Abstract: An attempt was made to study the biocontrol activity of Trichoderma atroviride against Phomopsis theae Petch, a causative agent of Phomopsis canker disease in tea plants. Among 78 isolates enumerated, six strains, each representing an agroclimatic zone was chosen for further studies and later it was identified it as Trichoderma atroviride. A range of in vitro assessment such as growth parameters, antagonist activity against P. theae and compatibility with fungicides were evaluated for selected T. atroviride isolates. The correlation of various edaphic and environmental factors with population density of Trichoderma spp. showed positive results. The radial growth measurement and mycelium dry weight of Tv1 isolate obtained from Valparai showed the utmost growth on Potato Dextrose Agar. The highest antagonist activity of 65.77, 91.25 and 69.17% was observed for Tv1 isolates in dual culture test, antibiosis for volatile and non-volatile antifungal compounds. The compatibility of T. atroviride isolates tested against various contact and systematic fungicides clearly showed that Tv1 isolate can able to tolerate the concentration to higher extent. Hence the results revealed that Tv1 isolates proved to be eminent strains in controlling Phomopsis canker disease in tea plants.
INTRODUCTION
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). This foliage crop tends to endure severe stress both physically and physiologically owing to the frequent harvest of shoot (Thomas et al., 2009). Among the various diseases that afflict tea plants, collar canker disease caused by the fungus Phomopsis theae Petch is the most common one. The consequence of disease has led to the replanted of infected bushes. The decreases in yield have been estimated around 10-15% in Southern Indian tea plantation (Ponmurugan and Baby, 2008). Despite of its economic impact, effectual preventive measures are unavailable other than pruning of healthy wood and application of copper fungicides on prune cuts (Ponmurugan et al., 2002). The application of fungicides is mostly toxic and pollutes the atmosphere by spreading out in the air and accumulating in the soil. Random and extreme use of chemical fungicides for seed and soil management has led to the increase of pathogen resistance (Daghman et al., 2006). 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 Ukhtede, 2003).
According to Daami-Remadi et al. (2006), several soil borne plant pathogens, such as Rhizoctonia solani, Sclerotium sclerotiorum, Pythium spp., Stereum purpureum, Botrytis cinerea, Phomopsis viticola and Fusarium spp. are being successfully controlled by Trichoderma spp. 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. According to Jegathambigai et al. (2009), indigenous Trichoderma spp. has shown more effective, enhanced adaptability and antagonist activity in controlling the phytopathogen. Kucuk and Kivan (2004) had demonstrated the involvement of volatile metabolites secreted by Trichoderma harzianum in the inhibition of G. graminis, F. culmorum and F. moniliforme.
A biological agent, in addition to being competent, must also be adaptable to modern crop protection practices, including use of fungicides. Hence it has become essential to verify the compatibility of the bioagent with chemical fungicides. Trichoderma spp. has an intrinsic or stimulated resistance to many fungicides; however, the level of tolerance may varies with the fungicide (Khan and Shahzad, 2007). With this concept the intention of this present study was to 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 samples collected 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 2 mm mesh sieves and stored at 4°C 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 by Walkely and Black (1934), total nitrogen Micro-kjeldahl and molybdenum blue methods by Jackson (1973), available phosphorus Molybdenum blue methods by Jackson (1973) and exchangeable K Digital flame photometer-Elico by Gammon (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 correlate the population density of Trichoderma spp. with edaphic and environmental factors and to analysis the seasonal influences over the population density of Trichoderma spp.
Isolation and characteristic features of Trichoderma spp.: Trichoderma spp. was isolated from the above soil samples using Trichoderma Selective Media (TSM). After incubation, the fungal colonies were identified as Trichoderma spp. by morphological characterization traits (Ponmurugan and Deepa, 2010). Depending upon on radial growth measurements, antagonist activity and cultural characterization (Grondona et al., 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 (MTCC 9461). 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 25°C. 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 (2007a). 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 (Fiddaman and Rossall, 1993) and non-volatile antifungal compounds (Dennis and Webster, 1971). The P. theae (IMI No.-384005) 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 h 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 h. 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 h 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 h, 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). 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.
Statistical analysis: The data obtained were subjected to Analysis Of Variance (ANOVA) and the significant means were segregated by Critical Difference (CD) at various levels and Standard Error (SE) was also calculated (Gomez and Gomez, 1984).
