IN-VITRO EVALUATION OF TRICHODERMA HARZIANUM AGAINST SOIL-BORNE FUNGI OF ECONOMIC IMPORTANCE
A total of 767 soil samples were collected from different crops viz. dhaincha (Sesbania rostata), wheat (Triticum aestivum) and marigold (Tagetes patula) from 42 locations of Bangladesh. Eighty four isolates of T. harzianum were obtained from the collected samples. Soil-borne plant pathogens Sclerotium rolfsii, Fusarium oxysporum and Rhizoctonia solani were isolated from infected vegetable seedlings or plant parts from different locations of Bangladesh. The isolates of both T. harzianum and soil-borne plant pathogens were purified and cultured on PDA slant or filter disc and preserved in the refrigerator at 5 0C for future use. The isolates of T. harzianum were tested to assess their antagonistic activities against soil-borne fungi S. rolfsii, R. solani and F. oxysporum following dual culture on potato dextrose agar (PDA) in the laboratory. The in-vitro results indicated that T. harzianum was strong antagonistic against three different soil borne pathogenic fungi viz. S. rolfsii, R. solani and F. oxysporum in dual culture assay. Some isolates of T. harzianum viz. TM7, TM11, TM12, TM14, TNA1, TNA2, TNA3, TCN3, TDG1, TDG2, TJA1, TJA2, TJA3, TKG1, TJP2, TMP1, TMP2, TKC2, TBO2, TRM1 and TB2 were found most antagonists that remarkably reduced the radial growth of all the tested pathogens.
Soil-borne fungal phytopathogens are the causal agents of many diseases of economic importance such as foot rot, root rot, damping-off, seedling blight and vascular wilts (Lichtenzveig et al. 2006). These pathogens become challenging due to their years together perpetuation in soil in various form viz.oospore, conidia, sclerotia, chlamydospores or others (Mondal et al. 1996 and Agrios 1997). The major soil-borne notorious pathogens, Sclerotium rolfsii, Rhizoctonia solani, Fusarium oxysporum etc. cause diseases of many crops of economic importance in most of the tropical and sub-tropical regions of the world (Punja 1985).
With the purpose of reducing the economic losses caused by these soil-borne diseases, generally utilization of chemical fungicides is considered as easy and attractive approach for the farmers. Due to their relatively low cost, ease of use, and effectiveness, fungicides has become the primary means to combat fungal diseases (Vinale et al. 2008, Sharma 2011, Dias 2012). However, intensive uses of fungicides become harmful to non-target organisms and also develop resistance in pathogens, and the possible carcinogenicity as well as hazardous to environment and growers (Vinale et al. 2008, Doley and Jite 2012). Besides, antagonistic microbes are an attractive, environmental friendly and economic option over chemical fungicides (El-Bramawy and El-Sarag 2012). Amongst the range of antagonists, different species of Trichoderma of phylum Ascomycota are most useful and frequently isolated soil fungi that exist in plant root ecosystem (Harman et al. 2004). They are opportunistic, avirulent plant symbionts, as well as being parasites of other plant pathogenic fungi (Harman et al. 2004).These filamentous fungi are very wide spread in nature, with high population densities in soils and plant litters. They are saprophytic, quick growing and easy to culture and they can produce large amount of propagules with long shelf life. These Trichoderma species (T. viride, T. harzianum, T. longibrachiatum, T. hamatum, T. koningii and T. longibrachiatum) are very promising against phytopathogenic fungi such as F. oxysporum, S. rolfsii, Rhizoctonia solani, Pythium ultimum and Sclerotinia sclerotium (Manczinger et al. 2002, Benítez et al. 2004, Vinale et al. 2008). Many Trichoderma species are regarded as growth promoter of plants by increasing fresh weight, height and flowering in plants while potentially inhibiting pathogen growth (Sharma 2011). Hence, Trichoderma spp. are extensively exploiting and seeking attention of scientists from all over the world, and are also being commercially marketed as biopesticides, biofertilizers and soil amendments (Harman et al. 2004, Vinale et al. 2008, Sharma 2011, El-Bramawy and El-Sarag 2012). The current study was conducted in vitro to assess the antagonistic activity of T. harzianum collected from different location of Bangladesh to be useful against three economically important soil borne fungal pathogens, S. rolfsii, F. oxysporum and R. solani causing diseases on various crops.
