Bioefficacy of Trichoderma isolates against soil-borne pathogens

The study of morphology and bioefficacy of Trichoderma was undertaken to select the effective isolates against soil-borne pathogens. Fifty one (51) isolates (23 isolates of Trichoderma virens and 28 isolates of Trichoderma harzianum) were morphologically characterised based on the growth characteristics on PDA medium, the size and shape of phialides and conidia.These isolates were screened for bioefficacy against soil borne plant pathogens (Fusarium oxysporum, Rhizoctonia solani and Sclerotium rolfsii) based on their percent inhibition observed during dual culture, volatile and non-volatile methods. Eight T. virens isolates and 12 T. harzianum isolates were proven to be potential isolates against the soil-borne pathogens tested. No correlation was found between bioefficacy and morphology in both species isolates.


INTRODUCTION
Trichoderma spp. is cosmopolitan and abundant fungi in soil in a wide range of ecosystems and climatic zones.They are characterized by rapid growth, capability of utilizing diverse substrates and resistance to noxious chemicals (Klein and Eveleigh, 1998).Their economic importance includes their role as primary decomposers, producers of antibiotics and enzymes as well as biocontrol agents against a wide range of plant pathogens (Hjeljord and Tronsmo, 1998;Kubicek and Penttila, 1998;Rossman, 1996;Sivasithamparam and Gisalberti, 1998).Trichoderma spp.may inhibit the phytopathogenic fungi either by inducing resistance and plant defence reactions or by direct confrontation through mycoparasitism and antibiosis as well as competition (Howell, 1998(Howell, , 2003;;Papavizas, 1985;Verma et al., 2007).
In the direct interaction between Trichoderma spp.and the phytopathogenic fungi, mycoparasitism is one of the mechanisms observed in which the antagonist coils around the hyphae of the pathogen, develops hook-like structures known as appressoria coupled with production of lytic enzymes and then penetrates the pathogen hyphae (Chet, 1987;Kubicek et al., 2001).Coiling of the phytopathogenic fungal hyphae by Trichoderma spp. is one of the parameters used to characterize the mycoparasitism (Howell, 2003;Rocha-Ramirez et al., 2002).Trichoderma spp.produces a plethora of secondary metabolites showing anti microbial activity (Vinale et al., 2008).The chemical composition of secondary metabolites depends on the strains and classified as volatile (water-soluble) or non-volatile (water-insoluble) compounds (Ghisalberti and Sivasithamparam, 1991).
The knowledge of mechanisms of interaction of Trichoderma spp. with phytopathogenic fungi and plant host is of utmost importance to enhance the practical application of these beneficial microorganisms.Trichoderma spp. is among the microorganisms most frequently used as antagonists against soil-borne pathogens (Hjeljord andTronsmo, 1998 andHyakumachi et al., 1996).Soil-borne phytopathogens are known worldwide for causing root diseases in diverse cultures (Ogoshi, 1996).
Taxonomy of Trichoderma is currently based largely on morphological characters such as mycelia growth, phialides shape and size and conidial shape and size.However, most species descriptions are based on examination of a limited number of strains where the morphological differences are clear but these differences become less clear as more strains are studied.This result suggests that there are no enough morphological and cultural characters to reliably define species level (Samuels et al., 2013).
The isolates were identified using morphological characters.In vitro bioefficacy tests (dual culture, volatile and non-volatile methods) were performed, against soilborne pathogens to understand the ability of these isolates to produce water-soluble metabolites or volatile inhibitors.This approach is useful in selecting some potential isolates of Trichoderma spp.against soil-borne pathogens.

Morphological characterisation of Trichoderma isolates
The cultural characteristics of 51 isolates of Trichoderma spp.were studied in potato dextrose agar (PDA).The identification was performed using an interactive key for strain identification (Rifai, 1969;Domsch et al., 1980;Bissett, 1991 a, b;Samuels et al., 2013) based on the growth characters on PDA along with microscopic observations of the isolates.Conidiophores branching and apex of the conidiophore disposition, shape and size of the phialides and conidia size and shape were recorded.The photographs were taken under 100x magnification (phialides size and shape and conidial size and shape) and under 10x (conidiophore branching) magnification were measured in micrometer by using ImageJ software.

Soil-borne pathogens
Soil-borne plant pathogens (Fusarium oxysporum, Rhizoctonia solani and Sclerotium rolfsii) were obtained from Indian Type Culture Collection, Division of Plant Pathology, Indian Agricultural Research Institute (IARI) and identified based on the morphological characters (Rangaswami, 1958) and maintained in the PDA slants by repeated subculturing throughout the study.

