3.1. Diversity of Endophytic Fungi in Dillenia indica L.:
The present study about the diversity of endophytic fungi from Dillenia indica L. is the first of its kind, as per our literature survey. There is a high diversity of endophytic fungi associated with different plant parts of Dillenia indica L. A total of three hundred and ninety-nine (798) fungal endophytes were isolated from the different surface-sterilized explants of Dillenia indica L. Some of these isolates were morphologically similar after macroscopic and microscopic examinations. Fungal endophytes with identical characteristics were marked as repeats, with only those isolates that differed morphologically being identified as different endophytic fungal isolates. As a result of this, only twenty-five (25) different fungal endophytes were isolated from Dillenia indica L., which comprises 25 different species, namely Curvularia lunata, Cladosporium cladosporioides, Alternaria alternata, Colletotrichum gloeosporioides, Bipolaris crotonis, Fusarium oxysporum, Chaetomium globosum, Trichoderma viride, Clonostachys rosea, Diaporthe phaseolorum, Lasiodiplodia theobromae, Schizophyllum cummne, Phomopsis sp., Colletotrichum gigasporum, Fomitopsis meliae, Fusarium brchygibbosum, Pseudofusicoccum adensoniae, Daldinia eschscholtzii, Nigrospora sphaerica, Xylaria longipes, Neopestalotiopsis clavispora, Alternaria tenuissima, Aspergillus fumigatus, Colletotrichum musae, and Colletotrichum boninense from fresh tissues of plant. Colletotrichum gloeosporioides, Cladosporium cladosporioides, Lasiodiplodia theobromae were the most frequently occurring species during the study (Fig. 1). All the isolated fungi belong to four classes i.e., Sordariomycetes, Dothideomycetes, Agaricomycetes and Eurotiomycetes (Fig. 2). Some genera (Curvularia, Fusarium and Alternaria), which are cosmopolitan, are also obtained as endophytes in the current study. These taxa have already been reported by various researchers from tropical plants (Ferreira et al., 2015). Similarly, Arora et al., 2019 isolated fungal endophytes from Glycyrrhiza
glabra L. and found that the genus Phoma had a higher affinity than Fusarium. Wang et al., 2015 isolated twenty-four culturable endophytic fungi from Oryza rufipogon Griff and screened for their antagonistic activity against phytopathogens. One hundred fungal endophytes were isolated from black seed (Nigella sativa L.), and Penicillium, Alternaria and Cladosporium were predominant genera. All the isolated fungi belong to the phylum Ascomycota (Gopane et al., 2021). Endophytic fungi from Gentiana rigescens exhibited good cytotoxicity activity against different cell lines (Xu et al. 2020). Jagannath et al. 2020 isolated two hundred and three endophytic fungi belonging to twenty-nine species from Baliospermum montanum and found that the colonization and isolation rate was higher in stem followed by seed, root, leaf and flower. These fungi were also screened for amylase, lipase, protease, cellulase and phosphatase activity.
Fungal endophytes isolated from various tissues of the plant were identified morphologically to generic/species level. The fungal endophytes which could not be identified morphologically were subjected to molecular characterization (Tables 1–2). The ITS sequences of different isolates obtained were subjected to BLAST analysis, which revealed their homology with similar species. The ITS sequences have been deposited in the GenBank database, for which accession numbers have been provided and phylogenetic tree was constructed (Fig. 16–17).
Table 1
The endophytic fungi identified based on morphological characteristics, isolated different parts of Dillenia indica L.
