Trichoderma species have often been used in the management of crop plant diseases. Trichoderma is a genus of asexually reproducing fungi that is present in all types of soils. Some soil borne fungi are difficult to eradicate because they produce resting structures like sclerotia, chlamydospores or oospores for their survival for a longer period of time under adverse environmental conditions (Baker and Cooke, 1974). Use of fungicides for the control of soil borne diseases is costly and also produces environment and health hazards to human and adversely affects the beneficial microorganisms in soil (Dluzniewska, 2003). This was diverted the attention of plant pathologists towards alternate methods for the control of plant diseases. Trichoderma spp is a potential biocontrol agent for the management of various plant diseases like, sapota (Wagh and Bhale, 2011; Bhale, 2013),Leafy vegetables (Rajkonda et al.,2012), spinach (Bhale, 2012). Trichoderma species are known to suppress infections of soil borne pathogens like Macrophomina phaseolina, Rizoctonia solani, Fusarium species and Pythium species on various crops (Benitez et al., 2004, Adenkule et al., 2001, Ehtesham et al., 1990; Lutchmeah and Cooke, 1875; Howell, 1982). Species of Trichoderma also have growth promoting capabilities that may or may not be integral to biological control (Dubey et al., 2007; Benitez et al., 2004, Yedidia et al., 1998). The combined use of biocontrol agent and chemical pesticides has attracted much attention in order to obtain synergistic or additive effects in the control of soil borne diseases (Locke et al., 1985). Reduced amount of fungicide can stress and weaken the pathogen and render its propogules more susceptible to subsequent attack by the antagonist (Heiljord and Tronsmo, 1998). Srinivas and Ramkrishnan (2002) have reported that integration of biocontrol agents and commonly used fungicides showed positive association by reducing the seed infection compared to fungicide and the fungal antagonists’ individually. Recently, biological control combined with chemical fungicide at lower concentration is applicable. It is being a part of IPM (Integrated Plant disease Management) strategy which was applied since chemical controls have used individually cause environmental pollution.Therefore the present study, Trichoderma species viz., T. viride, T. harzianum, T. koningii, T.pseudokoningii and T. virens were tested for compatibility with fungicides like Mancozeb and Captan.

 

Rhizospheric soils of irrigated and non irrigated plants were collected from different parts of Marathwada region of Maharashtra, India. From the rhizosphere soil samples, Trichoderma spp were isolated by using PDA and Trichoderma selective medium (TSM) by dilution plate technique (Johnson, 1957). Theisolated specieswere identified up to species level based on colony characters, growth, structure of mycelium, conidiophores, phialides and conidia (Kubicek and Harman, 2002). All Trichoderma spp were purified by hyphal tip technique (Tuite, 1996). The isolated Trichoderma spp were maintained throughout the study by periodical transfers on PDA and TSM slants under aseptic conditions to keep the culture fresh and viable.

 

a) Mancozeb 75% WP (1000 – 8000 μg/ml) is a broad spectrum contact fungicide with a protective action which belongs to the dithiocarbamates (Manganese ethylene bisdithiocarbamte) family of chemicals, which also includes maneb.

 

b) Captan 50% WP (100-700 μg/ml) (N-trichlorome- thylthio-4-cyclohexene-1, 2-dicarboximide) were used for in vitro.

 

Fungicides like Mancozeb and Captan was incorporated into the medium after sterilization. The fungicides in proportionate dosage were incorporated in to the molten Czapek Dox Agar (CZA) medium after sterilization and dispersed thoroughly by continuous shaking. This was poured in to 90 mm petridishes. Mycelial discs of 8 mm cut from the growing margin of 7 days old culture of Trichoderma species was inoculated at the centre of the petridish and incubated at 26 ± 2 C. The CZA plate without fungicide served as control. The diameter of the colony was measured after 7 days and compared with the control (Tronosmo, 1989).

 

The test fungi were isolated from naturally infected plants viz. leaf spot of brinjal (Solanum melongana L.) caused by Alternaria alternata, Fruit rots of sapota (Manilkar zapota L.) caused by Rhizoctonia solani, Aspergillus niger and Geotrichum candidum, leaf spot ofspinach (Spinacea oleraea L.) and fruit rot of ivy guard (Coccinia indica Wight & Arn.) caused by Macrophomina phaseolina.

