Fungal endophytes were isolated from medicinal plants
A total of 58 different fungal endophytes were isolated from various medicinal parts (Table 1, Fig. 1). Maximum fungal endophytes were recovered from Pinus sp. (31.0%) followed by C. tamala (27.5%) and R. arboreum (25.8%). However least fungal colonization was observed in C. verum (5.1%). The host tissue of each plant sample exhibited a variation in colonization of the endophytic mycoflora. Further, colonization in different parts of plants was estimated. It was observed that maximum fungal colonization was observed in stem (62.1%) followed by leaf (24.1%). However, bark and stem internal tissues were least colonized by endophytes (Fig. 2).
Table 1
Details of fungal endophytes along with assigned culture code, plant name, plant part and sample collection site recovered from different medicinal plants under study
S.NO. | CULTURE CODE | PLANT NAME | PART | PLACE |
1. | # 7 RALFB | R. arboberum | Leaf | Bhowali |
2. | # 18 RALFB | R. arboberum | Leaf | Bhowali |
3. | # 21 RALFB | R. arboberum | Leaf | Bhowali |
4. | # 25 RALFB | R. arboberum | Leaf | Bhowali |
5. | # 1 RASTB | R. arboberum | Stem | Bhowali |
6. | # 4 RASTB | R. arboberum | Stem | Bhowali |
7. | # 5 RASTB | R. arboberum | Stem | Bhowali |
8. | # 11 RASTB | R. arboberum | Stem | Bhowali |
9. | # 15 RASTB | R. arboberum | Stem | Bhowali |
10. | # 17 RASTB | R. arboberum | Stem | Bhowali |
11. | # 26 RASTB | R. arboberum | Stem | Bhowali |
12. | # 27 RASTB | R. arboberum | Stem | Bhowali |
13. | # 28 RASTB | R. arboberum | Stem | Bhowali |
14. | # 29 RASTB | R. arboberum | Stem | Bhowali |
15. | # 9 RASTITB | R. arboberum | Stem internal tissue | Bhowali |
16. | # 2a PSSTB | Pinus sabiniana | Stem | Bhowali |
17. | # 2b PSSTB | Pinus sabiniana | Stem | Bhowali |
18. | # 4 PSSTB | Pinus sabiniana | Stem | Bhowali |
19. | # 5 PSSTB | Pinus sabiniana | Stem | Bhowali |
20. | # 7 PSSTB | Pinus sabiniana | Stem | Bhowali |
21. | # 9 PSSTB | Pinus sabiniana | Stem | Bhowali |
22. | # 13c PSSTB | Pinus sabiniana | Stem | Bhowali |
23. | # 14b PSSTB | Pinus sabiniana | Stem | Bhowali |
24. | # 14c PSSTB | Pinus sabiniana | Stem | Bhowali |
25. | # 15a PSSTB | Pinus sabiniana | Stem | Bhowali |
26. | # 15b PSSTB | Pinus sabiniana | Stem | Bhowali |
27. | # 15c PSSTB | Pinus sabiniana | Stem | Bhowali |
28. | # 16 PSSTB | Pinus sabiniana | Stem | Bhowali |
29. | # 16a PSSTB | Pinus sabiniana | Stem | Bhowali |
30. | # 17c PSSTB | Pinus sabiniana | Stem | Bhowali |
31. | # 08 PSSTITB | Pinus sabiniana | Stem internal tissue | Bhowali |
32. | # 10 PSSTITB | Pinus sabiniana | Stem internal tissue | Bhowali |
33. | # 18a PSSTITB | Pinus sabiniana | Stem internal tissue | Bhowali |
34. | # 1 OTLFN | O. tenuiflorum | Leaf | Nainital |
35. | # 14 OTLFN | O. tenuiflorum | Leaf | Nainital |
36. | # 16 OTLFN | O. tenuiflorum | Leaf | Nainital |
37. | # 18 OTLFN | O. tenuiflorum | Leaf | Nainital |
38. | # 10 OTSTN | O. tenuiflorum | Stem | Nainital |
39. | # 15 OTSTN | O. tenuiflorum | Stem | Nainital |
40. | # 1 CTLFN | C. tamala | Leaf | Nainital |
41. | # 15 CTLFN | C. tamala | Leaf | Nainital |
