The possible inhibitory effects of Trichoderma atroviride against the phytopathogen Phomopsis theae, the causative agent of collar canker disease in tea workss, were studied. An effort was made to correlate the population denseness of Trichoderma spp. obtained from the dirt samples of assorted parts of southern Indian tea plantation with edaphic and environmental factors. All the restraints were positively correlated with the population denseness, except for temperature at the maximal degree. Dirts obtained from Valparai part showed higher population denseness ( 14.30 cfu – 10-3g/soil dry wt ) , followed by dirts in Munnar ( 13.33 cfu – 10-3g/soil dry wt ) and to a lesser extent, those in the Koppa part ( 11.67 cfu – 10-3g/soil dry wt ) . There was clear fluctuation observed in Trichoderma spp. population in different seasons. The maximal population denseness was recorded during the southwest monsoon. A sum of 78 isolates were enumerated among which six were chosen from each agroclimatic zone and was identified it as Trichoderma atroviride. The radial growing measuring of Tv1 isolate obtained from Valparai showed the maximal growing ( 45 millimeter ) within 72 hours on PDA. Similarly the maximum mycelium dry weight was recorded on the seventh twenty-four hours of incubation by the same isolate. The highest antagonist activity of Tv1 isolate observed in double civilization trial was 65.77 % of suppression upon the pathogen. This was followed by antagonist activities of Tc3 and Tm3 isolates which was isolated from tea dirt samples of Coonoor and Munnar parts severally. Antibiosis for volatile and non-volatile fungicidal compounds produced by adversary showed that suppression per centum of P.theae was 91.25 % and 69.17 % severally by Tv1 isolate. The compatibility of T.atroviride isolates tested against assorted contact and systematic antifungals clearly showed that Tv1 isolate can able to digest the concentration of antifungals to higher extent.
Cardinal words: Tea ; Collar canker ; Trichoderma atroviride ; Phomopsis theae ; Biocontrol agent ; Antifungals
The quest for biological control of works plagues and pathogens continues to incite research and development in legion Fieldss. This is particularly the instance in plantation harvests like tea ( Camellia sinensis ( L. ) O.Kunze ) . A big figure of works diseases were successfully controlled through bacterial and fungous adversaries. However, merely meager information is available on the biological control of tea diseases ( Ponmurugan and Baby 2006 ) . Among the assorted diseases that afflict tea workss, collar canker disease caused by the fungus Phomopsis theae Petch is the most common one. The disease is of great effect as the septic countries are being replanted. Crop loss due to this disease is estimated at 10-15 % in southern Indian tea plantations. Avoidance of pre-disposing factors and uprooting and combustion of the shrubs in terrible instances are besides recommended as control steps ( Baby 2001 ) . Despite its economic significance, effectual control steps are non available other than sniping to healthy wood and application of Cu antifungals on prune cuts ( Ponmurugan and Baby 2006 ) . The application of antifungals is largely toxic and pollutes the ambiance by distributing out in the air and roll uping in the dirt. The frequent usage of such chemicals may promote the development of chemical opposition in pathogen ( Naseby et al. 2000 ) . Using Biocontrol Agents ( BCAs ) could be an alternate to chemicals in the direction of fungous diseases. Several commercial BCAs including both bacteriums and Fungis have been registered and are available as commercial merchandises for control of assorted diseases ( Punja and Utkhede 2003 ) .
