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The potential inhibitory effects of Trichoderma atroviride against the phytopathogen Phomopsis theae, the causative agent of collar canker disease in tea plants, were studied. An attempt was made to correlate the population density of Trichoderma spp. obtained from the soil samples of various regions of southern Indian tea plantation with edaphic and environmental factors. All the constraints were positively correlated with the population density, except for temperature at the maximum level. Soils obtained from Valparai region showed higher population density (14.30 cfu - 10-3g/soil dry wt), followed by soils in Munnar (13.33 cfu - 10-3g/soil dry wt) and to a lesser extent, those in the Koppa region (11.67 cfu - 10-3g/soil dry wt). There was clear variation observed in Trichoderma spp. population in different seasons. The maximum population density was recorded during the southwest monsoon. A total of 78 isolates were enumerated among which six were chosen from each agroclimatic zone and was identified it as Trichoderma atroviride. The radial growth measurement of Tv1 isolate obtained from Valparai showed the maximum growth (45 mm) within 72 hours on PDA. Similarly the maximal mycelium dry weight was recorded on the 7th day of incubation by the same isolate. The highest antagonist activity of Tv1 isolate observed in dual culture test was 65.77% of inhibition upon the pathogen. This was followed by antagonist activities of Tc3 and Tm3 isolates which was isolated from tea soil samples of Coonoor and Munnar regions respectively. Antibiosis for volatile and non-volatile antifungal compounds produced by antagonist showed that inhibition percentage of P.theae was 91.25% and 69.17% respectively by Tv1 isolate. The compatibility of T.atroviride isolates tested against various contact and systematic fungicides clearly showed that Tv1 isolate can able to tolerate the concentration of fungicides to higher extent.
Key words: Tea; Collar canker; Trichoderma atroviride; Phomopsis theae; Biocontrol agent; Fungicides
The quest for biological control of plant pests and pathogens continues to instigate research and development in numerous fields. This is especially the case in plantation crops like tea (Camellia sinensis (L.) O.Kunze). A large number of plant diseases were successfully controlled through bacterial and fungal antagonists. However, only meager information is available on the biological control of tea diseases (Ponmurugan and Baby 2006). Among the various diseases that afflict tea plants, collar canker disease caused by the fungus Phomopsis theae Petch is the most common one. The disease is of great consequence as the infected areas 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 burning of the bushes in severe cases are also recommended as control measures (Baby 2001). Despite its economic significance, effective control measures are not available other than pruning to healthy wood and application of copper fungicides on prune cuts (Ponmurugan and Baby 2006). The application of fungicides is mostly toxic and pollutes the atmosphere by spreading out in the air and accumulating in the soil. The frequent use of such chemicals may encourage the development of chemical resistance in pathogen (Naseby et al. 2000). Using Biocontrol Agents (BCAs) could be an alternative to chemicals in the management of fungal diseases. Several commercial BCAs including both bacteria and fungi have been registered and are available as commercial products for control of various diseases (Punja and Utkhede 2003).
Trichoderma spp. are most frequently isolated soil fungi and present in plant root ecosystems (Harman et al. 2004). Many studies have proved the potential of Trichoderma spp. as biocontrol agents of several soil borne plant 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 antagonism for space and nutrients, secretion of enzymes, mycoparasitism and production of antimicrobial compounds. Kucuk and Kivanc (2004) had demonstrated the involvement of volatile metabolites secreted by Trichoderma harzianum in the inhibition of G.graminis, F. culmorum and F.moniliforme. The practical application of Trichoderma spp. as biocontrol agents integrated with chemical treatments requires selection of fungicide resistant isolates. A biological agent, in addition to being competent, must also be adaptable to modern crop protection practices, including use of fungicides. It has been reported that many Trichoderma spp. have an innate and/or induced resistance to many fungicides but the level of resistance varies with the fungicide (Khan and Shahzad 2007). Hence, the intention of this study was to correlate the edaphic and environmental factors with population density of Trichoderma spp. and screen the efficient indigenous isolate with prominent antagonist activity against P.theae and to ensure its compatibility with various fungicides.