42. | # 18 CTLFN | C. tamala | Leaf | Nainital |
43. | # 21 CTLFN | C. tamala | Leaf | Nainital |
44. | # 27 CTLFN | C. tamala | Leaf | Nainital |
45. | # 32 CTLFN | C. tamala | Leaf | Nainital |
46. | # 2 CTSTN | C. tamala | Stem | Nainital |
47. | # 4 CTSTN | C. tamala | Stem | Nainital |
48. | # 5 CTSTN | C. tamala | Stem | Nainital |
49. | # 6 CTSTN | C. tamala | Stem | Nainital |
50. | # 8 CTSTN | C. tamala | Stem | Nainital |
51. | # 10 CTSTN | C. tamala | Stem | Nainital |
52. | # 11 CTSTN | C. tamala | Stem | Nainital |
53. | # 13 CTSTN | C. tamala | Stem | Nainital |
54. | # 14 CTSTN | C. tamala | stem | Nainital |
55. | # 11 CTSTITN | C. tamala | Stem internal tissue | Nainital |
56. | # 5 CVBN | C. verum | Bark | Nainital |
57. | # 6a CVBN | C. verum | Bark | Nainital |
58. | # 18 CVBN | C. verum | Bark | Nainital |
Each endophytic fungal isolate was encoded based on the host plant, its part (LF for Leaf, ST for stem, STIT for stem internal tissue and B for Bark) and the place from where it was collected. For instance in #7 RALFB, #7 refers to the segment number, RA refers to the host medicinal plant scientific name, LF refers to leaf as explants for isolation and B refers to Bhowali the place of collection |
Screening Of Protease Producing Endophytic Fungal Isolates
In the preliminary screening studies, 31 endophytes out of 58 exhibited proteolytic activity (Table 2; Fig. 3). As per One-way ANOVA analysis [F (38,76) = 231.4, < 0.001] and Tukey’s post hoc analysis, #7PSSTB exhibited relatively highest protease production with zone size of 24 mm followed by #13CTSTN and #6CVSTN with the zone size of 21.6 mm and 20.6 mm respectively. Further, moderate level of activity was observed in #15bPSSTB and #18OTLFN and least activity was recorded in #1RASTB, #4PSSTB, #8PSTITB and #5CTSTN.
Table 2
in vitro enzyme production of 40 fungal endophytes (out of total 58 endophytes, 18 endophytes did not exhibited any activity)
S.NO. | CULTURE CODE | Average Zone size (in mm) |
PROTEASE | AMYLASE | CELLULASE | L-ASPARAGINASE |
1. - | # 21 RALFB | - | - | 12.3 ± 0.58 d | 17.6 ± 0.58 b |
2. | # 25 RALFB | 8.3 ± 0.58 jk | - | - | 20.0 ± 0.0 a |
3. | # 1 RASTB | 7.0 ± 0.0 jk | 13.0 ± 1.0 a | 8.0 ± 0.0 h | - |
4. | # 4 RASTB | 14.3 ± 0.58 defg | - | - | 19.6 ± 0.58 a |
5. | # 5 RASTB | 9.0 ± 0.0 ijk | 12.3 ± 0.58 ab | 10.0 ± 0.0 f | - |
6. | # 9 RASTB | - | - | - | 20.5 ± 1.52a |
7. | # 11 RASTB | - | - | - | 20.0 ± 0.0 a |
8. | # 17 RASTB | 12.0 ± 0.0 gh | - | 10.0 ± 0.0 f | 20.0 ± 0.0 a |
9. | # 26 RASTB | 13.3 ± 0.58 efgh | - | 8.0 ± 0.0 h | 15.3 ± 0.58 c |
10. | # 27 RASTB | 11.3 ± 0.58 hi | - | 8.0 ± 0.0 h | 16.0 ± 0.0bc |
11. | # 28 RASTB | 9.3 ± 0.58 ij | - | 9.0 ± 0.0 g | - |
12. | # 29 RASTB | - | - | 9.0 ± 0.0 g | - |
13. | # 2b PSSTB | 12.0 ± 0.0 gh | - | - | - |
14. | # 4 PSSTB | 6.67 ± 0.58 k | - | - | - |
15. | # 7 PSSTB | 24.0 ± 1.0a | 11.0 ± 1.0 c | 17.0 ± 0.0 a | - |
16. | # 9 PSSTB | 8.67 ± 0.58 jk | - | - | - |
17. | # 14b PSSTB | 15.67 ± 1.52 cde | 7.3 ± 1.1 d | - | - |
18. | # 14c PSSTB | 12.6 ± 1.15 fgh | - | - | - |
19. | # 15a PSSTB | - | - | - | - |