Trichoderma spp. are most often stray dirt Fungis and nowadays in works root ecosystems ( Harman et al. 2004 ) . Many surveies have proved the potency of Trichoderma spp. as biocontrol agents of several dirt borne works pathogens, such as Rhizoctonia solani, Sclerotium rolfsii, Pythium spp. , Drechslera tritici and Fusarium spp. ( Tronsmo 1991 ; Lorito et Al. 1993 ) . Diverse mechanisms have been recommended for their biocontrol activity, which comprises of hostility for infinite and foods, secernment of enzymes, mycoparasitism and production of antimicrobic compounds. Kucuk and Kivanc ( 2004 ) had demonstrated the engagement of volatile metabolites secreted by Trichoderma harzianum in the suppression of G.graminis, F. culmorum and F.moniliforme. The practical application of Trichoderma spp. as biocontrol agents integrated with chemical interventions requires choice of antifungal immune isolates. A biological agent, in add-on to being competent, must besides be adaptable to modern harvest protection patterns, including usage of antifungals. It has been reported that many Trichoderma spp. have an innate and/or induced opposition to many antifungals but the degree of opposition varies with the antifungal ( Khan and Shahzad 2007 ) . Hence, the purpose of this survey was to correlate the edaphic and environmental factors with population denseness of Trichoderma spp. and screen the efficient autochthonal isolate with outstanding adversary activity against P.theae and to guarantee its compatibility with assorted antifungals.
Materials and Methods
Sites of dirt sample aggregation and dirt analysis
The rhizosphere dirt samples were collected from 30 commercial tea seting estates covering six major territories of southern India viz. , Valparai, Coonoor, Munnar, Vandiperiyar, Gudalore and Koppa ( Table 1 ) . From each estate, dirt samples were collected indiscriminately, air dried in shadiness, gently alleted, sifted with 2mm mesh screens and stored at 4EsC for farther surveies. These dirt samples were subjected to assorted edaphic factors analysis such as pH ( digital pH meter-Elico ) , EC ( digital electrical conduction meter-Elico ) , organic C ( Dichromate oxidization method – Walkey and Black, 1934 ) , entire N ( Micro-kjeldahl and Mo blue methods – Jackson 1973 ) , available P ( Molybdenum blue methods – Jackson 1973 ) and exchangeable K ( Digital fire photometer-Elico – Ham and Nathan 1951 ) . The environmental specifics of the tea plantations such as lift, rainfall, temperature, comparative humidness and sunlight were besides gathered from the several estate to analysis seasonal influences over the population denseness of Trichoderma spp.
Isolation and characteristic characteristics of Trichoderma spp.
Trichoderma spp. was enumerated from the above dirt samples utilizing pour home base the method on Trichoderma Selective Media ( TSM ) by following the consecutive dilution method ( Elad and Chet 1983 ) . The home bases were incubated at 28EsC for 4 -7 yearss. After incubation, Trichoderma spp. was enumerated based on settlement morphology and later identified by morphological word picture traits ( Ponmurugan and Deepa 2010 ) . Depending upon on radial growing measurings, antagonist activity and cultural word picture ( Grondona 1997 ) six isolates from each agroclimatic zones were chosen for the present survey. Isolated Trichoderma spp. was sent to Microbial Type Culture Collection Center ( MTCC ) , Chandigarh, India for designation at species degree and farther identified as Trichoderma atroviride. The standard civilization T MTCC ( T.atroviride isolate – MTCC 2461 ) was besides acquired from MTCC for comparing.
In vitro rating selected T.atroviride isolates
Radial growing measurings and mycelial dry weight of T. atroviride isolates was studied utilizing Potato Dextrose Agar ( PDA ) and Potato Dextrose Broth ( PDB ) severally. Petri dishes ( 90 mm diameter ) incorporating PDA were centrally inoculated with a 5 millimeter of agar stoppers from 7 yearss old civilizations of T.atroviride isolates to find the radial growing measuring. Similarly, dry weight of the mycelium was determined by reassigning 5 millimeters actively turning civilizations into 100 milliliter of PDB and incubated at 25EsC. The mycelial mats were harvested at different intervals, and the dry weight of the mycelium was recorded. In order to analyze the growing wont of T.atroviride isolate in the tea ecosystem, tea works infusion agars and tea dirt agar medium were prepared by the method outlined by Ponmurugan and Baby ( 2007 ) . Different parts of tea workss such as root, root-bark, root wood, root bark, root wood and foliage infusions were used for the check. Soil agar medium was prepared individually for each territory and the radial growing of the adversary was measured.
