Fig 1.
Sample collection and processing for the isolation of fungal endophytes from the frankincense (Boswellia sacra) tree.
(A) Shows the hot and dry habitat of the frankincense tree; (B) resin emerging from the mature leaf of tree branches; (C) the milk-like resin oozing out from the lower epidermal layer of the tree; (D) the inner bark or the cortex region of the tree did not contain resinous ducts and was also evaluated for the presence of endophytes; (E) dried wounded outer bark in which the white milk was converted into crystalline gum (F); leaf pattern of the frankincense tree; (G) smaller parts of the tree (leaf, stem and bark parts) were used for isolation. A total of 208 10-mm tissue samples from the leaf and stem (bark) were used to isolate endophytic fungi. The black line is equivalent to 1 inch.
Table 1.
Endophytic fungal strains isolated from the stem and leaf parts of the Frankincense tree.
Fig 2.
Endophytic distribution and nucleotide homology.
Endophyte isolated from B. sacra, family-wise distribution (A) and comparison of nucleotide composition in the sequence with those from related strains in GenBank (B). A comparative assessment was made with the help of MEGA 6.0 [30].
Fig 3.
Phylogenetic analysis of endophytes.
Maximum Parsimony analysis of isolated endophytes based on the sequences obtained from the sequencing process of the internal transcribed spacer (ITS) region. The phylogeny was constructed using the homologous fungal sequences deposited in GenBank. The percentage of replicate trees in which the associated species clustered together in the bootstrap test (2000 replicates) are shown next to the branches. The analysis involved 84 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 296 positions in the final dataset. Evolutionary analyses were conducted in MEGA 6.0 [30].
Table 2.
Extracellular enzymes production ability of endophytes isolated from the different part of Frankincense tree.
Fig 4.
Principal Component Analysis (PCA) analysis.
PCA showing the correlation between different endophytic fungi and their enzyme production abilities. In PCA, a full cross-validation was used to validate the enzyme production ability among different species.
Fig 5.
Indole acetic acid (IAA) content in the culture filtrate of various endophytic fungal strains.
L-tryptophan-dependent Czapek media was used to grow fungal strains for 7 days. A standard in the same media was also read to yield a standard curve (R2 = 0.9985). A total of five replications (100 ml in each Erlenmeyer flask) were used to make sure the validation of IAA results. The bars representing different letters show that the mean values are significantly different from each other, as evaluated by the DMRT test (P<0.05).
Fig 6.
Chromatogram of the culture filtrate of BSS6 (Aureobasidium sp.) showing the correlation between the IAA peak and the standard IAA peak in the same fungal growth media and in water.
Fig 7.
Influence of endophyte inoculation on Host growth.
Effect of the application of endophytic fungus (Preussia sp. BSL10) on the growth dynamics and photosynthetic pigments of Boswellia sacra tree saplings. Each treatment included nine replications. The photosynthetic pigments were measured after 21 DAT. The graphs show the mean value of three replications with standard deviations. 0 DAT shows the readings without any treatments applied.