Isolation, Identification and Mass production of five Trichoderma spp. on Solid and Liquid Carrier Media for Commercialization

Trichoderma spp. are fast growing fungi, widely used as bio-control agents for controlling soil borne diseases of plants, as well as growth promoters. The present investigation was carried out to isolate and identified fast growing Trichoderma spp. and mass production using low cost widely available solid and liquid carrier materials. Several fungal spp. were isolated and colony and microscopic characterization were observed in order to identify Trichoderma to generic levels following identification keys. Fast growing Trichoderma spp. were confirmed up to species level following ITS-PCR and sequencing. Identified five Trichoderma spp. were inoculated in solid (coir dust, saw dust and parboiled rice) and liquid (coconut water and rice washed water) carrier materials separately under sterile conditions. Viability and stability of each Trichoderma spp. was tested for 180 days at 40 days intervals by calculating the colony forming units per milliliter (CFU/ml). Twelve Trichoderma isolates were identified up to generic level and five Trichoderma isolates were identified as fast growing species based on their growth rates. Following molecular characterization, identities of these five Trichoderma spp. were confirmed as T. viride, T. asperellum, T. virens, T. longibrachiatum and T. harzianum. Trichoderma spp. incubated in solid carrier materials showed higher (10 CFU/ml) values than the liquid carrier materials (10 CFU/ml) after 180 days and it was significantly different. The highest stabilization potential was recorded for parboiled rice (5 x 10 – 8 x 10 CFU/ml) from day 20 to180 for all five Trichoderma spp. Locally available solid and liquid substrates can be used for the mass multiplication of selected Trichoderma spp. to maintain the viability and virulence for long term commercial availability.


Introduction
Trichoderma species are free living, filamentous fungi that multiply rapidly on many substrates such as soil, decaying wood materials and as root colonizers (Grondona et al., 1997). The diverse metabolic capability and aggressively competitive nature help them to succeed in their habitats (Barari, 2016). When considering the morphological characterization such as conidia, they form mainly compact pustules which are typically in green shades, grey or brown (Sriram and Savitha, 2015). Use of colony morphological characters is the conventional method to identify Trichoderma species. Therefore, microscopic characteristics of Trichoderma isolates were studied in order to identify them at least up to generic level and to separate the different isolates from each other (Samuels et Research Article al., 2002). Information from microscopic studies are not specific to precisely identify Trichoderma species level because they have relatively few morphological characters and limited variation, that it may cause overlapping and misidentification of the isolates (Chennappa et al., 2017). Therefore, it is essential to use a molecular technique to compensate for the limitations of morphological characterization.
Trichoderma species have a high potential for reproduction and sporulation, as well as competitive ability and saprophytic survival (Howell, 2013). Trichoderma strains grow rapidly when inoculated in the soil, since they are naturally resistant to many toxic compounds, including herbicides, fungicides and pesticides (Seethapathy et al., 2017). The genus Trichoderma comprises of numerous strains that act as efficient Biological Control Agents (BCA). Several Trichoderma species are used as BCA against plant pathogenic fungi such as Sclerotinia, Verticillium, Rhizoctonia solani, Fusarium oxysporum, Colletotrichum gloeosporioides and Thielaviopsis paradoxa under both greenhouse and field conditions (Herath et al. 2015). The most effective species of Trichoderma are T. virens, T. viride, T. koningii, T. polysporum, T. hamatum and T. harzianum. Trichoderma species possess several control mechanisms to act against phytopathogenic organisms. These bio-control mechanisms include competition with plant pathogens, mycoparasitism, antibiosis, production of lytic enzymes and secretion of secondary metabolites (Woo et al., 2007). In addition to that, competition for nutrients or space, inducing resistance in host plants and inactivation of pathogen's enzymes are also utilized to control pathogens (Harman, 2000).
Trichoderma spp. control deleterious microorganisms which are cause a number of diseases and they produce growth stimulating factors such as plant hormones and growth factors . They increase the nutrient uptake through enhanced root growth or by promoting the availability of necessary nutrients. While also helping to reduce the concentrations of inhibitory substances of plant growth in soil (Veeken, et al., 2005). As most of the Trichoderma spp. are strong opportunistic invaders, fast growing, prolific producers of spores and powerful antibiotic producers, they are important in designing effective and safe bio-control strategies (Kumar et al., 2012).
Mass scale production of Trichoderma spp. would have great potential for commercial use. Mass production of Trichoderma spp. has become a focus of research, in the search for alternatives to pesticides and chemical fertilizers for control of plant diseases and growth promoters of plants (Parkash and Saikia, 2015). Many studies have reported on the use of conventional synthetic media like glucose, cellulose, soluble starch, and molasses to produce Trichoderma spp. However, the cost of these raw materials for commercial production of BCAs is one of the major limitations behind the restricted use. In recent years, considerable success has been achieved by the use of fungal bio control agents. But so far there is a lack of practicable and effective formulation of bio-agents to exploit it commercially (Subash et al., 2013).
Micropropagules of Trichoderma spp. in the form of conidia are preferred over chlamydospores and mycelial biomass because of the viability and stability in field application. Addition to that solid state fermentation is a cost effective system for sporulation of fungi (Cavalcante et al., 2008). In the investigation of Babu and Pallavi (2013) describe the way of use of household waste, vegetable waste and pother waste are good resources for formulation and stabilization of different Trichoderma spp. For commercial production, it is essential for the product to possess certain qualities. There is need for optimizing the operating parameters to increase sporulation or conidia production by adapting to cheaper and alternative substrates to achieve economically better yield. The dose requirement should be minimum, must have large shelf life, easy to apply, free from contamination, and should have economic feasibility with positive monetary return.
Therefore, stabilization and mass production of Trichoderma species as commercially available bio control agents in mostly available carrier material is very frequent in temperate countries and some tropical countries. There are no any published research work in Sri Lanka for the formulation and stabilization of Trichoderma species in different carrier materials in order to introduce them commercially except the investigations on their activities in research levels. There are only few published articles and in vitro experiments available for the formulation and stabilization of different Trichoderma spp. as bio control agents and plant growth promoters. So that the present investigation is carried out in an attempt to develop a better organic carrier material for the five species of Trichoderma (T. viride, T. asperellum, T. virens, T. longibrachiatum and T. harzianum) which is required by the Trichoderma spp. to maintain the viability and the bio controlling ability for a long duration. In order to introduce them into the market as bio control agents and plant growth promoters the knowhow of their formulation and stabilization in different carrier materials are significant.

