83
Pak. J. Phytopathol., Vol. 21(1): 83-88, 2009.
USEFULNESS OF DIFFERENT CULTURE MEDIA FOR IN-VITRO EVALUATION OF TRICHODERMA SPP. AGAINST SEED-BORNE FUNGI OF ECONOMIC IMPORTANCE
Azher Mustafa, M. Aslam Khan, M. Inam-ul-Haq, *M. Aslam Pervez and Ummad-ud-Din Umar
Department of Plant Pathology University of Agriculture Faisalabad
*Institute of Horticulture sciences University of Agriculture Faisalabad
ABSTRACT
Mycelial growth, conidal production and biomass yield of three different Trichoderma species (T. harzianum, T. viride, T. longibrachiatum) were examined on five different culture media includings Potato Dextrose Agar, Waksman agar, Agar-agar, Czepak’s agar and Corn Meal agar. The medium had a significant effect on growth rate and population of the three Trichoderma species. Potato Dextrose Agar was the best medium in terms of growth spore production and biomass yield. Trichoderma harzianum outclassed the three in terms of mycelial growth biomass yield and spore production. In-vitro, species of Trichoderma strongly antagonised six different seed borne pathogenic fungi viz. Fusarium moniliforme, Fusarium oxysporum, Rhizoctonia solani, Fusarium solani, Botryodiplodia theobromae and Alternaria alternata in dual culture assay. Trichoderma harzianum gave maximum inhibition of mycelia growth of all pathogenic fungi.
Key words: culture media, Trichoderma sp. seed-borne fungi.
INTRODUCTION
Biological control involves the use of one or more bioloigical organisms to control pathogens or diseases. The microbial inoculants as biocontrol agents are effective and attractive alternatives to prevent the deficiencies brought about by the exclusive reliance on chemicals (Nakkeeran et al., 2002).
The members of genus Trichoderma are free-living fungi that are common in soil and root ecosystems. They are opportunistic, avirulent plant symbionts, as well as being parasites of other 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, quickly growing and easy to culture and they can produce large amount of conidia with long shelflife. 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, Pythium ultimum and Sclerotinia sclerotium (Manczinger et al., 2002). These pathogenic fungi when associated with seeds of forest trees behave in different way and effect seed germination, seedling mortality, seedling growth (Mustafa et al., 2004).
Studies were conducted for the use of various culture media for isolation and growth of Trichoderma spp. (Elad et al. 1981 and Harman et al. 1990; 1991).Use of agar media has a useful
function in a preliminary selection of individual antagonists for subsequent tesing in pathogen management. Work was done to compare the efficacy of different culture media and to describe their strength and weakness in terms of fungal biomass yield and conidia production and investigated the antagonistic potential of different Trichoderma species against pathogenic fungi in- vitro.
MATERIALS AND METHODS
Isolation of Trichoderma species from soil:
Rhizosphere soil of healthy tree stand was collected from Forest Nursery Experimental Area, University of Agriculture Faisalabad and isolation was made through Dilution Plate Technique (Akhtar, 1966).
An aliquot(2ml) of 10-4 ml dilution was spread on sterilized PDA Petriplates added with chloremphenicol at 1g per liter after autoclaving the medium and incubated at 25 0C under 12 hours of light and darkness. The growing mycoflora was observed under compound microscope, identified with available literature Rifai and Webster (1969), Barnett (1998) and Bissett (1991a,b,c). The identified Trichoderma viride, Trichoderma harzianum and Trichoderma longibrachiatum spp.
were purified by hyphal tip culture technique and preserved in refrigerator at 5 C for further use (Tuti 1969).
84
Study of mycelial growth of fungi on different culture media: The mycelial growth of the isolates of three Trichoderma species was studied on five different media in one liter viz. Waksman agar medium (W.A.M.) (Agar (25gm),Glucose (10gm), Peptone (5gm), Monopotassium phosphate (1gm), Magnesiumsulphate (0.5gm); Potato dextrose agar medium (P.D.A.):Potato starch (20gm), Dextrose (20gm), Agar (20gm);Water agar medium (W.A.), Agar (20 gm); Cornmeal agar (C.M.A.),Cornmeal (20gm), Agar (5gm); Czepak’s agar (CZA), NaNO2
(2g), KH2PO4 (1g), MgSO4 (0.5gm), Glucose (20gm), Agar (15 gm). A 5mm block of each isolate was placed at the centre of five replicated petriplates containing different culture media and incubated at 25 oC with factorial arrangements. The mycelial growth was measured after five days of inoculation.
