Evaluation of Endophytic Fungi to Combat Drought Stress in Wheat (Triticum aestivum)

Abstract

The present work is based on the hypothesis that the fungal endo-mycobiome of Triticum aestivum L. (wheat) are the possible players in plant growth and fitness by playing an array of physiological roles from adaptation to biotic and abiotic stresses to stimulating plant growth. A total of 145 culturable endophytic fungal isolates were recovered from the different parts, viz. roots, internode, leaves, and spikes) collected from different growth stages of wheat (variety PBW 725) plants. 61 culturable endophytic fungal isolates were recovered from leaves, 37 from spikes, 30 from roots, and 17 were recovered from internode samples of wheat plants. Tentative identification of culturable endophytic fungal isolates was done by analyzing their morphological features and microscopic characteristics. Further, the total isolated endophytic fungi were grouped into different classes viz. Dothideomycetes (32%), Hyphomycetes (26%), Eurotiomycetes (21%), Sordariomycetes (6%), Zygomycetes (5%), and some isolates were not identified being non-sporulating in nature. All the culturable endophytic fungal isolates were screened for osmotic stress tolerance under in-vitro conditions. With the increase in the level of induced stress in the medium, the decline in the growth rate of culturable fungal endophytes was observed in the solid and broth media. At every growth stage of wheat plants (from germination to maturity stage), fungal isolates that exhibited the maximum drought stress tolerance were selected for further analysis. Based on statistical analysis, 10 potent isolates viz., #TAKR-1a, #2TAKR-6a, #3TAKR-1a, #4TKI-3a, #5TAKL 3a, #6TAKR-1a, #7TAKL-1b, #8TAKS-3a, #9TAKL-4a, and #9TAKR-1b exhibited more than 70% growth on agar plates, more than 30% induction in wet biomass and more than 50% increase in dry biomass in the presence of 20% PEG 6000. These 10 potent drought-tolerant endophytic fungi were used to analyze their various plant growth promoter properties and antioxidant activities. Potent fungal isolates produced auxin in the 138-710 μg/mL range in the presence of a precursor (L-tryptophan). However, similar isolates showed the potential to secrete auxin in the 30-187 μg/mL range without tryptophan. In the case of gibberellic acid production, all the isolates produced a varying amount of gibberellic acid ranging from 6.2 to 14 mg/mL in the Saborauds broth under shaking conditions. On Pikovskaya’s agar plates, the fungal isolates could solubilize phosphate with a phosphate solubilization index (PSI) in the range of 1.02–1.9. In Pikovskaya’s broth, maximum phosphate solubilization of 248±0.03 μg/mL was exhibited by the isolate #5TAKL-3a, while the fungal isolate #3TAKR-1a displayed a minimum of 77±1.1 μg/mL. Of the 10 isolates, 8 demonstrated ACC deaminase activity in the range of 0.20-2.25 α KB μmol per mg protein per h. Maximum ACC deaminase activity viz. 2.25±0.13 α KB μmol per mg protein per h was evinced by the drought-tolerant fungal isolate #7TAKL-1b, followed by #8TAKS-3a and #5TAKL-3a with activity of 1.56±0.1 and 1.54±0.05 μmol per mg protein per h, respectively. The least ACC deaminase activity of 0.20 ± 0.07 μmol per mg protein per h was shown by isolate #9TAKR-1b. Out of ten drought-tolerant fungal endophytes, eight isolates showed a brown colour change after adding the Nessler reagent, affirming ammonia production. A similar trend was observed in the potential of siderophore production by the drought-resistant endophytic isolates. In addition to the PGPR traits, three isolates, #5TAKL 3a, #8TAKS-3a, and #4TAKI-3a, showed the maximum ability to secrete extracellular enzyme followed by #2TAKR-6a and #9TAKL-4a. However, all the fungal endophytes showed negative results for the in-vitro toxicity test performed on blood agar plates. Antioxidant activity of the drought-tolerant endophytic isolates by DPPH assay ranged between 53-99 μg QE (Quercetin equivalent)/mg. Further, the methanolic extracts of endophytic fungi had f lavonoid content in the range of 55-643 μg QE/mg extract. The total phenolic content of the fungal extract varied significantly. However, the highest TPC exhibited by #5TAKL-3a was 397 μg GAE (Gallic acid equivalent) /mg extract, while the