Isolation and Identification of Trichoderma spp.
The soil samples with decomposing plant litter material were collected from Sinharaja, Hakgala and Kitulgala rain forests and surrounding areas in Sri Lanka. Trichoderma spp. were isolated into PDA plates using serial dilution and spread plate techniques. The plates were incubated at room temperature (28 °C) for 2 days. Suspected Trichoderma colonies were sub-cultured on new PDA plates to obtain N. Hewavitharana et al. (2018) Int. J. Appl. Sci. Biotechnol. Vol 6(4): 285-293 This paper can be downloaded online at http://ijasbt.org&http://nepjol.info/index.php/IJASBT pure cultures for further observation. By preparing sticky tape slides and using graticular calibrated phase contrast microscope, spore shape, spore size and mycelial width were measured and recorded. Identification was carried out according to the morphological and microscopic characteristics following the identification keys in Compendium of soil fungi (Domsch et al., 1980).

Growth Rates of Trichoderma Isolates
The growth rate of each Trichoderma isolate on PDA plates were studied (9cm diameter). Mycelial plugs (3 mm) from the margin of growing fungal colonies were cut and placed at the center of the agar plates. The diameter of each colony was measured at every 24 hour-intervals until the agar plates were fully colonized. The growth trial was repeated once and the average diameter of the colonies was taken from the three independent growth trials.

Molecular Characterization of Trichoderma spp.
Total DNA was isolated from 15 mg of each sample and 5μl of extracted DNA was subjected to the Polymerase chain reaction (PCR) using primers ITS1 and ITS4. Amplified DNA was subjected to DNA sequencing and the obtained DNA sequences were analyzed homologies NCBI BLAST (Basic Local Alignment Search Tool) data base compared with already existing DNA sequences in GenBank in order to identify the isolates into species level (Altzchul et al., 1990).

Stabilization and Mass Production of Trichoderma spp.
Mass production of selected Trichoderma spp. were done on naturally available solid substrates carrier materials (coir dust, saw dust and paddy seeds) and liquid carrier materials (coconut water and rice washed water) separately.

