The Endophytic Mycoflora of Bark, Leaf, and Stem Tissues of Azadirachta indica A. Juss (Neem) from Varanasi (India)

Microbial Ecology The Endophytic Mycoflora of Bark, Leaf, and Stem Tissues of Azadirachta indica A. Juss (Neem) from Varanasi (India) V.C. Verma1, S.K. Gond1, A. Kumar1, R.N. Kharwar1 and Gary Strobel2 (1) Mycopathology Laboratory, Department of Botany, Banaras Hindu University, Varanasi, India (2) Department of Plant Sciences, Montana State University, Bozeman, MT 59717, USA Received: 25 August 2006 / Accepted: 11 October 2006 / Online publication: 30 March 2007

Abstract

Introduction

A systematic study was made of the endophytes of Azadirachta indica A. Juss (the neem tree) growing in several of its natural habitats in India. A total of 233 isolates of endophytic fungi representing 18 fungal taxa were obtained from segments of bark, stem, and leaves of this tree. Hyphomycetes (62.2%) were the most prevalent followed by the Coelomycetes (27.4%) and Mycelia Sterilia (7.7%). As mathematically determined, the maximum species richness and frequency of colonization of endophytes appeared in leaf segments rather than stem and bark tissues from each location. Endophytic colonization frequency was also greater in leaves (45.5%) than bark (31.5%). The leaf samples from all locations were nearly constant in their endophytic composition, whereas bark samples showed maximum diversity at different locations. Inter-site comparisons for endophytic diversity, however, were not significantly different with Loc1 and Loc2 having a maximum of 66.67% Jc. The smallest similarity was between Loc2 and Loc3 of 54.17% Jc. The dominant endophytic fungi isolated were Phomopsis oblonga, Cladosporium cladosporioides, Pestalotiopsis sp., Trichoderma sp, and Aspergillus sp. Genera such as Periconia, Stenella, and Drechslera are reported here for the first time as endophytes from this host plant. This report illustrates the value of sampling different tissues of a given plant in several locations to obtain the greatest species diversity of endophytes. The rich and sizeable collection of endophytic fungi from this specific plant may represent a unique source of one or more of the interesting and useful bioactive compounds normally associated with A. indica such as the azadirachtins and related tetranortriterpenoids.

Originally, the term Bendophyte^ was introduced by De Barry [13] and was assigned to all those microbes that reside inside the living healthy tissues of the plants. Later, this term was expanded as Bfungi and bacteria including actinomycetes, which spend the whole or at least a part of their life cycle colonizing inter- or intracellularly, inside the healthy living tissue of the host plant, typically causing no apparent symptoms of disease [51, 56]. Until zrecently, extensive work has been done on endophytic fungi, mostly in the temperate regions of the world [35, 43]. However, new searches for endophytes with biological promise are being conducted in both temperate and tropical rainforests where there exists the largest terrestrial biodiversity of macro-life forms in the world [7, 25, 31]. Although most studies have been conducted on the mere presence and identity of endophytes in the young stem or leaf tissues [4, 22, 44, 47], a systematic and comparative approach to the specific location in the plant and identification of the endophytes is rarely done especially in tropical trees such as Azadirachta indica A. Juss (Meliaceae) [49]. A. indica A. Juss (Meliaceae) commonly known, as Bneem^ is one of the most effective medicinal plants in natural therapy and Ayurveda in India. Almost all parts of this tree including leaves, bark, and seeds contains useful substances that can be used to prepare natural and safe remedies for such aliments as dust allergies, fever, skin diseases, rheumatism etc. [5, 9]. Various constituents of this plant have already been reported as antibacterial [34], antiretroviral [54], antiarthritic, anti-inflammatory [33], and antiulcer [39]. Extracts of neem have also been evaluated against malaria [15], diabetes [14], and leukemia [38]. However, worldwide, the best-known use of neem is for its insecticidal activities [2, 24, 28]. On some occasions, an endophytic microbe may produce one or more of the same chemical substances

Correspondence to: R.N. Kharwar; E-mail: [email protected] DOI: 10.1007/s00248-006-9179-9