Average linear growth rate (ALGR) was calculated by the formula (Aneja, 1993 and Elad et al., 1981).
ALGR (mm/day) = (C5-C1)/4 Where
C5= Colony diameter in mm after Five days.
C1= colony diameter after one day of inoculation
Biomass production and yield of conidia of Trichoderma species: For biomass production Erlenmeyer flask (250ml) containing 100 ml of each medium was inoculated with three mycelial plugs (1cm in diameter) of the fungus taken from seven days old cultures on PDA. The flasks were plugged in aseptic conditions and placed in incubator at 30oC for 28 days. The culture was harvested finally from each replicate. The fungal biomass yield was assessed by collecting fungal biomass on pre- weighed filter paper. The dry weight was determined after 24 hours of oven drying at 60oC. Number of conidia per mg of the biomass were determined by dilution method with the aid of haemocytometer.
Antagonistic effect in terms of inhibition of mycelial growth of pathogenic fungi: In-vitro screening was done to obtain the suitable antagonist.
Different antagonists (T.viride, T.harzianum, T.longibrachiatum) were tested against six different pathogenic fungi associated with seeds of Dalbergia sissoo Roxb. following dual culture method (Dennis and Webster, 1971). Each petriplate/medium was inoculated with 5 mm diameter inoculums disc of antagonist positioned diametrically opposite to a 5 mm disc of the test pathogen. The inoculums discs were obtained from the margin of actively growing nine days old culture of antagonist and pathogenic fungi. The distance between discs was 6cm. The plates were then incubated at 25°C. The experiment was run in quadriplicate with CRD arrangements.
Pathogen growth was recorded every day and measurement taken on the day before contact or after 28 days if no contact occured between the colonies. Percentage inhibition of growth was calculated following the formula suggested by Sunder et al., (1995).
Percent inhibition= Y-Z X 100 Y
Where Y = Mycelial growth of pathogen alone (control)
Z = Mycelial growth of pathogen along with antagonist.
Statistical Analysis The data were analyzed subjected to analysis of variance (ANOVA) and the Duncan’s Multiple Range test at 5% level of probability was used to test the differences among mean values (Steel and Torrie, 1980).
.
RESULTS
Effect of different culture media on mycelial growth, conidial production and biomass yield of antagonistic fungi: For isolation, growth and physiological studies of antagonist, five different culture media were used. All the culture media used showed significant effect at (P=0.05) on mycelial growth, sporulation and biomass production of the Trichoderma species. The best medium was Potato Dextrose Agar (PDA) supporting maximum growth rate (45.43 mm) and minimum growth rate was observed on Corn Meal Agar medium (9 mm) for all the Biocntrol agents. Trichoderma viride showed best growth rate utilizing the all culture medium followed by Trichoderma longbrachiatum and T.
harziznum. Significant interaction showed that maximum growth rate was observed in case of T.
viride on PDA medium and minimum growth was in case of T. longibrachiatum and T. harzianum on Water agar medium (Table-1).
Similarly for spore production, maximum number of spores per 100 ml of culture media was produced by all the Trichoderma spp. on PDA medium and minimum spore production was observed on Corn Meal agar medium. The spore counts was maximum on PDA (18.05), Waksman agar medium (14.76), followed by CZA (12.23), water agar (10.73) and least number of spores obtained on Corn Meal Agar (5.64) for all the Trichoderma species.
Maximum spore production capacity was in case of T. virde utilizing all culture medium and other two antagonists T. harzianum and T. longibrachiatum responded in similar fashion in spore production (Table-2).