S.No. | Endophytic fungi | Spore characteristics |
1. | Curvularia lunata | Conidia 14–26 × 8.5–13 µm, 3–5 celled, asymmetrical to more or less curved at the third cell from the base, intermediate cells dark brown and usually smooth-walled. |
2. | Cladosporium cladosporioides. | Conidia solitary or catenate, in unbranched or branched acropetal chains, usually obovoid, ellipsoid, fusiform, 3.0–6.0 × 2–4 µm. |
3. | Alternaria alternata. | Conidia are pale to brown in colour, smooth or verruculose, usually 2–8 transverse and several longitudinal or oblique septa, 15–58 × 7.5–16 µm. |
4. | Colletotrichum gloeosporioides | Conidiophores cylindrical, hyaline to subhyaline, up to 30 µm long. Conidia were cylindrical, straight oval borne on hyaline conidiophores having 14–16 um in length and 5–6 um diameter. |
5. | Bipolaris crotonis | Conidia olivaceous brown to pale brown, smooth uniformly pigmented, broadly ellipsoidal, 6–11 (mostly 7–9) distoseptate, (48) 80–110 × 18–29µm. |
6. | Chaetomium globosum | Asci fasciculate, fusiform or clavate, spore-bearing part 25–40 × 5–10µm, stalks 15–20 µm long. Each ascus consists of eight biseriate to irregularly-arranged ascospores, which are olivaceous brown when mature, limoniform, usually biapiculate, bilaterally flattened, 7–8.5 × 5–8µm, with an apical germ pore. |
7. | Fusarium oxysporum | Asci fasciculate, fusiform or clavate, spore-bearing part 25–40 × 5–10µm, stalks 15–20 µm long. Each ascus consists of eight biseriate to irregularly-arranged ascospores, which are olivaceous brown when mature, limoniform, usually biapiculate, bilaterally flattened, 7–8.5 × 5–8µm, with an apical germ pore. |
8. | Clonostachys rosea | Conidiophore dimorphic i.e. verticillium-like and penicillate- like. Conidia globose to sub globose having size 3–8 × 2–4 µm. |
9. | Alternaria tenuissima. | Conidia are dilute tan to brown with dark walls mostly smooth, occasionally verruculose, 4–7 transverse and several longitudinal or oblique septa, overall length 22–75 × 8–15 µm thick in the broadest part swollen apex 4–5 µ wide. |
10. | Aspergillus niger | Conidiophore smooth, non-septate, thick walled, globose vesicle. Conidia globose to sub globose, black ,4–5 µm. |
11. | Trichoderma viridae. | Colonies are initially white later turn light green in colour. Conidiophore arise from undifferentiated aerial mycelium. Phialides are straight or sinus or hooked. The terminal phialide are whorl or solitary phialide are cylindrical. Conidia sub-globose to obovoid having 29 − 3.8 x 2.8–3.4. |
12. | Neopestalotiopsis clavispora | Colonies are white to pale yellow in colour, raised and fluffy, conidia 5 celled fusiform, basal cell is hyaline, 2–3 unbranched arise from the apical cell. |
Table 2
The endophytic fungi identified based on morphological characteristics, isolated different parts of Dillenia indica L.
S.No. | Fungal identification | Code | NCBI Accession no | Identity %age |
1. | Diporthe phaseoli | DLP21S4a1 | MK757156 | 97% |
2. | Schizophyllum communae | DSP22S3a1 | MK756215 | 99% |
3. | Phomopsis sp. | DLP24S2a1 | MK757169 | 98% |
4. | Fomitopsis meliae | DLP30S2a1 | MK757195 | 98% |
5. | Nigrospora sphaerica | DLP46S2a1 | MK757157 | 100% |
6. | Lasiodiplodia theobromae | DSP22S4a1 | MK644105 | 99% |
7. | Fusarium brachygibossum | DLP41S3a1 | MK757199 | 99% |
8. | Colletotrichum gigasporum | DSP26S2a1 | MK756322 | 99% |
9. | Xylaria longipilus | DLP41S2a1 | MK756123 | 99% |
10. | Pseudofussicoccum adansoniae | DSP42S2a1 | MK757196 | 99% |
11. | Daldinia eschscholzii | DSP40S2a1 | MN854982 | 99.6% |
12. | Colletotrichum gleosporides | DLP31S1a1 | MN855105 | 99.6% |
13. | Colletotrichum boninense | DSP32S3a3 | MW521131 | 99.87% |
There was a significant effect of host tissues on the colonization of fungal endophytes. A total of two hundred forty-nine (498) isolates belonging to 24 taxa were isolated from the leaves, one hundred fourteen (228) isolates having 16 species from the stem and thirty-six (72) isolates belongs to 6 species from fruits. The isolation rate was 5.85 %, 3.90 % and 1.21 found for leaves, stems and fruits, as shown below in Fig. 2. Colonization rate was found 55.6%, 25.6% and 08.8% for leaves, stems and fruits. The values of percentage frequency were 96% for leaves, 64% for stems and 20% for fruits. Thus, our results clearly show that fungal endophytes are more in number in leaves than stems and fruits. The colonization frequency was 61.2, 28.9 ad 9.01 for leaves, stems and fruits. The values of diversity indices i.e., Simpson Index, Shannon Index and evenness index, indicates that the tissues are highly diverse. The highest value of the Shannon index was observed in leaves i.e., 2.77, whereas the lowest value was 1.07 and was recorded in fruits. The present data also revealed the highest Shannon- Wiener diversity index in the leaves, which suggests that the leaves have a more diverse endophytic fungal community in contrast to the fruits and bark.