 

Antagonistic efficacy of Trichoderma spp viz., T. viride, T. harzianum, T. koningii, T. pseudokoningii and T. virens were tested against the isolated pathogenic fungi by dual culture experiment (Morton and Stroube 1955). Trichoderma spp and test fungi were inoculated 6 cm apart. Three replicates were maintained for each treatment and incubated at 28 ± 2°C for 7 days. Monoculture plates of both served as control. Seven days after incubation (DAI), radial growth of test fungi and Trichoderma spp were measured. Colony diameter of test fungi in dual culture plate was observed and compared with control. Percentage of radial growth inhibition (%RGI) was calculated by using the formula: 100 X [C - T / C], Where C = growth in control and T = growth in treatment (Vincent, 1947).

 

The degree of antagonism between each of the Trichodema species and test pathogens in dual culture was scored on scale of R₁-R₅ (Bell et al., 1982).

 

Statistics recitation in vitro compatibility was statistically analysed using the main factor fungicide i.e. Mancozeb and Capton and pathogenic fungi i.e. Alternaria alternate, Rhizoctonia solani, Aspergillus niger, Geotrichum candidum, Fusarium oxysporum f. sp. spinacae, Macrophomina phaseolina  and the sub-factors were the Trichoderma species. Arcsine transformation of biological control (Trichoderma species) percentage was calculated by using the following formula:

 

Y = arcsin=sin^-1

Where, p is the percentage of inhibition and Y is the result of transformation

 

Statistical analysis of the experiments was performed using the Handbook of Biological Statistics (McDonald, 2008).

 

Species of Trichoderma viz., T. viride, T. harzianum, T. koningii, T. pseudokoningii and T. virens were isolated from the rhizosphere soil of different crop plants. These isolates were deposited in Research laboratory, Department of Botany, Arts Science and Commerce College Naldurg. These isolates were maintained on PDA (Potato Dextrose Agar) media slants. Species of Trichoderma were amended with fungicides and results obtained following (Table 1&2; Figure 1).

 

 

 

b.  Trichoderma harzianum: Trichoderma harzianum grow at lower concentration of Mancozeb. The growth was inhibited when concentration of Mancozeb was more than 8000 μg/ml. However, increased concentration of Mancozeb decreased the radial growth. In media containing Captan@700 μg/ml, no growth was recorded. The growth of T. harzianum was observed at lower concentration from 100 to 400μg/ml.

 

c. Trichoderma koningii: Trichoderma koningii showed growth in Mancozeb@1000 to 5000 μg/ml. The growth was very low in concentration of 7000 μg/ml, no growth occurred when Mancozeb was used above the @8000 μg/ml. T. koningii showed growth at Captan @100, 200, 300 μg/ml. The growth was slow at Captan @ 500 and 600μg/ml. No growth was observed on Captan@700μg/ml.

 

d. Trichoderma pseudokoningii: Trichoderma pseudokoningii grow slow under media containing Mancozeb@1000 2000, 3000 and 4000 μg/ml, but it was inhibited completely at Mancozeb@5000μg/ml. The radial growth of T. pseudokoningii was also low at Captan@100, 200, 300 and 400μg/ml. But no growth was observed on Captan@500 μg/ml.

 

e. Trichoderma virens: Trichoderma virens grow readily under low concentration of Mancozeb(@1000 – 5000 μg/ml). The radial growth was gradually decreased at Mancozeb@5000 to 7000 μg/ml and no growth was recorded at 8000 μg/ml. Treatment of Captan @100 to 300 μg/ml, the radial growth of T. virens was recorded. However, growth was stopped on Captan @ 500 μg/ml.

 

Only, T.pseudokoningii was found susceptible at lower tested concentrations on both fungicides. The obtained results indicated that low concentration of Mancozeb and Captan does not affect the radial growth of mycelium of Trichoderma spp. However, increased concentration of fungicides were decreased the radial growth of mycelium.

 

Isolated plant pathogens i.e.Alternaria alternata, Rhizoctonia solani, Aspergillus niger, Geotrichum candidum, Fusarium oxysporum f. sp. spinacae and Macrophomina phaseolina were evaluated their antagonistic nature against Trichoderma species under in vitro condition. It was observed that growth of pathogenic fungi was reduced with respect to radial growth and sporulation. The mycelium of Trichoderma species when comes in contact with the test fungi it became fungistatic and the growth of test fungi were retarded (Table 3 & Figure 2). Alternaria alternata:Results indicated that Trichoderma species significantly inhibited the radial growth of Alternaria alternata incitant of leaf spot of brinjal (Solanum melangona). Maximum inhibition of A. alternata was observed with T. harzianum (90.4%) followed by T. koningii (77.7%), T. virens (74.4%) and T. viride (73.3%). However, minimum inhibition showed under the treatment of T. psudokoningii (71.1%).

