Effect of different substrates and carbon and nitrogen sources on growth and shelf life of Trichoderma pseudokoningii.
Trichoderma pseudokoningii is a hyperparasite on hyphae of many plant pathogens. However no reports on its mass production are available. Nine different organic substrates viz. rice grains sorghum grains wheat grains millet grains wheat straw rice husk cow dung sawdust and poultry manure were therefore evaluated for mass multiplication of T. pseudokoningii. Grains especially sorghum grains were found more suitable for growth and sporulation than wheat straw rice husk and cow dung. Sawdust and poultry manure were least effective. Sucrose and dextrose were found to be the most suitable carbon sources whereas ammonium nitrate was found to be the best nitrogen source for growth and sporulation of T. pseudokoningii on Czapek's Agar plates. Amendment of selected C and N sources to organic substrates resulted in greater growth and conidia production by T. pseudokoningii on wheat straw rice husk and millet grains but not on sorghum and rice grains. During studies on shelf life populations of T. pseudokoningii on different substrates attained the peck at 60-75 d interval and declined gradually thereafter. However even after 330 d the populations were greater than the population at 0- day. At 345-360 d interval populations were less than the initial populations at 0 d. Copyright 2014 Friends Science Publishers
Keywords: Trichoderma pseudokoningii; Mass multiplication; Carbon and nitrogen sources; Shelf life
The primary diseases threatening crop production are due to soil-borne plant pathogens that play a major role in the development of root rot disease complexes on many important field and horticultural crops which often result in the death of plants. Subsequently soil applied pesticides are costly and produce environmental hazards (Cook and Baker1983; Saleem et al. 2000; El-Katatny et al. 2000). Crop resistance to pathogens is the ideal means of controlling plant diseases but many crops have little or no resistance to certain plant pathogens. Use of microbial antagonists for the biological control of plant diseases is an alternative method for disease control which would also protect our environment from the use of hazardous chemicals (Larena et al. 2002; Harman et al. 2004).Trichoderma species are also known to produce plant growth promoting compounds that may or may not be essential for biological control (Omer and Shahzad 2007). Clarkson et al. (2004) observed that two isolates of T. viride and one isolate of T. pseudokoningii degraded up to80% sclerotia of four isolates of Sclerotium cepivorum in a silty-clay soil. Ferdousi et al. (2010) found that T.harzianum T. virens and T. pseudokoningii were highly effective against Alternaria fruit rot of chili and improved all the growth characters. Poliquit (1998) reported that T. pseudokoningii and T. viride inhibited the radial growth ofPhytophthora by 69.6% and 63.5% respectively. Taha and Salahuddin (1988) reported that T. harzianum T. pseudokoningii Penicillium pinophilum Bacillus cereus and Leuconostoc mesenteroides were effective antagonistic microorganisms against F. solani root rot disease of broad bean.There are reports where studies on mass production of T. harzianum T. viride Gliocaldium virens Paecilomyces lilacinus and Rhizobium meliloti have been made (Dawar and Ghaffar 2003; Pramod and Palakshappa 2009) but information on mass production of T. pseudokoningii is rather scanty. Fei et al. (2010) reported that wheat bran and corncob with a ratio of 8:2 H2O 55%~60% KNO3 0.1% calcium sulfate 1% and sucrose 1% supported good growth of T. pseudokoningii. The aim of present studies was to study the suitability of different substrates for mass production of T. pseudokoningii and effect of C+N sources on improvement in growth and sporulation of T. pseudokoningii.