MATERIALS AND METHOD
Four different experiments were conducted in the laboratory of Plant Pathology Division, Bangladesh Agricultural Research Institute, Gazipur during the years from 2011 to 2014.
Isolation of Trichoderma harzianum from roots
Rhizosphere soil and roots of healthy plant of dhaincha (Sesbania rostrata), marigold (Tagetes patula) and wheat (Triticum aestivum) grown in the field was collected from different location of Bangladesh. Roots were shaken gently to remove soil particles and washed thoroughly with tap water followed by surface sterilization with sodium hypochlorite solution (0.2%) and was rinsed with sterilized distilled water 3 times. Then, the cut pieces were transferred to 9 cm Petri dishes containing 10-15 ml acid potato-dextrose agar (APDA). The Petri dishes were incubated 4 days in the dark at 25±2 0C. After 4 days of incubation, plates were observed under microscope for Trichoderma colony. The identified isolates T. harzianum were purified by hyphal tip culture in water ager (WA) and preserved in the test tubes containing PDA at 25 0C.
Isolation of Trichoderma harzianum from soil
In case of soil, isolation was made through Dilution Plate Technique (Akhtar 1966). One gram of composite soil sample collected from the field was taken in a test tube containing 9 ml of sterilized water to make dilution as 1:10 and was thoroughly mixed with the PDA medium. Similarly a series of dilution process were continued until the samples were diluted to 1:10000. An aliquot (1ml) of each sample was placed in a Petri dishes containing PDA. The Petri dishes were incubated and allowed 5 days for growth at room temperature (25±2 0C). The growing mycoflora was observed under microscope and identified them following Rifai and Webster (1969) and Barnett and Hunter (1998). The identified T. harzianum was purified by hyphal tip culture in water agar (WA) and single spore isolation technique and preserved in the PDA slant and filter disc at 5 0C in the refrigerator for future use
Antagonistic effect of T. harzianum against pathogenic fungi
Antifungal activity of all the isolates of T. harzianum against the target fungal pathogens S. rolfsii, F. oxysporum and R. solani were assessed in vitro conditions following the dual culture technique of Rini and Sulochana (2007). Both T. harzianum and pathogenic fungi mycelial blocks (5mm) were cut from the periphery of 3-days old culture and were placed on PDA plate, facing opposite to each other. The distance between discs was approximately 6cm. Petri dishes were incubated at 27±2 °C for 3 days. The experiment was set up following Completely Randomized Design with five replications. Colony diameter was recorded after 48 hours of incubation for S. rolfsii, F. oxysporum and R. solani, and 72 hours of incubation for F. oxysporum and R. solani. The colony diameter in each Petri dish was measured at three places and average was calculated. Percent inhibition of growth was calculated using the following the formula as suggested by Sunder et al. (1995).
Where Y = Mycelial growth of pathogen alone (control) Z =Mycelial growth of pathogen along with antagonist.
RESULTS AND DISCUSSION
A total of 84 T. harzianum isolates were obtained from 767 soil and root samples (Table 1).
All T. harzianum isolates remarkably reduced the radial growth of S. rolfsii, F. oxysporum and R. solani on PDA in dual culture techniques (Table 2, 3 and 4). The T. harzianum isolates reduced 19.42-42.72%, 17.68-47.85% and 15.51-43.82% radial growth of S. rolfsii, F. oxysporum and R. solani, respectively at 48 hrs of incubation. Besides, at 72 hrs incubation T. harzianum isolates reduced 18.56-41.24% and 29.60-53.68% redial growth of F. oxysporum and R. solani, respectively (Table 3 and 4). Most of the T. harzianum isolates showed better performance in reducing the growth of S. rolfsii, F. oxysporum and R. solani that indicated the antagonistic effect of Trichoderma on the radial growth of the targeted pathogens. The T. harzianum isolate TNA3 suppressed 42.72% radial growth of S. rolfsii at 48 hrs followed by TM14, TJA2, TJA3, TNA1, TNA3, TKG1, TDG1, TJA1, TM7, TDG2, TJP2, TM2, TM3, TM11 and TM12 ranging from 34.62% to 40.66%. However, TCN1, TM1, TM8 and TM6 isolates reduced 19.42, 19.96, 20.88 and 21.79%, respectively of the growth of S. rolfsii. In case of F. oxysporum, T. harzianum isolate TJA3 reduced 47.85% radial growth followed by TNA3, TM14, TJP1, TKG1, TMP1, TJP2, TJA2, TM7, TJA1, TBO1, TM6, TNA2 and TDG2 where TMP3 reduced only 17.68% redial growth at 48 hrs incubation. The T. harzianum isolate TBO3 suppressed 45.02% vegetative growth of R. solani at 48 hrs incubation followed by TM14, TJA3, TKC1, TNA3, TMP1, TRM1, TB1 and TJA2 where TM1, TM2, TM3 and TM6 isolates reduced 15.51, 17.30, 17.98 and 18.43% redial growth, respectively. Therefore, among the isolates of T. harzianum TM7, TM11, TM12, TM14, TNA1, TNA2, TNA3, TCN3, TDG1, TDG2, TJA1, TJA2, TJA3, TKG1, TJP2, TMP1,TMP2, TKC2, TBO2, TRM1 and TB2 were found effective for suppressing the radial growth of pathogenic fungi S. rolfsii, R. solani and F. oxysporum in in-vitro study.