The study showed that the composition of fungal endophytes in Dillenia indica L. was influenced by the seasons. Monsoon (Rainy season) had the highest number of isolates (312 isolates), followed by summer (208 isolates), Winter (164 isolates) and Autumn (115 isolates). During the rainy season, the dominating taxa were Colletotrichum gloeosporioides, Neopestalotiopsis clavispora, Fusarium brchygibbosum and Daldinia eschscholtzii followed by Alernaria alternata, Fusarium oxysporum, Chaetomium globosum and Alternaria tenuissima. Species diversity was highest during the rainy season (19 species) and lowest during the Winter (12 species). Different numbers of fungal endophytes were obtained during different months of the year. The maximum isolates were obtained during September, followed by August, March and April month and the minimum during July month. Some taxa of the endophytic fungi were reported in only one or two seasons. For example, Colletotrichum boninense was exclusively reported during Winter, whereas the Colletotrichum gloeosporioides was reported in all the seasons. Schizophyllum commune was found in the Rainy and Winter seasons.
The highest colonization frequency was in the rainy season (37.89%), followed by Autumn (32.5), summer (30.3%) and Winter (25%). However, species diversity data show a somewhat correlation with the number of isolates observed during different seasons. The isolation rate ranges from 4.0 to 6.0%, and the percentage frequency varies from 48% to 76 % during different seasons. The colonization rate from 25.0–38.4% of endophytic fungi was obtained from in the present study (Fig. 3–9). It is important to note that the seasons have a direct effect on the composition of fungal communities. The seasonal variation in colonization patterns may be due to the seasonal activities of fungal endophytes. During rainy seasons, greater endophytic fungal diversity was observed in Terminalia arjuna twigs and bark (Tejesvi et al., 2005) and Centella asiatica leaves (Gupta and Chaturvedi, 2017). The rainy and summer seasons, as previously stated, support high endophytic fungi expression (Kim et al. 2013, Singh et al. 2016, Schulthess et al. 1998, Higgins et al., 2011). It has been reported that during the rainy seasons, high moisture and temperature support the growth and dispersal of fungal endophyte spores (Mishra et al., 2012). However, several studies have found that endophytic fungal diversity is substantially higher in the Winter months than in the rainy and summer months (Naik et al., 2008; Fang et al., 2013). The seasonal variation in fungal endophytic diversity may be due to secondary metabolite levels fluctuate throughout the year (Fang et al., 2013). Seasons influence endophytic antibacterial communities in medicinal plants (Barman and Dkhar 2020).
The majority of the isolated fungi belonged to Ascomycota (83.33%), which is identical to the findings of Goveas et al. (2011) from the threatened plant Coscinium fenestratum. Only 2% belonged to Basidiomycota. Sordariomycetes and Dothideomycetes were the main classes of Ascomycota in the endophytic assemblage, as revealed from the results of Li et al. (2016). The Shannon-wiener index for leaves, fruits and stems were 2.60, 1.598 and 1.077, while Simpson’s diversity index was 0.919, 0.776 and 0.892 for leaves, stems and fruits. The evenness index was 0.937, 0.892 and 0.980 for leaves, bark and fruits (Fig. 10–15). It was seen that only one or a few species dominated the endophytic community of the host, whereas the majority of them were rare (Petrini et al. 1992). Numerous factors such as biotic, abiotic, chemical composition and architecture of host tissues are involved in the deviation of colonization of endophytic fungi in specific tissues (Sanchez–Azofeifa 2012, Liu et al. 2012).
