6-guerrero-foliar fungal.pmd J.J.G. Guerrero et al. 37 SCIENCE DILIMAN (JANUARY-JUNE 2019) 31:1, 37-53 Fol iar Fungal Endophytes of Selected Medicinal Plants from the Province of Albay, Phil ippines Jonathan Jaime G. Guerrero* Mheljor A. General Jazzlyn T. Imperial College of Science Bicol University ABSTRACT F u n g a l e n d o p h y t e s w e r e i s o l a t e d f r o m t h e l e a v e s o f t h e 1 0 m o s t frequently used medicinal plants in the province of Albay, Philippines at three different locations: upland, lowland, and coastal areas. Their occurrence, frequency, and isolation rates were compared. A total of 120 i s o l a te s b e l o n g i n g to 1 7 s p ec i e s we r e i d e n t i f i ed. G l o m e r e l l a c i n g u l a t a (Stoneman) Spauld. & H. Schrenk and Colletotrichum gloeosporioides M.B. Dickman were the most frequent fungi occurring in 10 and nine plants, respectively. No signif icant difference in the total number of isolates, a s w e l l a s t h e t o t a l n u m b e r o f u n i q u e s p e c i e s f r o m a m o n g s a m p l i n g s i t e s , w a s d e t e c t e d . B l u m e a b a l s a m i f e r a ( L . ) D C h a r b o r e d t h e m o s t endophytes with 16 isolates, while banana leaves yielded the least with eight isolates. There were species of fungi that cut across all sampling sites, while a few occurred only in one site. The collection of additional s a m p l e s f r o m o t h e r s i t e s w i t h i n t h e p r o v i n c e a n d t h e t e s t i n g o f t h e biological properties of the isolates are recommended. Keyword s: Albay, endophytes, Glomerella cingulata, medicinal plants, upland ISSN 0115-7809 Print / ISSN 2012-0818 Online INTRODUCTION Traditionally, fungal endophytes are species of fungi residing within their hosts without causing apparent harm, emerging only during host-tissue senescence (Rodriguez et al. 2009) or when some physiological changes happen in the host tissue. Endophytism has long been regarded to be a form of mutualism. However, Saikkonen et al. (1998) strongly suggested that this forms a continuum of _______________ *Corresponding Author Foliar Fungal Endophytes of Selected Medicinal Plants 38 interaction, and that endophytism simply refers to where the fungus resides and not exactly how it interacts with its plant host. All plants, therefore, would host at least one fungal endophyte throughout its life cycle. The continuous documentation of fungal endophytes of plants emerges from numerous objective endpoints. It can be towards the practical application of their natural products because of the superiority of natural selection over combinatorial chemistry in novel substance discovery (Schulz et al. 2002). Moreover, an ecological standpoint examines the changing roles the fungi play in relation to its plant host (Carroll 1988; Saikkonen et al. 1998; Rodriguez et al. 2009). Diversity, taxonomy, and phylogeny also are important drivers for endophyte research because of their ubiquity and geographical distribution (Arnold and Lutzoni 2007). More importantly, endophytes are signif icant in estimating overall fungal diversity (Arnold et al. 2 0 0 0 ) . Regardless of the objective, it remains clear that endophyte documentation is a common denominator of nearly every, if not all, fungal endophyte research. Plant grouping is the basis for many processes, such as bioprospecting, ecological inferences, and diversity analysis. These groupings could be formal taxonomic grouping, or an informal grouping based on phenotypic, functional, or geographical similarities. The absence of species identif ication, or at least characterization, may be an obstacle and can substantially limit future prospects. Medicinal plants form an informal but relatively cohesive group. The extent of the members of this group is unclear as their medicinal uses vary from place to place. In the Philippines, plants with medicinal value were already documented as early as the 1950s. Comprehensive ethnobotanical surveys were also carried out in different parts of the country (Tantiado 2012; Abe and Ohtani 2013). Several research have elucidated various properties of medicinal plants, such as antibacterial (Penecilla and Magno 2011), anti-hyperglycemic (Villasenor and Lamadrid 2006) and antioxidant properties (Peteros and Uy 2010), among others. The diversity of plants and