LARGE SCALE PRODUCTION AND INCREASED SHELF LIFE OF TRICHODERMA HARZIANUM INOCULUMS IN SEMI SOLID MEDIUM

Trichoderma harzianum is a well-known bio control agent that is commercially produced to prevent development of several soil borne plant pathogen. In addition to control of plant diseases, T. harzianum also promotes plant growth. Solid and liquid state fermentation methods are commonly used for mass production of T. harzianum inoculums. Solid fermentation is expensive as it requires substrate for fermentation. Liquid fermentation is also undesirable due to increase of pH, chemical degradation and lower shelf life over the time of storage. Therefore, an alternative semi solid medium has been developed for large scale fermentation of T. harizanum inoculums. The newly developed medium showed more cell count of T. harizanum and less chemical changes (most probably oxidation) over time compared to solid or liquid media. Two to three days after fermentation, the newly developed semi solid medium showed less production of gas compared to other media. Therefore, the newly developed semi solid medium could be used to increase the quality and quantity of T. harizanum inoculums for large scale commercial production.


INTRODUCTION
Trichoderma was first classified as fungi in 1794 and observed as problematic and embraced alternative of several fungi like Puccinia, Mucor, Ascobolus and a few slime molds.This concept leaded Trichoderma which is identified as T. viride before 1969.Therefore, most of the identified taxa before 1969 are in all probability of misidentified since T. viride could be a comparatively rare species.
Based on a study, trichoderma harzianum is the bio-control agent especially used in foliar application, seed treatment, as well as soil treatment to protect from various plant pathogens that cause diseases [1][2][3].It is also used for manufacturing of enzymes.Currently, the plant diseases caused by Botrytis, Fusarium and Penicillium sp. are treated by the 3Tac which commercial biotechnological products of Trichoderma.According to research, to meet the increased demands of food and fiber, green revolution is essential in intensified agriculture of near future [4].In our modern food crop production system, the use of chemical pesticides and fertilizers has adverse effect on natural ecosystem resulted destruction of beneficial organism like honey bees, effect on non-target pests, chemical residues in food, feed and fodder [5].According to a scholar, T. harzianum is mainly used and produced to prevent the development of several soil borne plant pathogens that not only cause diseases but also compete for nutrients and space, do mycoparasitism, produce inhibitory compounds as well as secretes chitinolytic enzymes [6,7].
Trichoderma species are principally green-spore forming ascomycetes present in nearly every type of temperate and tropical soils.Based on a study, most of the Trichoderma species are found in decaying plants and within the rhizosphere of plants [8][9][10][11].Their diverse metabolic alteration and aggressive competitive nature help to colonize in their habits.
The antagonistic fungi T. harzianum has shown as a promising bio-control agent against soil borne plant pathogens.T. harzianum could be recommended not only for control the plant diseases but also as a growth promoter.According to several scholars, bacteria and fungi are multiplied by different fermentation techniques and sold in the different markets as to use for control product growth [12][13][14][15][16][17][18].Study showed the beneficial action of Trichoderma spp. is not only fighting for pathogens but also playing role for opportunistic plant symbionts [19][20][21][22].According to previous studies, this interaction with plants initially starts in the rhizosphere and continues to root proliferation, plant growth, and plant protection [23][24][25][26][27]. Hence, these fungi may be applied for redress of impure soil and water by the treatment to become acceptable for plants [28][29][30][31].Based on a study, the disease in Chili could be controlled effectively by T. harzianum.T. harzianum are soil borne within the field, green-spore ascomycetes which will be found everywhere in the planet [32][33][34][35][36].
This study is conducting the large-scale production of Trichoderma harzianum into the gel/semi gel media and increasing the shelf life, before this study we found only the production of T. harzianumis spp. in small amount and short time shelf life.The solid-state fermentation of T. harzinum is applying for the processes during which insoluble elements or fungi in water are used for growth of microorganism.Within the fermentative process, rice was not exceeding the capability of saturation stage for growth of T. harzinum.

Measuring the all ingredients
All ingredients were taken at right amount.The citric acid and ascorbic acid were used to lower pH (4.5 to 4.8) of medium to avoid production of excessive CO2 through fermentation; xanthan gum was added for gel formation and stabilization of the culture and other ingredients.

Mixing all the ingredients
First the water and xanthan gum were mixed by mixing machine and then the T. harzianum culture (solid fermented form) was added and mixed for 30 minutes.Then, all the rest of the ingredients were added and mixed properly for 5 hours for getting homogenized gel, with a shelf life of about 06 (Six) months.

RESULTS AND DISCUSSION
T. harzinum was successfully grown in the proposed semisolid medium and showed better result than the other media, (about 5.6 x 10 5 cfu, where x 10 3 was in previous media).Semi solid medium has been developed for large scale fermentation of T. harizanum inoculums.The newly developed medium showed more cell count of T. harizanum and less chemical changes probably oxidation) over time compared to solid or liquid media.Two to three days after fermentation, the newly developed semi solid medium showed less production of gas compared to other media.

CONCLUSION
This semi/gel solid medium has been developed for large scale fermentation of T. harizanum inoculums.The newly developed medium showed more cell count of T. harizanum (about 5.6 x 10 5 cfu) and less chemical changes (most probably oxidation) over time compared to solid or liquid media.Two to three days after fermentation, the newly developed semi solid medium showed less production of gas compared to other media.Before this study the growth rate of T. harzinum into a media is only 3.8 x 10 3 cfu, now in this media the growth of T. harzinum is more increase and found 5.6 x 10 5 cfu into a normal culture media (Plate Count Agar or Nutrient Agar media).

Figure 2 : 3 . 1
Figure 2: After 12 days fermentation at 35 o C to 37 o C3.1 Observation and Morphology of TrichodermaThis fungus is easy to reproduce at a low-cost cooked rice medium and its potential makes it deserve of the attention of people.The gel formation of T. harzinum are developed for the increasing shelf life and prevent the oxidation of liquid suspension of T. harzinum, liquid suspension form of T.

Table 1 :
Microbiological result of T.