Phylogeny and Taxonomical Investigation of Trichoderma spp. from Indian Region of Indo-Burma Biodiversity Hot Spot Region with Special Reference to Manipur

Towards assessing the genetic diversity and occurrence of Trichoderma species from the Indian region of Indo-Burma Biodiversity hotspot, a total of 193 Trichoderma strains were isolated from cultivated soils of nine different districts of Manipur comprising 4 different agroclimatic zones. The isolates were grouped based on the morphological characteristics. ITS-RFLP of the rDNA region using three restriction digestion enzymes: Mob1, Taq1, and Hinf1, showed interspecific variations among 65 isolates of Trichoderma. Based on ITS sequence data, a total of 22 different types of representative Trichoderma species were reported and phylogenetic analysis showed 4 well-separated main clades in which T. harzianum was found to be the most prevalent spp. among all the Trichoderma spp. Combined molecular and phenotypic data leads to the development of a taxonomy of all the 22 different Trichoderma spp., which was reported for the first time from this unique region. All these species were found to produce different extrolites and enzymes responsible for the biocontrol activities against the harmful fungal phytopathogens that hamper in food production. This potential indigenous Trichoderma spp. can be targeted for the development of suitable bioformulation against soil and seedborne pathogens in sustainable agricultural practice.


Introduction
The genus Trichoderma was widely studied due to its rapid growth, capability of utilizing diverse substrates, and resistance to noxious chemicals [1]. Trichoderma are often the predominant components of the mycoflora in soils of various ecosystems, such as agricultural fields, prairie, forest, salt marshes, and desert [2]. Several Trichoderma species are significant biocontrol agents against fungal phytopathogens and act as stimulators for plant health [3,4]. Trichoderma species produce diverse metabolites, most notably commercially important cellulose, hemicellulases, antibiotics, peptaibiotics, and the toxins (such as Trichodermamides) and Trichothecenes that display in vitro cytotoxicity [5][6][7][8].
Due to the ecological importance of Trichoderma spp. and its application as a biocontrol agent in the field, it is important to understand its biodiversity and biogeography. However, accurate species identification based on morphology is difficult because of the paucity and similarity of morphological characters [9,10] and increasing numbers of morphologically cryptic species [11,12]. This has already resulted in incorrect identification [13]. Therefore, with the advent of molecular methods and identification tools based on sequence analysis of multiple genes, it is now possible to identify every Trichoderma isolate and recognize it as a putative new species [9,14,15]. The current diversity of the holomorphic genus Hypocrea/Trichoderma is reflected in approximately 160 species, the majority of which have been recognized by molecular phylogeny of pure cultures and herbaria specimens [15,16].
The natural mechanisms promoting high fungal diversity have remained unclear, but it seems likely that differential preference for soil and climatic conditions and host plants play the key role [17]. [19], Egypt by Gherbawy et al., 2004 [20], and Central and South American region by Druzhinina et al., press. Their studies led to the identification of several new species [21,22] and furthermore revealed a unique species in all these regions. This could be the result of geographic/climatic bias of some species. In this study we intended to determine the occurrence and species diversity of Trichoderma collected from unique biodiversity hotspot region of NE India.

Geography of Sampling
Sites. Sampling was done from nine different districts of Manipur comprising four distinct agroclimatic zones, namely, (i) subtropical plain zone, (ii) subtropical hill zone, (iii) temperate sub-Alpine zone, and (iv) midtropical hill zone, which differ in their geographic location, altitude, and climate ( Figure 1). Subtropical plain zone comprises Imphal West (711 m above sea level; average rainfall, 1259. Trichoderma isolates investigated in this study were isolated from the total 90 soil samples collected from nine different districts of Manipur (10 samples from each district pooled from 5 spots).

