Vol. 9(24), pp. 1895-1905, 12 June, 2014 DOI: 10.5897/AJAR2013.7970 Article Number: 6CBE0D945413 African Journal of Agricultural ISSN 1991-637X Copyright © 2014 Research Author(s) retain the copyright of this article http://www.academicjournals.org/AJAR Full Length Research Paper Production of a novel bioformulation of Trichoderma/Hypocrea using biotechnological approaches Mohammad Shahid1*, Mukesh Srivastava1, Anuradha Singh1, Vipul Kumar1, Sonika Pandey1, Antima Sharma1, Smita Rastogi2, Neelam Pathak2 and A. K. Srivastava2 1Biocontrol Laboratory, Department of Plant Pathology, C. S. Azad University of Agriculture and Technology, Kanpur 208002, India. 2Department of Biosciences, Integral University, Lucknow 226026, India. Received 24 September, 2013; Accepted 7 April, 2014 Seven different strains of Trichoderma isolated from wilt infected legume fields of UP State were tested for their antagonistic activity against Fusarium (soil borne pathogen), which is expressed as a zone of inhibition in the culture plates. Seven strains were identified as Trichoderma viride, Trichoderma harzianum, Trichoderma asperellum, Trichoderma koningii, Trichoderma atroviride, Trichoderma longibrachiatum and Trichoderma virens. Upon successful identification, morphological description and sequencing of the isolated strains with the help of universal Internal transcribed spacer (ITS) primers, the sequences are submitted to NCBI and allotted with the accession numbers JX119211, KC800922, KC800921, KC800924, KC008065, JX978542 and KC800923, respectively. Mycoparasitism is an inherent ability of the genus Trichoderma that involves the role of cell wall degrading enzymes such as xylanase, glucanase, chitinase, and proteinase etc. and acting as a biocontrol agent. This study reports the xylanase enzyme activity for all the isolated strains on a media containing birchwood xylan as a carbon source where T. harzianum (Th Azad), could show maximum activity there upon acting as a strain of utmost importance to the farmers for protecting their crops against wilting. Genetic variability within different strains of the same species was also analyzed to develop a novel strain possessing competitive ability, growth promoting characters and inducing resistance in plants. A percentage of polymorphism in Simple sequence repeats (SSRs) is obtained within the seven strains of Trichoderma species, which is comparatively higher (>77%) than with Random amplified polymorphic DNA (RAPD) primers (~50%). The study aims at exploring Trichoderma species and then preparing a simple bioformulation that is cheap, easy to apply and readily accessible to the farmers. The shelf life of Trichoderma in the prepared bioformulation is even checked for 180 days and it is concluded that the number of propagules start declining from 30th day onwards when the bioformulation is prepared in talc as a carrier material. It was also found that seed treatment with the bioformulation T. harzianum (Th Azad) (5 g/kg seeds) could increase seed germination, root and shoot length and seedling vigour over untreated ones. Key words: Antagonism, bio-control agent, Trichoderma, shelf life, polymorphism, genetic variability. INTRODUCTION The genus Trichoderma has its own significance in the pathogens to acting as a provider of nutrition to the soil agricultural industry due to its varied activities ranging as well. Several scientists have worked on how this from being a valuable antagonist against the soil-borne genus acts as a potential biocontrol agent against a 1896 Afr. J. Agric. Res. range of pathogenic fungi. Harman et al. (2004) have virgin soils where there is no intervention of agricultural even reported Trichoderma as opportunistic, avirulent practices. As Trichoderma is an ecofriendly biological plant symbionts. They have explained the features of control agent (BCA) against other soil borne plant Trichoderma as to how it colonizes the roots that pathogens, it is necessary to grow it at suitable conditions eventually proves beneficial to the soil in terms of before it is used for commercial purposes. Different pH, nutrition and plant growth increasing crop productivity temperatures and agitation speeds have been tested in simultaneously. this study for a better growth of different isolates of The biocontrol activity of Trichoderma is of immense Trichoderma species. importance not only to agriculture and its crops but also Druzhinina and Kubicek (2005) studied and brought the environment as it does not accumulate in the food forth the species concepts and biodiversity in chain and thus does no harm to the plants, animals and Trichoderma and Hypocrea by aggregating the humans (Monte and Llobell, 2003). The genes and gene