Enhancement of Antagonism through Protoplast Fusion in Trichoderma spp

The filamentous fungi Trichoderma is used asmost important biocontrol agent. Because of the absence of sexual reproduction in this fungus, other methods of genetic improvement have been developed to enhance its bicontrol potential.In the present investigation protoplast fusion of four different Trichoderma spp. was evaluated for their antagonistic activity against the soil borne plant pathogen like Fusarium spp. which causes wilting diseases in many crops. In protoplast fusion technique, protoplast isolation by lysis enzyme at the concentration of 10 mg/ml, protoplasts were fused intra and inter specifically, with 30% (w/v) PEG solution and those fusants regenerated on osmatic stabilizer amended PDA media. Eight self fusants and eighteen fusants were selected for assessment of antagonastic activity against fusarium spp. in dual culture technique. The results revealed that among eight intra fusants, fusantTvdsf-2 (93.70%) was recorded highest per cent inhibition against F. o. f. sp. udumandTvssf-1 (86.70%) against the F. solani and inter specific fusants, the maximum per cent inhibition mycelia growth was observed in Tvs×Tas-1 (94.07%) and Tvd×Tas-1 (94.07%) against F. o. f. sp. udumand Tvd×Tas-1 (89.63%) against the F. solani. These outcomes indicated that protoplast fusion technique was successful tools to enhance the antagonistic effects of Trichoderma species against several fungal plant pathogens.

Soil borne pathogens have a broad host range and persist for longer periods in soil by resistant resting structures.Chemical control of soil borne pathogens provides certain degree of control but at the same time have adverse effects on environment affecting the beneficial soil microorganisms.Therefore, biological control of plant pathogens has been considered as a potential control strategy in recent years and search for these biological agents is increasing.Trichoderma is the most commonly used fungal biological control agent and have long been known as effective antagonists againstplant pathogenic fungi (Papavizas, 1985).
Soil borne diseases caused Fusarium spp.which was causes economical loss in many crops.Pesticide treatment is the mostusual method for controlling these diseases.However, these pesticides are expensive and areharmful on human health.Therefore, using biocontrol agents aremore effective, less expensive and safer for human health.Genus Trichoderma is one of the mostimportant filamentous fungi used as a biocontrolagent.Many species under this genus has been usedagainst diseases in a wide variety of economically important crops.Because ofthe absence of sexual reproduction, other methods of genetic improvement have beendeveloped in these fungi such as transformation andproptoplast fusion (Balasubramanian and Lalithakumari, 2008).
The present work aimed to apply protoplast fusion of four Trichoderma spp. to enhancement their bio-control abilities against some of important plant fungal pathogens (Fusarium spp.), which cause wilting diseases that attacking different crops.

MATERIAL AND METHODS
The mutation was carried out by using four Trichoderma species viz., Trichodermaharzianum, Trichodermavirens and Trichodermaviride isolates were collected from NBAIR (National Bureau of Agricultural Insects Resources) Bengaluru and Trichodermaasperellum from IIOR (Indian Institute of Oil Research) Hyderabad andFusariumoxysporum f. sp.udum and Fusariumsolani, the causal agent of wilt diseases in pigeon pea and acid lime were isolated from diseased plants.Those bio-agent and pathogenic fungi was sub cultured on PDA slants and allowed to grow at 28±1°C for ten days and such slants were preserved in a refrigerator.The procedure or protocol followed for the isolation of protoplast, fusion of protoplasts is given below.

Isolation of protoplasts from Trichoderma species
The parental strains of Trichoderma species like Trichoderma harzianum, Trichoderma virens, Trichoderma viride and Trichoderma asperellum were grown separately in 50 ml of PDB in 150 ml Erlenmeyer flasks by inoculating 1 ml conidial suspension of respective culture at H"51 0 6 conidia/ml.The flasks were incubated on a rotary shaker with a speed of 120 rpm at room temperature for 15 hours.Then cultures were harvested and the young mycelia were separated by filtration using Whitman No.1 filter paper.About 100 mg of fresh mycelium was washed with sterile distilled water followed by 0.1 M phosphate buffer (pH 5.8) and osmatic stabilizer (0.7 M KCl) then incubated with Lysing Enzymes (Sigma Chemicals Co.) at 10 mg/ml concentration prepared in phosphate buffer containing 0.7 M KCl as osmotic stabilizer.The mycelia-enzyme mixtures were incubated on a rotatory shaker with a speed of 75 rpm at room temperature and the lysis of fungal cell wall and the release of protoplasts were monitored at 30 min interval under a light microscope.After 4hr, the enzyme-protoplast mixture was filtered through a sterile cotton swab and centrifuged at 3000 rpm for 5 min at 4 0 C. The supernatant was discarded and the protoplast sediment was suspended immediately in buffer and osmotic stabilizer solution.These protoplasts were used for further fusion process.

