Isolation and identification of endophytic fungi
A total of 101 endophytic fungal isolates belonging to 18 genera and 35 fungal species were isolated from vegetative organs of 11 selected medicinal plants growing naturally on the north-western coast of Egypt (Table 1). The isolated genera namely, Alternaria, Phoma, Mucor, Scytalidium, Pythium, Ulocladium, Spegazzinia, Sarcinomyces Aspergillus, Cladosporium, Penicillium, Clafosporium, Rhizopus, Fusarium, Nigrosora, Trichocladium, Leptosphaeria and Aureobasidium.
Table 1
List of endophytic fungal species isolated from 11 selected medicinal plants growing naturally on the north-western coast of Egypt
Name of plants | Plant organs | Fungal code No. | Name of fungal isolates |
A- Scorpiurus muricatus L. | Fruits | 1 | Alternaria pluriseptata |
Stem | 2 3 | Phoma dennissii Alternaria pluriseptata |
Leaves | 4,5 6 7 | Alternaria phragmospora Alternaria alternaria Alternaria pluriseptata |
B- Faganiu cretica L. | Leaves | 8 9 10 11 16 | Alternaria pluriseptata Phoma glomerata Scytalidium japonicum Pythium nayoroense Ulocladium atrum |
Stem | 12 13 | Alternaria pluriseptata Alternaria chlamydospora |
Fruits | 14 15 | Mucor hiemalis Alternaria pluriseptata |
C- Mellilotus indicus (L.)All. | Leaves | 17 18 19 20 21 22 | Penicillium aurantiogriseum Clafosporium cladosporioides Rhizopus stolonifer Sarcinomyces phaeomriformis Alternaria pluriseptata Aspergillus niger |
Stem | 23 24 | Penicillium aurantiogriseum Penicillium marneffei |
Fruits | 25 26 27 28 29 30 31 | Alternaria pluriseptata Alternaria phragmospora Spegazzinia pakeri Sarcinomyces phaeomriformis Cladosporium tenuissium Penicillium expansum Penicillium roqueforti |
D- Carduus getulus Pomel | Stem | 32 33 34 | Alternaria pluriseptata Ulocladium alternariae Rhizopus oryzae |
Leaves | 35 36 | Alternaria pluriseptata Rhizopus microspores |
Fruits | 37 38 39 40 | Pythium elongutum Pythium nayoroerse Alternaria pluriseptata Pencillium expansum |
E- Pancratium maritimum L. | Pulp | 41 42 | Mucor hiemalis Aspergillus niger |
Leaves | 43 44 45 46 47 48 | Alternaria pluriseptata Alternaria alternaria Pythium nayoroerse Cladosporium nigrellam Cladosporium cladosporioides Penicillium marneffei |
F- Asphodelus aestivus Brot. | Leaves | 49 50 51 52 53 54 | Fusarium verticilloioides Cladosporium nigrellam Rhizopus stolonifer Alternaria pluriseptata Ulocladium alternariae Fusarium oxysporum |
Stem | 55 56 57 58 59 60 | Fusarium verticilloioides Fusarium oxysporum Alternaria pluriseptata Cladosporium nigrellam Rhizopus stolonifer Penicillium raistrickii |
G- Lotus polyphyllos E. D. Clarke | Stem | 61 62,63 | Alternaria pluriseptata Ulocladium alternariae |
Leaves | 64 65 66,67 | Ulocladium alternariae Alternaria pluriseptata Aspergillus niger |
H- Lycium europaeum L. | Leaves | 68 69 70 71 72 73 74 75 76 77 | Rhizopus stolonifer Trichocladium asperum Pythium nayoroerse Nigrosora sphaeria Mucor hiemalis Rhizopus stolonifer Penicillium marneffei Ulocladium atrum Alternaria chlamydospora Aspergillus niger |
Stem | 78 79 80 81 82 | Alternaria alternata Rhizopus stolonifer Alternaria pluriseptata Nigrosora sphaeria Ulocladium atrum |
J- Echium angustifolium Mill. | Leaves | 83 84 85 86 87 | Aspergillus terrus Coccidioides immitis Rhizopus stolonifer Alternaria pluriseptata Spegazzinia pakeri |
K- Nicotiana glauca Graham | Leaves | 88 89 90 91 92 | Cladosporium nigrellam Rhizopus stolonifer Cladosporium sphaerospermum Alternaria pluriseptata Spegazzinia pakeri |
Stem | 93 94 95 96 97 | Alternaria chlamydospora Alternaria pluriseptata Ulocladium atrum Leptosphaeria senegalensis Aureobasidium pellalanus |
L- Ononis vaginalis Vahl, Symb. | Leaves | 98 99 100 101 | Alternaria pluriseptata Ulocladium atrum Aspergillus niger Cladosporium nigrellam |
Endophytes frequency and diversity indices
A list of 101 isolates and their frequencies, relative frequencies were recorded in the investigated plant tissues. The detailed results summarized in Table 2, revealed that 56% isolates were obtained from plant leaf tissues, 27% from the stem, 15% from fruits and 2% from pulps. The dominant endophytic species recorded with high relative frequencies were Alternaria pluriseptata (20%) with 14 active isolates against the tested pathogens, Rhizopus stolonifer (8%) four of them were active, Aspergillus niger (6%), four of them were active, Ulocladium atrum, Cladosporium nigrellam, and Ulocladium alternariae share the same relative frequency (5%) and gave positive results for two, one and two as active strains respectively and Pythium nayoroense (4%) with 50% active isolates. The rest of the listed endophytic fungi were recorded with lower relative frequencies.
