Endophytic fungi of wild banana (Musa acuminata) at Doi Suthep Pui National Park, Thailand*

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1 Endophytic fungi of wild banana (Musa acuminata) at Doi Suthep Pui National Park, Thailand* Wipornpan PHOTITA 1, Saisamorn LUMYONG 1, Pipob LUMYONG 2 and Kevin D. HYDE 3 Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand Department of Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai, Thailand Centre for Research in Fungal Diversity, Department of Ecology and Biodiversity, The University of Hong Kong, Pokfulam Road, Hong Kong. wipornpan hotmail.com Received 27 September 2000; accepted 9 January Endophytic fungi were isolated from 7500 samples of wild Musa acuminata collected from five sites at Doi Suthep Pui National Park, Thailand during December 1998 to July Overall colonization rates from surface sterilized tissues were 56 5, 48 9, 48, 47 9 and 41 7% for the Medicinal Plant Garden, Ban Suthep, Queen Sirikit Botanic Garden, San Gu, and Montatarn waterfall sites respectively. Sixty-one different fungal taxa were isolated. Fewer isolates were recovered from younger than older samples. Xylariaceous taxa and Guignardia cocoicola were the most frequently isolated endophytes from leaves and were either absent or rare in midrib, petiole and pseudostem. Colletotrichum gloeosporioides, C. musae, Guignardia cocoicola, various sterile mycelia and xylariaceous spp. were common at all sites. The endophyte fungal communities at the five sites were found to differ. Deightoniella torulosa was the most frequent isolate at the Ban Suthep site and was either absent or rare at other sites. Colletotrichum species were most common in the midribs and petioles at all sites, while Pyriculariopsis parasitica and Dactylaria sp. were most common in the pseudostems. The endophyte communities isolated from M. acuminata in this study are compared with those from previous studies on tropical hosts. Several of the endophytes isolated are established pathogens of banana and provide support for the hypothesis that some endophytes are latent pathogens. The diversity of fungi on banana is discussed in relation to global estimates of numbers of fungus species. INTRODUCTION The study of endophytes of tropical plants has received much attention because endophytes are believed to be both diverse and to provide an excellent potential source of biologically active novel compounds (Dreyfuss & Petrini, 1984; Hyde, 2001). Studies in the tropics have included endophytes in bamboo (Umali, Quimio & Hyde, 1999; Lumyong et al. 2000) and palms (Rodrigues, 1994; Taylor, Hyde & Jones, 1999; Fro hlich, Hyde & Petrini, 2000). The only previous study on the endophytes of Musa sp. was conducted in Australia and Hong Kong and was mostly confined to cultivated plants (Brown, Hyde & Guest, 1998). The Musaceae is a monocotyledonous family comprising Musa and Ensete species. There are 37 species of Musa and seven species of Ensete (Price, 1995). This family has its highest species diversity in South East Asia with representatives occurring across the Old World Tropics. Due to the commercial and subsistence value of Musa species they have * Paper presented at the Asian Mycological Congress 2000 (AMC 2000), incorporating the 2nd Asia-Pacific Mycological Congress on Biodiversity and Biotechnology, and held at the University of Hong Kong on 9 13 July Corresponding author. been introduced throughout the tropics and subtropics, as well as in more marginal climates, such as those in Florida and Israel. Most fungal disease research has focused on the commercial cultivars. The present study was initiated to investigate the ecology and biodiversity of endophytic fungi in wild banana species at Doi Suthep Pui National Park, Thailand. This research was primarily motivated by the desire to establish whether the endophytes in banana differed from those in other tropical hosts. We also wanted to establish whether the endophytes in banana were particularly diverse and whether there were any evidence that at least some of the endophytes of banana are latent pathogens. MATERIALS AND METHODS Sample collection Healthy wild banana plants were sampled from four sites at Doi Suthep Pui National Park at Ban Suthep (site I, alt. 350 m), Montatarn waterfall (site II, alt. 600 m), Medicinal Plant Garden (site III, alt. 950 m) and San Gu Region (site IV, alt m) and one site at Queen Sirikit Botanic Garden (site V, alt. 600 m). Samples were collected from each site every three months from December 1998 to July 1999, so that in total 15