Bioefficacy between autochthonal T.atroviride and P.theae was studied by following the method of double civilization trial ( Huang and Hoes 1976 ) and antibiosis of volatile ( Fiddman and Rossall 1993 ) and non-volatile fungicidal compounds ( Dennis and Webster 1971 ) . The P.theae ( UPASI – MP ) strain was obtained from Plant Pathology Division, UPASI Tea Research Institute, Valparai, India. For double civilization experiments mycelial discs ( 5 millimeter in diameter ) of P. theae and T.atroviride were placed at diametrically opposite points on a petri dish incorporating PDA. After 48 hours of incubation clip, the per centum suppression of the pathogen by the adversary was determined. To analyze the antagonist consequence of volatile metabolites produced by T.atroviride isolates, each counter isolate was grown on a unfertile cellophane disc lying on PDA for 48 hours. The cellophane with the mycelium was removed in the same place in which the pathogen was made to turn. Radial growing of the pathogen was determined after 72 hours and was compared with the control. In order to analyze the efficaciousness of non-volatile compounds, the bottom palpebras of two PDA petri home bases were inoculated with mycelial phonograph record of T.atroviride isolates and P.theae individually. The two palpebras were so reversed, puting one above the other, and sealed air-tight through parafilm. After 96 hours, the settlement diameter of P.theae was measured.
The sensitiveness of the different T.atroviride isolates to assorted contact and systemic antifungals was evaluated by nutrient poisoned technique ( Adams and Wong 1991 ) . Required measure of antifungal was added to sterilise PDA medium to bring forth coveted concentrations. Unamended PDA serves as control. The contact antifungal such as Blitox ( Copper Oxychloride ) , Kocide ( Cu hydrated oxide ) , Mancozeb ( Dithane M-45 ) and Bordeaux mixture and systemic antifungal such as Bavistin ( carbendazim ) , Contaf ( hexaconzole ) , Calixin ( tridemorph ) and Baycor ( bitertanol ) were tested against T.atroviride isolates. The informations obtained were subjected to Analysis Of Variance ( ANOVA ) and the important agencies were segregated by Critical Difference ( Cadmium ) at assorted degrees of significance and standard mistake ( SE ) was besides calculated ( Gomez and Gomez 1984 ) .
Population denseness, morphological and physiological characteristics of Trichoderma spp.
The consequences on the edaphic factors showed that, the rhizosphere soils taken from different tea plantations were acidic in nature ( Table 1 ) . Tea dirts are normally acidic due to the drawn-out usage of nitrogen-bearing fertilisers such as urea and ammonium sulfate to increase harvest production in the ecological niche ( Nioh et al. 1995 ) . Least fluctuation was observed among the assorted territories for pH and EC. Organic C, N, P and K degrees continued to be higher in Valparai when compared with other territories. The population denseness of Trichoderma spp. enumerated from the six territories clearly showed that the Valparai part has the highest count of 14.30 – 10-3 cfu g-1 dirt dry wt when compared with other parts. The correlativity coefficients of edaphic and environment factors which influence the population denseness of Trichoderma spp. are presented in Table 2. Positive correlativity was observed among the environmental factors such as rainfall, temperature lower limit, RH factor and sunlight, with the population denseness of Trichoderma spp.
Seasonal fluctuations seemed to carry the denseness of Trichoderma spp. population to a big extent ( Fig. 1 ) . The rainy season resulted in higher population, followed by the winter and summer seasons. The southwest monsoon is extremely good for Valparai, Munnar, Coonoor and Gudalur. The staying parts benefit from the nor’-east monsoon. In these countries Trichoderma spp. attains its maximal population denseness during the months of June to September and ranges lower degree in the station monsoon season. Besides there was a drastic decrease in the population denseness in the winter, with the diminution go oning in the summer.