85
Table 1. Effect of different culture medium on mycelial growth of Antagonistic fungi Substrates
(Culture Media)
Mycelial growth mm/day
T. harzianum T. longibrachiatum T. viride Mean
PDA 41.30±0.86 c* 45.87±1.65 b 49.16±0.89a 45.43±2.18 A
CMA 10.88±0.63h 6.31±0.46 i 12.19±0.90 h 9.79±0.84D
WAM 24.62±0.74f 31.80±0.66 e 20.95±0.57 g 25.79±1.40C
WAGAR 5.43±0.12 i 6.26±1.70 i 9.94±0.23 h 7.21±0.78E
CZA 32.27±0.73 e 36.63±0.00 d 40.60±2.07 c 39.17±1.73B
Mean 23.70±3.31 C 28.18±4.48 B 29.97±4.66 A
DMR (P=0.05) BCA = 1.26 Media = 1.36 BCA x Media = 2.837
Table 2. Effect of different culture medium on Spore Production of Antagonistic fungi Substrates
(Culture Media)
Log No. of spores/100 ml of culture medium
T. harzianum T. longibrachiatum T. viride Mean
PDA 18.10±0.70 16.80±0.12 19.23±0.05 18.05±0.36A
CMA 4.94±0.17 5.34±0.65 6.62±0.61 5.64±0.35D
WAM 16.68±0.13 12.46±0.10 15.13±1.75 14.76±0.74B
WAGAR 10.39±1.07 10.49±0.54 11.28±1.70 10.73±0.64C
CZA 11.96±1.69 11.58±0.72 13.15±1.27 12.23±0.70C
Means 12.42±1.14 AB 11.34±0.86 B 13.09±1.08 A
DMR (P=0.05) BCA = 1.22 Media = 1.58
Table 3. Effect of different culture medium on biomass yield of Antagonistic fungi Substrates
(Culture Media)
Biomass yield mg/100 ml of culture medium
T. harzianum T. longibrachiatum T. viride Mean
PDA 4.13±0.13 4.03±0.16 4.17±0.12 4.11±0.07A
CMA 2.75±0.23 2.53±0.14 2.94±0.21 2.74±0.11C
WAM 3.64±0.25 4.13±0.49 3.68±0.29 3.81±0.19AB
WAGAR 3.44±0.19 3.68±0.24 3.09±0.22 3.40±0.13B
CZA 3.40±0.46 3.05±0.58 3.48±0.39 3.31±0.25B
Mean 3.47±0.15 A 3.48±0.20 A 3.47±0.14 A*
DMR (P=0.05) Media = 0.50
*Means followed by the same letter within a column are not significant different at (P>0.05) byDMRT
Fig-1: Trichoderma viride (A) and Trichoderma harzianum (B) on PDA medium
A B
86
Table 4:- Percent inhibition of mycelial growth of pathogenic fungi by different BCA in vitro
Treatments
% inhibition of mycelial growth Fuarium
oxysporum
Fusarium moniliforme
Rhizoctonia solani
Fusarium solani
Botrydiplodi a
theobronae
Alternaria alternate
Mean
T. viride 26.71±1.35 ij 34.77±1.32b 18.23±0.67 cd 29.18±3.65 hi 33.33±4.72hi 17.63±0.57kl 26.64±1.79C
T. harzianum 20.40±1.10 jk 26.81±0.93h 12.09±1.12kl 16.16±1.26 kl 19.28±3.21kl 28.92±0.70hi 20.61±1.33D
T. longibrachi atum
50.04±1.32fg 35.72±1.64ij 17.35±1.42 l 26.62±3.15 ij 45.83±4.69g 50.75±1.48fg 37.72±2.75B
Control 90.00±1.65 b 67.94±1.04h 56.20±0.39kl 62.29±1.76 de 90.00±3.57a 70.43±1.22c 71.97±8.29A
Mean 47.55±5.35 A 41.31±5.98 B 25.97±4.71 D 33.57±4.56C 47.11±4.10A 41.94 ±5.33 B
DMR (P = 0.05)
BCA = 2.748 Fungi = 3.365 BCA x Fungi = 6.71
Data are means of four replication. *Means followed by the same letter within a column are not significantly different at (P>0.05)by DMRT. Mean±S.E.
For biomass production, individual effect of BCA and media used were significant. Two way interactions of BCA and media were non-significant.
All the antagonists utilized the culture media efficiently and biomass yield was maximum on PDA and minimum on Corn Meal Agar. However, potential for production of biomass yield among the antagonist did not differ significantly. The performance of culture media revealed that biomass production was maximum mg/100ml of Potato Dextrose Agar (4.11) followed by Waksman agar (8.81), Water agar (3.40), Czepak’s Agar (3.31) and Corn Meal Agar (2.74) (Table-3).
Inhibition of mycelial growth of seed borne pathogenic fungi of Dalbergia sisooo (Roxb.):In dual culture plate assay the data recorded on percent decrease of radial mycelial growth of different seed borne fungi is presented Table-4. The results revealed the significant effect on the radial mycelial growth inhibition of all pathogenic fungi at (p=0.05) by Trichoderma species as compared to control.