Table 2
Isolated fungi, frequency (F), relative frequency (RF), isolation rate (IR %), Richness (S), Species diversity, Shannon index (H), Simpson diversity (1-D) and Evenness values (E) tissues.
NO | Endophyte species | (F) | R.F (%) | organs | No of active isolates |
Fruit | Leave | stem | pulp |
1 | Alternaria pluriseptata | 21 | 20 | 4 | 10 | 7 | 0 | 14 |
2 | Phoma dennissii | 1 | 1 | 0 | 0 | 1 | 0 | 1 |
3 | Alternaria phragmospora | 3 | 3 | 1 | 2 | 0 | 0 | 3 |
4 | Alternaria alternaria | 3 | 3 | 0 | 2 | 1 | 0 | 2 |
5 | Phoma glomerata | 1 | 1 | 0 | 1 | 0 | 0 | 1 |
6 | Scytalidium japonicum | 1 | 1 | 0 | 1 | 0 | 0 | 1 |
7 | Pythium nayoroense | 4 | 4 | 1 | 3 | 0 | 0 | 2 |
8 | Ulocladium atrum | 5 | 5 | 0 | 3 | 2 | 0 | 2 |
9 | Alternaria chlamydospora | 3 | 3 | 0 | 1 | 2 | 0 | 2 |
10 | Mucor hiemalis | 3 | 3 | 1 | 1 | 0 | 1 | 1 |
11 | Penicillium aurantiogriseum | 2 | 2 | 0 | 1 | 1 | 0 | 1 |
12 | Cladosporium cladosporioides | 2 | 2 | 0 | 2 | 0 | 0 | 1 |
13 | Rhizopus stolonifer | 8 | 8 | 0 | 6 | 2 | 0 | 4 |
14 | Sarcinomyces phaeomriformis | 2 | 2 | 1 | 1 | 0 | 0 | 2 |
15 | Aspergillus niger | 6 | 6 | 0 | 5 | 0 | 1 | 4 |
16 | Penicillium marneffei | 3 | 3 | 0 | 2 | 1 | 0 | 3 |
17 | Spegazzinia pakeri | 3 | 3 | 1 | 2 | 0 | 0 | 3 |
18 | Cladosporium tenuissium | 1 | 1 | 1 | 0 | 0 | 0 | 0 |
19 | Ulocladium alternariae | 5 | 5 | 0 | 2 | 3 | 0 | 1 |
20 | Rhizopus oryzae | 1 | 1 | 0 | 0 | 1 | 0 | 1 |
21 | Rhizopus microspores | 1 | 1 | 0 | 1 | 0 | 0 | 0 |
22 | Pythium elongutum | 1 | 1 | 1 | 0 | 0 | 0 | 1 |
23 | Cladosporium nigrellam | 5 | 5 | 0 | 4 | 1 | 0 | 2 |
24 | Fusarium verticilloioides | 2 | 2 | 1 | 1 | 0 | 0 | 2 |
25 | Fusarium oxysporum | 2 | 2 | 0 | 1 | 1 | 0 | 0 |
26 | Penicillium raistrickii | 1 | 1 | 0 | 0 | 1 | 0 | 1 |
27 | Trichocladium asperum | 1 | 1 | 0 | 1 | 0 | 0 | 0 |
28 | Nigrosora sphaeria | 2 | 2 | 0 | 1 | 1 | 0 | 1 |
29 | Aspergillus terrus | 1 | 1 | 0 | 1 | 0 | 0 | 1 |
30 | Coccidioides immitis | 1 | 1 | 0 | 1 | 0 | 0 | 1 |
31 | Cladosporium sphaerospermum | 1 | 1 | 0 | 1 | 0 | 0 | 0 |
32 | Leptosphaeria senegalensis | 1 | 1 | 0 | 0 | 1 | 0 | 1 |
33 | Aureobasidium pellalanus | 1 | 1 | 0 | 0 | 1 | 0 | 1 |
34 | Penicillium expansum | 2 | 2 | 2 | 0 | 0 | 0 | 0 |
35 | Penicillium roqueforti | 1 | 1 | 1 | 0 | 0 | 0 | 0 |
| Total no of isolates | 101 | 100 | 15 | 57 | 27 | 2 | 75 |
| Isolation rate (%) | | 15 | 56 | 27 | 2 | |
| Species richness (S) | 11 | 26 | 16 | 2 |
Species diversity | Simpson’s index of diversity (1 − D) | 0.93 | 0.95 | 0.92 | 1 |
Shannon index (H) | 2.14 | 2.55 | 2.59 | 0.7 |
| Evenness (E) | 0.89 | 0.78 | 0.93 | 1 |
On the other hand, the richness and the diversity of the isolated endophytic fungi were calculated; plant leaf tissues settled the highest richness value with 26 species followed by stem (16 species) and 11 species for fruits. Simpson diversity indices values were, 0.95 for leaf tissues, 0.93 for fruits and 0.92 for stem, consistently; Shannon index values (H = 2.55 in leaf tissues), (H = 2.59 in stem tissues) and (H = 2.14 in fruits) while recorded in pulps as the lowest value (H = 0.7). Evenness indice4s for the leaf (E = 0.78), for the stem (E = 0.93) and for fruits (E = 0.89)
