2.40 K avaka 35 : 11—20, 2007
Diversity and abundance of endophytic fungi in four plant species of Western Ghat forests in Goa, Southern India
M.A. D 'souza and D.J. Bhat Department of Botany, Goa University, Goa - 403 206, India
ABSTRACT Endophytic fungi associated with leaves of four plant species, viz. Calamus ihwaiiesiii, Careya arborea, Dendrocalamus sirictus and Saraca asoca of Western Ghats forests in Goa State, Southern India, were investigated during the pre-monsoon, monsoon and post:monsqon periods of 1999-2001. Features such as colony morphology and shape, size and colour of sporulating-structures were used for species identification and recognition of morphotypes. From a total of 1200 leaf segments; 865 isolates belonging to 44 taxa (Ascomycetes (3), Coelomycetes (7), Hyphomycetes (10) and Sterile mycelia (24)] were recognized. Higher number of endophytes was isolated during the post-monsoon period; sterile forms were found to be prevalent in all seasons. Statistical analysis showed that the expected number of species of endophytes in all plants is maximum in post-monsoon except in Saraca asoca in which it was during the monsoon season. Jaccard Similarity Coefficient revealed that each plant species hosted a distinct endophytic assemblage.
INTRODUCTION plant pests and pharmaceutical (Bills, 1995 Wagner and Lewis 2000; Benhamou et al. Fungal endophytes are characterized 2002; Stiele et a i, 1993; Lee et a i, 1995). by their ability to produce apparantely harmless infections in living plant tissues- While the role played by individual (Carroll, 1988; Bills, 1995). Endophytic fungus is said to be important, the fangi have been isolated from a diverse significance of endophytic communities in range of plants and plant parts and studies plant ecology is yet to be assessed. indicate that they are ubiquitous (Fisher and Endophytes associated with temperate tree Petrini, 1990; Petrini and Fisher, 1987, species have been studied since the middle 1988; Siber, 1989; Azevedo et ai, 2000). of 1970’s but not much is known on While some are widespread (Petrini, 1986), diversity, ecology and biology of those from many of them have a restricted host range the tropics (Bills, 1995). Rodrigues (1994) (Carroll and Carroll, 1978; Sherwoodpike et and Rodrigues and Samuels (1990) studied a i, i 986). the foliar fungal endophytes of two tropical Although endophytic fungi are palm species, viz. Euterpe oleraceae Mart., recognized as dormant saprobes (Chapela in Brazil and Licuala ramsayi (Muell.) and Boddy, 1988) or latent pathogens Domin., in Australia. The endophyte (Carroll, 1986). properties such as pest assemblage of Stylosanthes guianens'is deterrence, herbivory (Lactch et a i, 1985; Swart, a-Brazilian pasture legume (Periera et Bacon et a!., 1986), release of growth a i, 1993), Gynoxis oleifolia Muchler, a tree promoting stimuli and increased competitive species of Ecuador (Fisher et a i, 1995) and ability in the host (Clay, 5986’) have been Parthenium hysterophorus (Asteraceae), a attributed to them. They are now known to tropical herb (Romero et al., 2001) and produce metabolites useful in biocontrol of several tropical plant species from India 12 M.A- D'SOUZA AND D.J BHAT
(Suryanaryanan et a i, 1998; Suryanarayanan Sampling and Isolation and Kumai‘esan, 2000; Suryanarayanan and From each plant, a set of five fresh Vijaykrishna, 2001; Suryanarayanan and and disease-free young (from leaf bud), Thennarasan, 2004), have been studied. middle-aged (mature) and old (nearing senescence) leaves was considered as a The Western Ghat forests in sample. The samples were transported to the Southern India, one of the major laboratory in collection bags and processed biodiversity hotspots in the world, harbour a within 24 h of collection. wide variety of tropical plant species. The The 3-step surface sterilization present work was undertaken to recover a process described by Fisher and Petrini maximum number of endophytic fungi from (1987) was followed. The leaves were four of the plant species and to estimate washed thoroughly under running tap water to remove any adhered debris from the their diversity in this region. surface, surface-sterilized by sequential MATERIALS AND METHODS immersion in 70% ethanol (1 min), 4% Study season, site and plants sampled sodium hypochlorite (3 min) and 70% The study was carried out