Materials and Methods
Sites of soil sample collection and soil analysis
The rhizosphere soil samples were collected from thirty commercial tea planting estates covering six major districts of southern India viz., Valparai, Coonoor, Munnar, Vandiperiyar, Gudalore and Koppa (Table 1). From each estate, soil samples were collected randomly, air dried in shade, gently alleted, sifted with 2mm mesh sieves and stored at 4ËšC for further studies. These soil samples were subjected to various edaphic factors analysis such as pH (digital pH meter-Elico), EC (digital electrical conductivity meter-Elico), organic carbon (Dichromate oxidation method - Walkey and Black, 1934), total nitrogen (Micro-kjeldahl and molybdenum blue methods - Jackson 1973), available phosphorus (Molybdenum blue methods - Jackson 1973) and exchangeable K (Digital flame photometer-Elico - Gammon and Nathan 1951). The environmental particulars of the tea plantations such as elevation, rainfall, temperature, relative humidity and sunshine were also gathered from the respective estate to analysis seasonal influences over the population density of Trichoderma spp.
Isolation and characteristic features of Trichoderma spp.
Trichoderma spp. was enumerated from the above soil samples using pour plate the method on Trichoderma Selective Media (TSM) by following the serial dilution method (Elad and Chet 1983). The plates were incubated at 28ËšC for 4 -7 days. After incubation, Trichoderma spp. was enumerated based on colony morphology and subsequently identified by morphological characterization traits (Ponmurugan and Deepa 2010). Depending upon on radial growth measurements, antagonist activity and cultural characterization (Grondona 1997) six isolates from each agroclimatic zones were chosen for the present study. Isolated Trichoderma spp. was sent to Microbial Type Culture Collection Center (MTCC), Chandigarh, India for identification at species level and further identified as Trichoderma atroviride. The standard culture T MTCC (T.atroviride isolate - MTCC 2461) was also acquired from MTCC for comparison.
In vitro evaluation selected T.atroviride isolates
Radial growth measurements and mycelial dry weight of T. atroviride isolates was studied using Potato Dextrose Agar (PDA) and Potato Dextrose Broth (PDB) respectively. Petri dishes (90 mm diameter) containing PDA were centrally inoculated with a 5 mm of agar plugs from 7 days old cultures of T.atroviride isolates to determine the radial growth measurement. Similarly, dry weight of the mycelium was determined by transferring 5 mm actively growing cultures into 100 ml of PDB and incubated at 25ËšC. The mycelial mats were harvested at different intervals, and the dry weight of the mycelium was recorded. In order to study the growth habit of T.atroviride isolate in the tea ecosystem, tea plant extract agars and tea soil agar medium were prepared by the method outlined by Ponmurugan and Baby (2007). Different parts of tea plants such as root, root-bark, root wood, stem bark, stem wood and leaf extracts were used for the assay. Soil agar medium was prepared separately for each district and the radial growth of the antagonist was measured.
Bioefficacy between indigenous T.atroviride and P.theae was studied by following the method of dual culture test (Huang and Hoes 1976) and antibiosis of volatile (Fiddman and Rossall 1993) and non-volatile antifungal compounds (Dennis and Webster 1971). The P.theae (UPASI - MP) strain was obtained from Plant Pathology Division, UPASI Tea Research Institute, Valparai, India. For dual culture experiments mycelial disks (5 mm in diameter) of P. theae and T.atroviride were placed at diametrically opposite points on a petri dish containing PDA. After 48 hours of incubation time, the percentage inhibition of the pathogen by the antagonist was determined. To study the antagonist effect of volatile metabolites produced by T.atroviride isolates, each antagonistic isolate was grown on a sterile cellophane disk lying on PDA for 48 hours. The cellophane with the mycelium was removed in the same position in which the pathogen was made to grow. Radial growth of the pathogen was determined after 72 hours and was compared with the control. In order to study the efficacy of non-volatile compounds, the bottom lids of two PDA petri plates were inoculated with mycelial discs of T.atroviride isolates and P.theae separately. The two lids were then reversed, placing one above the other, and sealed air-tight through parafilm. After 96 hours, the colony diameter of P.theae was measured.
The sensitivity of the different T.atroviride isolates to various contact and systemic fungicides was evaluated by food poisoned technique (Adams and Wong 1991). Required quantity of fungicide was added to sterilize PDA medium to produce desired concentrations. Unamended PDA serves as control. The contact fungicide such as Blitox (Copper Oxychloride), Kocide (copper hydroxide), Mancozeb (Dithane M-45) and Bordeaux mixture and systemic fungicide such as Bavistin (carbendazim), Contaf (hexaconzole), Calixin (tridemorph) and Baycor (bitertanol) were tested against T.atroviride isolates. The data obtained were subjected to Analysis Of Variance (ANOVA) and the significant means were segregated by Critical Difference (CD) at various levels of significance and standard error (SE) was also calculated (Gomez and Gomez 1984).