Extracellular enzymes productions:
The endophytes produce a variety of bioactive molecules, which include enzymes; microbial enzymes help in hydrolysis and biodegradation processes and facilitate their colonization inside the host plant tissues. Endophyte obtains nutrition from plant tissues and helps plants against biotic and abiotic stress tolerance. (Sunitha et al., 2013).
The isolated endophytic fungi were screened for extracellular enzymes like amylase, lipase, protease, asparaginase, cellulase and ligninolytic enzymes by the agar plug method. All the fungi can produce one or other extracellular enzymes. The results showed that 73%, 40%, 33%, 73%, 46%, 40% of the isolates were positive for amylase, lipase, protease, Asparaginase, cellulase and ligninolytic enzymes respectively (Table 3 and Fig. 18). Fomitopsis meliae exhibited maximum amylase activity whereas Phomopsis sp., Curvularia lunata, Schizophyllum commune, Daldinia eschschozia exhibited significant activity. The highest activity for cellulase and lignin was shown by Schizophyllum commune, Daldinia eschschozia, Colletotrichum gigasporum and Schizophyllum commune showed significant activity. The highest activity for protease and lipase was shown by Lasiodiplodia theobromae and Schizophyllum commune. Most of the isolates showed asparaginase activity. The results indicate that the fungi from Dillenia indica L. are a promising source of extracellular enzymes having an immense value in pharmaceutical and industrial applications. Similarly, Uzma et al. (2016) screened 112 fungal endophytes for different enzymes such as cellulase, laccase, pectinase, amylase and asparaginase. From them, 29%, 28%, 18%, 40% and 0% were showed positive activity for cellulase, laccase, pectinase, amylase and asparaginase activity. Thus, these enzymes help to invade the plant tissues. These enzymes also suppress plant pathogens. Endophytes might behave as latent saprophytes; when the host dies, they use these enzymes to degrade the plant tissues to obtain nutrients (Vazquez de Aldana et al., 2013). Aspergillus sp. isolated from corneal ulcers/ keratitis produces protease, lipase, DNase, elastase, and keratinase (Sangeetha 2020).
Table 3
Extracellular enzymatic activity of isolated endophytic fungi.
S.No. | FUNGUS | ENZYMES |
Amylase | Lipase | Protease | Asparaginase | Cellulase | Ligninolytic |
1. | Curvularia lunata | - | - | - | ++ | - | - |
2. | Daldinia eschscholtzii | ++ | +++ | ++ | +++ | ++ | ++ |
3. | Lasiodiplodia theobromae | +++ | ++ | +++ | + | + | - |
4. | Schizophyllum commune | +++ | - | - | - | +++ | +++ |
5. | Cladosporium cladosporioides | - | + | + | ++ | - | - |
6. | Colletotrichum gigasporum | + | | - | + | ++ | ++ |
7. | Diaporthe phaseolorum | ++ | - | - | + | +++ | +++ |
8. | Colletotrichum gleosporides | + | - | +++ | +++ | + | - |
9. | Xylaria longipilus | + | - | ++ | ++ | ++ | + |
10. | Trichoderma viride | ++ | ++ | + | ++ | + | - |
11. | Fomitopsis meliae | ++ | + | + | +++ | ++ | + |
12. | Fusarium oxysporum | - | - | - | - | - | - |
13. | Neopestalotiopsis clavispora | ++ | - | - | ++ | + | - |
14. | Chaetomium globosum | - | + | - | ++ | + | - |
15. | Nigrospora sphaerica | ++ | +++ | ++ | +++ | - | - |
- No activity, + less activity, ++ good activity. +++ significant activity. |