RESULTS
Population density, morphological and physiological features of Trichoderma spp.: The results of the edaphic factors showed that the rhizosphere soils taken from different tea plantations were acidic in nature (Table 1). Values of pH and EC were almost similar to all the regions were Trichoderma spp. were isolated. Organic carbon, phosphorus and potassium levels continued to be higher in Valparai when compared with other districts and showed significant difference when compared with Vandiperiyar, Gudalur and Koppa regions. The population density of Trichoderma spp. enumerated from the six districts clearly showed that the Valparai region has the highest count of 14.30x10-3 cfu g-1 soil dry wt when compared with other regions. No significant difference was observed for population density of Trichoderma spp. within the districts, however, the significant difference was observed between the Valparai and Koppa 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, Relative humidity and sunshine, with the population density of Trichoderma spp. Correlation of population density with potassium gave positive results (r = 0.910, p<0.01). Other parameters such as available phosphorus and total Nitrogen content also gave positive results with population density by the correlation values of r = 0.686, 0.602 at p<0.05, respectively. The above results justify that edaphic and environmental factors were favorable for the growth of Trichoderma spp. This might be the reason for increased population density at Valparai regions than compared to other districts. However, negative correlation was observed for minimum temperature (r = -0.372, p<0.05).
| Table 1: | Effect of edaphic and environmental parameters on the population density of Trichoderma spp. in Southern Indian tea plantations |
| Values in the parentheses indicate the elevation of particular place in meter above mean sea level. x Isolates were designated based on name of the place; T: represents Trichoderma; Number denotes field number of the tea estate. OC: Total Organic Carbon (%); N: Total Nitrogen content (ppm); P: Available Phosphorus (ppm); K: Exchangeable Potassium (ppm); Temp: Temperature (°C), RH: Relative humidity (%); Sun shine-Mean Sunshine period (h day-1); z Population density of Trichoderma spp. (cfu x 10-3 g/ soil dry wt); yAverage values for four years (2006-2010). Means within a column followed by the same superscript letter(s) are not significant | |
| Table 2: | Correlation coefficient of edaphic and environmental parameters on the population density of Trichoderma spp. in Valparai tea plantations |
| *Correlation is significant at the 0.05 level (2 tailed). **Correlation is significant at the 0.01 level (2 tailed). OC: Total Organic Carbon (%); N: Total Nitrogen content (ppm): P: Available Phosphorus (ppm); K: Exchangeable Potassium (ppm); Temp: Temperature, RH: Relative humidity; Sun shine: Mean Sunshine period (h day-1), bPopulation density of Trichoderma spp. (cfu x 10-3 g/ soil dry wt). cAverage values for four years (2006-2010) | |
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 were benefited 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.
Characteristic features of T. atroviride were described in Table 3. Individual variations were not observed between the isolates. The appearance of the colonies 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. However, there was no growth observed with ammonium oxalate as the carbon source. The isolates showed positive response at 37°C but were unable to grow over 4 and 40°C temperature which coincided with the nature of environmental factors. This clearly verifies that all isolates obtained from different agro climatic regions and selected for the present study belongs to similar species of T. atroviride.
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 radial growth measurement for isolates show vast divergence ranged and the lowest value ranged from 30 mm day-1 for T MTCC to the highest value of 38.20 mm for Tv1 isolates on 3rd of incubation. Significant difference was observed between Tv1 and other isolates at p≤0.05. However, there was no significant difference observed between Tc3, Tm3, Tvan4, Tg2 and Tk2 isolates. The percentage of dry weight of T. atroviride isolates between fifth and seventh days did not show much difference for all the isolates. The Tv1 isolate had a higher percentage of 95.02 and the standard culture obtained from MTCC showed around 85.32% in its dry weight. The above results illustrate that significant difference was not observed between Tv1, Tc3, Tm3 and Tg2 isolates but however increased in biomass for Tv1 isolates on 7th day of incubation showed significant difference between Tvan4, Tk2 and T MTCC isolates.
| Fig. 1: | Seasonal variation of population density of Trichoderma spp. in Southern Indian tea plantations |
| Table 3: | Characteristic features of T. atroviride isolates |
| * Standard culture used for comparison purpose, +: Presence, -: Absence | |
| Table 4: | Assessment of growth parameters of different isolates of T. atroviride |
| *Standard culture used for comparison purpose, Means within a column followed by the same superscript letter (s) are not significant; Each value is the mean of five replicates | |
The radial growth of T. atroviride isolate on media amended with various extracts of tea root, stem, leaf and soil was clearly specified in Table 5. Vast significant difference was observed between the isolates. Among the tea plant extract agar medium tested, tea root and stem extract agar supported the maximal growth for Tv1 isolate of 42.33 mm followed by the soil extract agar medium of 41.67 mm with five days of incubation. Tc3 isolate also showed better results of 40.33 mm radial growth in tea stem bark, tea stem wood and tea soil extract agar medium. 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 when compared to all other isolates. Tk2 and T MTCC isolate showed the least measurement of 44.33 and 41 mm followed by Tg2 isolates. No significant difference was observed between Tvan4, Tg2 and Tk2 isolates. Hence the dual culture experiment clearly demonstrated that Tv1 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.