Isolation and Storage of Pure
Cultures. Rose Bengal agar [23] was used as a selective medium for the isolation of Trichoderma species, using soil dilution plating method. 1lt. of Trichoderma selective medium comprises MgSO 4 7H 2 O (0.2 g), K 2 HPO 4 (0.9 g), KCl (015 g), NH 4 NO 3 (1.0 g), Glucose (3.0 g), Rose Bengal (0.15 g), and agar (20 g). Putative Trichoderma colonies were purified by two rounds of subculturing on potato dextrose agar (PDA). Pure cultures were maintained in mineral oil at 4 ∘ C and also at −20 ∘ C by suspending the fungal spores in 10% (w/v) skim milk incorporated with silica powder. At the same time the cultures were lyophilized and stored in ampules.

Morphological Analysis.
For morphological analysis, strains were grown on PDA at 27 ∘ C for 7-8 days. Growth rates were determined at 20, 25, 30, 35, and 40 ∘ C for 72 h on PDA [24]. Microscopic observations were done using trinocular microscope (Carl Zais Axio ImageM2, Germany). Conidiophore structures and morphology were examined on macronematous conidiogenous pustules or from fascicles when conidia were matured. Conidial morphology and size were recorded after 14 days of incubation. Trichoderma species were identified according to Gams and Bissett [25] and Samuels et al. [26,27].

DNA Extraction and
Amplification. The extraction of genomic DNA was performed with minor modification as described by Hermosa et al. [28]. The ITS region of the nuclear small-subunit rRNA gene was amplified in an automated thermocycler (Bio-rad-C1000 Thermal Cycler) using the primers ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC) as described by White et al., 1990 [29]. The PCR reactions were performed in a total volume of 50 L, containing 1x standard PCR incubation buffer, 0.

Phylogenetic Analysis.
Sequence alignment was conducted with the CLUSTAL W program [31]. All characters were equally weighted and alignment gaps were treated as missing data. Relative support for specific clades represented in the tree was estimated by bootstrap analysis of 1000 replicates [32]. Nucleotide divergences were estimated using Kimura's two-parameter method. Sequence data analysis was carried out by a stepwise approach.

Results
A total of 193 Trichoderma spp. were isolated from the cultivated soil of Manipur using Rose Bengal agar medium. This region consists of 4 different agroclimatic conditions with varied soil types and topographical identity (Table 1 and Figure 1). Out of the total collection, 65 Trichoderma isolates were selected based on their morphological identification and their genomic DNA was amplified using the ITS 1 and 4 primers of the rDNA region.

ITS-RFLP.
The phylogenetic diversity of 65 Trichoderma strains was analyzed using ITS restriction fragment length polymorphism (ITS-RFLP) of the ribosomal spacer (rDNA) region. The amplified rDNA fragment length ranged from 548 to 607 bp. The ITS region provided greater resolution for distinguishing isolates of Trichoderma spp. ITS-RFLP carried out by using MboI and Hindf1 illustrated distinct bands to differentiate among the groups as compared to the samples treated with TaqI ( Figure 2). Trichoderma harzianum and Trichoderma aureoviride exhibited a high level of intraspecific polymorphism.

Phylogenetic
Inference. The phylogenetic tree obtained by sequence analysis of ITS region of 65 Trichoderma strains is represented in Figure 3. The ITS sequence was chosen for this analysis because it has been shown to be more informative with various sections of the genus Trichoderma [36,37] Figure 4. The identification, origin, and NCBI Genebank accession numbers and isolation details are given in Table 1.