 

 

 

b. Aspergillus niger: Antagonistic activity of isolated Trichoderma species against Aspergillus niger were evaluated under in vitro condition and results were recorded. Trichoderma koningii exhibited maximum antagonism against A. niger (57.7%). However, T. harzianum also showed antagonism against A. niger (54.4%). Radial growth of A. niger was also inhibited by T. viride (44.4%), T. virens (44.4%) and T. pseudokoningii (42.2%). It was observed that the isolated Trichoderma species showed moderate inhibitory effect over A. niger.

 

c. Geotrichum candidum: Results revealed that antagonistic effect of Trichoderma species over Geotrichum candidum causal agent of fruit rot of sapota (Manilkar zapota). Maximum inhibition against G. candidum was recorded by T. viride (74.4%), followed by T. pseudokoningii (72.2%), T. virens (68.9%), T. koningii (66.7%) and T. harzianum (61.1%). The isolated Trichoderma species exhibited significant antagonism against G. candidum.

 

d. Fusarium oxysporum f. sp. spinacae: In vitro antagonistic nature of Trichoderma species against Fusarium oxysporum f. sp. spinacae causal agent of wilt of spinach (Spinacea oleracea)was tested. It was cleared that Trichoderma species inhibited the mycelial growth of F. oxysporum f. sp. spinacae. Among Trichoderma species, maximum inhibition showed by T. virens (70.0%) followed by T. harzianum (66.6%). T. pseudokoningii (62.2%) and T. viride (55.5%).

 

e. Macrophomina phaseolina: Antagonistic effect of Trichoderma species against Macrophomina phaseolina incited fruit rot of ivy guard (Coccinia indica) was recorded and the results showed that Trichoderma species were significant in reducing the radial growth of mycelium of the test fungus. The inhibitory effect of T. viride (84.4%) found maximum followed by T. harzianum (83.3%) and T. pseudokoningii (81.1%). The antagonistic effects by T. virens (57.7%) and T. koningii (55.5%) over M. phaseolina were recorded minimum but these were also significant.  

 

According to modified Bell’s scale, T.harzianum overgrew beyond 90 percent (R₁ scale).  In case of R.solani, only T.koningii overgrew beyond 60 percent (R₃ scale).All Trichoderma species were failed to

progress beyond 60 percent (R₃ scale) in A.niger. In G.candidum, T.pseudokoningii and T.viride overgrew at least two third of pathogen (R₂ scale) but others were beyond 60 percent (R₃ scale). In case of F.oxysprum f. sp.spinaceae, T. virens overgrew at least two-third pathogens (70% over growth). Except T.koningii and T. virens other Trichoderma species were overgrew at least two third of pathogen (R₂ scale) in M. phaseolina (Table 4)

 

 

Experiments were also conducted to determine the compatibility of biocontrol agents with commercially effective chemicals against white rot of apple (Dematophora necatrix) in India (Gupta and Sharma, 2004). They concluded that Carbendazim was inhibitory to all fungal antagonists whereas Mancozeb and Phorate were least inhibitory at 200ppm. Radial growth of mycelium of Trichoderma spp was inhibited at Carbendazim @ 1ppm and Triphonate Methyl @ 10ppm (Malathi et al., 2002). Inhibitory action of Isofenphos – methyl on T. harzianum was strong and fungistatic rate was 64.37% under treatment with 200μg/ml (Ching et al., 2008). The fuingicide Iprodione and T. harzianum were not found to be compatible (Sumitra and Madhuban, 2006). The T. harzianum was least sensitive to Procymidone and Captan and most sensitive to Mancozeb, Tebuconazole and Thiram (Mclean et al., 2001). Inhibitory effect of Carbendazim and Thiophanate Methyl on T. harzianum while Captan and Thiram recorded least inhibitory effect on T. harzianum (Gowdar et al., 2006). Recently, the fungicide Bordeaux mixture 1% found highly inhibitory to Trichoderma as compared to Copper oxychloride and Mancozeb (Suseela and Joseph, 2010). Some strains of Trichoderma show compatibility with fungicides as they are tolerant of fungicides and successfully used in IPM strategy (Dutta and Chatterjee, 2004; Hetong et al., 2008). Trichoderma viride was not compatible with Dithane, Bavistin and Ridimil in any level of selected concentration (Tapwal et al., 2012)