Solid carrier media
Two kilograms of coir dust, saw dust and paddy seeds were washed thoroughly and then the paddy seeds were soaked for 24 hours in water and boiled, until husk was split. Coir dust and saw dust were sieved using a 3 mm mesh. To each carrier material 2.5 g of dry poultry manure and 2.5 g of sugar was added (Table 1). The moisture level of each medium was adjusted to 40% by adding distilled water. The prepared media were put in polypropylene autoclave bags (15 x 20 cm), and five replicates of each treatment was prepared. All media were prepared and autoclaved at 121 o C (15 psi) for 25 minutes. Tetracycline (500 mg) was added into 250 g of each medium to inhibit bacterial growth. Afterwards, each Trichoderma spp. grown on PDA mat (2cm ×2 cm) was inoculated separately in to all carrier media under sterile conditions and incubated at room temperature (28 o C) for 180 days.

Liquid Carrier Media
Potato dextrose broth, coconut water and rice washed water were tested as liquid carrier material for mass production of Trichoderma spp. Coconut water and rice washed water were adjusted by adding sieved dry cow dung (2.5 g per 250 ml) ( Table 2). Prepared liquid carrier media were filled into 250 ml of Erlenmeyer flask with screw cap individually. Five replicates of each treatment were prepared. All media were sterilized by autoclaving at 121 o C (15 psi) for 25 minutes and cooled to room temperature before inoculation. Tetracycline (500 mg) was added to each flask to inhibit bacteria growth. Five Trichoderma spp. grown on PDA (2cm ×2 cm) were inoculated into all liquid media under sterile conditions and incubated at room temperature (28 o C) on a shaker at 60 rpm for 180 days. 3 -M.P.C Parboiled rice (245 g) + Dry poultry manure (2.5 g) + Sugar (2.5 g) Dry poultry manure (5 g) + Sugar(5 g)

Determination of the Viability of Trichoderma spp. in the Formulated Products
Viability and stability of each Trichoderma spp. was tested for 180 days at 40 days intervals by calculating the number of colony forming units per one milliliter (CFU/ml) of carrier material. From each solid carrier material 1g was mixed thoroughly in 10 ml of sterile distilled water and was allowed to stand. From the supernatant, a serial dilution was prepared up to 10 -5 . From the 10 -4 and 10 -5 dilutions, 1 ml of suspension was taken without disturbing the surface of the medium and spread separately on plates using a spreader. For liquid carrier media, same procedure was followed using 1ml of carrier material and CFU/ml was calculated. Three replications were maintained for each formulation. Trichoderma inoculated petri dishes were incubated at room temperature (28ºC) for three days and CFU were calculated using following formula (Khan et al., 2011).

Isolation and Identification of Fast Growth Trichoderma spp.
A total of 24 fungal cultures were successfully isolated from different soil and decomposition litter samples. Based on the preliminary screening and colony morphology on PDA plates, 12 fungal isolates were identified as Trichoderma species. Besides macroscopic characteristics and growth rate, microscopic features of Trichoderma isolates are also important morphological keys in the identification of Trichoderma species (Gams and Bissett, 2002). The microscopic features that are frequently studied include the shapes and sizes of conidia, the branching patterns of conidiophores, the shapes and sizes of phialides (Samuels et al., 2002). Different terms have been used to describe the shapes of the conidia because no systematic rule has been established to define the shapes of the conidia. Also, the description of the shapes of conidia may be subjective. Therefore, the descriptions alone of the shapes of conidia are not a good criterion in identifying most of the isolates. However, the measurements of spore and phialide sizes are useful in identification of possible Trichoderma species. Using the identification key in the Compendium of soil fungi (Domsch et al., 1980) and online identification key provided by Samuels et al, (2002). Isolates were identified up to generic level both macroscopic and microscopic characteristics. Conformity of the isolates was obtained which revealed that all 12 isolates belonged to Trichoderma species.
Almost all Trichoderma isolates grown in PDA plates showed confluent growth within 4-5 days because of their higher growth rates with slight variations among some of the isolates. Among the twelve Trichoderma isolates, five Trichoderma species were identified as fast growing species based on their growth rates. Molecular identification techniques were utilized to identify the isolates up to species level, since colony characteristics were not sufficient to do so. In this study, of 5.8S rRNA gene was amplified using primer pair ITS1 and ITS4. The two spacers, together with the 5.8S gene, are normally referred to as the ITS region. This is a very convenient tool of species identification because rRNA genes are universally conserved, while the ITS region is highly variable. The ITS region evolves rapidly and even within a genus it may vary among species (Kannangara et al., 2016). Therefore, the sequences of these regions can be used for identification of closely related species. Restriction digestion of the amplified DNA using AluI enzyme was performed to differentiate the Trichoderma isolates depending on their genomic variability. At present, RFLP is recognized as a good technique to differentiate species, because of the cleavage of DNA molecules at specific sites (Sharma et al., 2009). Therefore, the molecular characterization was also applied during the current study, in order to differentiate the isolated Trichoderma species.