Antimicrobial activity of isolated endophytic fungi
Based on the similarity measure between the different endophytes activity, the antimicrobial activity data against eight selected significant pathogens were subjected to two-way clustering analysis. The resulted dendrogram revealed seven contiguous groups of fungi: The superimposed groups are discriminated at high resolution at the cut level of 0.65. Hierarchically, the resulted groups are arranged on the figure (1) and the pattern of each assemblage is clarifying as following:
Group I
this group separated firstly at the base of the tree with 14 isolates and showed high activity range against E. coli (mean 23 mm clear zone), Table 3. This group divided into three subgroups. The first subgroup; Alternaria pluriseptata (35), Alternaria phragmospora(5), Rhizopus stolonifer (51 and 68), Penicillium marneffei (48), Aspergillus niger (77), Cladosporium nigrellam (88), and Ulocladium alternariae (53) isolated from leaf tissues and revealed high activity only against E. coli. Cladosporium cladosporioides (18), Penicillium aurantiogriseum (23), Alternaria phragmospora (26) isolated from Mellilotus indicus and inhibited Aspergillus flavus, E.coli, and Pseudomonas argenosa were included in the second subgroup. The third subgroup represented by Scytalidium japonicum (10), Cladosporium cladosporioides (47), and Alternaria pluriseptata (98), isolated from leaf tissues and showed varied activity against E. coli, Pseudomonas argenosa, Bacills subtilus, Aspergillus flavus, and Penicillium expansum.
Table 3
Two-way cluster groups (I - VII), No. of isolated endophytes in each group, inhibition zone (main/mm) and the most pathogens affected by groups
Groups | No of isolates | inhibition zone main (mm) | Most affected pathogens |
I | 14 | 23 | E. Coli |
II | 10 | 20 | Staphylococcus aureus |
III | 10 | 13 | Candida albicans |
IV | 15 | 17 | Pseudomonas argenosa |
V | 28 | 0 | Not active |
VI | 18 | 21 | Bacillus subtilus |
VII | 6 | 20 | Bacillus subtilus, Staphylococcus aureus, Penicillium expansu, Aspergillus flavus, Pseudomonas argenosa and E.coli |
Group II
Ten fungal strains assembled in group II, inhibited mainly and strongly Staphylococcus aureus with mean inhibition zone of 20 mm. Table 3. Mucor hiemalis (14), Ulocladium alternariae (63), Pythium nayoroense (38), Alternaria pluriseptata (43 and 21), Fusarium oxysporum (56) included in active subgroup against only Staphylococcus aureus. Fusarium verticilloioides (55) appeared in the same group but showed activity against Candida albicanus in addition to its activity against Staphylococcus aureus. The second subgroup represented by two isolates of Alternaria pluriseptata (12 and 15) and one isolate of Sarcinomyces phaeomriformis (20) was multiactive strains against E. coli, Staphylococcus aureus and Candida albicans.