Materials and Methods
Multiplication of T. pseudokoningii
Culture of T. pseudokoningii present in the Pest and Disease Research Lab (PDRL) Department of Agriculture and Agribusiness Management University of Karachi was used during the present studies. Pure cultures of T.pseudokoningii were produced on potato sucrose agar (PSA) medium. Ten mL sterilized water was added to one week old culture plate of the fungus and the conidia separated from the conidiophores by gently rubbing the agar surface with a sterilized spatula. The contents were poured in a sterilized beaker and the process was repeated to most of all the conidia in suspension. Two drops of Tween twenty were added to the conidial suspension to suspend the hydrophobic conidia in water. The population of conidia per mL of suspension was determined by a haemocytometer using the following formula:Cfu mL-1= No. of conidia in large square A- dilution factor A- 1.25 A-106The population of T. pseudokoningii adjusted to1.2A-107 conidia per mL using N1V1 = N2V2 formula. This population was used in all the experiments.Sorghum grains rice grains millet grains wheat grains wheat straw saw dust rice husk poultry manure and cow dung were used for mass multiplication of T. pseudokoningii. The substrates were soaked in water for two h squeezed with hands to remove excess moisture incontainers and 50 g of a substrate was transferred in eachpolyethylene bag. The bags were sealed and then sterilized in an autoclave at 15 psi with 121C for 20 min. After cooling the substrates in each bag were inoculated by injecting 2 mL conidial suspension of T. pseudokoningii containing 1.2A-107 conidia per mL. There were three replicate for each treatment. The inoculated substrates were stored at 302C and their population determined with thehelp of haemocytometer after 15 d intervals.
Effect of Carbon Sources on In Vitro Growth on T. Pseudokoningii
Different carbon sources viz. Sucrose Maltose Dextrose Glucose Starch and Cellulose were added to Czapek's Dox Agar (CzDA) medium without nitrogen sources to get final concentrations of 0 10000 20000 30000 40000 and50000 ppm before the medium was autoclaved. CzDA without carbon and nitrogen sources served as control. Media were sterilized for 20 min at 15 psi with 121C. Penicillin @100000 U L-1 and streptomycin @ 0.2 g L-1 were added to sterilized stock media just before pouring to inhibit the bacterial growth. The media were poured in 9 cm dia. Petri plates @ 10 mL per plate. There were three replicate for each treatment. After solidification a 5 mm dia inoculum disc of T. pseudokoningii was placed in center of each Petri plate. The plates were incubated at 282C and dia of the growing colonies were recorded daily.
Effect of N on In Vitro Growth of T. pseudokoningii
Different Nitrogen sources viz. NPK Urea DAP ammonium nitrate and sodium nitrate were used separately to the CzDA medium to get the final concentrations of 01000 3000 5000 7000 9000 and 10000 ppm. No carbon source was added to the medium. There were therereplicates for each treatment. The effect of nitrogen sources on in vitro growth of T. pseudokoningii analyzed by the method described above for carbon source.
Combined Effect of Selective Carbon and NitrogenSource on In Vitro Growth on T. pseudokoningii
Sucrose @ 30000 ppm and ammonium nitrate @ 3000 ppm were used as selected carbon and nitrogen sources. The carbon and nitrogen sources were mixed in the PSA medium whereas PSA without added carbon and nitrogen sources served as control. There were there replicates for each treatment. Effect of carbon and nitrogen sources on in- vitro growth and conidia production by T. pseudokoningii was recorded by the methods described above.
Effect of Carbon and Nitrogen Sources on Growth andSporulation of T. pseudokoningii on Organic Substrates
Five selected substrates viz. rice grains sorghum grains millet grains (most suitable substrates) wheat straw and rice husk (less suitable substrates) were used for multiplication of T. pseudokoningii. The substrates were soaked in water for two h in containers squeezed with hands to remove excess moisture and 50 g of a substrate was transferred in a polyethylene bag. Sucrose @ 1.5 g per 50 g substrate and ammonium nitrate @ 0.15 g per 50 g substrate were mixed with the substrates as selected carbon and nitrogen sources. Carbon and nitrogen sources were also used separately. Substrates without carbon and nitrogen sources served as control. There were there replicates for each treatment. Growth and population of T. pseudokoningii was determined using the methods described above.
Shelf-life of T. pseudokoningii
The shelf-life of T. pseudokoningii was evaluated on sorghum grain millet grains rice grains wheat straw and rice husk. Polyethylene bags filled with 50 g of each substrate were inoculated with 2 mL conidial suspension of the test antagonist containing 1.2 A- 107 conidia per mL. Each substrate was evaluated with and without selected carbon and nitrogen sources. The bags were stored at room temperature and populations of the microbial antagonist were determined from 0 to 360 d with 15 d interval. Three replicate bags of each treatment were used for determination of population at each time interval.Experiments were carried out in complete randomized block design. Data were analyzed by ANOVA using Statistix 8.1 software. Least significant difference (LSD) were calculated at pless than 0.05 level.