Among the three antagonists used T. harzianum was the most effective in inhibiting the mycelial growth of pathogenic fungi followed by T. viride and T.
longibrachiatum. Among the different pathogenic fungi, maximum percent inhibition of mycelial
growth on average was observed in case of Botryodiplodia theobromae (47.11) and Fusarium oxysporum (47.55%) followed by Fusarium moniliforme (41.31%) and A. alternate (41.94%) and least inhibition of growth was observed in case of Rhizoctonia solani (25.97%). Significant interaction showed that Trichoderma harzianum inhibits the maximum growth of Rhizoctonia solani among all the pathogenic fungi.
DISCUSSION
For isolation of antagonist soil should be collected whenever possible from the rhizosphere of the host to be protected rather than from the soil mass (Baker et al 1979). Species of Trichoderma are ubiquitous and are relatively easy to isolate. The medium used in this study had significant effect on the growth rate, conidial production and Biomass yield of the Trichoderma spp. For isolation and growth studies antagonistic fungi and as for various laboratory tests selection of media is an important and critical factor.
The choice of medium is essential and important stage in the development of successful laboratory experiment pilot scale development and eventual manufacturing process. The candidates’ fungi
87
Trichoderma species was first screened and evaluated in terms of growth rate on different culture media and for antagonistic ability. Media used was commonly available, relatively cheaper and supporting both mycelial growth and spore production of the entire antagonist and because of wide spread adoption in studies of this kind. Potato Dextrose Agar (PDA) medium was found best for all types of growth of Trichoderma species. The difference in growth and population was observed of three Trichoderma species. This was due to inherent ability of the Trichoderma spp. to utilize the different culture media and difference in their composition and to their natural ecological behaviour. Wang et al. (1999) described the fast growth rate of Trichoderma species. The growth and morphological features of Trichoderma species was studied on different culture media Corn Meal agar, Oat Meal Agar, Potato Dextrose Agar,Czapek’s Dox agar and Special Nutrient Media as reported (Elad et al., 1981; Shalini et al. 2006; Harman et al. 1991).
These results have implications for the use of such in-vitro test as a part of general screen for biological efficiency of different biocontrol agents.
In biocontrol living micro-organisms are employed as antagonist parasites or predators. In the present investigation inhibition of mycelial growth seed borne fungal pathogens by Trichoderma species clearly showed their antagonistic property in wide variation in growth inhibition of six pathogenic fungi. Maximum growth inhibition was done by Trichoderma harzianum. Different workers reported the antagonistic activity of different Trichoderma isolates against different phtyopathogenic fungi such as R. solani, F. oxysporum and Sclerotium rolfsii (Deshmukh and Raut, 1992; Xu et al., 1993; Askew and Laing, 1994). Different mechanism are said to be involved i.e. competition, production of antibiotics inhibiting fungal growth by producing volatile and non- volatile compounds as reported by (Michrina et al., 1995; Calistru, 1997).
Mycoparsitism of fungal hyphae, endoconidia and chlamydospores and finally lyses of protoplasm was observed in case of Trichoderma harzianum and T.
viride against pathogenic fungi (Ramanujam et al., 2002). These results indicate that these Trichoderma species are potential biocontrol agents and can be used against seed borne pathogenic fung.
REFERENCES
Akhtar, C. M. 1966. The isolation of soil fungi-I, A simple method of isolating fungi from soil. The needle Method. W. Pak. J. Agri. Res., 4: 122- 131.
Aneja, K. R. 1993. Measurement of growth of microorganisms and factor influencing growth.
Experiments of Microbiology, Plant Pathology and Tissue Culture. Department of Botany, Kurukshetra University, Kurukshetra. PP:168- 169.
Askew, D. J. and M. D. Laing. 1994. The in-vitro screening of 118 Trichoderma isolates for antagonism to Rhizoctonia solani and an evaluation of different environmental sites of Trichoderma as sources of aggressive strains.
Plant and Soil 159(2): 227-281.
Baker, K. F., R. J. Cook and S. D. Garret, 1979.
Selecting soil as a source of antagonist.
“Biological Control of Plant Pathogens”. Pub.
S. Chand & Company Ltd. Ram Nagar, New Delhi-110002. pp: 108-110.
Barnett, H. L. and B. B. Hunter, 1998. Illustrated genera of imperfect fungi 4th Edition. St. Paul, MN, APS Press. pp:223.
Issett, J., 1991a. A revision of genus Trichoderma,II.Infrageneric classification.
Canadian J. Bot., 69:2357-2372.
Bissett, J. 1991b. A revision of genus Trichoderma,III.Section Pachybasium.
Canadian J. Bot., 69:2372-2417.