lowest was 19 μg GAE/mg extract exhibited by #3TAKR-1a. Selected ten endophytic fungal isolates exhibited a total antioxidant activity ranging between 120-890 μg AAE (Ascorbic acid equivalent)/mg extract). The maximum ferric reduction ability was shown by fungal isolate #5TAKL-3a with a value of 167±0.24 μg AAE/mg extract, and the least was displayed by fungal isolate #9TAKL-4a with a value of 10±0.12 μg AAE/mg extract. The Nitric Oxide (NO) radical scavenging activity was maximum in #5TAKL-3a with the value of (5.3 ±1.1 mg GAE/mg extract) and minimum value of 0.96 ± 0.1 mg GA/mg extract was exhibited by the fungal isolate #9TAKR-1b. One-way ANOVA showed a significant difference (p<0.05) in the in-vitro anti-bacterial and anti fungal activities among the different drought-tolerant endophytic fungi. Based on morphological characters and multi-locus phylogenetic analysis of the nuclear rDNA internal transcribed spacer region (ITS1-5.8S-ITS2 = ITS), β-tubulin (TUB 2), and RNA polymerase II second largest subunit (RPB2) genes, potent two fungal isolate #5TAKL-3a and #8TAKS-3a was identified as Penicillium citrinum and Talaromyces purpureogenus respectively. However, the other three were identified as Rhizopus oryzae (#TAKR-1a), Edenia gomezpompae (#4TAKI-3a), and Fusarium begoniae (#9TAKR-1b) based on rDNA-ITS gene-based sequencing method. Penicillium citrinum (#5TAKL-3a) and Talaromyces purpureogenus (#8TAKS-3a) were found to possess the highest capability to tolerate drought stress under in-vitro conditions. Hence, based on the PGP attributes and antioxidant potential, Penicillium citrinum and Talaromyces purpureogenus isolates were further selected for further bioinoculant studies through pot trials. The stress group (control+10% PEG) of wheat seedlings (10 days old) showed a decrease in fresh shoots, root weight, shoot and root length, relative water content (RWC), chlorophyll, and carotenoid content in comparison with control (culture un-treated) group. The biochemical parameters in the stressed/test group exhibited increased proline content, MDA level, protein content, total soluble sugars, and reducing sugars. Wheat seedlings treated with Penicillium citrinum (control + #5TAKL-3a group), Talaromyces purpureogenus culture (control+ #8TAKS-3a), and with the bio-consortium (Penicillium citrinum #5TAKL-3a and Talaromyces purpureogenus #8TAKS-3a in 1:1) displayed the enhancement in fresh shoot, root weight, shoot, root length, RWC, and the photosynthetic pigments, viz. chlorophyll and carotenoid content. However, a significant (p<0.05) reduction in protein content, MDA content, total soluble sugars, and reducing sugars was observed. Further, Penicillium citrinum, Talaromyces purpureogenus, and their bio-consortium were encapsulated using sodium alginate and carboxymethyl cellulose (Alg-CMC) and transferred into the pots containing sterile soil and wheat seeds were allowed to grow in pots under normal conditions. At the grain-filling stage, wheat plants were exposed to drought stress induced by 10% and 20% PEG significantly. To analyze the efficacy of culture treatment, various physio biochemical properties such as fresh shoot weight, shoot height, fresh root weight, root length, relative water content, total chlorophyll content and carotenoid content and increased the level of proline, MDA content, total soluble sugars, and reducing sugar and antioxidant enzymatic activities (superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and peroxidases (POX)) were observed. Results indicate that the prepared bio-consortium of fungal isolates (1:1) showed maximum potential to enhance the growth of wheat plants under normal and drought conditions as compared to control (culture un-treated) as well as encapsulated individually culture (Penicillium citrinum and Talaromyces purpureogenus) treated wheat plants. The findings of this study generate ample interest to extend this research to the next level by using a bio-consortium of potent fungal endophytes that have broad applicability across disciplines. This bio-consortium will produce healthy food crops for a better future by lowering the usage of chemicals and groundwater. Thus, it can be utilized as an inexpensive, widely available, and environmentally friendly replacement, leading to sustainable agricult