Growth of T. pseudokoningii on Different Substrates Generally cereal grains were found more suitable for mass production of T. pseudokoningii as significantly maximumpopulations were recorded on cereal grains as compared to other substrates (Fig. 1). However the highest population of T. pseudokoningii was observed on sorghum grains (58.7A-108 cfu g-1) followed by millet grains (54.53A-108 cfu g-1). The poultry manure was least suitable substrate and produced significantly lowest T. pseudokoningii population (1.06A-108 cfu g-1) followed by cow dung (2.10A-108 cfu g-1) and saw dust (2.27A-108 cfu g-1). Rice grains wheat grains wheat straw and rice husk performed moderately and produced 25.23A-108 23.06A-108 17.76A-108 and 15.56A-108 cfu g-1 of T. pseudokoningii respectively (Fig. 1).
Effect of Different Carbon Sources on In-vitro Growth of T. pseudokoningii
In all carbon sources the colony growth of T. pseudokoningii increased with increasing concentrations except in case of cellulose and starch where maximum colony growth occurred at lowest concentration (Fig. 2). Significantly highest colony growth was recorded on sucrose followed by dextrose glucose and maltose amended media (Fig. 2). However Dextrose glucose and maltose amended media produced less conidia but more superficial and fluffy mycelial growth of T. pseudokoningii as compared to the sucrose amended medium which support abundant conidial production of test antagonistic fungus. Sucrose was therefore used as a selected carbon source in further experiments.
Effect of Different Nitrogen Sources
Urea appeared to be the most toxic nitrogen source showed significant growth suppression at all concentrations. Sodium nitrate was the second least affective N-source. NPK gave good growth at 10000 ppm and the growth declined with decrease in concentration. Petri plates containing DAP amended media were filled within 4 d but mycelial growth was very scanty and submerged with no conidia production at all. Maximum growth and conidia production was observed where the media were amended with Ammonium nitrate @ 3000 ppm or more (Fig. 3). Ammonium nitrate @3000 ppm was therefore selected as the best nitrogen source for further experimentation.
Combined Effect of Carbon and Nitrogen Sources
The combined use of best carbon and nitrogen sources acted positively on the mycelial growth and conidial production of test antagonistic fungus as significantly higher colony growth of T. pseudokoningii was recorded on medium amended with sucrose @ 30000 ppm + ammonium nitrate@ 3000 ppm as compared to control (Fig. 4).
Effect of Carbon and Nitrogen Sources on theSporulation of T. pseudokoningii on Organic Substrates
In case of sorghum grains and rice grains the addition of carbon and nitrogen alone or in combination actednegatively on the sporulation of T. pseudokoningii as in both substrates significantly very high population of T. pseudokoningii recorded on un-amended (control) substrates as compared to carbon-nitrogen amended substrates (Fig. 5). Among all the treatments highest population of T. pseudokoningii was recorded on un-amended sorghum grains (50A-109 cfu g-1) and rice grains (30A-109 cfu g-1). However the addition of carbon and nitrogen significantly enhanced the conidial population of T. pseudokoningii in C and N amended wheat straw rice husk and millet grains as compared to un-amended substrates (Fig. 5). In case of wheat straw the conidial population of the test fungus increased from 13A-109 cfu g-1 (in un-amended substrate) to24A-109 cfu g-1 (in C+N amended substrate). Similarly in rice husk the number of conidia of T. pseudokoningii wereincreased from 8A-109 cfu g-1 (in un-amended substrate) to24A-109 cfu g-1 (in C+N amended substrate) (Fig. 5).