their medicinal properties may mirror the diversity of fungal endophytes residing within them, and may even extend to the potential properties of the fungi themselves. Huang et al. (2008) for instance, have shown that certain fungal taxa were more likely to coexist with plants producing certain phenolic compounds. Radu and Kqueen (2002) posed the question of whether medicinal properties are produced by plants themselves or are consequences of J.J.G. Guerrero et al. 39 the fungal-plant association. From a biological standpoint, this may be hard to answer because growing a plant without any endophyte may be challenging. However, the fungi, even without the plant, may be harnessed to produce the same benef its as that of its host. Therefore, this research is an initial species assessment of the fungal endophytes of ten medicinal plants located in the province of Albay, Philippines based on a previous medicinal plant survey by Mirandilla and Abalon (2013). The fungal e n d o p h y t e s o f t h e m e d i c i n a l p l a n t s u s e d i n t h i s r e s e a r c h h a v e m i n i m a l documentation. For instance, cassava (Manihot esculenta Crantz.) and coconut (Cocos nucifera L.) only record bacterial endophytes (Melo et al. 2009; Rajendran et al. 2015), while research on the fungal endophyte of mango was limited only to Colletotrichum spp. (Vieira et al. 2015). Likewise, literature comparing the occurrence of foliar fungal endophytes in upland, lowland, and coastal areas are limited. The ten medicinal plants included in this study are the ten most used in the province, and thus, their occurrence in the sampling sites are guaranteed. MATERIALS AND METHODS Med icinal plants Ten medicinal plants were collected in this study based on the previous assessment of their diversity and use in the province of Albay, Philippines (Mirandilla and Abalon 2013). The 10 medicinal plants shown in Table 1 are the most commonly used in the province of Albay. Common Name Scientific Name Code Used in this Study Papaya Carica papaya L. CP Cassava Manihot esculenta Crantz. M E Mango Mangifera indica L. MI Jackfruit Artocarpus heterophyllus Lam. AH Gabi Colocasia esculenta (L.) Schott. CE Coconut Cocos nucifera L. CN Malunggay Moringa oleifera Lam. M O Oregano Origanum vulgare L. OV Banana Musa paradisiaca L. MP Sambong Blumea balsamifera (L.) DC BB Table 1. Med icinal plants in Albay, Phil ippines used in this study Foliar Fungal Endophytes of Selected Medicinal Plants 40 Sampl ing sites Sampling was performed from August to September 2016. Four sampling areas were selected within the province of Albay to represent sites with adjacent upland, lowland, and coastal areas, namely the cities of Ligao, Legazpi, Tabaco, and the municipality of Pio Duran. Sites were qualitatively classif ied as upland, lowland, and coastal based on the characteristics of the area and on elevation. Upland areas refer to sites with hilly or inclined terrains, lowland areas refer to those close to residences and human settlements, and coastal areas refer to those proximate to the sea. One individual of each medicinal plant from each area was chosen. Leaf samples were collected from each of the medicinal plants and were taken immediately to the laboratory for plating. For those with big leaves, such as banana and coconut, a random portion of the entire compound leaf was cut out and placed in a sterile plastic bag. Isolation of fungal endophytes Ten mature leaves without apparent symptoms of disease, such as necrosis, chlorosis, and presence of external wounds and deformities, were selected from the fresh samples. The leaves were washed thoroughly with running water to remove any debris on the surface. After blot drying, a 0.64-cm diameter puncher was used to create one circular leaf disc per leaf at the mid-section of the blade not including the midrib. Each disc was sterilized following the methods of Torres and dela Cruz (2015) with modif ications. Leaf discs were soaked in 95% ethanol for 1 minute, then transferred to a 0.5% NaOH (Zonrox®) solution for 2 minutes, and washed twice in sterile distilled water. Leaf discs were then blot dried prior to plating. Leaf discs were plated on potato dextrose agar (PDA, Himedia). A leaf print, done by touching the leaf disc on the PDA, was made to ensure only endophytes were isolated. Plates with growth on