This thesis is an outcome of efforts made on studies on the diversity, ecology and

Transcription

1 Chapter V SUMMARY

2 This thesis is an outcome of efforts made on studies on the diversity, ecology and activities of microfungi associated with some medicinal plants of the forests of Western Ghats in Goa, India. For the diversity studies, a total of 15 medicinal plants were considered, of which 11 were further analyzed from ecological stand point. Three major fungal microhabitats in the plants, viz. the leaf surface (with phylloplane fungi), the internal tissues of leaves, stem, root and bark (with endophytic fungi) and the fallen litter (with saprotrophic fungi), were investigated. Fungal isolation techniques such as moist chamber incubation, particle-plating, 3-step sterilization and leaf-washing were employed for the study. Samples were collected over a period of two years from two sites in Goa, viz. Colem and Mashem, and in two seasons, i.e. 'wet' (June to October) and 'dry' (December to May). This enduring exercise was carried out to understand the effect of location and seasonality on diversity and colonization pattern of fungi. Besides, fungal diversity in the internal tissues from various plant parts such as leaf, stem, bark and/or root were compared to determine tissue preference by the endophytes. The fungi isolated were identified down to species level based on conventional morphology-based taxonomy. RAPD was employed using a single primer to detect genetic polymorphism in 3 isolates of Phomopsis stipata obtained from different plants. One isolate of Phyllosticta sp. which appeared morphologically distinct from the commonly occurring species in the genus was subjected to sequence analysis. A number of isolates were subjected to preliminary antimicrobial screening in order to determine the potential of the isolates. The study resulted with documentation of a total of 392 distinct isolates which included 214 identified species [Zygomycotina (3), Ascomycotina (30), Basidiomycotina (6), Deuteromycotina (181D and 178 nonsporulating morphotypes. The diversity of fungi -272-

3 and their host specificity was not very significant, except for a few new and rare fungi. This observation was similar to the work done earlier on medicinal plants in India and elsewhere but different from those carried out on some common forest plants of the region. The fungi reported in the presented study mostly belonged to some of the common saprophytic genera such as Acremonium, Aspergillus, Chaetomium, Cladosporium, Curvularia, Emericella, Eurotium, Fusarium, Gliomastix, Nucor, Myrothecium, Nigrospora, Paecilomyces Penicillium, Pestalotiopsis, Phoma, Phyllosticta, Rhizopus, Scopulariopsis, alaromyces, Trichoderma and Verticillium. This observation was in agreement with previous similar studies done elsewhere. Besides, the common and rare species, the study resulted with nine new taxa of fungi. These included Acremoniula microspora, Alternaria goanensis, Dictyochaeta microsporae, Echinosphaeria macrospore, Harpophora konkanensis, Phaeoacremonium emblicae, P. indicum, Rachicladosporium indicum and Vermiculariopsiella endophytica. Many of them were recovered as endophytes indicating that amongst the endophytic fungi there could be many more new species. Hyphomycetous fungi were the most dominant group encountered during the study, followed by non-sporulating morphotypes, Coelomycetes and Ascomycetes, in the order of abundance. Basidiomycetous and Zygomycetous fungi occurred occasionally during the investigation. This observation is in confirmation with similar hitherto studies. The present effort led to the recognition of several anamorph-teleomorph connections such as Ascotricha chartarum-dicyma chartarum, Emericella nidulans- Aspergillus nidulans, Eurotium chevalieri- Aspergillus penicilloides, Guignardia bidwelli- Phyllosticta ampelicida and Guignardia citricarpa-phyllosticta citricarpa. Holomorphic -273-

4 connection between Vermiculariopsiella endophytica- Echinosphaeria macrospora was established for the first time through this study. Observations on RAPD analysis showed that Phomopsis stipata isolates displayed polymorphism, proving RAPD be a confirmatory method and OPK-19 an adept primer in detecting intra-specific DNA polymorphism in Phomopsis stipata isolates. Sequence analysis confirmed the holomorphic connectivity between Guignardia and Phyllosticta. In absence of sufficient sequence data at the Genbank and obscurity in morphology-based identification, the identity of the isolate remained uncertain. Preliminary antimicrobial screening of the isolates indicated that a good number of cultures are bioactive and a few of which with promising activities can be developed further. Similar observations were made earlier by others who had screened endophytes obtained from medicinal plants for bioactivity. In the present study, phylloplane and saprotrophic fungi were also screened, along with the endophytes. The results indicated that, similar to the endophytes, the phylloplane and saprotrophic fungi associated with medicinal plants are also creative. On comparing the strains of similar fungi from different sources for bioactivity, it was concluded that the bioactivity pattern of the fungus was influenced by combined effect of the host, substrate, tissue or geographical location from where the fungus was collected. When the creativity index of sporulating and non-sporulating fungal isolates was compared, it was observed that there was not much of variability in their activity pattern suggesting that the taxonomically anonymous group of fungi such as the non-sporulating ones are also equally creative and should not be overlooked in bioprospecting exercise. In the ecological studies, the following observations were made. When the diversity of fungi in each medicinal plant was compared, it was observed that the type of fungi the plant harbors does not necessarily depend on the narrow taxonomic groupings -274-

5 of plants. Rather, the diversity is influenced by higher taxonomic groupings such as Classes (Monocotyledonae or Dicotyledonae). When 3 microhabitats within the plants were compared for the pattern of fungal diversity, a proportionate number of fungi was found to overlap between the internal plant tissue and external saprophytes. This is in conformity with previous observations and based on the understanding that fungi occurring as endophytes occur as latent pathogens at the senescent stage of the plant part and later occur as saprophytic fungi. Non-overlap in the other species may not be a fact but an artifact of highly diverse fungal assemblages. Low and inconsistent overlap between phylloplane and endophytes and between phylloplane and saprotrophs was observed as reported in previous studies. The saprophytic fungal diversity was found to be less compared to other studies such as that on the monocots. This could be due to physico-chemical properties of the substrate that are known to regulate fungal flora ad colonization patterns of litter. It was observed that for all the 4 tissue types studied, viz. leaf, stem, root and bark, the colonization frequency of each fungus varied, in most cases, with the wet season having more than the dry one. The variations in location did not exert a recognizable influence on the species diversity and colonization rate of fungi suggesting that no site can be considered to have higher colonization rates than the other. The overall colonization rates were comparable to other studies carried out in tropics. The study revealed that there are numerous singletons, appearing apparently due to their limited growth and colonization of the internal plant tissue. It was observed that, be it individual tissue types or the host on the whole, wet season supported higher endophytic diversity than the dry. This was statistically significant and further inferred that precipitation encouraged increase in endophytic diversity, as reported previously elsewhere

6 Species diversity varied with locations. This suggested that every site had its distinct assemblage of fungi and the diversity of each of this assemblage, i.e. the probability that two fungi taken at a random would belong to different species was high in all plants studied. The diversity of fungi in each of the internal tissue was found to be equally high unlike that observed in previous studies. When the overlap in diversity within tissues was studied it was observed that in most of the cases endophytic fungi exhibited a strict tissue preference. This adaptation could be aimed to minimize competition within them and maintain balanced relation between fungi and plants