during the ethanol (0.5 min), repeatedly rinsed in pre-monsoon (Feb-May), monsoon sterile distilled water and cut in to segments of approximately 0.2-0.5 cm with a sterile (June-Sept.) and post-monsoon (Oct-Jan.) blade. Three hundred segments of each plant seasons of 1999-2001. These seasons are species were Hated on 2% Malt F.xtract unique in this part of the tropical world. The Agar (ME A) plates incorporated with pre-monsoon is warm and humid (temp. antibiotics (0.1 g Penicillin G, 0.1 g 28 - 37°C; humidity (70 - 90%). The Streptomycin, 0.1 g Neomycin, 0.1 g monsoon or rainy season has precipitation Bacitracin and 0.1 g Polymyxin in 10 ml ranging' between 200-350 cm, humidity sterile distilled water and added to 500 ml MEA). The plates were incubated at 22°C always remaining 100% and temperatures for 7-28 days. Fungal hyphae emerging seldom going above 23° C. T he from within the leaf segments were post-monsoon season has temperature range transfeiTed to fresh 2% ' MEA plates. from 17-28°C and humidity between Dissimilar colonies were considered as 40-70%. possible taxonomic entities and transfeiTed Four widely distributed native plant to slants. Sporulating fungi were identified species, namely Calamus thwaites'n Becc. & usinj* standard taxonomic keys. Non-sporulating isolates were characterized Hook. (Arecaceae), Dendrocatamus si rictus in to different morphotypes based to cultural (Roxb.) Nees. (Poaceae), Saraca asoca characters such as growth rate, surface (Roxb.) de Wilde (Casesalpinaceae) and texture, margin shape and hyphal Careya arborea Roxb. (Lecythidaceae) were pigmentation. _ examined. Of these, Saraca asoca and Statistical Analysis Calamus thwaitesii were from Bondla Wildlife Sanctuary and Careya arborea and ■ The frequency of colonization (%) of Dendrocatamus strictus from Mollem each endophyte species was calculated by the method of Fisher and Petrini (1987) as, Wildlife Sanctuary, two sampling sites 30 the number of colonized segments divided km apart north-south and located along the by the total number of segments plated Western Ghats in Goa State. x 100. The proportion of species belonging DIVERSITY AND ABUNDANCE OF ENDOPHYTIC FUNGI to either the Ascomycetes, Coelomycetes, sterile forms) were isolated. Of these, 9 Hyphomycetes or the Mycelia steriiia in a were recovered from Calamus thwaitesii, 11 single host plant species was calculated by firom Saraca asoca, 16 from Careya the formula (N/N, x 100), w here yv, is the arborea and 8 from Deiidrocalamus strictus number of fungal species belonging to a (Table 1). However, only 30 were isolated- group and N t is the total number of species more than once from the four plant species. from a single host species. The jaccard Similar studies conducted earlier Similarity Coefficient was calculated for al! indicated that the abundance of endophytic possible pairs of hosts to compare the fungi can be affected by season (Petrini, endophytf' .jm-ernbtejes,- io • the - I95i; Koungues 1994). In the present work, following formula: species such as Acremoniella sarcinellae, Similarity Coefficient = C/(A + j{ + C), Cochtiobolus ' lunatus, Ramichloridium where A and B are the total num ber of fasciculatum, Volutella roseola and several fungal species isolated from any two hosts nonsporulating forms were isolated only in and C the number of fungal species found in one season and it is possible that these are common {Sneath and Sokai. 1973). The of ‘one-season participation’. Further, results were expressed as percentages. Rainidiloridiwti faciculatum, Velu'elta roseola and nonsporulating forms 3, 10, 14, To estimate the expected number of 19 and 20 appeared only once in the entire species. | E(Sn)\ from the isolates study period and this 'one-time appearance’ obtained in the pre-monsoon, monsoon and by these fungi probably is very unique. post-monsoon season for each plant species, rare faction index was performed following Maximum number of endophytes the method of Ludwig & Reynolds (1988) were isolated from Careya arborea (145) as follows. during the post monsoon season (Table 2). -The least number of isolates were W -
BOO Fig !: Rarefaction curve for for curve Rarefaction !: Fig i 2 Rrfcin uv for curve Rarefaction 2: Fig .. ’OZ AD . BHAT D.J AND D’SOUZA M.A. aau tliwailesii Calamus aaa asoca Saraca DIVERSITY AND ABUNDANCE OF ENDOPHYTIC FUNG!