Trichoderma isolates
Bioefficacy of Trichoderma isolates against three soil-borne plant pathogens, viz., F. oxysporum, R. solani, and S. rolfsii were evaluated using dual culture technique and production of volatile and non-volatile antibiotics (Dennis and Webster, 1971a, b).

Dual culture method
Trichoderma isolates were tested for their potential to antagonize in vitro against three soil-borne pathogens (F.oxysporum, R. solani, and S. rolfsii) using dual culture method.The test fungus and Trichoderma isolates were grown on PDA at 28±2°C for a week.A disc of 5 mm of the target fungus cut from periphery of the mycelium was transferred to Petri plate with PDA.Trichoderma was transferred aseptically to the same plate.Each plate received two discs, one of Trichoderma mycelium and other of the test pathogen, placed 7 cm away from each other.The plates were incubated at 28±2 0 C and observed after eight days for growth of antagonist and test fungus, index of antagonism as percent growth inhibition of test pathogens was calculated (Morton and Stroube, 1955) (Figures 3  and 4).

Volatile method
The volatile test was carried out to observe the production of volatile inhibitors by Trichoderma isolates.The upper lid of PDA plates was inoculated with agar 5 mm disc of Trichoderma isolates and the lower lid was inoculated with soil-borne pathogens simultaneously.The two lids were taped together with adhesive tape (Dennis and Webster, 1971b) and incubated at 28±2°C for eight days.The growth of soil-borne pathogens was recorded after 72 h.In the control, soil-borne pathogens were cultured in the same way but without Trichoderma isolates in the bottom plate (Dennis and Webster, 1971a) (Figures 3 and 4).

Non-volatile method
The non-volatile test was carried out to find the production of water soluble inhibitors by the Trichoderma isolates against soil-borne pathogens (Dennis and Webster, 1971b).The isolates of Trichoderma culture filtrate concentration 7.5 and 15% (v/v) was inoculated in 100 ml sterile potato dextrose broth in 250 ml conical flasks.Inoculated flasks were incubated at 28±2°C for 15 days.The culture was filtered through Whatman No.42 filter papers and filtrate was collected in a sterile flask.The culture filtrate was added to molten PDA medium to obtain a final concentration of 10% (v/v).The medium was poured into the Petri plates at 15 ml/plate and 5 mm discs of pathogens were inoculated after solidification.Control plates were maintained without amending the culture filtrate.Petri plates were sealed with parafilm tape and incubated at 28±2°C for 8 days.Radial growth of soil-borne pathogens was recorded (Figures 3 and 4) and percent inhibition was calculated as per formulae adopted by Garcia (1991) as: where R1 is the farthest radial distance grown by the pathogen in the direction of the antagonist (control) while R2 represents the distance grown on a line between inoculation positions of the pathogen and the antagonist.

Morphological identification of Trichoderma isolates
Morphological characters such as growth characteristics, phialides disposition, shape and size and conidial shape and size of Trichoderma isolates were studied through microscopy and 23 isolates were confirmed as T. virens (dark coloured, conidiation effuse, covering the entire plate to green flat pustules concentrated near the margin and 28 isolates T. harzianum (pea coloured loosely aggregated flat pustules spread throughout the plate was observed in most of the isolates.Colourless to dark brown colour was observed at the reverse side of the plate) based on key given by Giddens et al. (1958), Rifai (1969) and Bissett (1984Bissett ( , 1991a, b) , b) (Figures 1 and 2) Concerning T. virens, the highest cultural growth was observed in the isolate V-19 (82.50 mm) and lowest in the isolateV-8 (47.50 mm).The length, width at the middle and width at the base of phialides in isolates of T. virens studied were from 5.80 (V-10) to 11.21(V-22) µm, 1.40 (V-20) to 3.07 (V-19) µm and 0.99 (V-20) to 2.38 (V-19) µm, respectively.Phialides shape was ampulliform in all T. virens isolates studied.Length and width of conidia of isolates were significantly varied ranging from 5.00 (V-1) to 6.59 (V-6) µm and 4.04 (V-1) to 5.26 (V-6) µm, respectively.The length/width (L/W) ratio of conidia ranged from 1.08 (V-22) to 1.31 (V-7) µm.Conidial shape of was obvoid to broadly ellipsoidal in all the T. virens isolates (Table 1).