Population density, morphological and physiological features of Trichoderma spp.
The results on the edaphic factors showed that, the rhizosphere soils taken from different tea plantations were acidic in nature (Table 1). Tea soils are usually acidic due to the prolonged use of nitrogenous fertilizers such as urea and ammonium sulphate to increase crop production in the ecological niche (Nioh et al. 1995). Least variation was observed among the various districts for pH and EC. Organic carbon, nitrogen, phosphorus and potassium levels continued to be higher in Valparai when compared with other districts. The population density of Trichoderma spp. enumerated from the six districts clearly showed that the Valparai region has the highest count of 14.30 - 10-3 cfu g-1 soil dry wt when compared with other regions. The correlation coefficients of edaphic and environment factors which influence the population density of Trichoderma spp. are presented in Table 2. Positive correlation was observed among the environmental factors such as rainfall, temperature minimum, RH factor and sunshine, with the population density of Trichoderma spp.
Seasonal variations seemed to persuade the density of Trichoderma spp. population to a large extent (Fig. 1). The rainy season resulted in higher population, followed by the winter and summer seasons. The southwest monsoon is highly beneficial for Valparai, Munnar, Coonoor and Gudalur. The remaining regions benefit from the northeast monsoon. In these areas Trichoderma spp. attains its maximum population density during the months of June to September and reaches lower level in the post monsoon season. Also there was a drastic reduction in the population density in the winter, with the decline continuing in the summer.
There were no individual variations observed between the isolates. However, the appearance of the colony was similar with young whitish conidia with restricted concentric rings which turn to greenish with compact conidiophores throughout when it becomes older. Formations of chlamydospores were abundant within seven days and T. atroviride can be easily distinguished by the presence of coconut odor. The diffusing pigment in culture medium was not observed. None of the isolates studied showed any change in the pH of the medium with glucose as the carbon source. Growth and sporulation were seen on citric acid, lactic acid, urea and nitrite amended medium (Table 3). However, there was no growth observed with ammonium oxalate as the carbon source. The isolates showed positive response at 37ËšC but were unable to grow over 4 ËšC and 40 ËšC temperature which coincided with the nature of environmental factors.
Efficacy of T.atroviride isolates over P.theae
The results of radial growth and dry weight of mycelium of T.atroviride isolates are presented in Table 4. The mycelium of Tv1 isolate had covered the entire plate within five days of incubation. The mean of average linear growth 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 significant difference was observed between isolates. The percentage of dry weight of T.atroviride show vast divergence and the mass of mycelium between the fifth and seventh days did not show much difference for all the isolates. The Tv1 isolate had a higher percentage of 95.22 and the standard culture obtained from MTCC showed around 85.32% in its dry weight.
Significant differences were observed between the radial growth of T. atroviride isolate on media amended with various extracts of tea root, stem, leaf and soil (Table 5). Among the tea plant extract agar medium tested, tea root and stem extract agar followed by the soil extract agar medium supported the maximal growth. Indigenous isolates obtained from the tea soil sample showed vast divergence in radial growth when compared with standard culture obtained from MTCC.
All the six indigenous T.atroviride isolates and T MTCC produced different percentages of inhibition on P.theae, ranging from 51.11% to 65.77 % (Table 6) in dual culture test. Strain Tv1 showed the highest linear growth of 74.33 mm which was statistically significant compared to all the other isolates. T MTCC isolate showed the least measurement of 41 mm followed by 52% shown by Tk2 and Tg2 isolates. Hence the dual culture experiment clearly demonstrated that T. atroviride isolates completely inhibited the growth of P.theae with colony degradation ranging from 7 to 9 days, it achieved this by visible penetration with the formation of small tufts thereby crumpling and distorting the pathogen hyphae.
Antibiosis, the production of antimicrobial compounds, and mycoparasitism, the feeding on a fungus by another organism, are mechanisms whereby Trichoderma spp. provides protection to plants against plant pathogens [Chet et al. 1998; Howell 2003; Harman et al. 2004). The results of antagonists, Tv1 isolate registered higher antibiosis than other isolates of 91.25% of inhibition, which was followed by Tc3 and Tm3 of 81.08 % and 76.25 % respectively (Table 6). The isolates studied here varied greatly in their abilities to produce antimicrobial compounds inhibitory to P.theae and in their abilities to parasitize the pathogen.