Bissett, J., 1991c. A revision of genus Trichoderma,IV.Section Longibrachiatum Sec.
Nov. Canadian J. Bot.,69:924-31.
Calistru,C., M. Mclean and P.Berjak. 1997. In-vitro studies on the potential for biological control of Aspergillus flavus and Fusarium moniliforme by Trichoderma species; A study of the production of extracelluar metabolites by Trichoderma species. Mycopathologia, 137(20:115-124.
Elad, Y., I. Chet and Y. Henis. 1981. A selective medium for improving quantitative isolation of Trichoderma spp. from soil. Phytoparasitica, 9(1): 59-67.
Das, B. C., S. K. Roy and L. C. Boro. 1997. Mass multiplication of Trichoderma species on different media. Ind. J. Agri. Sci., 10(1):95- 100.
Dennis, C. and J. Webster, 1971. Antagonistic properties of species groups of Trichoderma.
II. Production of volatile antibioitics. Trans.
Br. Mycol. Soc. 57(1):41-48.
Deshmukh, P. P. and J. G. Raut. 1992. Antagonism by Trichoderma spp. on five plant pathogenic fungi. New Agriculturist. 3(2):127-130.
Harman, G. E., C. R. Howell, A. Viterbo, I. Chet and M. Lorito. 2004. Trichoderma species- opportunistic, avirulent plant symbionts, Nat.
Rev. Microbiol. 2(1): 43-56.
a
88
Harman, G. E., X. Jin, T. E. Stasz, G. Peruzzotti, A.
C. Leopold and A. G. Taylor. 1991. Production of conidial biomass of Trichoderma harzianum for biological control. Biological Control.
1(1):23-28.
Harman, G. E., X. Jin, T. E. Stasz, G. Peruzzotti, A.
C. Leopold and A. G. Taylor. 1990.
Development of media to produce conidial biomass of Trichoderma harzianum for biological control. Phytopathology, 80:992.
Manav, M. and R. S. Singh. 2003. Shelf life of different formulations of mutant and parent strain of Trichoderma harzianum at variable temperatures. Plant Dis. Res., 18(2):144-146.
Manczinger, L., Z. Antal and L. Kredics. 2002.
Ecophysiology and breeding of Mycoparasitic strains (a review). Acta Microbiol. Immunol.
Hungarica, 49(1):1-14.
Michrina, J., A. Michalikova, T. Rohaic and R.
Kulichova. 1995. Antibiosis as a possible mechanisim of antagonistic action of T.
harzianum against F. culmorum. Ochrana- Rostlin, 31(3):177-184.
Mustafa, A., S.M. Khan and A.Rehman. 2004. Fungi associated with Shisham (Dalbergia sissoo Roxb.) seed and their control. Pak. J.
Phytopathol. 16(2): 73-75.
Nakkeeran, S., A. S. Krishnamoorthy, V.
Ramamoorthy and P. Renukadevi. 2002.
Microbial inoculants in plant disease control. J.
Ecobiol., 14(2):83-94.
Ramanujam, B., K. K. N. Nambiar, I. Rohini and R.
Iyer. 2002. Hyphal interaction studies between Thielaviopsis paradoxa and its antagonistic fungi. Ind. Phytopathl., 5(4):516-518.
Rifai, M. A. and Webster. 1969. A revision of the genus Trichoderma spp. Mycol. Paper series 116:1-56, CMI, Kew, Surrey, England
Shalani, K. P. Narayan, Lata, A. S. Kotasthane.
2006. Genetic relatidness among Trichoderma isolates inhibiting a pathogenic fungi Rhizoctonia solani. African J. Biotech., 5(8):580-584.
Steel R. G. D. and J. H. Torrie. 1980. Principles and Procedures of Statistics.2nd Ed. Mcgraw Hill BooknCo., New York, USA. pp.377-398.
Wang, W., J. Chen, Y. Sun, Z. Zeng, X. Y. Shi and X. Y. Shi. 1999. A preliminary study on the growth inhibiton effects of Trichoderma viride on six species of soil borne plant pathogenic fungi. Chinese J. Bio Control. 15(3):142-143.
Xu, T. and J. P. Zhong and D. B. Li. 1993.
Antagonism of Trichoderma harzianum T82 and Trichoderma species NF9 against soil and seed borne pathogens. Acta. Phytopathol. Ca.
Scinica, 23(1)63-67.
Pakistan Journal of Phytopathology, Published By:
Pakistan Phytopathological Society www.pakps.com
