Screening for thermotolrance of endophytic isolates
The fungal endophytes isolated from Artemisia and xerophytic plants of Western Himalayan cold desert and preserved at School of Ecology and Conservation Laboratory, University of Agricultural Sciences, Bangalore-560065. The 22 isolates were procured and rejuvenated on potato dextrose agar (PDA) for the present study. The endophytic isolates were screened for temperature tolerance. Isolates were cultured in PDA plates and incubated at different temperature (28 oC, 30 oC, 32 oC, 34 oC, 36 oC, 38 oC and 40 oC) for five days. Fungal growth was measured by radial diameter of colony on fifth day of incubation.
Molecular identification of thermotolerant endophytic isolates
The endophytic isolates of genomic DNA were extracted by Cetyltrimethylammonium bromide (CTAB) method (Vainio et al. 1998). The internal transcribed spacer (ITS) region of genomic DNA was amplified using universal primer ITS1-F (5՛ TCCGTAGGTGAACCTGCGG 3՛) and ITS4-R (5՛ TCCTCCGCTTATTGATATGC 3՛) by polymerase chain reaction (PCR). PCR amplification was performed using Master cycler (Eppendorf, Germany) with a 20µl reaction mixture that comprised 2µl 1X taq buffer with MgCl2 (1.5mM), 2µl dNTP’s (10mM), 0.5 µl each primer (10pmol), 0.3µl Taq DNA polymerase (3U) and 1µl template DNA (100ng). The PCR was carried out with an initial denaturation at 94 °C for 4 min, followed by 35 cycles at 94 °C for 30s, 55 °C for 1 min and 72 °C for 30s, and a final extension at 72 °C for 12 min. The PCR amplified products were sequenced by SciGenome labs, Cochin, Kerala, India. The nucleotide sequences were queried in the NCBI GenBank database using a Basic Local Alignment Search Tool (BLAST). Sequences of each fungal species and corresponding reference sequences from GenBank were subjected to ClustalW analysis. The phylogenetic tree was constructed through maximum likelihood method and Tamura- Nei model, using MEGA X. The recognized sequences were placed in GenBank with accession number.
Interaction of fungal endophytes with Rice under heat stress
Evaluation of fungal endophytes on their ability to impart heat tolerance in rice (variety IR-64) was carried out in plant growth chamber at Indian Institute of Horticulture Research (ICAR-IIHR), Hesaraghatta, Bangalore. There were two sets of experiments. 1. Heat stress (45 oC for 7h per day for 10 days) and 2. Without heat stress (normal temperature conditions, 30±0.5 oC). Each set comprised with following treatments. 1. Control (uninoculated plants) 2.Ceriporia lacerate 3. Endomelanconiopsis endophytica and 4. Penicilium funiculosum. Rice seeds were surface sterilized using 3 % sodium hypochlorite followed by 70 % alcohol. The surface sterilized seeds were repeatedly washed with sterile water and soaked for overnight. The pre-germinated seeds were sown in pots filled with soil and FYM (1:1w/w). Three seedlings per pot were maintained and grown for fifteen days. The thermotolerant endophytes were inoculated by stem prick method (Bhunjun et al. 2020) and allowed to colonize for 10 days. After colonization, set-1 seedlings were exposed to heat (45 oC) for 10 days in growth chamber. Observations for plant height, number of tillers, number of leaves, root volume, fresh and dry weight of roots were recorded after 10 days of heat exposure. Similarly, observations for plants grown under normal conditions (set-2) were recorded.
The data generated during experimentation was analyzed by one-way analysis of variance and means were separated by Duncan’s Multiple Range Test (DMRT) using the software XL STAT. The 3-D plot of stress tolerance index (STI) of biomass was constructed according to Fernandez (1992) model using iPASTIC online tool kit (https://manzik.com/ipastic/).