 

Many workers in the discipline of plant pathology suggested that growth of plant pathogenic fungi were inhibited by the Trichoderma species because of some factors produced and these substances may be volatile and non volatile (Reusser, 1967).The growth inhibition of pathogenic fungi may be due to antibiotic secretion like trichodermin, trichoviridin, dermadin and sesquiterpene heptalic acid (Nakkeeran et al., 2002), nutrient impoverishment and pH alteration in the medium (Maheshwari et al., 2001). Such type of variability in antagonistic potential of Trichoderma species against plant pathogens has been also reported (Saha and Pan, 1996; Bell et al., 1982). Mathew and Gupta (1998) also reported that T. harzianum also exhibited maximum antagonistic activity causing 58% inhibition followed by T. viride (46%) and T. virens (45%). Isolates of T. viride, T. harzianum and T. virens were evaluated for their antagonism against Pythium aphanidermatium causing damping off of tomato and found that many isolates inhibited the growth of P. aphanidermatum (Kumar and Hooda, 2007).Trichoderma viride is economically important because of their mycoparasitic ability which makes them suitable for the application as biocontrol agent against soil borne plant pathogenic fungi (Manczinger et al., 2000). The effective in vitro screening test of T. viride was carried out against Rhizopus oryzae and Aspergillus flavus pathoges of post harvest cassava (Manihot esculents Crantz.), root rot and reported that T. viride was most promoting candidate for the biocontrol (Ubalua and Oti, 2007). Growth of Rhizoctonia solani, a pathogen involved in cotton seedling disease was inhibited by the strains of T. harzianum and T. longibracheatum (Arsan-Amal et al., 2005). Strains of T. koningii were used for their antagonistic nature against Rhizoctonia solani under in vitro condition and inhibited mycelial growth by producing toxic metabolites (Melo and Faull, 2000). In the dual culture experiment evaluated by Hajieghrari et al. (2008), T. virens and T. harzianum inhibited the growth of soil borne pathogenic fungi such as R. solani, M. phaseolina, Phytophthora cactorum and Fusarium graminearum forming inhibition zone without physical contact between them. In vitro antagonistic potential of T. viride against Alternaria alternata, Ulocladium botrytis, Cladosporium harbarum, Cephalosporium madurae, Penicillium chrysogonum,Fusarium oxysporum and Humicola grisa were tested and found that significant inhibition of radial growth of these fungi in dual culture experiment (Abou-Zeid et al., 2008).

 

Rajendiran et al. (2010) evaluated antagonistic effects of T. viride on post harvest pathogens of fruit and vegetables such as Aspergillus niger, A. flavus, A. fumigatus, Fusarium sp and Penicillium sp. Trichoderma viride inhibited the radial growth of A. niger (55%), A. flavus (51%), A. fumigates (52%), Fusarium sp (64%) and Penicillium sp in dual culture. The impact of isolates of T. viride, T. harzianum and T. virens on soil borne fungal pathogens such as R. solani, S. rolfsii and Sclerotinia sclerotiorum were evaluated and inhibitory effects were reported (Amin et al., 2010). Reports on antagonistic potential of T. harzianum over Fusarium oxysporum f. sp. vanilla the stem rot pathogen of vanilla was showed by Naik et al. (2010).The isolates were found fully overgrown on all corm rot pathogens of saffron (Hassan et al., 2011). Trichoderma viride was found to exhibit effective antagonistic potentiality against R. solani (Giagole et al., 2011).

 

Trichoderma species can be used together with compatible fungicides in the integrated disease management towards the control of crop plants and soil borne pathogens. It is possible to develop Trichoderma tolerant of chemical fungicides without lack of antagonistic activity. The antagonistic nature of Trichoderma species against pathogenic fungi were evaluated under in vitro condition. It was observed that growth of pathogenic fungi was reduced with respect to radial growth and sporulation. The mycelium of Trichoderma species when comes in contact with the test fungi it became fungistatic and the growth of test fungi were retarded. The antagonism was exhibited with respect to secretion of extra cellular enzymes, antibiotics and competition related food and space. Pathogenic fungi and Trichoderma species created competition and the latter found to be dominant over the pathogenic fungi. Mycoparasitic properties of Trichoderma species was found to be the main reason responsible for their antagonistic nature.

 

Authors are thankfully acknowledged to UGC, New Delhi for financial assistance of major research project.

 

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