2 E F F E collections were made, i.e. three from each site. Five randomly selected plants were sampled per site on each collection date. Two pseudostem parts (outer and inner pseudostem) and two leaves were removed from each plant: one old leaf, defined as either the third or fourth most recently opened leaf, and one young leaf, which was always the youngest opened leaf. The samples were taken to the laboratory and processed within 24 h. Isolation of endophytes The leaves were first washed in running water. Leaf discs (3 mm diam) were cut to include the vein (10 samples) and intervein tissues (10 samples) from each leaf sample, using a sterile cork borer. Ten segments (5 5 5 mm) were cut from each of the petiole, midrib and two pseudostem samples using a sterile razor blade. All leaf discs and segments were surface sterilized in 75% ethanol for 1 min, 1% sodium hypochlorite for 3 min and 95% ethanol for 0 5 min and then dried in sterilized paper. This protocol was found to be the optimum triple sterilized procedure for isolating endophytes from M. acuminata following a pilot experiment (Photita et al. 1999). Five surface sterilized leaf discs and segments were evenly spaced in Petri dishes (9 cm diam) containing 2% (w v) malt extract agar (MEA) with added Rose Bengal (30 mg l) to slow down fungal growth and streptomycin sulfate (50 mg l) to suppress bacterial growth. Fungi growing out from the plant tissues were transferred to test tubes containing corn meal agar slants and incubated at room temperature ( 25 C) to promote sporulation. Non-sporulating cultures were transferred to incubation chambers comprising a screw top glass jar (5 5 cm diam x 10 5 cm high), containing 1 cm of solid potato dextrose agar, and an 8 cm vertical strip of banana petiole. In a second protocol the banana petiole to induce sporulation. In this way it was possible to assign many of the sterile mycelia to xylariaceous taxa. Statistical analyses The results from the three collections for each site are combined for analysis. Overall colonization rates (CR) were determined as described by Petrini, Stone & Carroll (1982). Colonization rate G Total number of leaf discs petiole midrib pseudostem segments in a sample yielding 1 isolate H G Total number of leaf discs petiole midrib pseudostem segments in that sample H Relative frequency of isolation was used for species abundance and calculated as the number of discs colonized by a given fungus divided by the total number of discs infected, and expressed as percentages. A Chi- squared (χ ) goodness-of-fit test was performed to test whether the colonization rates of five trials were statistically different. A Kruskal Wallis test was used for multisample analysis, such as the investigation of the number of isolates recovered from different tissue types at each site. Identification Sporulating isolates were identified at the genus and where possible to species level. xylariaceous taxa produced stromatalike structures, but did not sporulate. They were sorted into different taxa based on stromatal and colony morphology. Non-sporulating isolates were sorted into morphospecies on the basis of colony surface texture, hyphal pigmentation, exudates, and growth rates, as described by Fro hlich et al. (2000). Voucher slides and cultures are held in the Department of Biology, Chang Mai University. RESULTS AND DISCUSSION Overall colonization Sixty-one different fungal taxa were isolated from Musa acuminata (Table 1). The most frequently isolated species were xylariaceous taxa, Colletotrichum musae, C. gloeosporioides, Guignardia cocoicola and various sterile mycelia (Table. 