Inhibition of radial growth of P.theae in the presence of volatile compounds ranged from 30.83% to 69.17%. The Tv1 isolate showed the highest percentage of inhibition which was statistically significant when compared to other isolates. T MTCC and Tg2 correspondingly showed least inhibitory result on P.theae, at 30.83% and 37.50%. All the fungicides in their different concentrations significantly inhibited the growth of T. atroviride isolates (Table 7). The response of T.atroviride varied with the fungicides used. None of the isolates grew when Carbendazim and Calixin were used at concentration of 100ppm. The Tv1 isolate showed better compatibility with various fungicides used than compared to other isolates and is able to tolerate the Blitox, Kocide and Bordeaux mixture to a large extent, when compared with Contaf and Baycor. The inhibitory effect of all fungicides on mycelium growth increases with an increase in the concentration. However the growth of the mycelium colony obtained when treated with fungicide was meager and scanty when compared with the control.
It is best to limit or avoid the use of chemicals in agriculture, particularly in plantation crops. The most capable way to accomplish this is to use of BCAs. These not only control the phytopathogen but also could easily provide growth enhancement for crops. Soil biodiversity plays a key role in the sustainability of agriculture systems and indicates the level of health of the soil. This is especially so when we consider the richness of microorganisms that is involved in biological control of soilborne diseases (Vargas 2006). The present study clearly indicates that edaphic and environmental factors highly favor the Trichoderma spp. population obtained from tea soil samples. Mineral nutrition is essential for growth and, within a narrower range, for stimulating fungal secondary metabolism (Duffy et al. 1997). High total Nitrogen availability increased sporulation, production of antifungal anthroquinone pigments and hyphal growth rate (Fargasova 1992). This highly correlated with Valparai soil samples, where edaphic factors were favorable for Trichoderma spp. than compared with other regions. The population density of Trichoderma spp. isolated from soil samples during different seasons evidently shows that rainy season favors the growth of Trichoderma spp., whereas summer influences a reduction in its populations. This relates with the work done by Panda et al. (2009) which concluded that higher moisture content of the soil in the rainy season and higher soil temperature in the summer might be the reason for causing such fluctuation.
Tea soils contained different types of Trichoderma spp. (Baby and Chandramouli 2002), but we have selected only T.atroviride for the present study to assess the performance against the disease. Various studies have reported that T. atroviride acts as a biocontrol agent for a wide range of economically important aerial and soilborne plant pathogens (Brunner 2005) and has been found to be effective against the myxomycete Polymyxa betae (Jakubikova et al. 2006). According to Vinale et al. (2004) the ability of T.harzianum T22 and T. atroviride P1 to improve the growth of lettuce, tomato and pepper plants under field conditions was investigated. These findings evidently showed the unrivaled activity of T.atroviride against different pathogens which perhaps also acts as a successful biocontrol agent against P.theae.
The main objective of the present study was to screen the efficient indigenous isolates for controlling Phomopsis canker diseases in tea plants. Out of 78 isolates obtained, six were chosen based on rapid growth rate, antagonist activity and morphological similarity for further studies. Subsequently it was identified as T.atroviride by MTCC. In this work, the Tv1 isolate obtained from tea soil sample of Valparai district proved to be remarkably competent in growth than rate and biomass production of other indigenous isolates and the standard culture obtained from MTCC. Additionally the capacity of this isolate to utilize the various tea plant extract medium clearly showed its capability to act as the best antagonist against tea phytopathogen P.theae. The antagonist activity of Tv1 isolate against P.theae in dual culture and antibiosis clearly shows its competent nature and proved to be a pre-eminent strain in controlling the Phomopsis canker disease. This highlights the conclusion 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 check the compatibility of biocontrol agent with frequently used fungicides in tea plants by in vitro is quiet essential. This helps to ensure the survival of the antagonist in the soil after the application in tea fields. Hence, with this approach the compatibility of both systemic and contact fungicides tested gave positive results and proved the efficacy of Tv1 as the best antagonist to be applied in the field. This highly correlates with the work done by Bagwan 2010, which shows the capable nature of Trichoderma spp. in resisting the fungicides used. The present study endows researches with primary data. Further research is being conducted to asses the practical application of this species as biocontrol agents in the field against P.theae.