Effect of media on growth and antagonistic activity of selected Trichoderma strains against Ganoderma

1 Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia. 2 Biotechnology Research Institute, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia. 3 Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia. 4 Laboratory of Marine Biotechnology, Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia.


INTRODUCTION
Trichoderma sp. as a biocontrol agent of plant diseases has been recognized since the early 1930s (Weindling, 1934).Weindling described the mycoparasitic action of Trichoderma on Rhizoctonia and Sclerotinia and its beneficial effects on plant disease control.Subsequently, many studies have shown that Trichoderma sp. is effective biocontrol agents for managing plant disease (Schubert et al., 2008;Howell, 2003).Trichoderma sp. has been proven to be highly effective for controlling Ganoderma boninense infection in oil palms (Izzati and Abdullah, 2008;Sundram et al., 2008;Sariah et al., 2005;Susanto et al., 2005;Abdullah et al., 2003).Kashem et al. (2011) also reported that Trichoderma spp.control the foot and foot rot of lentil (Lens culinaris Medik) caused by Fusarium oxysporum.Trichoderma can be found in nearly all agricultural soils.Most species grow rapidly, produce abundant conidia, and have a wide range of enzymes including cellulases.They have the hallmarks of ruderals species (Mathivanan et al., 2000).Trichoderma is able to grow in soils having a pH range of 2.5 to 9.5, although most prefer a slight to moderately acidic environment.The species that prefer the more acidic soils are usually regarded as having a more stresstolerant growth habit and are less aggressive.All species can produce colonies which have either white to yellow to green mature fruiting areas.Colonies can have floccose and elliptical conidia, or tufted non-floccose globulose conidia.
Different species of Trichoderma have shown antagonistic activity on other species of fungi via several processes.These include production of soluble antibiotics (peptides), volatile and non-volatile antibiotics, or by direct parasitism.This is achieved when they coil around the hyphae of other fungi in a process called mycoparasitsm limits the growth and activity of plant pathogenic fungi (Chet et.al., 1998).However, the biocontrol activity depends upon biomass production (Singh et al., 2007).Currently, there is a limited knowledge available with respect to biomass production of Trichoderma sp. on different media in in vitro studies.Hence, the present study was conducted to evaluate growth rate and antagonistic activity of T. harzianum (T1132) and T. virens (T128) on potato sucrose agar (PSA) and potato dextrose agar (PDA) media.The growth rate was evaluated based on spore counts and the antagonistic activity was determined by PIRG analysis (percentage inhibition radial growth).

Preparation of PDA and PSA media
Potato dextrose agar (PDA) media was prepared by adding 19 g of PDA to 500 ml distilled water and the mixture was homogenized in the microwave and autoclaved at 121°C for 15 min.The Potato sucrose agar (PSA) media was prepared by conventional method.Briefly, 300 g peeled potato was cut into small pieces and then boiled to produce a homogenate.The homogenate was then sieved using a cloth net.The potato broth was poured into 1000 ml of distilled water containing 21.3 g agar and 20 g sugar.The mixture was autoclaved at 121°C for 15 min.

Collection of Ganoderma and Trichoderma spp.
Ganoderma mycelium was isolated from the innermost contextual tissues of Ganoderma, also called fruiting body of Ganoderma.Pieces of tissues measuring 3 × 3 × 2 mm each were cut out from the contextual layer and surface sterilized in 3% NaOCl (Clorox) for 1 min and again shaken with sterilized distill water for 15 s to remove the Clorox.A single piece was picked with a sterilized scalpel and placed in the centre of a Potato Dextrose Agar (PDA) culture plate.The agar media had earlier been steam-sterilized at 121°C for 15 min, following treatment of media with sterilized 0.5 g streptomycin.The samples were seeded into the Petri dishes for 4 days to grow the mycelia.Next, Ganoderma was cultured on slides and the morphology with clam connection which is a characteristic of Ganoderma was observed under the light microscope.After ensuring the selection of Ganoderma, then, fresh mycelium was sub-culture in PSA and PDA media and mycelia growth was observed.Mycelia growth in PSA media observed to be faster than in the PDA media.The mycelia were again sub-cultured on PSA contained plate for the wood decay test with the isolated Ganoderma sp.T. harzianum (strain 1132) and T. virens (strain 128) were obtained from the slant stock culture of the Mycology Laboratory, Department of Biology, Universiti Putra Malaysia (UPM).

Wood decay test of isolated Ganoderma
Wood decay test of Ganoderma was performed using rubber wood blocks.Freshly cut rubber wood blocks (2.5 × 2.5 × 5 cm 3 ) were used to prepare Gano-wood block inocula as described in (Naher et al., 2011).Briefly, the blocks were soaked in distilled water for overnight.After overnight socking, the blocks were placed in autoclavable polypropylene plastic bags (10 × 32 cm) and autoclaved at 121°C for 45 min.After that, 100 ml of PSA medium was transferred into each block, and the blocks were again autoclaved at 121°C for 45 min.Subsequently, the sterilized rubber wood blocks were cooled in a laminar flow and turned every 2 min to ensure that they absorbed the PSA medium properly.Seven-day old Ganoderma sp.cultures were cut into small pieces and transferred into the plastic bags containing the sterilized blocks; 100 ml of PSA medium were added, and the blocks were incubated for 10 to 12 weeks at room temperature (Abdullah et al., 2001).The control blocks were prepared without Ganoderma inoculation.Similar weights of wood blocks were selected for control and Ganowood block preparation.The control and Gano-wood blocks were weighed every month by using an electric balance for three months to determine whether Ganoderma was consuming or decaying the tissue.