& Volume 54, 119–125 (2007) & * Springer Science + Business Media, LLC 2007

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that are known from its host plant. Such is the case of fungal taxol, a potent anti-breast cancer drug, being produced by the endophytic fungus on Taxus brevifolia– Taxomyces andreanae [48]. Thus, to learn if any endophytic microbes may be producing one or more of a plethora of bioactive substances known from A. indica, it would first be critical to do a systematic study on the endophytes of this plant and to learn of their biology and distribution both within the plant and in plants from several locations. Ultimately, a collection of such endophytes would then serve as a library to begin more comprehensive fermentation, screening, and chemical characterization studies. Although there are a few fragmentary reports on endophytes from A. indica, no comprehensive work has been done on comparative tissue studies on A. indica much less how location may affect the population of its endophytes [32, 40]. This report shows the relative importance of tissue preference of endophytes of neem and their biodiversity. It represents an important initial step in beginning to understand the role of endophytes to this important plant that will eventually begin the processes involved in sorting out some of the biologically interesting compounds that are made by these microorganisms. Materials and Methods Collection of Samples. Three different locations were selected for sampling and were designated as location 1, the campus of Banaras Hindu University, Varanasi, India (relatively free of atmospheric pollution with acceptable irrigation, (Az1 = Loc1); location 2, the Sarnath Excavation Area,Varanasi, (having exposure to air pollution) (Az2 = Loc2); and location 3, Rajghat, (near the banks of the river Ganga with high humidity and water availability) (Az3 = Loc3) Varanasi, respectively. Leaves, bark, and stem were collected from individual plants at each location. Samples were labeled and collected, and each was assigned a code (AzB for bark, AzL for leaf, AzS for stem, followed by a slash and a number denoting the location). All samples were immediately brought to the laboratory in an icebox, and the tissues were screened for endophytic fungi. Screening, Identification, and Preservation of EnAll the samples were washed properly in dophytes.

running tap water followed by double distilled water before processing. The samples were cut into small pieces. Bark and stem samples were cut into 1.0  1.0 cm pieces; leaves were cut into small discs using a sterile pinch cutter. To eliminate epiphytic microorganisms, all the samples were initially surface treated [37]. The samples were immersed in 70% ethanol for 1–3 min and then sterilized with aqueous sodium hypochlorite (4% available chlorine) for 3–5 min and then rinsed in 70% ethanol

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for nearly 2–5 s, before a final rinse in sterilized double distilled water. Each sample was then dried under aseptic conditions. Segments (a total of 200 at four to six segments per Petri plate) of each sample were placed on potato dextrose agar (PDA) amended with streptomycin (100 mg/l). The Parafilmi-sealed petri dishes were then incubated in a Biochemical Oxygen Demand (BOD) incubator with humidity (L K Scientific Inc.) for 25 days at 12-h light/dark cycle at 27 T 2-C [52]. The plates were checked on alternate days, and hyphal tips of actively growing fungi were then subcultured. The endophytic fungi were identified according to their macroscopic and microscopic characteristics such as the morphology of fruiting structures and spore morphology. Standard taxonomic manuals were used to identify the fungal genera [1, 3, 16, 17, 41]. All isolated and identified endophytic fungi were assigned specific code numbers (MCPL: 001-233) and maintained in cryovials on PDA layered with glycerol (15%, v/v) and also in a lyophilized form at _20-C in a deep freezer (Blue Star). All the samples were deposited in the Department of Botany, Banaras Hindu University, India. Analysis of Data. The relative frequency (percent CF) of colonization of endophytic species was calculated as the number of segments colonized by a single endophyte divided by the total number of segments observed  100 [23]. This is expressed as %CF = (Ncol / Nt)  100; where Ncol = the number of segments colonized by each fungus, and Nt = the total number of segments. The dominant endophytes were calculated as the percentage colony frequency of a given endophyte divided by the sum of the percentage of colony frequencies of all endophytes  100 [29]. Utilizing the data of percentage colony frequency in bark, stem, and leaves of different locations, Simpson_s Diversity indices (1Dominance) and Shannon–Wiener indices were calculated, whereas the Simpson_s Diversity = 1 _ S (pi)2. The Shannon–Wiener diversity = _S s (pi log2 pi), where pi = proportion of frequency of colonization of the ith species in a sample. Species evenness was calculated with the formula as per Whittaker [55] Species evenness = S / ln(Ni) _ ln(Ns) Where S = the total number of species present in a sample. (Ni) = the number of species with the highest presence. (Ns) = the number of species with the least presence.

