Twenty four isolates of Trichoderma species were obtained from apple rhizosphere of Jammu and Kashmir, a North-western region of India and identified on the basis of morpho-cultural characters followed by their molecular confirmation using sequencing of ITS, EF1-α1 and RPB2 genes following different protocols [11, 15, 28, 29].
Morpho-cultural characterization of 24 isolates of Trichodema species was studied based on colony characters like texture, margins, colour, shape, diameter and growth rate, spore characteristics, shape of phialides, and mycelial dry weight. Based on colony texture, three groups comprising of Group I (flat cottony growth), Group II (fluffy growth) and Group III (flat granular growth) were formed accommodating 33.33, 41.67 and 25.00 per cent of isolates, respectively. Grouping of the isolates based on colony margins was also carried out and distributed 24 isolates into two groups. Group I with uniform margins accommodated 87.5 per cent of isolates and Group II with irregular margins comprised of 12.50 per cent isolates. Similarly, grouping based on colony colour distributed the isolates into four groups, Group I with whitish to light green colony color, Group II (white to dark green), Group III (white to black green) and Group IV (dull white to green) accommodating 20.83, 45.83, 16.67 and 16.67 per cent isolates, respectively. Similarly, based on colony shape, 4 groups were formed namely Group I, II, III and IV accommodating 16.67, 16.67, 25 and 41.66 per cent isolates, respectively. Present results were in close agreement with the observations of different workers around the world [30, 31, 32] who also found that the colonies of various Trichoderma isolates were flat cottony, fluffy or raised and flat granular in texture having regular or irregular margins on PDA medium. Sharma and Singh [33] also reported that the majority of Trichoderma isolates showed different colony colour like dark green (12 isolates), whitish green (9 isolates), light green (6 isolates) and rest of the isolates (3) had yellowish green colonies. Kamaruzzaman et al. [34] also observed that the colony colour of 10 selected isolates of Trichoderma varied from dark green to light green or black green or whitish green. Similarly, Amaresh et al. [35] also reported colour variation from dark green or light green to yellowish green with flat to raised growth pattern. These observations were in close agreement with our results. The colony shape varied in different isolates of this fungus. Production of green spore mass was observed either at centre of the Petri plates containing PDA medium making 1–2 concentric rings or spread over the entire plate with yellowish or whitish aggregated spore mass or white mycelium with green conidia towards the margin of the plate. Some isolates formed white mycelium with limited green conidial formation in concentric rings near the centre or dispersed along the margin. The results of colony shape were in close agreement with the observations made by other workers [30, 36].
Distinct variation was found in spore characteristics viz., spore colour, shape, ornamentation, size, and shape of phialides in different isolates. Based on conidial colour, four groups such as Group I with olive green coloured spores, Group II (yellow green), Group III (pale green) and Group IV (bright green) were formed accommodating 4 (16.60%), 5 (20.80%), 5 (20.80%) and 10 (41.40%) isolates, respectively. Likewise, grouping of isolates on the basis of spore shape resulted in four groups viz., Group I with globose shaped conidia, Group II (globose to sub-globose), Group III (globose to sub-cylindrical) and Group IV (sub-globose to obvoid condia) accommodating 4 (16.60%), 5 (20.80%), 5 (20.80%) and 10 (41.40%) isolates, respectively. Grouping on the basis of spore ornamentation, isolates were distributed into two groups namely Group I with smooth walled conidia accommodated 83.33 per cent isolates and Group II (rough walled) comprised of 16.67 per cent isolates. Based on shape of phialides, four groups such as Group I with flask shaped phialides, Group II (slender), Group III (elongated) and Group IV (swollen) phialides were formed accommodating 4 (16.60%), 5 (20.80%), 5 (20.80%) and 10 (41.40%) isolates, respectively. Similar observations were recorded by different workers all over the world [27, 30, 36, 37, 38,, 39, 40, 41], and Kabir et al. [42] who also found that the shape and ornamentation of various Trichoderma isolates as globose, globose to sub-globose, globose to sub-cylindrical and sub-globose to obvoid in shape having smooth and rough ornamentation. Similarly, Gams and Bisset [28] found that conidial colour varied from typical green to olive green or grey or brown or sometimes colourless. Sriram et al. [43] also reported that the conidia in Trichoderma species were olive green or dark green in colour. Grouping on the basis of phialide shape distributed different isolates into four groups viz., Group I with flask shaped phialides, Group II (slender), Group III (elongated) and Group IV (swollen) accommodating 4 (16.60%), 5 (20.80%), 5 (20.80%) and 10 (41.40%) isolates, respectively. Rifai [9] and other workers [30, 31, 40, 41] from time to time also found that the shape of phialide of various Trichoderma isolates were flask to slender, elongated or swollen confirming the results obtained from the present study.