Intra and inter-specific protoplast fusion
Intra and Inter-specific protoplast fusion was carried out by the method given by Prabavathy et al. (2006).Intra specific fusion was done within the species, such as, Trichoderma harzianum (Th×Th), T. virens, (Tvs×Tvs), T. viride (Tvd×Tvd) and T. asperellum (Tas×Tas).In case of inter specific fusion, on different combination of Trichoderma was fused with the another species of Trichoderma (Th×Tvs, Th×Tvd, Th×Tas, Tvs×Tvd, Tvs×Tas and Tvd×Tas).
Polyethylene glycol (PEG), (MW 6000, Himedia company) prepared in STC buffer contained 0.6 M Sorbitol, 10 mM Tris-HCl and 10 mM Calcium chloride (pH 7.5) was used as fusogen.One ml of protoplasts suspension (1×10 6 protoplasts/ml) was mixed with an equal volume of 30% PEG solution and the fusion mixture was incubated at room temperature.After 10 min, the mixture was diluted with 1 ml of STC buffer.The protoplast fusion was observed under microscope (Axiozeiss Company) and microphotographs were taken.
The fusion mixture was serially diluted with the osmotic stabilizer for regenerationand plated on regeneration medium (PDA amended with osmotic stabilizer) and controls were maintained separately for non-fusions.Observations were made every day on regeneration of fusion protoplasts on agar media.After regeneration of protoplasts on media, all the growing fusants were selected based on the fast growth comparing with the parent isolates as check and grown separately on the PDA plates, Selected regenerated intra and inter specific fused protoplasts were enlisted as below.
The antagonistic effects of each intra and inter specific protoplast fusions of different Trichoderm spp.and their parental Trichoderma spp.isolates were evaluated against Fusarium oxysporum f. sp.udum and Fusarium solani were evaluated in dual culture plate technique.The percent inhibition of the growth of the pathogen was calculated by using the formula suggested by Vincent (1947) Percent inhibition in mycelial growth/conidial germination = C -T × 100 C Where, C = Mycelial growth/conidial germination in control T = Mycelial growth/conidial germination in treatment

RESULTS AND DISCUSSION
Protoplast fusion has rapidly become acceptable tools for the fungal genetics with the development of new techniques of gene transfer such as protoplast fusion and enhancement of antagonistic ability of Trichoderma spp.byprotoplast fusion.These experiments included protoplast isolation, regeneration andintra and inter specific fusions.In addition, evaluation of obtained fusants by dual culture experiment.

Protoplast isolation fusion and regeneration
Isolation of protoplast, first completely lysed the young mycelium and release of protoplasts were observed after 3 hour (Fig. 1 to 4).Initially released protoplasts were smaller in size, but later enlarged and formed spherical structures.The protoplasts yield significantly affected by the concentrations of lysing enzymes and specific conditions including mycelium age, temperature, medium, etc..The conditions for releasing the protoplasts were similar as reported by EL-Bondkly (2006).
The lysis of fungal mycelium was confined only to the tip portion, 10 mg/ml concentrations of lysing enzymes with 0.7 M KCl as osmotic stabilizer was optimal for the release of protoplasts.Peåer and Chet (1990) obtained highest protoplasts yield from T. harzianum using Novozyme 234 at 10 mg/ml with 0.6 M KCl.The isolated protoplasts were subjected to intra and inter specific protoplast fusion with the use of 30% PEG 6000.The protoplasts were attracted to each other's and pairs of protoplasts were observed when PEG solution was added to the protoplasts.Later, the plasma membrane at the place of contact dissolved and protoplasmic contents fused together that may half union and fully fusion (fig 9 and 10), then followed by nuclear fusion in most cases.
The fused protoplasts became single, larger and round or oval-shaped structure representing in fig 10.As similar concentration of PEG was reported as optimum for inter-specific fusion of protoplasts between T. harzianum and T.      Finally regeneration the fused protoplasts on PDA medium supplemented with osmatic stabilizer was done.Out of number of regenerated colony forming units on PDA media, based on fast growth, eight intra specific and 18 inter specific protoplast fusions were selected.They were subjected to their antagonistic activity evaluation along with the parent isolates against the soil borne pathogens like F. oxysporum f. sp.udum and F. solani.

Table 1 .
In vitro evaluation of intra specific fusions against Fusarium oxysporum f. sp.udum *Arc sine values

Table 2 .
In vitro evaluation of intra specific fusions against Fusarium solani *Arc sine values

Table 3 .
In vitro evaluation of inter specific fusions against F. o. f. sp.udum *Arc sine values

Table 4 .
In vitro evaluation of inter specific fusions against Fusarium solani *Arc sine values