Results and Discussion

Endophytic fungi from bark, stems, and leaves of A. indica were isolated and evaluated with the procedures described. Specific plants occurring in three different locations (Az1, Az2, and Az3) were the sources of the

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endophytic fungal diversity among them. A total of 233 isolates belonging to 18 fungal taxa were obtained from 200 segments studied (Table 1). Collectively, among the total endophytic isolates recovered from neem in this study, there were 62.2% Hyphomycetes, 27.4% Coelomycetes, and 7.7% Mycelia Sterilia. The maximum of 38.6% endophytes of the total in this study were recovered from location Az1, while the least, at 33.5%, were recorded from location Az2 (Fig. 1). Among 233 isolates, Az1 yielded 90 isolates (36 from bark and 25 from stem tissues) followed by Az2 with 87 (30 from leaf and 12 from bark tissues), and finally, Az3 with 76 isolates (32 from leaf and 15 from bark tissues) (Fig. 2). The percent colonization of tissue samples by endophytic fungi at location Az1 was 45.0% higher than the others (Table 2). Significant fluctuations in the recovery of endophytic fungi were also recorded while analyzing individual bark samples from each location (Table 1). The dominant fungi observed were Aspergillus, Cladosporium, Curvularia, Pestalotiopsis, Phomopsis, and Trichoderma. It was interesting that three fungal genera not

Figure 1. The percentage recovery of different endophytic groups from three locations.

previously reported from A. indica, Drechslera, Periconia, and Stenella sp., were also obtained from A. indica. Generally, the leaf samples from each location showed more diversity of endophytic fungi than the bark and stem samples (Table 3). The site AzL/1 harbored maximum endophytic diversity with 15.4%, whereas there was only 6.4% recovered from site AzL/3. Trichoderma viride was observed as the dominant endophytic fungus in all samples of A. indica, which is in agreement with another

a Table 1. Occurrence and identification of endophytic fungi from barks, leaf, and stem pieces of Azadirachta indica growing at three different locations near Varanasi, India

Loc1a Endophytic fungi Coelomycetes Pestalotiopsis sp. Pestalotia macrotricha Phomopsis oblonga Phoma eupyrena Hyphomycetes Acremonium sp. Alternaria alternata Cladosporium cladosporioides Cladosporium acaciicola Curvularia lunata Curvularia catenulata Drechslera sp. Verticillium albo-atrum Trichoderma viride Nigrospora oryzae Stenella sp. Periconia sp. Aspergillus flavus A. niger A. oryzae Penicillium sp. Fusarium oxysporum F. moniliformae F. soloni F. chlaydosporum Gliomastix sp. Mycelia Sterilia Total isolates

Loc2a

Loc3a

AzBb

AzLb

AzSb

AzBb

AzLb

AzSb

AzBb

AzLb

AzSb

03 – 05 –

– – 07 05

01 – 03 02

02 – 01 02

05 – 02 –

03 01 04 –

01 – – 03

02 01 02 02

– – 03 04

05 – 02 – 01 – – – 07 01 01 – 02 03 – – 05 – – – – 01 36

– 01 01 – – – – – 01 – – – 03 – 01 01 01 01 02 03 – 02 29

03 – – – 01 – – 01 – – – – 04 01 – – 01 02 01 05 – – 25

– – 02 – – – – – – 01 01 – 01 – – – – – – 01 – 01 12

03 – 03 01 – 01 – 01 05 – – – 01 – 03 – 02 – – – 01 02 30

– – 01 – 04 – – – 01 05 – – 02 02 – – 01 – – – – 01 25

– 01 – – 02 – 01 – – – – 01 02 – – – 01 – – – – 03 15

– – 03 – – – – – – 06 – – 02 03 – – 02 01 – 03 – 05 32

04 – 04 – 01 – – – 03 – – – 01 05 – – 01 – – – – 03 29

a Locations selected for study: Loc1, Banaras Hindu University campus; Loc2; Sarnath Excavation Region; Loc3, Gangetic basinVthe belt of the River Ganga; total taxa = 18; total isolates = 233, total segments plotted = 200. b AzB Bark, AzL leaf, AzS stem