Shelf-life of T. pseudokoningii
On sorghum grains the conidial populations of T. pseudokoningii were 46A-109 cfu g-1 on un-amended and26A-109 cfu g-1 on C+N amended substrates after 15 d of incubation and reaches to highest i.e. 76A-109 cfu g-1 on un- amended and 53A-109 cfu g-1 on C+N amended substrates at60 d. After then the conidial population of T. pseudokoningiigradually decreased in both the treatments. After 180 d storage the populations of T. pseudokoningii declined to18A-109 and 14A-109 cfu g-1 on un-amended and C+N amended substrates respectively. Populations in 360 d old inocula further declined to 0.09A-109 and 0.015A-109 cfu g-1 on un-amended (control) and C+N amended sorghumgrains respectively (Fig. 6).Populations on rice grains also showed similar trend. The conidial populations were 34A-109 and 19A-109 cfu g-1 on un-amended and C+N amended rice grains respectively after 15 d growth. Greatest conidial populations i.e. 73A-109 cfu g-1 on un-amended and 48A-109 cfu g-1 on C+N amended rice grains were observed after 60 d. The populations declined gradually thereafter and after 360 d incubation only 0.057A-109 and 0.006A-109 cfu g-1 were recorded in un- amended (control) and C+N amended rice grains respectively (Fig. 6).In case of millet grains the conidial populations of T. pseudokoningii were 40A-109 cfu g-1 on C+N amended and21A-109 cfu g-1 on un-amended substrates after 15 d of incubation. The maximum conidial populations wasachieved after 60 d of incubation that were 65A-109 cfu g-1 on C+N amended and 50A-109 cfu g-1 on un-amended millet grains. After that the conidial populations of T.pseudokoningii reduced gradually and only 0.058A-109and 0.002A-109 cfu g-1 were recorded on C+N amended and un-amended millet grains respectively after 360 d growth (Fig. 6).After 15 d incubation populations of T. pseudokoningii on wheat straw were 1.4A-109 cfu g-1 in C+N amended and 1.1A-109 cfu g-1 in un-amended treatments. The populations reached to maximum i.e. 3.7A-109 and 1.3A-109 cfu g-1 substrates in C+N amended and un-amended treatments after 105 d incubation respectively. Thereafter the T. pseudokoningii populations on both types of substrates declined gradually and after 6 month of incubation 2.5A-109 and 0.062A-109 cfu g-1 of T. pseudokoningii were recorded on C+N amended and un- amended substrates respectively. The conidial populations of T. pseudokoningii after 360 d of incubation reduced to0.002A-109 cfu g-1 on C+N amended and 0.0015A-109 cfu g-1on un-amended wheat straw (Fig. 6).In case of rice husk the conidial populations of T. pseudokoningii were 1.1A-109 cfu g-1 on C+N amended and0.065A-109 cfu g-1 on un-amended substrates. The conidial population achieved its peaks after 105 d incubation where3A-109 and 1.3A-109 cfu g-1 were recorded in C+N amended and un-amended substrates respectively. The population ofT. pseudokoningii was slowly declined on both types of substrates and after 6 month of incubation 0.045A-109 and0.025A-109 cfu g-1 were recorded on C+N amended and un-amended rice husk respectively. No population wasrecorded after 315 d incubation on C+N amended rice husk and after 285 d on un-amended rice husk (Fig. 6).
Despite of their effectiveness the main difficulty in the widespread application of bio-control agent like T. pseudokoningii is their large-scale availability for field use. For this purpose several workers have tried different substrates such as sugarcane bagasse sugarcane ash rice grain sorghum grain millet grain cotton cake mustard cake wheat straw rice straw saw dust farmyard manure (FYM) and wheat bran were used for mass multiplication of bio-control agents (Sharma and Singh2004; Sangle and Bambawale 2005; Sharma et al. 2005). During the present studies sorghum grains followed by millet grains appeared to be the most effective substrates in which highest population of T. pseudokoningii was recorded. Our findings are in confirmation to those reported by Tiwari et al (2004) who found millet as the greatest indigenous substrate for mass production of T. viride with sporulation of 8A-109 spores after 15 d incubation. Malik and Dawar (2003) and Dawar and Ghaffar (2003) also reported that sorghum grains produced significantly more population of T. harzianum than other substrates. The reports on mass multiplication of T. pseudokoningii are not available. It seems that present work is the first work on mass multiplication of