The results of antagonists, Tv1 isolate registered higher antibiosis for non-volatile compounds of 91.25% of inhibition than other isolates 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 against P. theae and their capability to parasitize the pathogen. Tv1 isolate showed vast significant difference between other isolates but however no significant difference was observed between Tc3 and Tm3 isolates. 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%.
The average growth of T. atroviride isolates against different contact and systemic fungicides in various concentrations was described in Table 7. The response of T. atroviride isolates varied with the fungicides used. The Tv1 isolate showed better compatibility than compared to other isolates and it was able to tolerate the Blitox, Kocide and Bordeaux mixture to a large extent, when compared with Calixin, Carbendazim and Dithane M-45. The inhibitory effect of all fungicides on mycelium growth increases with an increase in the concentration.
| Table 5: | Effect of tea plant extract and tea soil extract agar medium on the radial growth of T. atroviride |
| *Standard culture used for comparison purpose. **Trichoderma selective media used for comparison purpose; Means within a row followed by the same superscript letter(s) are not significant; Each value is the mean of three replicates | |
| Table 6: | Hyperparasitism of different isolates of T. atroviride on the growth of P. theae |
| * Standard culture used for comparison purpose, Values in parentheses indicate the percentage of inhibition of P. theae by T. atroviride isolates; x Linear growth of T. atroviride isolates on 5th day (mm); y Radial growth of P. theae on 5th day (mm); Means within a column followed by the same superscript letter (s) are not significant; Each value is the mean of three replicates | |
| Table 7: | Effect of various fungicides on the growth of T. atroviride isolates |
| * Standard culture used for comparison purpose; x Contact fungicides; other fungicides are systemic in nature. yAverage values obtained for 10, 50 and 100 ppm concentration of different fungicides used; Means within a row followed by the same superscript letter (s) are not significant; Each value is the mean of three replicates | |
The significant difference was observed between indigenous and standard isolate used. However, the growth of the mycelium colony obtained when treated with fungicide was meager and scanty when compared with the control.
DISCUSSION
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 biocontrol agents. These not only control the phytopathogen but also could easily provide growth enhancement for crops. The biocontrol agent in crop protection and pest management will have the prospective to enhance crop yield quality and quantity (Oyekanmi et al., 2008).
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 (Gil et al., 2009). The present study clearly indicates that edaphic and environmental factors highly favor the Trichoderma spp. population obtained from tea soil samples. Tea soils are usually acidic due to the prolonged use of nitrogenous fertilizers such as urea and ammonium sulphate to increase crop production (Nioh et al., 1995). Although, similarity was observed for pH and EC values, other essential nutrient showed variation between each agro climatic zones. 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. and this strain showed more antagonists when compared to other isolates. 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 study 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.
In the present study T. atroviride was selected to evaluate the performance against the Phomopsis canker disease. Various studies have reported that T. atroviride acts as a biocontrol agent for wide range of aerial and soilborne plant pathogens (Brunner et al., 2005). Jakubikova et al. (2006) have reported that T. atroviride was found to be effective against Polymyxa betae. According to Vinale et al. (2006) the ability of T. harzianum and T. atroviride 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. From recent findings carried out by Osman et al. (2011) revealed that T. harzianum was found efficient antagonist in controlling of root rot disease in soy bean caused by R. solani. Above studies goes along with the present research that indigenous T. atroviride isolates were highly effective in controlling stem canker diseases. Apart from enhance biocontrol activity T. harzianum was also found to be an efficient mobilize of organic phosphorous when compared to other fungi (Yadav et al., 2011). Hence, usage of Trichoderma spp. for biocontrol of P. theae in tea field will surely enhance the tea productivity also.
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. Present study, the Tv1 isolate obtained from tea soil sample of Valparai district proved to be remarkably competent in growth rate and biomass production than other indigenous isolates and standard culture. Additionally the capacity of this isolate to utilize the various tea plant extract medium clearly showed its ability to survive in tea soils and 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 finding of Ponmurugan and Baby (2007b) that Trichoderma spp. could act as an efficient biocontrol agents against root diseases and collar canker 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 to the fungicides used. According to Ayed et al. (2007), various biological fungicides such as Biocont-T, Funga stop and Polyversum has proved to effectual in control of vascular wilt disease in potato caused by F. oxysporum.
CONCLUSION
The current study highlights with the findings that Tv1 T. atroviride isolated from Valparai tea soil samples showed higher efficiency in growth parameters and antagonist activities. Moreover, this isolate has proved to be a competent strain with common fungicides used in tea plantation. The present study endows researches with primary data. Further research is being conducted to analysis the integrated approach of this species as biocontrol agents along with common fungicides in the tea field against P. theae.