Effect of Trichoderma virens on radial growth of the soil-pathogens
Dual culture method: To select the effective bio-agents of Trichoderma isolates against soil-borne plant pathogens viz., F. oxysporum, R. solani and S. rolfsii (Figure 4), dual culture technique was used.All isolates of T. virens inhibited the mycelial growth of the soil-borne plant pathogen significantly over control.Among 23 isolates of T. virens, isolate V-9 inhibited the growth of F. oxysporum up to 82.31% which was significantly superior over all other isolates, while the isolates V-12 (53.88%) and V-5 (54.85%) showed the lowest inhibition.Isolate V-21 showed the highest percent inhibition of R. solani growth (81.76%) as compared to other isolates studied, while the isolate V-4 (42.93%) and V-18 (50.03%) showed the lowest inhibition.Isolate V-8 showed the highest percent inhibition of S. rolfsii growth (87.39%) which was significantly superior to all other isolates, while the isolates V-12 (50.23%) and V-6 (51.47%) showed the lowest inhibition (Figure 3 and Table 3).
Results (Tables 5 and 6) of mycelial growth percent inhibition for screening of Trichoderma isolates against soil-borne pathogens revealed that there was a clear difference within the isolates of T. virens and T. harzianum with respect to their percent inhibition against the pathogens tested in different methods used.It appeared that large numbers of T. virens and T. harzianum isolates fell under the category of Group 2 and very few numbers fell under category of Group 1. From the grouping, eight high potential (V-7, V-8, V-9, V-17, V-19, V-21, V-22 and V-23) and two low potential (V-4 and V-18) isolates from T. virens and twelve high potential (H-2, H-3, H-7, H-9, H-10, H-11, H-12, H-16, H-18, H-21, H-26 and H-28) The few morphological characters available are variable to some degree with respect to variable climatic and geographic locations, leading to overlap among species.
In the present finding, eight high potential and two low potential isolates from T. virens and 12 high potential and 2 low potential isolates from T. harzianum were selected as a promising isolates.The high and low potential isolates showed highest and lowest percent inhibition in the three methods used and against the three soil-borne pathogens tested.The possible explanation of this result may be due to their inherent potentiality to adapt well in introduced conditions (Papavizas, 1985;Bae and Knudsen, 2005), though it rarely occurs (Whipps, 2001).Higher growth rate ability of the selected strains are indicative of their better antagonistic potential.Mathur and Sarbhoy (1978) reported that T. viride and T. harzianum inhibited the growth of S. rolfsii by 88 and 86%, respectively.Mathew and Gupta (1998) showed that T. harzianum exhibited maximum antagonistic activity causing 58.3% inhibition of F. oxysporum.f. sp.lycopersici, R. solani and S. rolfsii followed by T. hamatum, T. viride and T. virens inhibition by 48.3, 46.1 and 44.9%, respectively.Recently, Noveriza and Quimio (2004) reported that Trichoderma spp.were able to cause 66.36% growth inhibition of F. oxysporum.f. sp.lycopersici, R. solani and S. rolfsii through dual culture technique and were also significantly inhibited by Trichoderma spp. in vitro (Lozoya-  et al., 2006;Choudhary et al., 2007;Kumar and Hooda, 2007;Pan andBhagat, 2007, 2008).

Conclusion
The objective of the present study was to inves-tigate morphology and identification of Trichoderma spp.before conducting bioefficacy test in vitro as well as in vivo.Bioefficacy helps to select some promising isolates of Trichoderma species against soil-borne plant pathogens.Fifty one Trichoderma isolates obtained from Indian Type Culture Collection were morphologically characterised and identified as T. virens (23 isolates) and T. harzianum (28 isolates) on the basis of the literature reported.These isolates were tested for their bioefficacy using 3 methods (dual culture, volatile and non-volatile) against soil-borne pathogens viz., F. oxysporum, R. solani and S. rolfsii.Out of 51 isolates, 8 isolates of T. virens and 12 isolates of T. harzianum were proved as potential biocontrol agents.

Figure 3 .
Figure 3. List of high and low potential isolates of Trichoderma virens selected from the bioefficacy methods.

Figure 4 .
Figure 4. List of high and low potential isolates of Trichoderma harzianum selected from the bioefficacy methods.

Table 1 .
Morphological characters used for the identification of T. virens isolates.

Table 2 .
Morphological characters used for the identification of T. harzianum isolates.

Table 6 .
Grouping of Trichoderma harzianum isolates based on percent inhibition against soil-borne pathogens.