Production of Different Cell Wall Degrading Enzymes.
Each of the representative species of 65 Trichoderma strains was determined for the production of different enzymatic activities, namely, chitinase, protease, and -1,3-glucanase. These enzymatic activities for each one of the representative strains of 22 different Trichoderma spp. are given in Table 2.
Chitinase enzyme that can degrade chitin, a major component of structural polysaccharide of the fungal pathogen cell wall, was evaluated using the chitin detection medium. The diameter of the purple color zone formation indicates the presence of chitinase activity and its zone diameter ranged from 41 to 79.66 mm in which T67 (T. aureoviride) showed the highest chitinase activity. The production of extracellular protease enzyme was determined for all the strains in skim milk agar plate. The diameter of halo zone formation ranged from 10.33 to 42.66 mm, in which T176 (T. spirale) was the highest producer of protease enzyme. -1,3 glucanase which hydrolyzes the O-glycosidic linkages of -glucan chains in the fungal cell wall is one of the important defense mechanisms exhibited by Trichoderma to fight against fungal pathogens. All the 22 representative strains of Trichoderma spp. exhibited -1,3-glucanase activity with a clearance zone diameter ranging from 11.33 to 53.66 mm. T12 (T. harzianum) produced the highest -1,3-glucanase enzyme activity.
Diagnosis. Fast growing with optimum growth temperature between 20 ∘ C to 25 ∘ C. Colonies uniformly flat and velvety, colony color cool white, yellow pigment produced. Hyphae form uniform lawn over the white colony, mycelia aerial comprising short hyphae in the form of a uniform lawn over the colony. Conidiophore arises from substrate hyphae or from aerial hyphae, 50-100 m long, smooth, typically branched along the length in a verticillate fashion. Conidia L/W 3.5-5 × 2.5-3 m, green, clavate to ellipsoidal or subglobose shape, often with a truncate or slightly protuberant base smooth, held in drops of pale green to colorless. Stromata solitary to gregarious, 1-5 mm diameter circular to elliptic in outline, centrally but broadly attached, at first yellow but becoming slightly rufous with age. Ascospore L/W 3.5-4.0 × 3.3-3.7 m, green, more or less monomorphic and subglobose, thick walled. Pigment intense yellow, no distinct odor detected ( Figure 5).

Teleomorph. Hypocrea atroviridis Dodd.
Diagnosis. Colony characteristics uniformly dispersed not pustulate or in conflict, sharply delimited and more or less dense central disk within which most conidia form, colony color green after sporulation, colony radius 42-60 mm in three days incubation. Hyphae white, sharply delimited with a more or less dense central disk within which most conidia form, mycelia formed uniform mat. Conidiophores branching typically unilateral although paired branches are common, branches typically arouse at 90 ∘ or less with respect to the branch above the point of branching, paired branching systems, phialide 6.0-9.7 m long, straight or sinuous, sometimes hooked, whorls of 2-4, often cylindrical and narrow neck, conidia L/W 1.0-1.3 m, subglobose to ovoidal, chlamydospores abundant within 7 days, globose to subglobose, terminal or intercalary, stromata L/W 0.9-2.4 m in diameter, solitary to gregarious, adjacent stromata often fused, ascospores L/W 4.3-4.4 × 3.9 × 4.0 m dimorphic, hyaline, thick walled, finely spinulose, distal part globose to subglobose, sweet (coconut) odor typically noticed ( Figure 5).
Diagnosis. Colonies grow very fast filling the Petri plate within 1 week up to 90 mm diameter, dark green hyphae dense cottony with aerial mycelium, conidiophore formed around the margin of the colony in a more or less continuous, 2.0-4.0 m wide cottony pustules, with a discernible main axis, conidia abundant in the aerial mycelium formed concentric ring with dark green color, broadly ellipsoidal to ovoidal, smooth, phialides L/W 6.0-9.7 × 1.8-3.5 m, legeniform and somewhat swollen in the middle to cylindrical, straight, rarely slightly hooked or sinuous, stromata at first semieffused, 0.5-10 mm in diameter brownish orange to light brown with a white margin (0.5-10 mm diameter), chlamydospore not observed, ascospore L/W 4.3-4.4 × 3.9 × 4.0 m, hyaline, finely spinulate, dimorphic, distal part subglobose, proximal part wedge-shaped to oblong or slightly ellipsoidal. No diffusing pigments and no distinct odour were detected ( Figure 5). Diagnosis. Colony grew moderately, reaching up to 5 cm in diameter after 3-day incubation, very white and often grew densely, producing some aerial mycelium which is fluccose in nature, produce disperse cushion shaped. Hyphae white with dense central disk, mycelia grew mostly close to the agar, conidiophore compact tufts with large color variation, pale yellow and greenish yellow to greyish green, phialides densely clustered on wide main axis, conidia L/W 4.2-5.0 × 2.7-3.0 m, green ellipsoidal, 2.7-3.0 m, smooth, chlamydospores terminal and intercalary, subglobose to globose with 10-13 m diameter, 48-53 mm colony radius at 25 ∘ C-30 ∘ C, not growing at or above 35 ∘ C. No diffusing pigments and no odor produced ( Figure 6).
Diagnosis. Colony grew moderately forming up to 5 concentric rings of dense conidial production, hyphae formed lawn, mycelia sparse and grew close to the agar, aerial mycelium lacking, conidiophore regularly branched and typically paired, phialide straight, conidia L/W 1.0-1.7 m, green to dark green, cushion shaped tufts, subglobose or ovoidal, finely spinulose. Chlamydospore abundant within one week, terminal or infrequently intercalary, hyphae, subglobose to ovoidal, smooth, and pale green. No distinct pigments and no distinct odour were detected ( Figure 6).
Diagnosis. Colony continuous, confluent pulvinate aggregates, colony radius 65-70 mm within three days of incubation, conidial mass dark green, sometimes mottled with white flecks, conidia formed within 24 h at 30 ∘ -40 ∘ C tending to form concentric rings, hyphae sometimes mottled with white flecks and often with inconspicuous wefts of yellow hyphae on the surface of the conidial mass. Conidiophore consists of a strongly developed central axis, often paired; the main axis was 2.2-3.2 m wide, phialides L/W 4.8-8.5 × 2.5-3.5 m, solitary, rarely in verticils, intercalary. Conidia ellipsoidal to oblong, green in color. Chlamydospores generally abundant, terminal and then subglobose to globose or intercalary. Pigment yellow diffusing through the agar, no distinct odor detected ( Figure 6).

