Morpho-cultural and molecular characterization of Trichoderma species from apple rhizosphere of North-western Himalayan temperate region of India
Authors: S. Sana B. , H. Sumaira , Dar M. S. , Padder Bila A. , Khan Imran , M. Misbah , M. Maheen , K. Sehla , Nabi Asha , Mir Snober S. , Shah Mehraj D. 
Published: January 2023

Background
The pesticide based disease management has many disadvantages including soil, water and air pollution besides adverse impact on humans and environment. Therefore, an alternative strategy is to identify and use ecofriendly management strategy like biological control. The identification of locally available bio-agents such as Trichoderma species is essential for successful disease management. To eliminate the mis-identification of Trichoderma species, a combined strategy of conventional (morphological and cultural) and molecular (multigene sequencing) approaches to characterize Trichoderma isolates have been recommended for their identification.
Methods and Results
A combined morpho-cultural and molecular approach including multigene sequencing based on internal transcribed spacer (ITS), alpha elongation factor (EF1-α1) and RNA polymerase B II (RPB2) were used to characterize 24 Trichoderma isolates from apple rhizosphere of Jammu and Kashmir. A significant variation was observed in morpho-cultural characteristics of different isolates of Trichoderma which grouped the Trichoderma isolates into four groups viz., Group I, II, III and IV accommodating 4 (16.67%), 4 (16.67%), 6 (25.00%) and 10 (41.66%) isolates and identified as T. harzianum, T. koningiopsis, T. viride and T. hamatum, respectively. The concreated sequence data set based on three genes resulted them into 03 independent clades as Clade I, II and III based on ITS, EF1-α1 and RPB2. The sequencing results obtained from ITS region grouped them into five sub-clades identified as T. harzianum (Clade I), T. viride (Clade II), T. asperelloides (Clade III) and T. koningiopsis (Clade VI) accommodating 4 (16.67%), 6 (25.00%), 5 (20.83%) and 4 (29.16%), isolates, respectively and two independent lineages by 2 (8.33%) isolates. However, two genes EF1-α1 and RPB2 genes grouped 24 Trichoderma isolates into two separate clades based on EF1-α1 and RPB2 genes, respectively, and grouped 24 isolates into six sub-clades identified as T. asperelloides (Clade I), T. asperellum (Clade II), T. hamatum (Clade III), T. viride (Clade IV), T. koningiopsis (Clade V) and T. harzianum (Clade VI). The morpho-cultural and molecular characterization alone could not produce similar results. Thus, the final identification of 24 Trichoderma isolates was obtained based on a combined morpho-cultural and molecular approach, and grouped them into six groups (I-VI) (T. koningiopsis, T. viride, T. asperellum, T. asperelloides, T. hamatum and T. harzianum) accommodating 5 (PTi1, PR3, NT1, NT2, Z2), 5 (PNi2, SS, TB1, NT3, Z3), 3 (Psh1, PNi3, TB3), 4 (PTi3, PNi1, PR1, TB2), 3 (PR2, SR, SG) and 4 (Psh2, Psh3, PTi2, Z1) isolates, respectively, indicating presence of six species of Trichoderma in apple rhizosphere in temperate region of Jammu and Kashmir, India.
Conclusion
Individual method alone could not identify these isolates, thus, the combined morpho-cultural and molecular approach was employed for final identification of 24 Trichoderma isolates and identified them as T. koningiopsis, T. viride, T. asperellum, T. asperelloides, T. hamatum and T. harzianum isolated from apple rhizosphere in Jammu and Kashmir, India.