a. Pre-monsoon Dendrocalamus strictus b. Monsoon c. Post-monsoon
Fig 4: Rarefaction curve for Dendrocalamus strictus M.A. D'SOUZA AND D.J. BHAT
Table 1: Frequency of occurrence of endophytic fungi in four plant species during different seasons
Calamus Fungi / Plant Dendrocaiamus thwaitesii Saraca asoca Careya arborea species sirictus Pre Mon Post Pre Mon Post Pre Mon Post Pre Mon Post Hyphomycetes Acrernoniella 4 _ _ ■ sarcinella
Acrernoniella sp. - - _ _ - _ 1 I Aspergillus heslrictus - - 38 _ Ctadosporium - ___ 2 1 herbarum
Corynospora smithii 12 - __ _ Corynospora 6 17 — _ cassicola
Kumbhamaya indica - - - - __ __ 13 25 46 Ramichloridum __ _ _ j fasciculatum
Bharalheeya -- 16 4 _ aoanensis
Volulella roseola -__ _ 1 j Coelomycetes
Pestalotiopsis -__ _ _ 1 4 versicolor
Macrophoma sp. - -- 17 19 _ Vermiculariopsielta -- 1 3 12 2 2 21 31 elegans Undetermined _ 5 2 sporodoehial from 1 Undetermined _ 1 1 sporodoehial from 2 Undetermined 1 I pycnidial form Undetermined 7 : _ acervular form Ascomycetes
Cochiiobolus lunaius -- - 9 4 19 86 s Guignardia sp. ! -- - 15 _ 20 5 22 1 9 Xylaria sp. 1 3 3 - 19 2 2 Non-Sporulating NSM 1 3 I 4 _ ■ NSM 2 -_ 15 _ 1 NSM 3 -- - __ _ 10 3 NSM 4 - _ 3! _ 1 NSM 5 - - - 8 ------DIVERSITY AND ABUNDANCE OF ENDOPHYTIC FUNGI 17 Table 1 contd.,
t 'alamti s Fungi / Plant Dendrocatamus tfmaites it Saraca asoca Careya arborea species strictus Pre Mon Post Pre Mon Post Pre Mon Post Pre Mon NSM 6 Post - - - - - 2 ---_ , — NSM 7 __ - - - - - 23 --__ NSM 8 — - - - - - 16 - - _ _ NSM 9 “ - - - - - 2 --_ — _ NSM 10 ~ - - ~ - - - 1 -_ _ . _ NSM 11 ” ------9 __ _ NSM 12 ------4 - _ . __ NSM 13 ~ - - - - - ■- " 2 _ _ . NSM 14 — ’ ------1 -- _ _ NSM 15 ------96 -_ NSM 16 • ------7 --_ NSM 17 ------3 - _ NSM 18 — ------2 _ _ NSM 19 ' ------1 - _ ' _ NSM 20 ------1 - _ _ NSM 2! ------2 _ NSM 22 ~ ------5 _ NSM 23 ------_ 2 _ NSM 24 - - - -- 15 - __ _ Total no. of 36 27 63 76 71 96 65 111 145 16 individuals 67 92 Total no of species 7 5 8 5 7 8 5 9 10 4 9 10 Note: Pre: Pr Post: Post—Monsoon [October—January]
1 able 2: Total number of isolates and- species - | ------— “ V.of viiuujiujutendophytic 11fungi recorded from fourfn n r plantr\In«* speciesM \nntAn duringJ ___ i ____i-m' different seasons Season Plant species Total Total Asco- Coelo-. Hypho- Myce- Isolates Species niycetes mycetes mycetes liasterilia Calamus lliwaitesii Pre 36 7 1 2 3 1 Mon 27 5 1 2 ■ t 1 Post 63 8 2 2 | 3 Saraca asoca Pre 76 5 1 2 1 1 Mon 71 7 3 2 1 1 Post 96 8 2 3 1 2 Careya arborea Pre 65 5 1 1 0 3 Mon 111 9 2 2 0 5 Post 145 10 1 0 1 g Dendrocalamus Pre 16 4 1 0 2 j strictus Mon 67 9 I 2 3 3 Post 92 10 2 3 3 3 18 M.A. D’SOUZA AND D.J. BHAT restrictus (38% in Calamus thwaitesti and studied. This was in accordance with Petrini 54% in Saraca asoca) showed the highest (1986) who showed that some fungi are frequency of occurrence in the widespread in their occurrence. post-monsoon