(XVII) Trichoderma spirale Indira & Kamala
Diagnosis. Colony formed more or less distinct concentric rings. Pustules typically formed, pulvinate to subglobose, gray-green (0.5-1.5 mm), compact, colony color yellowish green. Hyphae formed uniform lawn, mycelia cool white fluorescent light with pustules formed around the periphery of the colony and a synanamorph forming abundantly in the aerial mycelium. Conidiophore formed a sterile hair from the base from which arise short, broad fertile branches. Phialides L/W 8.8-9.2 × 4.0-4.2 m, arise singly, directly from any of the branches, or they arise in whorls at the end of branches, phialides often doliform then clustered in grape-like fashion, when not densely clustered they are ampulliform. Conidia L/W 3.5-4.5 × 2.5-3.0 m, green in color, oblong to narrowly ellipsoidal (2.5-3.0 m), smooth. Chlamydospores typically abundant, intercalary, often formed in chains of several globose to subglobose (7.0-15.0 m) diameter. A yellow pigment tending to diffuse through the agar within 48 h ( Figure 6).  Figure 6).

(XXII) Trichoderma erinaceum Indira & Kamala
Diagnosis. Colony formed flat lawns in concentric rings with some tendency to form flat pustules reaching colony radius 60-65 mm within 3-day incubation and dark green in color. Hyphae formed flat lawns, mycelia concentric rings with some tendency to form flat pustules, conidiophore branches arising at angles of 90 ∘ C or less with respect to the main axis, the main axis of the conidiophore (2.2-3.0 m wide). Phialides arose from branches near the base or in whorls of 2 or 3, nearly cylindrical to swollen in the middle (6.0-8.0 m long), conidia 1.3-1.5 (L/W), ellipsoidal to broadly ellipsoidal (1.3-1.5), smooth, sometimes yellow associated with conidia in pustules. Chlamydospore terminal to intercalary, globose to subglobose (10.0-13.0 m). No diffusing pigment was detected. More or less strong odour detected ( Figure 6).