The majority of the T. harzianum isolates collected from different location of the country exhibited variable antagonistic activity against the S. rolfsii, R. solani and F. oxysporum in dual cultures. The antagonistic activity of Trichoderma against S. rolfsii was reported by many authors (Sultana et al. 2012, Shaigan et al. 2008, Yaqub and Shahzad 2005, Khattabi et al. 2004, Howel 2003, Yogendra and Singh 2002, Virupaksha et al. 1997). The fungus F. oxysporum was also reported to be suppressed by Trichoderma (Devi et al. 2013, Meraj and Nandkar 2012, Sharma 2011, John et al.2010, Ramezani 2010, Nikam et al. 2007, Kaur et al. 2003). Similarly the antagonistic activity of Trichoderma against R. solani was also reported by many investigators (Tapwal et al. 2014, Seema and Devaki 2012, Prasad and Kumar 2011). The inhibition of radial growth of two interacting organisms i.e. pathogenic fungi and T. harzianum in dual culture might be attributed to non-volatile and volatile inhibitory substance e.g. harzianic acids, tricholin, viridian, glisoprenins, massoilactone gliovir and heptelidic acid etc. released by one or both organisms in the growth medium that probably hinder the pathogen colonization (Rini and Sulochana 2007). Mishra et al. (2011) and Amin et al. (2010) observed the inhibitory activity of volatile and non-volatile compounds of Trichoderma species on the growth of Rhizoctonia, Fusarium, Alternaria, Colletotrichum etc. Comparatively fast growth of T. harzianum than the pathogenic fungi might be indication of competition efficiencies of antagonists for space and nutrients (Devi et al. 2013). Wide variation in the antagonism of a population of T. harzianum was reported by Davet (1986). Similarly, Tapwal et al. (2011) and Prasad and Kumar (2011) reported that Trichoderma as potential antagonist against the phytopathogen like R. solani and F. oxysporum in dual culture technique. Nagamma and Nagaraja (2015) also reported that T. harzianum effectively inhibited the mycelial growth of S. rolfsii under in-vitro conditions. Fakhrunnisa et al. (2006) found T. harzianum to be highly effective against F. oxysporum. The present study concluded that T. harzianum was the best biocontrol agent against economic important soil borne fungal pathogens S. rolfsii, R. solani and F. oxysporum.
Table 1. Collection of soil samples from different locations of Bangladesh and isolation of Trichoderma harzianum
|Sl. No.||Location||Number of soil samples tested||Number T. harzianum isolates obtained|
|26||Faridpur and Madaripur||20||3|
Table 2 In vitro suppression of radial growth of Sclerotium rolfsii by Trichoderma harzianum isolates at dual culture
|Average radial growth of S. rolfsii at 48hrs (cm)||Growth suppression (%)||Accessions (T.harzianum)||Average radial growth of S. rolfsii at 48hrs (cm)||Growth suppression (%)|
Table 3 In vitro suppression of radial growth of Fusarium oxysporum by Trichoderma harzianum isolates at dual culture
|Accessions (T. harzianum)||Average radial growth of F. oxysporum at 48hrs (cm)||Growth suppression (%)||Average radial growth of F. oxysporum at 72hrs (cm)||Growth suppression (%)|
Table 4 In vitro suppression of radial growth of Rhizoctonia solani by Trichoderma harzianum isolates at dual culture
|Accessions (T. harzianum)||Average radial growth of R. solani at 48hrs (cm)||Growth suppression (%)||Average radial growth of R. solani at 72hrs (cm)||Growth suppression
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