1). The number of taxa isolated is similar to the 63 endophytic taxa isolated from palms (Rodrigues, 1994), but higher than many previous studies, e.g. Rodrigues & Samuels (1990), 12 taxa on palms, Pereira, Azovedo & Petrini (1993), 13 taxa on Stylosanthes, Fisher et al. (1994), 23 taxa on Opuntia stricta, Brown et al. (1998), 25 taxa on Musa sp. and Umali et al. (1999), 37 taxa from bamboo leaves. The overall colonization rates (CR) for the 5 sites did not differ greatly. They were 56 5% for Medicinal Plant Garden, 48 9% for Ban Suthep, 48% for Queen Sirikit Botanic Garden, 47 9% for San Gu Region, and 41 7%, for Montatarn Waterfall (Fig. 1). These results were within the range of other tropical endophyte studies. A great variation in colonization rates has been reported for palm endophytes, from as low as 12 5% (Rodrigues & Samuels, 1990) to as high as % (Fro hlich et al. 2000). In some studies, in which leaflets only were examined, the colonization rates were 21 30% on palms (Rodrigues, 1994) and 20 3% in palms (Southcott & Johnson, 1997). Colonization rates reported in a previous study on banana endophytes were % (Brown et al. 1998). Site effect The most common species isolated varied from one site to another. Banana samples from Ban Suthep were most frequently colonized by Deightoniella torulosa. Colletotrichum musae, xylariaceous taxa and Guignardia cocoicola were more frequently isolated from banana samples collected at Medicinal Plant Garden. Cordana musae was the most common species isolated from banana samples collected at Queen Sirikit Botanic Garden. The most common species isolated by Brown et al. (1998) from Musa sp., which is the only comparable study, were Colletotrichum gloeosporioides, Pestalotiopsis palmarum and Nigrospora oryzae in Hong Kong and Colletotrichum gloeosporioides, Glomerella cingulata and Phoma sp. in Australia. Endophytic species abundance also varied according to the site within each location. Several studies have shown that site specific factors may influence the level of infection (Carroll, 1995). This variation is probably a reflection of the range of

3 Table 1. Relative frequency (%) of endophytic fungi on young and old tissues of Musa acuminata at five sites. Taxa Young Site I Site II Site III Site IV Site V Site I Site II Site III Site IV Site V Old Ascomycete. sp Chaetomium sp. 1 3 Cladosporium sp Colletotrichum gloeosporioides Colletotrichum musae Cordana musae Curvularia sp Dactylaria sp Deightoniella torulosa Fusarium sp Fusarium sp Fusarium sp Guignardia cocoicola Helminthosporium sp Hyphomycete sp Hyphomycete sp Hyphomycete sp Hyphomycete sp Nigrospora oryzae Periconiella musae Pestalotiopsis sp Phialophora sp Phoma sp Phoma sp Phomopsis sp Phomopsis sp Pyriculariopsis parasitica Stachybotrys sp Sterile mycelium sp Sterile mycelium sp Sterile mycelium sp Sterile mycelium sp. 4 2 Sterile mycelium sp Sterile mycelium sp Sterile mycelium sp. 7 4 Sterile mycelium sp Sterile mycelium sp Verticillium sp Xylariaceous taxa Rare isolates (less than 1% relative frequency) in each site: Site I (Young): Deightoniella torulosa, Nigrospora oryzae and Constantiniella sp. Site I (old): Arthrinium sp., Drechslera sp., Glomerella sp., Dactylaria sp., Hyphomycete sp. 12, and 14 Site II (Young): Colletotrichum gloeosporioides, Nigrospora oryzae, Hyphomycete sp. 1, and Sterile mycelium sp. 5 Site II (old): Fusarium sp. 2, Drechslera sp., Pestalotiopsis sp., Phoma sp. 2, Verticillium sp., Sterile mycelium sp. 14, and 18 Site III (Young): Fusarium sp. 1, Phoma sp. 1, Hyphomycete sp. 1, Cordana musae, and Verticillium sp. Site III (old): Fusarium sp. 2, Drechslera sp., Glomerella sp., Periconia digitata, Periconiella musae, Pestalotiopsis sp., Phoma sp. 2, Phomopsis sp. 3, Sterile mycelium sp. 6, 9, 16, and 17, and Hyphomycete sp. 1 Site IV (Young): Cladosporium sp., Curvularia sp., Fusarium sp. 3, xylariaceous spp., and Sterile mycelium sp. 3 Site IV (old): Phoma sp. 2, Scytalidium sp., Sterile mycelium sp. 3, and Constantiniella sp. Site V (Young): Pyriculariopsis parasitica, Stachybotrys sp., Sterile mycelium sp. 3 and Constantiniella sp. Site V (old): Fusarium sp. 1, Glomerella sp., Nigrospora oryzae, Phoma sp. 2, Sagenoma sp., Stachybotrys sp., Sterile mycelium sp. 8, Hyphomycete sp. 5, and 6 environmental conditions under which the banana plants grew. Differences in environmental factors included humidity, temperature, rainfall and potential inoculum sources. For example, Musa plants at sites I-IV are natural stands underneath mixed deciduous forest, while site V is a wild banana plantation in Queen Sirikit Botanic Garden. Site I is located near to a village where there may be an effect due to human activity. The canopy above sites II and IV is less dense than at other sites and humidity and thus potential inoculum is probably lower. Site III is deep in the forest where the humidity is high (80 90%) all year round, and is likely to have a higher inoculum potential. Age effect There were significantly more endophyte isolates obtained from old than from young banana tissues (P 0 05) (Fig. 2). This result is in agreement with previous studies (Brown et al. 1998, Espinosa Garcia & Langenheim 1990, Fisher et al. 1995, Hata & Futai 1993, Rodrigues 1994, Taylor et al. 1999). With