Discussion
The present study on the occurrence and diversity of Trichoderma spp. from Manipur was carried out for the first time from this region. The samples were collected from different subtropical agroclimatic zones comprising both hills and plains and were allowed to grow in Trichoderma specific media. Despite the fact that Trichoderma spp. are major group of organisms found from the mycoflora in tropical forest and cultivated soil, their actual distribution, presence, and association with different plants and soils have not been fully investigated. The results from this study stress the importance of the use of molecular identifications tools to describe the occurrence of Trichoderma diversity from this region. Detection of polymorphism using PCR-RFLP analysis of the rDNA ITS region has been successfully used for identifying several species of fungi [38,39]. In this study, from the total 193 Trichoderma isolates obtained from 9 different districts, 65 Trichoderma isolates were selected according to their morphological identification and they were grouped using ITS-RFLP using three restriction enzymes, namely, Taq1 In this study, we also detected a remarkable diversity of genetically sibling species from the Harzianum clade in nearly all soil samples [45,46]. The second abundant species identified in the present study was Trichoderma aureoviride. Major interrelated factors affecting microbial diversity in soil include physicochemical properties of soil. The relative effects of these factors differ in different soil types, horizons, and climatic zones [46]. The diversity and occurrence of Trichoderma species reported from four different agroclimatic zones of Manipur, namely, (i) subtropical plain zone, (ii) subtropical hill zone, (iii) temperate sub-Alpine zone, and (iv) midtropical hill zone, clearly indicate that the climatic topography and soil type are a major factor in the species distribution of Trichoderma. The subtropical plain zone comprises four districts, namely, Imphal East, Imphal West, Thoubal, and Bishnupur district. A total of 11 different species of Trichoderma occurred in this zone, namely, T. atroviride, T. ovalisporum, T. album, T. tomentosum, T. harzianum, H. intricata, T. hamatum, H. rufa, T. aureoviride, T. inhamatum, and T. amazonicum, and common soil types which occur in this region were alluvial, clay loamy, red gravelly sandy, and loamy soil. Subtropical hill zone covers two main districts, namely, Churachandpur and Chandel. In this district a total of 10 different types of Trichoderma species were found to occur, namely, T. harzianum, T. longibrachiatum, T. piluliferem, T. petersenii, H. virens, H. rufa, H. nigricans, T. aereoviride, T. koningiopsis, and T. erinaceum, with the occurrence of alluvial and loamy soil types. The temperate sub-Alpine zone comprises two districts, namely, Senapati and Ukhrul. In this type of zone only few types of Trichoderma variety were present, namely, T. harzianum and T. aureoviride, having soil type of lateritic black regur and red ferruginous, whereas, the midtropical hill zone covered only one district, namely, Tamenglong district, part of Ukhrul, and some portion of Imphal and Tamenglong district with the report of occurrence of 5 types of species, namely, T. atroviride, T. album, T. koningiopsis, T. gamsii, and T. spirale. The soil type mainly comprises alluvial soil. The occurrence of highest genetic diversity of Trichoderma species was reported from Bishnupur district with a total of eleven types of Trichoderma spp. followed by Chandel district with nine spp. and Tamenglong and Imphal East districts with five and four spp., respectively.
All the 22 representative strains of Trichoderma were found to produce three important enzymes, namely, chitinase, protease, and -1,3-glucanase. The production of chitinase enzymes by these 22 representative strains which were represented by the purple color zone formation ranges from 41 to 79.66 mm in diameter. Harman et al. [3] described the types of chitinase detected from T. harzianum, T. atroviride, and T. virens. Howell [47] tested the role of chitinases in mycoparasitism and believed that chitinase is a key enzyme in this process. The protease activity ranged from 10.33 to 42.66 mm clearance zone diameter in skim milk agar medium. Benitez et al. [48] demonstrated that protease from T. harzianum plays an important role in biological control. Szekeres et al. [49] reported the role of protease in the mycoparasitism and have reinforced with the isolation of new protease overproducing strains of T. harzianum. -1,3-glucanases have been found to be directly involved in the mycoparasitism interaction between Trichoderma species and its host [50]. Production of four -1,3-glucanases and their role of hydrolyzing the O-glycosidic linkage of -1,3-glucan chains in the fungal cell wall by T. harzianum have been described by Kitamoto et al. [51]. This work on diversity analysis of Trichoderma strains will provide a better identification of Trichoderma spp. with biocontrol mechanisms which can be used for the development of suitable bioformulation in sustainable agriculture.

Disclosure
The Institute of Bioresources and Sustainable Development, Imphal, is an autonomous Research and Development Institute under Department of Biotechnology, Ministry of Science and Technology, Government of India.