morphological, physiological and genetic studies and products involved in the biocontrol mechanism of presented an update on the taxonomy and phylogeny of Trichoderma provide a vast array of research to the a number of taxa. This helped us in understanding that scientists in biotechnology and bioinformatics as well. the identification of Trichoderma only on the basis of Various strains of Trichoderma have been isolated and morphology is not of high precision. Thus, molecular identified till date in an attempt to investigate the identification and characterization comes under morphology, molecular characterization, phylogeny, investigation that would help in evaluating the genetic taxonomy, mode of action, enzymatic activity, diversity between the species. antagonistic activity, bioformulation, etc. which has Kumar et al. (2011), Shahid et al. (2012a) and Sagar et opened the gates of new research on this topic. Most al. (2011) focused on the molecular identification and ongoing research is aimed at developing new techniques analysis of the genetic variability of a specific strain of or strains that can prove ecologically beneficial to the Trichoderma based on antagonistic and Random agriculture. amplified polymorphic DNA (RAPD) analysis in some The intrageneric classification by Bisset (1991) shows leguminous crops (pigeonpea, chickpea and lentil) significant morphological similarities between produced in Uttar Pradesh (India). RAPD analysis with a Trichoderma and Hypocrea and have defined genus set of 20 OPA primers was carried out on 5 isolates of Trichoderma to include the anamorphs of Hypocrea. the same species (Trichoderma longibrachiatum) The morphology of Trichoderma sp. is very interesting collected from different soil samples of pigeon pea. This to study as there are a finite number of morphological resulted in a significant amount of genetic variability descriptors to study and disseminate the genus and its where more than 50% of the amplified fragments in each features (Gams and Bissett, 1998; Gams and Meyer, case were polymorphic. Thus, it was concluded that there 1998). It is believed that the identification of any was good genetic variability among the isolates under microorganism becomes quite easy by a careful study. morphological observation; hence, a detailed Based on the genetic variability studies done earlier, morphological description of some of the commercially the study now was focused upon developing a strain- important strains of Trichoderma has been carried out in specific molecular marker solely for the identification of this study. Samuels (2006) described the systematics, Trichoderma species. rRNA based analysis is thought to the sexual stage and the ecology of Trichoderma and be the best one to explore the microbial diversity and mentioned in his study that the morphology of identify new strains (Shahid et al., 2013). Trichoderma is not only limited to a few characters but The genus Trichoderma is well known among the many species may be included in this genus due to their microbial world as BCA and this is due to the presence of geographical distribution. certain specific cell wall degrading enzymes such as Isolation, characterization and morphological chitinases, glucananses, proteinases, xylanases, etc. to description of Trichoderma species are important before name a few. Each enzyme has its own role in further dissemination is done leading to the biomass mycoparasitism thus, it becomes important to study the production at different environmental and cultural activity of such enzymes produced by Trichoderma conditions. An attempt has been made to grow different species. The induction of xylanase from T. harzianum (Th species of Trichoderma at varying pH, temperature and Azad) by using different carbon sources was studied by agitation speeds in order to reveal all the relevant and Singh et al. (2012b). Highest xylanase activity (5.8 ±0.01 favorable parameters. IU/ml) was observed at 120 h with 1% birchwood xylan The isolates from the soils of legume fields are more as a carbon source. adaptive to the tested pH ranges than the isolates from The study also includes the behavior of these BCAs *Corresponding author. E-mail: [email protected] Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Shahid et al. 1897 against fungal wilt pathogens affecting leguminous crops xylan as a substrate. The reaction mixture contained 1ml of 1% (lentil, pigeonpea and chickpea). The strains distribution xylan solution (in 0.1M, pH 5 sodium citrate buffer) and 2 ml of in several genotypes could also support the idea of enzyme was added to the reaction tubes and incubated at 40°C. The amount of reducing sugar in the reaction tubes was measured developing