Group III
Ten strains represented this group; seven isolate from leaf tissues, two other from stem and one from fruit tissues. Three different patterns of activity for these 10 endophytes with moderate inhibitory zones (main = 13 mm). Alternaria pluriseptata (8), Alternaria chlamydospora (13), Aspergillus niger (22), Rhizopus stolonifer (85) were similar in their activity against Pseudomonas argenosa, Staphylococcus aureus and Aspergillus flavus. While Fusarium verticilloioides (49) and Alternaria pluriseptata (61) isolated from Asphodelus aestivus and Lotus polyphyllo respectively showed high activity against Pseudomonas argenosa, Staphylococcus aureus, Aspergillus flavus and Candida albicans in a different pattern. Cladosporium nigrellam(58), Alternaria alternaria (44), Phoma glomerata (9), and Spegazzinia pakeri (27) isolated from leaf tissues of Asphodelus aestivus, Pancratium maritimum, Fagonia cretica and Mellilotus indicus respectively, mostly inhibited Candida albicans and Aspergillus flavus in third pattern.
Group IV
Fungal isolates in group IV characterized by distinct antimicrobial activity against Pseudomonas argenosa with main inhibition zone of 17 mm. table (3). Fifteen strains were incorporated into this group (graph 1) mostly isolated from investigated leaf tissues and assembled in two subgroups. Rhizopus stolonifer (19), Penicillium raistrickii (60), Aspergillus niger (66), Nigrosora sphaeria (71), Rhizopus oryzae (34), Spegazzinia pakeri (87), Ulocladium atrum (82), Pythium elongutum (37), Alternaria pluriseptata (57), Aspergillus niger and Coccidioides immitis (84) aggregated in a subset and behaved in a similar manner against one pathogen, Pseudomonas argenosa. The other subgroup, Alternaria phragmospora (26), Ulocladium atrum (75), Penicillium expansum (30) and Penicillium marneffei revealed high antimicrobial activity against two pathogens, Candida glabrata and Pseudomonas argenosa.
Group V (Inactive group)
Twenty-six strains assembled together in group V that gave negative effect against all tested pathogens. Plant species, six inactive strains obtained from Lycium europaeum, four isolates from Carduus getulus, and three inactive strains from both Asphodelus aestivus and Nicotiana glauca. Other plant species represented by at least one inactive strain. Alternaria phragmospora (4) and Alternaria pluriseptata (76) grouped as closely related to group V However these two strains aere active against Candida glabrata and Penicillium expansum, respectively graph (1).
Group VI
This group contained 18 strains isolated from all plant species, shared the ability of inhibiting Bacillus subtilis with inhibition zone (main = 21mm) table (3).and differentiated from each other in terms of antimicrobial activity against other tested pathogens. Group VI divided into smaller six subsets based on the number of pathogens affected by its endophytic fungi. Four isolates, Aspergillus niger (100), Sarcinomyces phaeomriformis (28), Rhizopus stolonifer (59) Cladosporium nigrellam (101) were active against four different pathogens. While, Rhizopus stolonifer (73), Nigrosora sphaeria (81), Pythium nayoroense (45) revealed Antimicrobial activity against three pathogens. Phoma dennissii (2), Cladosporium nigrellam (46), Ulocladium atrum (99) and Alternaria pluriseptata (65) gave positive effect against one pathogen while the rest of the isolates in group VI activated positively against two pathogens.
Group VII (Multi-active group)
six isolates classified into two subgroups identified multivariate responses against all pathogens with wide inhibition zones (main = 20 mm). Two strains of Alternaria pluriseptata (6 and 7) isolated from Scorpiurus muricatu leaf tissues and one strain of Rhizopus stolonifer isolated from the leaf tissues of Nicotiana glauca revealed high antimicrobial activity against all tested pathogens except for C. albicanus. On the other hand; Penicillium marneffei (24) isolated from Mellilotus indicus, Alternaria pluriseptata (32) isolated from Carduus getulus and Spegazzinia pakeri (92) isolated from Nicotiana glauca revealed strong activity against all tested pathogens except E. coli.