20. | # 15b PSSTB | 18.0 ± 0.0 c | - | - | - |
21. | # 15c PSSTB | 9.0 ± 1.0 ijk | - | - | - |
22. | # 16 PSSTB | - | 12.0 ± 0.0 b | 11.6 ± 0.58 de | - |
23. | # 16a PSSTB | - | - | 15.6 ± 0.58 b | - |
24. | # 8 PSSTITB | 7.0 ± 0.0 jk | - | - | - |
25. | # 10 PSSTITB | 8.0 ± 0.0 jk | - | 9.0 ± 0.0 g | - |
26. | # 18a PSSTITB | 15.0 ± 2.0 def | - | - | - |
27. | # 14 OTLFN | 12.0 ± 0.0 gh | - | - | - |
28. | # 18 OTLFN | 18.0 ± 1.0c | - | - | - |
29. | # 10 OTSTN | - | - | - | - |
30. | # 15 OTSTN | 7.3 ± 0.58 jk | - | - | - |
31. | # 1 CTLFN | 15.0 ± 1.0 def | - | - | 8.0 ± 0.0 d |
32. | # 32 CTLFN | 16.0 ± 1.0 cd | - | - | - |
33. | # 5 CTSTN | 7.0 ± 0.0 jk | - | - | - |
34. | # 6 CTSTN | 20.6 ± 0.58b | - | 14.6 ± 0.58 c | - |
35. | # 8 CTSTN | 14.0 ± 0.0 defg | - | - | - |
36. | # 10 CTSTN | - | - | 10.0 ± 0.0 f | - |
37. | # 13 CTSTN | 21.6 ± 0.58ab | - | 14.6 ± 0.58 c | - |
38. | # 14 CTSTN | 15.6 ± 0.58cde | - | - | - |
39. | # 5 CVBN | 12.3 ± 2.08 gh | - | - | - |
40. | # 6a CVBN | 12.0 ± 0.0 gh | - | 11.0 ± 1.0 e | |
*Data presented as mean ± standard deviation of three replicates. Means with different superscript letters are different by Tukey’s post-hoc test (p < 0.05). |
Screening Of Amylase Producing Endophytic Fungal Isolates
In the amylolytic screening assay, only 5 endophytes exhibited the amylase producing potential. As per One-way ANOVA analysis [F (38,76) = 469.2, < 0.001] and Tukey’s post hoc analysis, #1 RASTB was found to be potent amylase producer with zone size of 13.0 mm followed by #5 RASTB with zone size of 12.3 mm (Fig. 4a-b). However, least activity was observed in #14b PSSTB.
Screening Of Cellulase Producing Fungal Endophytic Isolates
Cellulose is imperative in leather, detergents and food processing industries. In the cellulolytic activity, 15 endophytes out of 58 showed positive results. As per One-way ANOVA analysis [F (38,76) = 1473, < 0.001] and Tukey’s post hoc analysis, maximum activity was recorded in #7 PSSTB with a zone size of 17.0 mm followed by #16a PSSTB with zone size of 16.3 mm. However least activity was recorded in #26RASTB and #27RASTB with zone size of 8.0 mm (Fig. 4c-f).
Screening Of L-asparaginase Producing Fungal Endophytes
The isolated endophytic fungal isolates were screened for the production of L-asparaginase enzyme. In the L-asparaginase screening assay, 9 endophytes were found to be potent L-asparaginase producer. One-way ANOVA analysis [F (9,18) = 130.3, < 0.001] and Tukey’s post hoc analysis revealed maximum L-asparaginase production in #9 RASTB with a zone size of 20.3 mm followed by #11 RASTB and #17 RASTB with zone size of 20 mm (Table 2; Fig. 5).
Bioactivity Screening Of Partially Purified Protein
Further the protein purification was done from the endophytic fungal isolates using salting out method and evaluated for enzyme activities. The partially purified protein of #7PSSTB, opted due to consistent best results in all the three screenings, was again subjected for bioactivity profiling. In protease, amylase and cellulase activity assay, zone size of 16 mm, 12 mm and 16 mm respectively was obtained (Fig. 6).