Effects of Different Solid Carrier Media On Growth of Trichoderma spp.
There was no significant difference among T. viride, T. asperellum, T. harzianum, T. longibrachiatum and T. virens with respect to the number of CFU/ml produced in the selected solid carrier materials (coir dust, saw dust and parboiled rice). All the five Trichoderma spp. grown in three different carrier materials showed more or less similar pattern of stabilization after 180 th days ( Figure 01). Following the CFU/ml values, the highest stabilization potential was recorded for parboiled rice after 180 days for all five Trichoderma spp. (a) T. viride; 6.2 x 10 6 CFU/ml (b) T. asperellum; 5.2 x 10 6 CFU/ml (c) T. harzianum; 5.4 x 10 6 CFU/ml (d) T. longibrachiatum; 6.8 x 10 6 CFU/ml and (e) T. virens; 6.8 x 10 6 CFU/ml, respectively (Fig. 1). The used carrier materials were rich in lignocellulosic organic compounds and contained nutrients essential for fungal growth such as carbon, nitrogen and mineral salts. In preparations of carrier materials, sugar was added as the carbon source and cow dung or poultry manure were also added as nitrogen source, since Trichoderma require different carbon and nitrogen sources for their growth and development .
Results obtained for the CFU/ml values of T. viride, T. asperellum, T. hazianum, T. longibrachiatum and T. virens inoculated in parboiled rice at room temperature were higher after 180 days compared to the saw dust and coir dust. It may be due to the availability of higher carbon contents, higher nutrients, optimum C: N ratio and the neutral pH value in the parboiled rice that enhanced the production of fungal biomass of Trichoderma spp. According to Khan et al. (2011) water absorption capacity, texture and ability to form hydrogen bonds of the carrier materials also affect the production of high amount of fungal biomass. As per the results obtained, parboiled rice was considered as the best carrier material for the five Trichoderma spp. tested than coir and saw dust media.
In present study, survival and viability was determined up to 180 days, because the colony count gradually increased and started to reduce to constant level within that time.
Maximum growth and higher survival stabilization of N. Hewavitharana et al. (2018) Int. J. Appl. Sci. Biotechnol. Vol 6(4): 285-293 This paper can be downloaded online at http://ijasbt.org&http://nepjol.info/index.php/IJASBT Trichoderma can change after 180 days, therefore further study is required. Additionally, coir dust and saw dust based formulations were found suitable for growth and viability of Trichoderma spp. during storage. Trichoderma is a cellulotic fungus, therefore lignocellulosic substrates is a better source for sporulation, growth and development for long periods. Maximum growth and higher survival rate during storage in carrier material formulation is a major advantage for marketing these bio-control agents at commercial level.
Considering parboiled rice mass production, initial stabilization process slows down (low CFU/ml in initial stage) due to rigid rice husk. With time CFU/ml of Trichoderma increased due to stabilization, which resulted in an increment in mass. Instead of rice, other alternative substrates can be used for the mass production of Trichoderma. Parkash and Saikia (2015) used wheat straw, sorghum grains, lignite and stillage, molasses and brewer's yeast, and wheat bran medium for mass production of Trichoderma spp. In order to enhance growth rate and sporulation, various cellulosic residues were supplemented with chickpea flour as organic nitrogen supplement at two and four percent. According to the results, supplementation of all the substrates with chickpea flour enhanced growth and sporulation, and 4% chickpea flour supplementation gave maximum response in all the cases. In addition, paddy straw, sugarcane baggase and wheat straw, each supplemented with 4% chickpea flour, were found to be promising substrates for mass multiplication of T. harzianum (Parkash and Saikia, 2015).