season. It may be said that in The jaccard Similarity Coefficient the tropics, the post-monsoon is the best showed that the composition of endophytes season for maximum recovery of endophytic recovered did not overlap by more than 18% fungi colonii.ing the plant species in their in Calamus thwaitessii and Saraca asoca annual occurrence. Maximum colonization and by 21% in Careya arborea and of endophytic fungi during the monsoon and Dendrocalamus strictus though these four post monsoon in the tropics may be due to plant species were practically exposed to the high moisturfe content (humidity). similar environmental conditions and fungal Carroll and Carroll (1978) and inoculum (Table 3). This observation Sherwood-Pike et al. (1986) observed that indicated that each plant species exhibited the plant species were specific with regard certain degree of distinctness in its to the number and type of endophytes endophyte composition. harboured. This was found true in the present study.' Kutnbhamaya itidica, Of the different groups of originally reported as an endophyte from endophytic fungi colonizing the plant young leaves of Carissa conjesta in India species, in Careya arborea the highest (Jacob and Bhat, 200), was consistenly percentage of fungi were the nonsporulating ico!afpH from Dendrocalamus strictus during forms (72.72%) and the lowest were the the period of investigation irrespective of coelomycetes In contrast, in Saraca asoca, the seasons. Vermiculariopsielta elegans the proportion of hyphomycetes, and an undetermined ascomycete form were coelomycetes and ascomycetes were found common to all plant species. uniform (Table 4). The fungal endophytes Macrophoma sp. and Vermictdariopsiella colonize the plant parts by adapting to its elegans were widespread in all plant species internal environment. Studies carried out Table 3: Jaccard Similarity Coefficient for the endophytes isolated from four plant species (expressed as %)
Calamus Dendrocalamus Plant species Saraca asoca Careya arborea thwaitesii strictus Calamus thwaitesii 100 18.18 12.9 15.38 Saraca asoca 100 12.9 10.71 Careya arborea 100 21.21 Dendrocalamus strictus 100 Table 4: Percent occurrence (%) of different groups of fungi as endophytes in four plant species
Host Plant Hyphomycetes Coelomycetes Ascomycetes Mycelia Steriiia Calamus thwaitesii 46 IS..1) 15.5 23 Saraca asoca 23 23 23 31 Careya arborea 14 4 9 72.72 Dendrocalamus 46.66 0 20 33.33 strictus DIVERSITY AND ABUNDANCE OF ENDOPHYTIC FUNQI 19 simultaneously on fungal colonization on C a r r o l , G .C ., a n d C a r r o l , F E . 1978. Studies leaf litter of these plant species on the incidence of coniferous needle (unpublished) in our laboratory, have shown endophytes in the Pacific Northwest. that fungi such as Pestalotiopsis versicolcr, Canadian Journal Botany 56: 3034-3043.
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