There were no single fluctuations observed between the isolates. However, the visual aspect of the settlement was similar with immature milky conidiospore with restricted concentric rings which turn to greenish with compact conidiophores throughout when it becomes older. Formations of chlamydospores were abundant within seven yearss and T. atroviride can be easy distinguished by the presence of coconut olfactory property. The spreading pigment in civilization medium was non observed. None of the isolates studied showed any alteration in the pH of the medium with glucose as the C beginning. Growth and monogenesis were seen on citric acid, lactic acid, urea and nitrite amended medium ( Table 3 ) . However, there was no growing observed with ammonium oxalate as the C beginning. The isolates showed positive response at 37EsC but were unable to turn over 4 EsC and 40 EsC temperature which coincided with the nature of environmental factors.
Efficacy of T.atroviride isolates over P.theae
The consequences of radial growing and dry weight of mycelium of T.atroviride isolates are presented in Table 4. The mycelium of Tv1 isolate had covered the full home base within five yearss of incubation. The mean of mean additive growing rates for isolates ranged from the lowest value of 13.5 mm/day for T MTCC to the highest value of 15.8 mm/day for Tv1 isolates. A important difference was observed between isolates. The per centum of dry weight of T.atroviride show huge divergency and the mass of mycelium between the fifth and 7th yearss did non demo much difference for all the isolates. The Tv1 isolate had a higher per centum of 95.22 and the standard civilization obtained from MTCC showed around 85.32 % in its dry weight.
Significant differences were observed between the radial growing of T. atroviride isolate on media amended with assorted infusions of tea root, root, foliage and dirt ( Table 5 ) . Among the tea works infusion agar medium tested, tea root and root infusion agar followed by the dirt infusion agar medium supported the maximum growing. Autochthonal isolates obtained from the tea dirt sample showed huge divergency in radial growing when compared with standard civilization obtained from MTCC.
All the six autochthonal T.atroviride isolates and T MTCC produced different per centums of suppression on P.theae, runing from 51.11 % to 65.77 % ( Table 6 ) in double civilization trial. Strain Tv1 showed the highest additive growing of 74.33 millimeter which was statistically important compared to all the other isolates. T MTCC isolate showed the least measuring of 41 millimeter followed by 52 % shown by Tk2 and Tg2 isolates. Hence the double civilization experiment clearly demonstrated that T. atroviride isolates wholly inhibited the growing of P.theae with settlement debasement runing from 7 to 9 yearss, it achieved this by seeable incursion with the formation of little tussocks thereby crumbling and falsifying the pathogen hyphae.
Antibiosis, the production of antimicrobic compounds, and mycoparasitism, the eating on a fungus by another being, are mechanisms whereby Trichoderma spp. provides protection to workss against works pathogens [ Chet et Al. 1998 ; Howell 2003 ; Harman et al. 2004 ) . The consequences of adversaries, Tv1 isolate registered higher antibiosis than other isolates of 91.25 % of suppression, which was followed by Tc3 and Tm3 of 81.08 % and 76.25 % severally ( Table 6 ) . The isolates studied here varied greatly in their abilities to bring forth antimicrobic compounds inhibitory to P.theae and in their abilities to parasitize the pathogen.
Inhibition of radial growing of P.theae in the presence of volatile compounds ranged from 30.83 % to 69.17 % . The Tv1 isolate showed the highest per centum of suppression which was statistically important when compared to other isolates. T MTCC and Tg2 correspondingly showed least repressive consequence on P.theae, at 30.83 % and 37.50 % . All the antifungals in their different concentrations significantly inhibited the growing of T. atroviride isolates ( Table 7 ) . The response of T.atroviride varied with the antifungals used. None of the isolates grew when Carbendazim and Calixin were used at concentration of 100ppm. The Tv1 isolate showed better compatibility with assorted antifungals used than compared to other isolates and is able to digest the Blitox, Kocide and Bordeaux mixture to a big extent, when compared with Contaf and Baycor. The repressive consequence of all antifungals on mycelium growing increases with an addition in the concentration. However the growing of the mycelium settlement obtained when treated with antifungal was meager and pantie when compared with the control.