T. pseudokoningii.During the present studies also revealed that the addition of carbon and nitrogen sources significantly improved the conidial population of T. pseudokoningii in wheat straw rice husk and millet grains as compared with un-amended carbon and nitrogen substrates. In this respect Abdullah et al. (2005) reported that mycelial yield of T. harzianum was significantly enhanced when the medium was supplemented with sucrose or glucose as a carbon source. Monga (2001) also observed that sucrose and glucose as a carbon sources provided best sporulation of T. koningii and T. harzianum. He also reported that these two species produced maximum biomass on maltose and glucose.Among the five different nitrogen sources i.e. urea sodium nitrate ammonium nitrate DAP and NPK used during the present studies maximum colony growth of T. pseudokoningii were observed on ammonium nitrate followed by DAP. Jayaswal et al. (2003) also reported that growth and sporulation of T. viride was favored more by ammonium form of nitrogen as compared to nitrite or nitrate forms. Jayaraj and Ramabadran (1998) also assessed the effect of seven different nitrogen sources on in vitro growth and sporulation of T. harzianum and observed that ammonium nitrate ammonium sulphate and sodium nitrate provided maximum growth and sporulation of theantagonistic fungus. Gashe (1992) reported that nitrogen inthe form of KNO3 was better than NH4Cl or urea for thegrowth of Trichoderma species.On the basis of these results different combinations of carbon and nitrogen sources were tried and it was found that culture medium amended with sucrose @ 30000 ppm and ammonium nitrate @ 3000 ppm provided the best mycelial growth of the tested T. pseudokoningii. Jayaswal et al. (2003) found that the growth and sporulation of T. viridewas greatly influenced by various carbon and nitrogensources. They observed the best growth and sporulation of T. viride on sucrose peptone and trehalose supplemented medium. Growth and sporulation both were favored by ammonium forms of nitrogen compared to nitrite or nitrate forms.The results of our experiments on the effect of C and N sources on growth and sporulation of T. pseudokoningii on different substrates showed that amendment of carbon and/or nitrogen sources in sorghum and rice grains substrates failed to produce any positive effect on conidia production by T. pseudokoningii. It means sorghum and rice grains substrates performed better and produced higher population of T. pseudokoningii when used without the addition of carbon and/or nitrogen sources. However in case of millet grains wheat straw and rice husk significantly increased populations of T. pseudokoningii were recorded on C+N amended substrates as compared to the un-amended substrates.During experiments on shelf life of the T. pseudokoningii on different substrates with and without amendment with extra nutrients it was observed that on sorghum rice and millet grains (either amended with C+N or not) the population of T. pseudokoningii attained the peek after 60 d of incubation then gradually decline and reached to lowest at 360 d. In case of rice husk and wheat straw the population of T. pseudokoningii reached to maximum after 60 d of incubation in case of un-amended substrates and after 45 d in case of C+N amended substrates. On wheat straw the population of these antagonistic fungi was lowest after 360 d however on rice husk the conidia of T. pseudokoningii lost their viability after 285 d and 315 d when multiplied on un-amended and C+N amended substrates respectively.These studies exposed that shelf lives of the fungus varied greatly with the type of substrate or medium and temperature during storage (Prasad et al. 2002; Singh et al.2007). Khan et al. (2011) reported that vermi-compost de- oiled caster cake and farm yard manure formulations reserved shelf life of T. viride for 220 190 and 180 d respectively as compared to the gypsum and talc powder where the cfu g-1 declined after 80 d of storage. Our studies also indicated that nutritionally rich substrates supported greater shelf life as compared to the nutritionally poor substrates. However in view of the greater cost of the grains enhancement of T. pseudokoningii population of rice husk what straw and millet grains by C+N amendment is quite encouraging since it could provide a cheap means for mass production of the biocontrol agent that however need further elucidation.