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Number of Isolates

40

30

20

10 N=

3

3

3

1.00

2.00

3.00

Location Selected for study report on A. indica endophytes (Table 2) [32]. In contrast, others have observed Fusarium avenaceum as the dominant endophytic fungus in A. indica [40]. Chaetomium crispatum and Chaetomium globosum also have been recorded as representatives of ascomycetous genera existing as endophytes in A. indica, but neither of these organisms appeared as endophytes in this study [40]. The dominance of endophytes recovered from bark samples (AzB/2 _5.1% and AzB/3 _6.4%) was only about one-third that of AzB/1 (15.4%) (Table 3). Furthermore, among the bark samples from all three locations, samples from Az1 had the most diverse endophytic assemblage (Table 3). Contrary to bark samples, the leaf samples had a stable endophytic diversity. Leaf samples AzL/3 with 13.7% recovery had the greatest diversity of endophytes followed by AzL/2 (12.8%) and AzL/1 (12.4%). However, AzL/1, despite the low recovery of endophytes, exhibited maximum species richness (13). Leaf samples from each location harbored the greatest number of endophytes as compared to the stem and bark of the same location. This finding was in accordance with the report of endophyte diversity in leaves, twigs, and roots of Gynoxis oleifolia (Table 3) [19]. Endophytes from AzB/1 had the maximum Shannon–Wiener diversity index (3.29), and this is significantly higher than the value (2.30) obtained from bark endophytes of Terminalia arjuna [53] (Table 4). The lowest endophyte diversity was from AzB/2 (1.14). On the other hand, the Simpson_s

Figure 2. A box plot showing the distribution of fungal endophytes according to locations Loc1, Loc2, and Loc3. The line between the boxes shows the median value, the bar lines above and under the boxes the show maximum and minimum values of endophytes recovered at each location, while the box itself represent the semi-quartile range.

diversity index ranged between 0 to 1, whereby, the larger index value proportionally reflects the greater species diversity. For instance, AzL/3 had the maximum Simpson_s index, i.e., a greater diversity for the leaf samples from location 3. The evenness of all the samples was also calculated in which AzB/2 (12.98) was comparatively more uniform than the rest of the samples. Endophytes of AzB/2 had greater diversity than the rest of the bark samples. Despite significant variations in the specific recovery of the endophytic community from plant tissues in each location, the inter-site comparisons, as a whole, were not significant. Similarity between Loc1 and Loc2 was found to be a maximum of 66.67% Jc, while the relationship of Loc2 and Loc3 was 54.17% Jc (Table 5). Certain environmental conditions may account for these relationships. For instance, Loc1 and Loc2 both have moist and humid conditions that actually favors establishment of endophytic colonization, but Loc3 has prevailing dry conditions that may tend to limit the rate of endophytic colonization. Overall, it appears that each location of A. indica seems to possess a common Bcore endophytic community^ (Tables 1 and 2). However, the Bodd or rare incidental^ endophytic species does occur, especially in the more humid conditions, which is a major factor for tissue specific fluctuations in the recovery of endophytes. Various workers have studied distribution patterns of endophytes within plant tissues, and in most cases,

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Table 2. The percentage colonization and dominance of fungi from all endophytic isolates obtained from the total segments studieda

Endophytic fungi

Total isolates Percentage Dominance of each fungus (%) CF b of fungi

Coelomycetes Pestalotiopsis sp. Pestalotia macrotricha Phomopsis oblonga Phoma eupyrena Hyphomycetes Acremonium acremonium Alternaria alternata Cladosporium cladosporioides Cladosporium acaciicola Curvularia lunata Curvularia catenulata Drechslera sp. Verticillium albo-atrum Trichoderma viride Nigrospora oryzae Stenella sp. Periconia sp. Aspergillus flavous A. niger A. oryzae Penicillium sp. Fusarium oxysporum F. moniliformae F. soloni F. chlaydosporum Gliomastix sp. Mycelia Sterilia

8.5 1.0

7.33 0.86

Loc2

27 18

13.5 9.0

11.63 7.76

Loc3

15

7.5

6.47

02 15

1.0 7.5

0.86 6.47

01

0.5

0.43

09 01

4.5 0.5

3.88 0.43

01 02

0.5 1.0

0.43 0.86

17 13 02 01 18 14 04 01 14

8.5 6.5 1.0 0.5 9.0 7.0 2.0 0.5 7.0

7.33 5.60 0.86 0.43 7.76 6.03 1.72 0.43 6.03

04 03 12 01 18

2.0 1.5 6.0 0.5 9.0

1.72 1.29 5.17 0.43 7.76

Based on 200 segments plotted. Colonization frequency (see Materials and Methods section for details).

b

Table 3. Dominant endophytes, their species richness and percentage colonization of A. indica at each location

Loc1 Loc2 Loc3

a

Total Percentage Dominance isolates (%) CF of fungi AzB AzL AzS AzB AzL AzS AzB AzL AzS

36 29 25 12 30 25 15 32 29

Loc1

17 02

a

Sampling a location

Table 4. Different diversity indices for each location

Sampling a location

18.0 14.5 12.5 06.0 15.0 12.5 07.5 16.0 14.5

15.45 12.44 10.72 05.15 12.88 10.73 06.44 13.73 12.44

Species richness 12 13 12 09 13 11 09 12 10

Locations selected for study: Loc1, Banaras Hindu University campus; Loc2, Sarnath Excavation Region; Loc3, Gangetic basin?the belt of River Ganga. The data presented are on the basis of 200 segments plotted.