It is best to restrict or avoid the usage of chemicals in agribusiness, peculiarly in plantation harvests. The most capable manner to carry through this is to utilize of BCAs. These non merely command the phytopathogen but besides could easy supply growing sweetening for harvests. Soil biodiversity plays a cardinal function in the sustainability of agribusiness systems and indicates the degree of wellness of the dirt. This is particularly so when we consider the profusion of micro-organisms that is involved in biological control of soilborne diseases ( Vargas 2006 ) . The present survey clearly indicates that edaphic and environmental factors extremely favor the Trichoderma spp. population obtained from tea dirt samples. Mineral nutrition is indispensable for growing and, within a narrower scope, for exciting fungous secondary metamorphosis ( Duffy et al. 1997 ) . High entire Nitrogen handiness increased monogenesis, production of fungicidal anthroquinone pigments and hyphal growing rate ( Fargasova 1992 ) . This extremely correlated with Valparai dirt samples, where edaphic factors were favourable for Trichoderma spp. than compared with other parts. The population denseness of Trichoderma spp. isolated from dirt samples during different seasons obviously shows that rainy season favors the growing of Trichoderma spp. , whereas summer influences a decrease in its populations. This relates with the work done by Panda et Al. ( 2009 ) which concluded that higher wet content of the dirt in the rainy season and higher dirt temperature in the summer might be the ground for doing such fluctuation.
Tea dirts contained different types of Trichoderma spp. ( Baby and Chandramouli 2002 ) , but we have selected merely T.atroviride for the present survey to measure the public presentation against the disease. Assorted surveies have reported that T. atroviride acts as a biocontrol agent for a broad scope of economically of import forward pass and soilborne works pathogens ( Brunner 2005 ) and has been found to be effectual against the myxomycete Polymyxa betae ( Jakubikova et al. 2006 ) . Harmonizing to Vinale et Al. ( 2004 ) the ability of T.harzianum T22 and T. atroviride P1 to better the growing of boodle, tomato and Piper nigrum workss under field conditions was investigated. These findings obviously showed the matchless activity of T.atroviride against different pathogens which possibly besides acts as a successful biocontrol agent against P.theae.
The chief aim of the present survey was to test the efficient autochthonal isolates for commanding Phomopsis canker diseases in tea workss. Out of 78 isolates obtained, six were chosen based on rapid growing rate, adversary activity and morphological similarity for farther surveies. Subsequently it was identified as T.atroviride by MTCC. In this work, the Tv1 isolate obtained from tea dirt sample of Valparai territory proved to be unusually competent in growing than rate and biomass production of other autochthonal isolates and the standard civilization obtained from MTCC. Additionally the capacity of this isolate to use the assorted tea works infusion medium clearly showed its capableness to move as the best adversary against tea phytopathogen P.theae. The antagonist activity of Tv1 isolate against P.theae in double civilization and antibiosis clearly shows its competent nature and proved to be a pre-eminent strain in commanding the Phomopsis canker disease. This highlights the decision reached by Ponmurugan and Baby ( 2006 ) that Trichoderma spp. could be profitable used as biocontrol agents against primary and secondary root diseases and collar canker caused by Phomopsis theae in tea plantations.
To look into the compatibility of biocontrol agent with often used antifungals in tea workss by in vitro is quiet indispensable. This helps to guarantee the endurance of the adversary in the dirt after the application in tea Fieldss. Hence, with this attack the compatibility of both systemic and contact antifungals tested gave positive consequences and proved the efficaciousness of Tv1 as the best adversary to be applied in the field. This extremely correlates with the work done by Bagwan 2010, which shows the capable nature of Trichoderma spp. in defying the antifungals used. The present survey endows researches with primary informations. Further research is being conducted to asses the practical application of this species as biocontrol agents in the field against P.theae.