Abdullah F. J. Nagappan and N.H. Sebran 2005. Biomass production ofTrichoderma harzianum (Rifai) in palm oil mill effluents (Pome).Int. J. Bio. Biotechnol. 2: 571575Clarkson J.P. A. Mead T. Payne and J.M. Whipps 2004. Effect ofenvironmental factors and Sclerotium cepivorum isolate on sclerotial degradation and biological control of white rot by Trichoderma. Plant Pathol. 53: 353362Cook R.J. and K.F. Baker 1983. The nature and practice of biological control of plant pathogens. Amer. Phytopathol. Soc. Minnesota. pp:539 Minnesota USADawar S. and A. Ghaffar 2003. Screening of substrates for massproductionof biocontrol agents. Pak. J. Bot. 35: 409414El-Katatny M.K. W. Somitsch. K.H. Robra. M.S. ElKatatny and G.M.Gubitz 2000. Production of chitinase and 13 glucanase byTrichoderma harzianum for control of the phytopathogenic fungusSclerotium rolfsii. Food Technol. Biotechnol. 38: 173180Fei S. C. Kaoshan and Z. Pengying. 2010. The Study of Solid StateFermentation of Trichoderma pseudokoningii Spores with wheatBran and Corncob. Chin. Agric. Sci. Bull. 26: 236239Ferdousi M.B. M.A. Rahman and M.F. Alam 2010. Biological control of Alternaria fruit rots of chili by Trichoderma species under field conditions. Mycobiology 38: 113Gashe B.A. 1992. Cellulase production and activity by Trichoderma sp.A001. J. Appl. Microbiol. 73: 7982Harman G.E. C.R. Howell A. Viterbo I. Chet M. Loreto 2004.Trichoderma species opportunistic avirulent plant symbiontsNature Rev. Microbiol. 2: 4356Jayaraj J. and R. Ramabadran 1998. Effect of certain nitrogenous sourceson the in vitro growth sporulation and production of antifungal substances by Trichoderma harzianum. J. Mycol. Plant Pathol. 28:2325Jayaswal R.K. R. Singh and Y.S. Lee 2003. Influence of physiologicaland environmental factors on growth and sporulation of an antagonistic strain of Trichoderma viride RSR 7. Mycobiology. 31:3641Khan S. N.B. Bagwan M.A. Iqbal and R.R. Tamboli 2011. Massmultiplication and shelf life of liquid fermented final product ofTrichoderma viride in different formulations. Adv. Bioresour. 2:178182Larena I. P. Melgarejo and A.De Cal 2002. Production survival andevaluation of solidsubstrate inocula of Penicillium oxalicum abiocontrol agent against Fusarium wilt of tomato. Phytopathology92: 863869Malik G. and S. Dawar 2003. Biological control of root infecting fungiwith Trichoderma harzianum. Pak. J. Bot. 35: 971975Monga D. 2001. Effect of carbon and nitrogen sources on sporegermination biomass production and antifungal metabolites by species of Trichoderma and Gliocladium. Ind. Phytopathol. 54:43543Omer M.K. and S. Shahzad 2007. Screening of Trichoderma species fortolerance to fungicides. Pak. J. Bot. 39: 945951Poliquit E.S. 1998. Screening of five fungal isolates for biological controlof Phytophthora on coconut. Phil. J. Crop Sci. 23: 42Pramod K.T. and M.G. Palakshappa 2009. Evaluation of suitablesubstrates for on farm production of antagonist Trichodermaharzianum. Karnataka J. Agric. Sci. 22: 115117Prasad R.D. R. Rangeshwaran C.P. Anuroop and P.R. Phanikumar 2002.Bioefficacy and shelf life of conidial and chlamydosporeformulations of Trichoderma harzianum Rifai. J. Biol. Cont. 16:145148Saleem A. K. Hamid A.H. Tariq and F.F. Jamil 2000. Chemical controlof root and collar rot of chilies. Pak. J. Phytopathol. 12: 15Sangle U.R. and O.M. Bambawale 2005. Evaluation of substrates for massmultiplication of Trichoderma spp. Ind. J. Plant Prot. 33: 298300Sharma R. and U. Singh 2004. FYM based Trichoderma harzianumformulations and their role in plant growth promotion. Adv. PlantSci. 17: 557560Sharma S. A. Aggarwal V. Parkash and D. Sharma 2005. Massproduction of VAM fungi using different substrates and hosts. J.Mycopathol. Res. 43: 5156Singh A. S. Srivastava and H.B. Singh 2007. Effect of subtracts on growthand shelf life of Trichoderma harzianum and its use in biocontrol ofdiseases. Biol. Res. Technol. 98: 470473Taha M.E. and Salahuddin 1988. Biological control of root rots of broadbean. National Agricultural Documentation Center Information Center IAEC (Iraq) IAEC P.O. Box 765 Tuwaitha Baghdad 132 IraqTiwari A.K. K. Kumar V.K. Razdan and T.R. Rather 2004. MassProduction of Trichoderma viride on indigenous substrates. Ann.Plant Prot. Sci. 12: 7174
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|Author:||Khanzada, Abdul Qayoom Rajput Muhammad Ali; Shahzad, Saleem|
|Publication:||International Journal of Agriculture and Biology|
|Date:||Oct 31, 2014|
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