Shannon– Wiener indices AzB AzL AzS AzB AzL AzS AzB AzL AzS

3.290 2.007 2.302 1.143 1.717 2.526 2.083 2.331 2.162

Simpson_s diversity indices

Evenness

0.579 0.878 0.884 0.883 0.853 0.879 0.531 0.903 0.884

6.185 6.701 7.500 12.98 8.079 6.836 8.196 6.700 6.215

a Locations selected for study: Loc1, Banaras Hindu University campus; Loc2, Sarnath Excavation Region; Loc3, Gangetic basin?the belt of River Ganga. The data presented are based on 200 segments plotted.

foliar endophytes were examined [31, 42, 57]. The species composition of the endophytic assemblage and frequency of infection varied according to host species, site characteristics such as elevation, exposure, associated vegetation, tissue type [20, 21, 45], and tissue age [18, 42]. On the other hand, for large woody perennials, growth stage and position in the canopy may also affect the distribution of endophytes [26]. Generally, the assemblage of foliar endophytes for a given host comprises a relatively consistent group of fungal genera and species, characterized by a few dominant species, and this study also corroborates this conclusion [11]. Similar findings were obtained in case of Sequoia sempervirens [46]. More recently, rDNA sequence analyses are being used to determine the effects of different ecological factors on the distribution of endophytic fungi [12]. Variations in endophytic assemblage from a single host in different study sites is usually attributed to the recovery of rare and incidental species with a more disjunct distribution; otherwise, a core group of fungal taxa are recovered in a relatively constant proportion from all samples [8]. It would be more contextual to say that the occurrence of the rare and incidental species, which were occasionally represented once or twice in several hundred samples, was also observed significantly in our study as well. The genera such as Drechslera, Periconia, and Stenella, sp. were recovered as incidental species for the first time. Periconia is of special interest as an isolate of it

Table 5. Jaccord_s similarity (JC) coefficients for all three locations

Samplinga location

Loc1

Loc2

Loc3

Loc1 Loc2 Loc3

100

66.67 100

65.22 54.17 100

a Locations selected for study: Loc1, Banaras Hindu University campus; Loc2, Sarnath Excavation Region; Loc3, Gangetic basin?the belt of River Ganga. The data presented are based on 200 segments plotted.

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from China is known to produce taxol [30], and it produces the anti-bacterial diterpenes?the periconicins [27]. The occurrence of rare and incidental species is usually proportional to the intensity of the sampling procedures and the sites chosen, rather than the host itself [36]. Our results strongly indicate that the sampling methods were adequate and thorough for rare species. Except for the occurrence of rare species as we have discussed earlier, there were no significant location-based differences in species recovery, and more or less, all locations had a similar type of fungal endophytic community (Tables 1, 2, 3). This is in spite of the fact that the tree samples existed in different environmental conditions. Nearly all endophytes studied, so far, produce bioactive compounds [50], and sometimes, these compounds have an enormous pharmaceutical, agricultural, and industrial potential. Phomopsis sp. was one of the dominant taxons in our study, and it produces phomopsichalasin, an isoindolone along with tremorgenic mycotoxins- paspalitrem A and C [6]. Similarly, Drechslera sp. produces Bpestasol,^ in liquid culture [10]. Although only fungal endophytes were specifically targeted in this study, the neem most likely is also host to a bacterial endoflora, and the selective medium used in this study precluded the isolation of these microbes. Nevertheless, the extensiveness of the fungal collection, represented by this study, is rich enough for some serious work to begin on the isolation and characterization of the secondary products of these organisms. In fact, this is probably the most comprehensive collection of endophytic fungi from neem in the world (MCPL 001-233). It seems apparent that there is a role for the endophytes in the biology of the neem plant, as they are present in each of the plant tissues that were sampled (Table 1); however, this will be sorted out only when the variables are significantly reduced to determine the specific roles of one or several endophytes through inoculation studies under greenhouse conditions. Furthermore, it is likely that there are intriguing interactions between and among endophytes existing in this plant, which only tends to further complicate such matters. The results of this study set a nice example for other studies on endophytic microbes given the fact that multiple sites, multiple plant tissues, and mathematical analyses have helped define how maximum biodiversity may be found in a fungal endophyte population in a given plant species. Acknowledgements

