reduction was recorded by C. viscosum and X. strumarium respectively. A. calamus and A. indica are also showing similar trend of disease control. The findings of both 1st and 2nd year trials are presented in Table 2.
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Table 2 Field evaluation of plant extracts in controlling Fusarium dieback disease Note: CD = Critical difference; CV = Critical variance |
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Application of herbal extracts in plant disease management has already been reported. A. indica at 20% concentration was recorded as most effective against brinjal wilt (F. solani f. sp. melongenae) (Babu et al., 2008). Crude extracts of A. indica and Tagetes minuta are observed as effective in controlling F. oxysporum Schl. f.sp. phaseoli under field evaluation (Obongoya et al., 2010). The popularity of biopesticide is increasing recently and some of the plant products are being used globally as green pesticides. There is enormous potential of plant derived products against microbial pathogens since the former is known to produce several secondary metabolites including antifungal compounds as well as phenolic derivatives. The existence of antimicrobial compounds in plant species like A. indica, A. calamus, C. viscosum and X. strumarium are reported. Phytochemical screening revealed the presence of glycosides, polyphenolic compounds, mucilage, volatile organic compounds (VOCs), alkaloids, eugenol etc. in A. calamus. The aerial parts of X. strumarium contain a mixture of alkaloids, antibacterial substances and sesquiterpene lactones that may create allergic reactions and toxicity to phytopathogens (Kaur et al., 2015). Phenolics and flavonoid compounds in C. viscosum may contribute to overall antimicrobial activity against pathogens.
2.3.3 Field experiment during 1st year with microbials
Post treatment observation was recorded in the microbial treated plots and thereby calculated the percent reduction of disease over untreated control. Among the tested microbials, T. viride is recorded as superior to B. subtilis in reducing the disease. At 5% and 10% concentrations, T. viride reduced the disease up to 66.6% and 71.1% respectively while the disease was controlled up to 48.8% and 59.1% when B. subtilis was used.
2.3.4 Field experiment during 2nd year using microbials
In the second year the microbial, T. viride and B. subtilis were applied at 2%, 5% and 10% concentration. The post treatment observation on percent disease reduction was recorded. The result showed the better efficacy of T. viride over B. subtilis and reduced the disease up to 86.9% at 10% concentration. The results of both 1st and 2nd year field trials were presented in Table 3. The results were statistically significant at p<0.05 level. In Table 2 and Table 3, the efficacy of herbal extracts and microbial biocides in controlling dieback disease of tea were presented in terms of percent reduction of disease over control.
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Table 3 Effect of Microbials in controlling Fusarium dieback disease in tea Note: CD = Critical difference; CV = Critical variance |
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2.4 Practical utility
In tea the cropping season starts from March-April and continues up to October-November. The infection of Fusarium incidence is also severe during the period. Two rounds of foliar treatment at 15 days interval reduce the severity of infection throughout the year. However, due to changing climate, specific disease incidence may not prevail throughout the cropping season or continue in the next year. Thus disease management in a cropping season seemed to be quite effective and economically viable in tea cultivation.
The plants selected for screening under field evaluation have also some other advantage as these are tropical weed plants of North-East India and easily available in most of the tea gardens. The plants can be incorporated as safe, suitable and cost effective alternative means of disease management. Applications of these herbal extracts against phytopathogens are less hazardous since, the plants are in traditional use for remedy of several human diseases since time immortal (Dutta and Nath, 1998).
The treatments are concentration dependent. Maximum reduction of the disease was recorded in higher concentrations, which seems to be an important issue regarding the standardization of the dose under field application. Field evaluation indicates almost similar findings on disease reduction at 5% and 10% concentration. This suggests the application of 5% concentration of herbal extracts in the field which will be cost effective with desired control.
Study reveals that foliar application of BCAs in Fusarium infected tea areas recorded effective disease reduction compared with the control, hence foliar application of the BCAs both microbials and herbal extracts are one of the effective way of disease control in tea. Foliar application of BCAs was reported as one of the efficient delivery in reducing plant pathogens (Maketon et al., 2008). The simplified extraction, preparation as well as application technology of BCAs adopted in the study by using hand operated back pack sprayers is one of the common adapting practice in the tea gardens during management of pest and diseases. However, this is the first report on BCAs to control dieback disease in tea. Thus, the use of aqueous extracts of A. calamus, A. indica, C. viscosum, X. strumarium and homogenial suspension of B. subtilis, T. viride may be recommended in controlling F. solani effectively.
3 Conclusion
The present investigation proves the potentialities of native botanicals as well as putative microbials isolated from the tea plantations have important and significant application in disease control. In the present investigation, aqueous extracts of plant species like, A. calamus, A. indica, C. viscosum and X. strumarium at 5% and 10% concentration can control dieback disease in tea caused by F. solani. Microbial biocontrol agents like B. subtilis and T. viride can also be used against Fusarium infestation in tea both in organic and conventional tea estates as a component of integrated disease management (IDM) schedule. Control of the disease in a cropping season by application of two rounds of herbal extracts and microbials are quite effective and economically viable in tea cultivation. The outcome of the study is helpful in formulating IDM schedule effectively in tea cultivation, which may lead the tea industry to reduce the load of toxic chemicals. The biological agents used in the present investigation are cost effective. These are locally available and thereby easily accessible from the point of customer demand, and significant to develop an eco-friendly environment.
Authors’ contributions
Satya Ranjan sarmah (corresponding author): substantial contributions to conception and design, conducting and monitoring the experiments, observation and collection of data, analysis and interpretation of data, manuscript writing. Pradip Kumar Baruah (co-author): contributed in interpretation and analysis of data, critical revision. Suresh Chandra Das (co-author): contributed by suggesting in design of the experiments and in drafting the article for important intellectual content.
Acknowledgments
The authors are thankful to the Director, Tocklai Tea Research Institute (TTRI), Tea Research Association (TRA), Jorhat-785 008, Assam, India for providing the necessary research facilities during the course of investigation.
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