Effects of Different Liquid Carrier Media On the Growth of Trichoderma spp.
All the five Trichoderma spp. grown in three different liquid carrier materials (PDB; potato dextrose broth, CWB; coconut water broth, and RWB; rice washed broth) showed more or less similar stabilization patterns over 180 th days ( Figure 02). There was no significant difference among the CFU/ml values of the three carrier materials for all Trichoderma spp. Among the CFU/ml values, PDB showed the highest stabilization potential (higher CFU/ml values) for all five Trichoderma spp. for multiplication from 20 to 180 th days (Fig. 2).
In the laboratory, PDB used for multiplication of fungal colony showed higher CFU/ml compared to the other two liquid carrier materials. However, there was no significant difference in CFU/ml, among the three carrier materials for all Trichoderma spp. Coconut water, rice washed water and waste materials in household can be used as cost effective carrier materials instead of PDB.
Emerson and Mikunthan (2015) have used locally available substrates to improve the growth of T. viride. Results of this study also showed that locally available liquid substrates such as black gram soaked water, coconut water and rice mill effluent can be used for the mass multiplication of T. viride by small scale farmers. Similarly, in the present investigation, coconut water and rice wash water are identified as locally available liquid waste from domestic kitchens. There is a good potential for the use of these waste carrier materials for the mass production of Trichoderma spp. Panahian et al. (2012) have used formulated sugar beet molasses, broken corn seed, wheat bran, soybean meal, crushed potatoes, sugar beet pulp culture medium as carrier materials for T. harzianum and T. virens.  have used vegetable waste, fruit juice waste, sugarcane baggase and rotten wheat grains for mass multiplication of different Trichoderma spp. and have reported sugarcane baggase as the best carrier material that yielded the highest amount of mycelia, spores and higher CFU/g. Babu and Pallavi (2013) recorded similar results when Trichoderma were tested for linear growth rates at different temperature levels (5-25 ºC). Optimum bio-mass was produced at temperatures between 20-30 ºC. Trichoderma spp. were found to be best grown at a temperature range of 25 to 30 ºC. Singh et al. (2014) showed that the temperature optimum of xylanase production was 28 ºC. In another study,  tested the effect of different temperatures on the mycelial growth of Trichoderma isolates. They showed that mycelial growth differed statistically with respect to the tested temperatures. Optimum temperature 25 ºC supported the highest mycelial growth of T. harzianum and T. hamatum, whereas the mycelial growth of T. virens was highest at 30 ºC. The present study reveals that, Trichoderma can sustain and survive the room temperature (28 0 C) to obtain sporulation, higher stabilization and mass production. Niranjana et al. (2009) demonstrated that pH can play a role in the regulation of extracellular enzyme, β-1, 6-glucanase production in T. harzianum. Biomass and conidia production by T. harzianum were examined in a defined medium at different pH values. Biomass production was observed within pH 3.5-9.5 with maximum growth between pH 5.5-7.5. Subash et al. (2013) had observed highest growth rates at pH 4.0 in a study where pH dependence of the linear mycelial growth and activities of extracellular enzymes of Trichoderma strains was tested.  reported that growth of Trichoderma spp. was maximum at pH 6 whereas, the growth was minimum at pH 4. Therefore, it is important to examine the effects of pH on mycelial growth and on the in vitro activities of extracellular enzymes contributing for competition and mycoparasitism of Trichoderma spp. with bio control potential. Considering the observations of previous studies, selected carrier materials were incubated at room temperature 28 o C and at 6-7 pH, for high bio mass production.
According to the results obtained for the five Trichoderma spp. on stabilization in solid and liquid carrier materials, contradictory results (CFU/ml) were obtained. Trichoderma spp. incubated in solid state carrier materials showed higher (10 6 CFU/ml) values than the liquid state carrier materials (10 4 CFU/ml) after 180 days and it was significantly different. This can be due to several characteristics of carrier materials ie; availability of higher carbon contents, optimum C: N ratio and the neutral pH value that enhance the production of higher fungal biomass of Trichoderma spp.
As described in the Panahian et al. (2012) propagules of Trichoderma can be produced in mass scale in both liquid and solid fermentation methods. As low cost materials to maximize the yield, crop residue, household waste, livestock waste and industrial waste can be used. As the cost of synthetic media for the mass multiplication at any biocontrol agent is high, mass multiplication with the use of organic waste materials was found to be economically advantageous and indicates the fact that these media are cost effective and hence support commercialization of Trichoderma spp. According to  CFU of Trichoderma should be a minimum of 2 x 10 6 CFU per ml or gram in selective medium of Trichoderma formulations as per the standards. Pathogenic contaminants should not be present and other microbial contaminants should not exceed a number of 1 x 10 4 count ml/g. The viability of the Trichoderma formulations should be maintained for a considerable period of time.

Conclusion
Selected Trichoderma spp. were successfully mass produced in naturally available solid carrier materials (coir dust, saw dust and parboiled rice) and liquid carrier materials (coconut water and rice washed water). Among the three solid substrate carrier materials, parboiled rice was identified as the most effective organic carrier material which maintained the viability and virulence of all Trichoderma spp. throughout the study period (180 days).