Microfungi on Leaves of Licuala bidentata (Arecaceae) from Sarawak, Malaysia

A microfungal survey was carried out on the living leaves and litters of Licuala bidentata in Kubah National Park in Sarawak, Malaysia. A total of 400 leaf segments (20 0 segments for each leaf type) were plated on two i solation media (water agar and malt extract agar) for endophytic a nd saprophytic fungal isolation. Forty-three microf ungal species were obtained from both leaf types, 31 species iden tifi d from living leaves and 18 species from litte rs. Only six species were common to both leaf types, with 25 and 12 spec ies exclusively identified from living leaves and l itters, respectively. New records of fungi from this host p lant and for the genus Licuala include Isthmotricladia laeensis, Chloridium sp., Mucor sp., Oidiodendron sp., Kinochaeta sp., Cryptophiale sp., Chrysosporium merdarium and Circinotrichum fertile. This study constitutes the first report on microf ungal community on L. bidentata. Implications of this new report in comparison with the microfungal species on other plant species in the genus Licuala are discussed.


Introduction
Microfungi are important components of our ecosystem that are involved in the breakdown of organic matter and recycling of nutrients for plants' use. They are also important in other aspects of human life such as drug production, biocontrol agents, and production of industrial chemicals [1]. The diversity and distribution of microfungi are still not fully understood, especially in the tropical regions of the world, where there are many unstudied areas, substrates and host plants. Adequate knowledge of global fungal diversity and distribution will not only enrich our biodiversity database, it also will improve the accessibility, usage, and conservation of our fungal natural endowments. This also will assist policy makers in prioritizing conservation programs in relation to specific areas and specific plant species.
Licuala bidentata is a member of the palm family Arecaceae, the only family in the order Arecales. Plant species in the genus Licuala are highly diverse in the forests of southeast Asia [2], with 141 species mainly found in the Malesian region [3]. However, this plant genus is not well studied for their microfungal communities, especially in Malaysia. Licuala bidentata is native to Asia and one of the palm species found in Kubah National Park in Sarawak, Malaysia, with characteristic leaves that split into very narrow leaflets.  [4]. On studies using another palm species, Eleiodoxa conferta, 462 fungal taxa were found [5]. Also, from the work of Mckenzie et al. [6] on two species of Nikau palms, 197 fungal taxa were isolated from different types of leaf materials. This study aimed to assess the fungal communities on living leaves and litters of L. bidentata from Kubah National Park in Sarawak. The outcome of this study will be a contribution to the microfungal communities from the palm family Arecaceae, to the genus Licuala, and also to Sarawak.

Materials and Methods
Sample collection and processing. Living leaves and leaf litters were collected from Kubah National Park in Sarawak (N 01 o 36' 721", E 110 o 11' 743 ") at an elevation of 163 m above sea level in March 2014 and September 2014. Kubah National Park is located 22 km from Kuching, the capital city of the state of Sarawak [7]. The park is covered with lowland mixed dipterocarp forest, a hilly terrain, and an elevation ranging from 20 m to 799 m above sea level. Each sample collection was recorded, noting the date of collection and type of leaf collected, either living leaves or leaf litters. The samples were put in plastic bags and transported to the laboratory for processing.
Isolation and identification of fungi. The leaves were prepared for the isolation of both endophytic and saprophytic fungi and plated on two different types of isolation media, water agar (WA) and malt extract agar (MEA).
The protocol for endophytic fungal isolation using the method of Rakotoniriana et al. [8] was followed with little modifications whereby hydrogen peroxide was used as a disinfectant instead of calcium hypochlorite. The leaves were then plated on WA and MEA in petri dishes with 5 segments equidistant to each other. The plates were sealed with parafilm and incubated at room temperature under alternating light and dark conditions. Observation and isolation of the growing fungi start from the third day of incubation on MEA and four weeks on WA.
For isolation of saprophytic fungi, the leaf samples were washed with double sterilized distilled water [9], cut into 1 cm 2 into a 500 mL conical flask, washed again with sterile distilled water 5 times, and then blotted on sterilized filter paper. The leaf segments were then plated as done for the endophytic fungi. A total of 400 leaf segments were plated, 200 each for living leaves and leaf litters.
Data on the occurrence of fungi on the leaf segments were recorded. The presence of microfungi on each leaf segment was recorded, observing the characteristics of each microfungus using a Motic stereo microscope model MZ-168 and an Olympus compound microscope model CX31. Micrographs were taken with a Samsung handheld camera model ES91. Identification of the observed microfungi were made to the genus level and, wherever possible, to species level based on the morphological characters. The fungal isolates were preserved as pure cultures and semipermanent slide specimens and deposited in the Mycology laboratory culture collections at Universiti Malaysia Sarawak.
Data analysis. Multiple colonies of the same fungal taxa growing on a single leaf segment were counted as one occurrence. The frequency of occurrence of each microfungal taxa observed was calculated and the frequency of isolation was determined according to [10,11]  Species accumulation curves (SACs) were plotted and species richness estimated with the nonparametric estimators, abundance-based coverage estimator (ACE), and Chao1 for microfungal abundance data using the EstimateS diversity software version 9.1.0 released June 2013 [12].

Results and Discussion
A total of 43 fungal taxa were identified from 400 leaf segments observed both on living leaves and leaf litters of L. bidentata, comprising 3 Ascomycetes, 1 Basidiomycete, 1 Zygomycetes, 5 Coelomycetes, and 29 Deuteromycetes and 4 sterile mycelia (Table 1). Thirtyone fungal species were identified on living leaves, and 18 species were on litters ( Figure 1). The Shannon and Simpson's diversity indices for fungal community on living leaves, 2.88 and 13.43, respectively, were higher than that of the litters, 2.48 and 9.1, respectively ( Table  2).
The higher number of fungal taxa observed from leaves as compared to litters indicates that the leaves contain more supporting nutrients for the microfungal communities, which depletes rapidly on decomposition of 164 the leaf. This is also evident from the feeble nature of the litters of L. bidentata.
The number of fungal taxa recovered from both leaf types was lower than those recorded from other palm species [4][5][6]. This could be attributed to the fact that only living leaves and litters were used in this study while other different plant tissues, such as trunks, petioles, and rachides were used in other studies. The most dominant microfungi on both leaf types include Isthmotricladia laeensis, black sterile mycelia, white sterile mycelia, Graphium penicillioides, Cercospora sp., Pestalotiopsis sp. 2, Beltraniella sp., Rhinocladium sp., Pestalotiopsis sp. and Thielavia sp. (Figure 2).
In conclusion, 43 microfungal taxa were identified from both leaves and litters of Licuala bidentata with living leaves harboring more taxa than the leaf litters. Isthmotricladia laeensis was one of the dominant species on both leaf types, which indicates a microfungal similarity with other palm host plants. New records of microfungi occurring on L. bidentata and on palm species were recorded for the first time. The result from this study contributes to knowledge of fungal diversity and distribution occurring on leaves of L. bidentata and on palms (family Arecaceae).