Growth and antagonistic activity of Trichoderma spp. strains cultured on different media
The growth of T. harzianum strain 1132 and T. virens strain 128 were determined by measuring spore counts of these two fungi using haemocytometer under the microscope.The following formula was used for calculating the production of Trichoderma spores: Where, 0.0025 mm 3 = total volume of haematocytometer, and 10 3 = spores in ml The antagonistic activity o of f T. harzianum strain 1132 and T. virens strain 128 against Ganoderma sp. was investigated in a dual culture experiment.A 6 mm diameter agar disc was taken from the edge of an actively growing pure culture of Ganoderma and placed 1 cm inside the edge of the two different Petri dishes containing PSA and PDA, respectively.The samples were allowed to grow for 3 days, by which time the colony had grown to ~2 cm in diameter.Next, a 6 mm diameter disc was taken from the T. harzianum 1132 or T. virens 128 culture and placed on the opposite side of the Petridish containing the Ganoderma sp.The control plates contained only Ganoderma.The experiment was run in triplicate.Trichoderma is a faster growing fungus, and hence three days later Trichoderma was placed on the Ganoderma culture in the Petri dishes.The attached time of T. virens 128 or T. harzianum 1132 with Ganoderma was approximately 76 to 78 h, in the PSA media and 83 to 86 h in the PDA media (Figure 2).The experiment was extending for another 2 days.At this time the percentage inhibition of radial growth (PIRG) of Ganoderma caused by T. harzianum 1132 and T. virens T128 respectively, was calculated as follows (Figure 2):

R1 -R2 PIRG = × 100 R1
Where, PIRG= percentage inhibition of radial growth, R1= the radial growth of the Ganoderma colony in the absence of the T. harzianum 1132 or T. virens T128, and R2= the radial growth of the Ganoderma colony in the presence of the T. harzianum 1132 or T. virens T128.

RESULTS AND DISCUSSION
Trichoderma sp. are generated well recognized biocontrol agents in the management of plant diseases (Vinale et al., 2008;Verma et al., 2007) and they have been previously shown to have a high efficacy for controlling Ganoderma (Abdullah et al., 2003).The present study was designed to evaluate the biocontrol activity of Trichoderma sp.cultured on two different medium against the wood decay fungus Ganoderma.A wood decay test was also carried out to determine the decaying ability of the isolated Ganoderma.The results showed that Ganoderma completely colonized the wood within three months with weight losses of around 6 gm, which was significant (p< 0.05) compared to the control blocks (Figure 1).The findings indicated that this was a strong wood decaying fungus.Hence this Ganoderma sp. was then used in subsequent experiments.
The growth rates of T. harzianum (strain 1132) and T. virens (128) as determined based on spore production showed that the growth rates of T. harzianum (1132) and T. virens (128) cultures were significantly different in the two media.The growth rates of T. harzianum (1132) on PDA and PSA media were 5.62 × 10 9 and 8.58 × 10 11 , respectively, while the growth rates of T. virens (128) on PDA and PSA media were 6.82 ×10 10 and 9.12 × 10 10 , respectively.A dynamic spore density was observed on PSA media for both tested fungi, but the growth rate was higher in T. virens strain 128 compared to T. harzianum strain 1132.The resulting data indicated that Trichoderma sp.growth was higher in PSA media for both tested fungi, which also showed that Trichoderma sp.cultivation was better with sucrose than dextrose or glucose augmentation.A previous study had however shown that growth of Trichoderma sp.growth was better in glucose media (Rodrigues et al., 2009).It was not clear that why this study data is not similar than other study, whereby it might be that the sucrose and potato were better carbon sources for growth of Trichoderma sp. in the PSA medium compared to glucose alone in the PDA medium.
Antagonistic activity based on the dual culture experiment showed that T. harzianum strongly inhibited the growth of Ganoderma (Figure 3).The PIRG rate of Ganoderma was found to be 59% with the T. harzianum strain 1132 on PDA and 65% on PSA, while with the T. virens strain T128 PIRG was 62% on PDA and 66% on PSA contained Petri dish (Figure 3).Perello et al. (2003) reported that Trichoderma sp.inhibited Drechslera triticirepentis colony growth with PIRGs of 50% and 74% in dual culture experiments.Sariah et al. (2005) had also described T. harzianum as a good biocontrol agent for controlling Ganoderma.However, the present study demonstrated the significant potential of PSA as an enhancing media for more effective control of Ganoderma.
In conclusion the results of this study data indicated better growth of T. virens and T. harzianum on PSA medium, which also resulted in higher inhibition of Ganoderma.Thus, PSA might therefore serve a beneficial carrier medium for culturing Trichoderma sp.used in the biocontrol of Ganoderma species and possibly other pathogens as well.

Figure 1 .Figure 2 .
Figure 1.Wood decay test of Ganoderma boninense (the test was carried out over three months).

Figure 3 .
Figure 3. PIRG analysis on Ganoderma using Trichoderma spp.cultured on PSA and PDA media.