The maximum conidial length and breadth was observed in isolate SR whereas the minimum was observed in isolate Z1. The maximum length and breadth ratio was observed in isolate PNi2, whereas the minimum length and breadth ratio was again observed in isolate Z1. Conidial size was reported to vary from of 2–5 × 2–4 µm in different Trichoderma isolates [28]. Similar observations were also recorded by Soesanto et al. [44], who found variability in spore dimensions of four Trichoderma isolates. Sriram et al. [43] recorded the conidial size in 30 different Trichoderma isolates and reported that the conidial size and L/W ratio varied significantly in T. viride and T. asperellum.
Significant variation was found in Trichoderma isolates with respect to colony diameter on PDA medium. The highest colony diameter was recorded in PR2 isolate and the least in Psh2 isolate. Similar results were also obtained by different workers in terms of colony diameter of Trichoderma isolates on PDA [33, 34, 36, 45, 46, 47]. Distinct variations were also observed in average growth rate of different isolates of Trichoderma on PDA medium. The highest growth rate was observed in Psh1, PNi2, NT2, Z1 isolates and the least growth rate was observed in TB2 isolate. Variations in terms of growth rates of different Trichoderma isolates has been reported in Trichoderma species by different workers [47, 48, 49]. A high level of variation was also observed in terms of mycelial dry weight in Trichoderma isolates. The highest mycelial dry weight was recorded in PR2 isolate and the least in PTi3 isolate. A significant variation in terms of mycelial dry weight of different isolates of Trichoderma was also reported by different workers [32, 49, 50, 51, 52].
Thus, based on morpho-cultural characterization, the 24 isolates of Trichoderma were identified up to species level and grouped them in four species. The isolates namely Psh2, Psh3, PTi2 and Z1 belong to T. harzianum, whereas the isolates PNi2, SS, TB1, NT1, NT3 and Z3 belong to T. viride as described by Bisset [25, 26] and others [27, 28, 30, 39, 41]. Isolates namely PTi1, PR3, NT2 and Z2 belong to T. koningiopsis and 10 isolates Psh1, PTi3, PNi1, PNi3, PR1, PR2, SR, SG, TB2 and TB3 belong T. hamatum as described by Rifai [9], Bissett [26] and Nagamani et al. [41].
Molecular Characterization Based On Multiple Gene Sequencing
The phylogenetic analysis of sequence based concreted data set of three genes namely internal transcribed spacer (ITS), alpha elongation factor (EF1-α1) and RNA polymerase B II (RPB2) genes grouped 24 isolates of Trichoderma species in to three distinct clades as clade A, B and C, respectively. Clade A based on internal transcribed spacer (ITS) sequences grouped the 24 isolates into five sub-clades viz., Clade I, II, III, IV and V accommodating 8.33, 16.67, 25.00, 20.84 and 29.16 per cent isolates, respectively (Table 6; Fig. 4). The isolates in clade I were identified as Trichoderma species (2 isolates), Clade II as T. harzianum (4 isolate), Clade III as T. viride (6 isolate), Clade IV as T. asperelloides (5 isolate) and Clade V as T. koningiopsis (7 isolate). Kindermann et al. [53] for the first time attempted to study the phylogeny of whole Trichoderma genus based on sequencing of ITS region, and reported it as a powerful tool for authentic identification of Trichoderma species being highly conserved within the species but varies from species to species. Other researchers also reported the importance of ITS sequence in Trichoderma species identification [54, 55, 56, 57]. However, the results obtained from the ITS were not in congruence with the morpho-cultural data. This could be possible due to the reason that few morphological characters with limited variation may lead to an overlap and misidentification of the isolates/strains also described by various workers from time to time [29, 58, 59, 60].
The phylogenetic analysis of sequences of 24 isolates based on alpha elongation factor (EF1-α1) (clade B) and RNA polymerase B II (RPB2) (clade C) genes revealed the similar results (Fig. 4). Twenty-four isolates of Trichoderma species based on EF1-α1 and RPB2 genes were grouped into six sub- clades viz., Clade I, II, III, IV, V and VI. The isolates in clade I were identified as T. koningiopsis, Clade II as T. viride, Clade III as T. asperellum, Clade IV as T. asperelloides, Clade V as T. hamatum and Clade VI as T. harzianum accommodating 20.83, 20.83, 12.50, 16.67, 12.50 and 16.67 per cent isolates, respectively. Other researchers have also reported the importance of alpha elongation factor and RNA polymerase B II genes sequencing in identification and phylogenetic analysis of Trichoderma species [11, 61, 62, 63, 64]. However, the results obtained from EF1-α1 and RPB2 genes were slightly different from results obtained from ITS region being highly conserved across the species. Therefore, this can be the probable reason for not differentiating the some species leading to mis-identifications, may be due to the presence of non-orthologous copies of ITS in these isolates as also reported by Hoyos-Carvajal et al. [15, 65, 66, 67]. Alpha elongation factor and RNA polymerase B II genes were also explored to distinguish Trichoderma species because of their variability, therefore able to reflect species differences within and among the groups of closely related species [14]. Due to their presence as a single copy in the genomes of all eukaryotes and slow rate of divergence, these genes are considered as very useful for the higher level of phylogenetic reconstruction [64, 66, 67, 68].