4 Colonization Rate Site I Site II Site III Site IV Site V Fig. 1. Overall colonization rates of endophytic fungi of Musa acuminata in Doi Suthep Pui National Park. Colonization Rate Young Old Site I Site II Site III Site IV Site V Fig. 2. Colonization rates of endophytic fungi on young and old samples of Musa acuminata. Colonization Rate Intervein Vein Midrib Petiole Pseudostem Fig. 3. Colonization rates of endophytic fungi from Musa acuminata tissues. Table 2. The most frequency isolated fungal endophytes from different tissues of Musa acuminata. Taxa Tissue Internal vein vein midrib petiole pseudostem Xylariaceous fungi Deightoniella torulosa Guignardia cocoicola Cordana musae Colletotrichum musae C. gloeosporioides Pyriculariopsis parasitica Dactylaria sp the exception of Ascomycete sp. 1, Chaetomium sp. and Constantiniella sp. found in young samples, all fungal taxa isolated were present on old samples. Most endophytes are believed to enter a plant when a spore lands on a leaf surface and grows into the plant through the stoma, or penetrates the host directly (Fro hlich et al. 2000). The increased incidence of endophyte taxa on the older leaves provide indirect evidence for this because the older leaves would have had more time to accumulate endophytes from the environment. Older leaves would also have more time to accumulate vertically transmitted colonisers that enter from the petiole (Fro hlich et al. 2000). Effect of the tissue type With the exception of site III, the results of the Kruskal Wallis test indicate that there were no significant differences between the numbers of isolates recovered from vein, intervein, midrib, petiole and pseudostem tissues. In site III, the number of isolates from the pseudostems were significantly lower (P 0 032) than in other tissues. Bar graphs were used to compare the colonisation rates of taxa against tissue types (Figs 3) and were: petiole (60 8%), pseudostem (57 6%), midrib (49 5%), intervein (39%), and vein (36 7%), respectively. There were, however, differences in the colonization rates of taxa isolated from tissue types, which indicates that some taxa have an affinity for different tissue types. Xylariaceous taxa and Guignardia cocoicola were the most frequently isolated from leaves. Pyriculariopsis parasitica and Dactylaria sp. were most common in the pseudostem. Colletotrichum musae and C. gloeosporioides were most common in the midribs and petioles (Table 2). These results are in agreement with many studies that have found differences in species abundance between plant tissues, such as bark, stems and leaves (Fisher et al. 1994), between midrib and laminar tissue (Rodrigues 1994, Fisher et al. 1995, Brown et al. 1998). Previous studies have indicated that endophytes may exhibit tissue specificity (Luginbuhl & Mu ller, 1980; Rodrigues & Samuels, 1990; Clay, 1992; Rodrigues, 1994; Fro hlich et al. 2000). Differences in endophyte assemblages in different tissue types might be a reflection of tissue preferences of individual dominating taxa (Luginbuhl & Mu ller, 1980; Widler & Mu ller, 1984; Rodrigues & Samuels, 1990; von Halmschlager, Butin & Donaubauer, 1993), and might reflect their capacity for utilizing or surviving within a specific substrate (Rodrigues, 1994). The factors that may be important in this respect, include the weathering of the leaf cuticle, tissue texture and changes in the tissue physiology and chemistry (Petrini & Carroll, 1981; Stone, 1987). Taxonomic composition of the endophytes In this study we identified 61 taxa of endophytes from Musa acuminata in Doi Suthep Pui National Park. This comprised eight identified species, 34 taxa identified to the generic level and separated based on spore and colony morphology, five xylariaceous taxa based on stromatal structure and colony morphology and 14 sterile mycelia based on colony