The authors are thankful to the Head of the Department of Botany, Banaras Hindu University, Varanasi India, for providing the necessary facilities. The authors also extend their thanks to CSIR/UGC, New Delhi for providing financial assistance in the form of JRF/SRF. GAS

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expresses his appreciation to the Montana Agricultural Experiment Station and the Montana Board of Research and Commercialization Technology for their support of this work. References 1. Ainsworth, GC, Sparrow, FK, Sussman, AS (1973) The Fungi? advanced Treaties, Taxonomic Review with Keys (Vol. IV A). Academic Press, New York, USA 2. Ascher, KRS (1993) Nonconventional insecticidal effects of pesticides available from the neem tree Azadirachta indica. Arch Insect Biochem Physiol 22: 433–449 3. Barnett, HL, Hunter, BB (1998) Illustrated Genera of Imperfect Fungi. (4th ed.) Mac Millan Publ. Co. ISBN: 0-89054-192-2, New York 4. Bettucci, L, Saravay, M (1993) Endophytic fungi of Eucalyptus globulus: a preliminary study. Mycol Res 97: 679–682 5. Bhargava, AK (1989) Neem skinment for the management of wounds. Intern. Conf. Recent Advances Medicinal Aromatic and Spice Crops. Abstract, New Delhi, India, p 65 6. Bills, GF, Giacobbe, RA, Lee, SH, Pelaez, F, Tracz, JS (1992) Tremorgenic mycotoxins Paspalitrems A and C from tropical Phomopsis. Mycol Res 96: 977–983 7. Bills, GF, Pollishook, JD (1991) Micro fungi from Carpinus caroliniana. Can J Bot 69: 1477–1482 8. Bills, GF, Pollishook, JD (1992) Recovery of endophytic fungi from Chamaecyparis thyoides. Sydowia 44: 1–12 9. Board on Science and Technology for International Development, National Research Council (1992) Report of an Ad Hoc Panel on Neem, A Tree for Solving Global Problems. National Academic Press, Washington DC, pp 60–113 10. Bunkers, GF, Kenfield, D, Strobel, GA (1991) Production of petasol by Drechslera gigantia in liquid culture. Mycol Res 95: 347–351 11. Carroll, GC (1995) Forest endophytes: pattern and process. Can J Bot 73(Suppl. 1): S1316–S1324 12. Collado, J, Platas, G, Paleaz, F (2001) Identification of an endophytic Nodulisporium sp. from Quercus ilex in central Spain as the anamorph of Biscogniauxia mediterranea by rDNA sequence analysis and effect of different ecological factors on distribution of the fungus. Mycologia 93: 875–886 13. De Bary, A (1866) Morphologie und physiologie der plize, Flechten, und Myxomyceten (Hofmeister_s Hand Book of Physiological Botany. Vol. 2) Leipzig 14. Dixit, VP, Sinha, R, Tank, R (1986) Effect of neem seed oil on the blood glucose concentration of normal and alloxane diabetic rats. J Ethnopharmacol 17: 95–98 15. Ekanem, OJ (1978) Has Azadirachta indica (Dogonyaro) any antimalarial activity. Niger Med J 8: 8–10 16. Ellis, MB (1971) Dematiaceous Hyphomycetes, Commonwealth Mycological Institute, Kew, Surrey England 17. Ellis, MB (1976) More Dematiaceous Hyphomycetes, Commonwealth Mycological Institute, Kew, Surrey England 18. Fisher, PJ, Anson, AE, Petrini, O (1986) Fungal endophytes in Ulex europaeus and U. galli. Trans Br Mycol Soc 86: 153–156 19. Fisher, PJ, Petrini, LE, Sutton, BC (1995) A study of fungal endophytes from leaves, stem, and root of Gynoxis oleifolia Muchler (Compositae) from Ecuador. Nova Hedwig 60: 589–594 20. Fisher, PJ, Petrini, O (1990) A comparative study of fungal endophytes in xylem and bark of Alnus sp. in England and Switzerland. Mycol Res 94: 313–319 21. Fisher, PJ, Petrini, O, Petrini, LE, Sutton, BC (1994) Fungal endophytes from the leaves and twigs of Quercus ilex L from England, Majorca and Switzerland. New Phytol 127: 133–137

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