Potential of Phytohormones producing endophytic fungi as biofertilizers

Abstract

Being a good source of phytohormones fungal endophytes have been considered as potent plant growth promoters. The present report elucidates the isolation of endophytic fungi from medicinal as well as crop plants. In current study 46 endophytic fungal isolates were isolated from medicinal (Cannabis sativa, Chenopodium album, Oxalis corniculata, Euphorbia helioscopia, Amaranthus viridis, And Parthenium hysterophorus) and crop (Triticum asativum, Brassica compestris) plants. Culture filtrated (CF) and biomass was initially screened on maize in order to find the plant growth promoting endophytic fungi and for indole acetic acid. Among these isolate culture filtrate of 13 strains showed promotary effect on maize seedling. The isolates produced IAA via L-tryptophan-independent pathway in rage of 1.254ug/mL to 26.73ug/mL and tryptophan dependent pathway in the range of 0.301ug/mL to 35.434ug/mL. Other important secondary metabolites including phenolic, flavonoid and sugars were also detected in the culture media of these endophytes. The isolated endophytes also supported growth of maize seedlings grown in fungal biomass supplemented soil. Based on their plant growth promoting potential, the endophytes CSR1, CSR3, TS2, CSL1, CHS1, EUR1, TS1 and BRL1 were selected for further study. Culture filtrates of the selected endophytes were screened for gibberellins (GAs) by using GAs deficient mutant rice Waito-C. The results revealed that different plant growth characteristic such as chlorophyll content, root-shoot length, and biomass production of Waito-C rice was significantly promoted during endophyts treatment showing the presence of GAs in their culture filtrate. Majority of these endophytes produced iron chelating siderophores and solubilized phosphate which is among the known phytostimulant tools of the endophytes. These morphologically representative strains were identified further by means of molecular phylogenetic analysis based on ITS and D1/D2 regions. Molecular identification using ITS and partial 28S ribosomal DNA (D1/D2) sequences from the isolates revealed 4 genera including Aspergillus, Fusarium, Bioplaris and Curvularia. These fungi are curvularia lunata TS2, Aspergillus fumigatus TS1, Aspergillus terreus EUR1, Aspergillus flavus CHS1, Aspergillus niger CSR3, Fusarium proliferatum x

BRL1, Fusarium oxysporum CSR1 and Bipolaris sp CSL1. The isolated strains belonged to division Ascomycetes and among these 2 species belong to order Pleosporales, 2 belong to hypocreals and 4 belong to eurotiales. The selected endophytes were further characterized by determining phytohormones including IAA and gibberellins (GAs) using GC/MS SIM. This study is the first report related to curvularia lunata, Aspergillus terreus and Fusarium proliferatum to produce IAA. This growth promotion was due to various types of GAs and presence of IAA in endophyte culture filtrate. The gas chromatography/mass spectrometry (GC/MS) analysis showed the presence of different gibberellins in various quantities (ng/ml). Nine kinds of GAs tested were biologically active: GA1, GA3, GA4, GA7, GA8, GA9, GA12, GA20 and GA24. Biologically active gibberellins include GA1, GA3, GA4, and GA7 were identified in almost all fungi. Moreover, GA1 was detected in high quantity in CSR1 (0.638±0.019 ng/mL), followed by BRL1 (0.392±0.007ng/mL), CSL1 (0.753±0.005 ng/mL) and CHS1 (0.133±0.012ng/mL) respectively. Similarly, GA3 was found in high concentration in two fungi EUR1 (0.352±0.012ng/mL) and TS1 (0.324±0.077ng/mL). Notably, GA4, important bioactive GA, was detected in almost all fungal cultures in significant amount, However, the highest concentrations were found in CSL1 (0.943±0.081ng/mL), CSR3 (0.479±0.01ng/mL) and CHS1 (0.435±0.016ng/mL). Similarly, the fungal strains CSL1, CHS1, EUR1, and BRL1 produced 0.638±0.012, 0.476±0.082, 0.423±0.003 and 0.492±0.005 ng/mL of GA7. Furthermore, Inactive types of GA present in the FCF were GA8, GA9, GA12, GA20 and GA24. The ABA contents were also checked in these isolates and among these fungal strains, CSR1 had the highest concentration of ABA (0.0903ng/ml) in its CF. Upon inoculation, these endophytes contributed significant amount of GAs to the endogenous pool of Waito-C rice. Among these endophytes, CSL1 increased GA1, GA3, GA4 and GA12 concentration up to several fold peaking at 18.37ng/g DW, 11.37ng/g DW, 33.23ng/g DW and 25.534ng/g DW respectively. The isolate CSR3 enhanced endogenous level of GA4, GA7 and GA12 by up to 20.34ng/g DW, 28.264ng/g DW and 34.23ng/g DW as compared to the non-endophytes seedlings. Similarly, TS2 significantly increased the concentration of endogenous GA4 and GA12 of the mutant rice. Along with GAs, endogenous ABA level was also significantly enhanced in Wiato-C rice inoculated with the isolated endophytes than the control. The percent increase by different strains TS2, TS1, BRL1, CSR1, EUR1, CHS1, CSL1 and CSR3 is, 14%, 46.6%, 6.5%, 14%, 67%, 39%, 60.7%, 77.9% respectively. Contrary to this, endogenous concentration of JA dropped significantly in the endophytes associated seedlings indicating involvement of fungal GA and IAA. The percent decrease of JA in treated seedlings as compared to non-treated Waito-C rice seedlings is TS2 (27%), TS1 (53.8%), BRL1 (23.9%), CSR1 (33.5%), EUR1 (61%), CHS1 (49.9%), CSL1 (56%), CSR3 (68%). Furthermore, RT-PCR confirmed the presence of GA and IAA pathways genes (P50-1, P450-4, ggs2, des and iaaH) in the selected endophytic strains by positive expression. Moreover, the application of these fungal spore suspensions with uniconazole and yucasin on maize seedling revealed that like exogenous GA3 and IAA, the endophytic fungal strains CF application mitigated the inhibitory effect of both yucasin and uniconazole and promote growth attributes of maize seedling. Also, secondary metabolites of maize seedlings associated with the endophytic fungi were significantly enhanced recovering the seedlings from the suppressing effects of both uniconazole and yucasin. Besides, the selected strains were checked on cucumber cotyledon to check its cytokine like activity. In these strains some increase chlorophyll contents as compare to control which indicate the presence of cytokinin like compound in them. These finding suggest that these gibberellins and IAA producing endophytic strains plays important roles in plant growth promotion, which could be used for the improvement of crop growth under diverse environmental condition to mitigate the stresses.