5 morphology. The numbers of endophytic taxa found in this study are similar to those found on other tropical hosts, e.g. Livistona palm (Guo, Hyde & Liew, 1998), Licuala palm (Fro hlich et al. 2000), bamboo (Umali et al. 1999; Lumyong et al. 2000). However, as most endophytic species cannot be identified to species level and often are only labeled as sterile mycelia, it is hard to speculate whether many endophytes are specific to an individual host. Five of the species identified in this study, Deightoniella torulosa, Cordana musae, Colletotrichum musae, Guignardia musae and Pyriculariopsis parasitica, are only known from Musa sp. (Photita et al. 2001a). Their presence as endophytes in banana is interesting as they may be latent pathogens. There are 37 species of Musa and seven species of Ensete (Price, 1995) and most of these banana species have yet to be studied for saprobes or endophytes. Some of the taxa we identified from M. acuminata are also likely to be specific at the family level. Some of the endophytes isolated from healthy tissue in this study are established pathogens of banana. Cordana musae causes leaf blotch, while Deightoniella torulosa causes leaf spots (Ellis, 1976). Guignardia musae causes freckle, Colletotrichum gloeosporioides and C. musae also cause anthracnose of fruits (Holliday, 1980). Brown et al. (1998) alluded to the possibility that banana pathogens may spend part of their life as endophytes. The results from this study are certainly supportive of this, although it is not apparent whether all strains isolated as endophytes are capable of producing disease. Collections from diseased tissues of the same plants as examined for endophytes in this study have revealed the presence of several pathogens such as C. gloeosporioides, C. musae and Deightoniella torulosa and saprobes such as Nigrospora oryzae, Periconiella musae and Pyriculariopsis parasitica (Holliday, 1980). We have also artificially infected detached leaves of Musa acuminata with endophytic strains of Deightoniella torulosa in moist chambers and disease symptoms were produced. The percentage of colonies that did not sporulate (sterile mycelia) identified is typical of other endophyte studies in the tropics (Lodge, Fisher & Sutton, 1996; Brown et al. 1998; Umali et al. 1998; Lumyong et al. 2000). Guo et al. (1998) isolated mycelia from palm petioles in a conical flask and obtained better sporulation, and were able to identify two species that were saprobes of Livistona chinensis. The evidence of Guo et al. (1998) suggest that some of the sterile mycelia isolated in endophyte studies may be specific to that host or host family. Guo, Hyde & Liew (2000) have also sequenced representative sterile mycelia and identified some to genus, some to family, and most to order. One group belonged in the Xylariales, but were not xylariaceous species. There are many xylariaceous-like species that occur on palms (e.g. Arecophila, Capsulospora, Oxydothis), that are members of the Xylariales. It would be interesting to find such taxa as endophytic sterile mycelia on palms, especially if they are host specific. Although we used the same incubation method (Guo et al. 2000), we were only able to get xylariaceous taxa to produce stromata in culture. Brown et al. (1998) list 46 pathogens that are probably unique to Musa species, while in this study five endophytes were identified that are probably unique to Musa species. Photita et al. (2001a) list 46 saprobes from Musa species in Hong Kong and 6 of these are most probably unique to Musa species. In a list of fungi on plants in the USA (Farr et al. 1989), there are 47 fungi that occur on Musa species. As a conservative estimate there are probably more than 200 fungi that are presently known only from Musa species (Farr et al. 1989; Brown et al. 1998; Photita et al. 2001a, b). The ratio of 6 fungi to each plant which is used amongst other things to estimate global fungal species numbers (Hawksworth, 1991), therefore appears to hold for Musa species, with a ratio of 200:37or5 4:1. ACKNOWLEDGEMENTS This research was supported by The Royal Golden Jubilee PhD Program (4BCM41D1) and the Biodiversity Research and Training Program (BRT ). A. Nuangmek is thanked for help with collecting samples. The Multiple Cropping Center, Faculty of Agriculture, Chiang Mai University, is thanked for laboratory facilities. REFERENCES Brown, K. B., Hyde, K. D. & Guest, D. I. 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