the leaf print were discarded and not used in the study. Identification of fungal endophytes Identif ication of isolates were f irst based on morpho-cultural characteristics and compared to existing taxonomic keys, specif ically that of Watanabe (2010). Isolates similar in cultural and morphological characteristics were grouped and a reference isolate was duplicated in tubes. Isolates or groups that exhibited differences in J.J.G. Guerrero et al. 41 morpho-cultural characteristics were separated into subgroups, and one reference isolate per subgroup was also sent for the sequencing of the ITS gene. Extraction of the genomic DNA from the culture and the sequencing of the ITS gene was performed by Macrogen, Korea. The primers ITS 1 5'-TCCGTAGGTGAACCTGCGG-3' and ITS 4 5’-TCCTCCGCT TATTGATATGC-3' were used for the amplif ication of the ITS gene, which is the universal DNA barcode marker for fungi (Schoch et al. 2012). The PCR reaction was performed with 20 ng of genomic DNA as the template: initial denaturation at 95ºC for 2 minutes; 35 cycles of 95ºC for 1 minute, 55ºC for 1 minute, and 72ºC for 1 minute; and a f inal extension at 72ºC for 10 minutes. The resulting nucleotide sequences were then cleared of noises and aligned using ChromasPro and Mega7 (Kumar et al. 2015). The identities of the isolates were determined by homology against the National Center for Biotechnology Information (NCBI) database using Basic Local Alignment Search Tool (BLAST) (Altschul et al. 1990). Only hits with homology greater than 97% were considered. For isolates which obtained hits with less than 97% homology, their identities were assigned based on their morpho-cultural characteristics. All isolates are maintained at the Bicol University College of Science Department of Biology with duplicate deposits at the University of the Philippines – Los Baños Museum of Natural History Microbial Culture Collection. Data analysis The isolation rate (IR) of each fungal species was calculated using the following equation: % 100   % 100  (1) (2) where [plant–site] is def ined as the number of medicinal plants sampled multiplied by number of locations (upland, lowland, and coastal). Differences in the number of unique isolates per location were statistically analyzed by the nonparametric Kruskall-Wallis test using the R programming software (R Core Team 2013). Signif icant difference was determined at an alpha value of 0.05. Foliar Fungal Endophytes of Selected Medicinal Plants 42 RESULTS A total of 120 isolates belonging to 17 fungal species were identif ied. Representative isolates are listed in Table 2 and are shown in Figure 1. Because of some morpho- cultural differences observed during the growth stage of the isolates, some species have numerous reference cultures. The counts of the isolates were then combined when appropriate to produce the succeeding tables in this study. REF-ISO 1 KP132299.1 Hormographiella aspergillata strain IHEM 14649 97 Hormographiella aspergillata REF-ISO 2 KF679356.1 Fusarium solani strain IHB F 2353 99 Fusarium solani REF-ISO 3 JX535014.1 Fusarium solani strain MML4012 97 Fusarium solani REF-ISO 4 KP724996.1 Coprinopsis cinerea isolate 21L06I2 99 Coprinopsis cinerea REF-ISO 5 KP900278.1 Colletotrichum gloeosporioides strain SS1-CJS12 99 Colletotrichum gloeospioides REF-ISO 6 JF710555.1 Colletotrichum gloeosporioides isolate OMC4 18S 98 Colletotrichum gloeospioides REF-ISO 7 JX902434.1 Colletotrichum gloeosporioides isolate OORC30 99 Colletotrichum gloeospioides REF-ISO 8 KT342872.1 Colletotrichum gloeosporioides strain DS01 18S 97 Colletotrichum gloeospioides REF-ISO 9 FJ172224.1 Colletotrichum gloeosporioides isolate CG0305 99 Colletotrichum gloeospioides REF-ISO 10 KF706658.1 Aspergillus unguis strain FS95 99 Aspergillus unguis REF-ISO 11 FJ654485.1 Aspergillus sp. SV/09-11 18S 98 Aspergillus sp.1 REF-ISO 12 KP686465.1 Aspergillus sp. BAB-4665 98 Aspergillus sp.1 REF-ISO 13 JF436891.1 Aspergillus sp. YMCA 11 97 Aspergillus sp.2 REF-ISO 14 KP686456.1 Aspergillus sp. BAB-4649 1 96 Aspergillus sp.3 REF-ISO 15 KF923855.1 Glomerella cingulata isolate UOM P 99 Glomerella cingulata REF-ISO 16 JX868760.1 Glomerella cingulata strain LVPEI.B431_11 99 Glomerella cingulata REF-ISO 17 KF848941.1 Phomopsis sp. FZ100 98 Phomopsis sp. REF-ISO 18 KT208382.1 Phomopsis sp. GXGLB-3 99 Phomopsis sp. REF-ISO 19 GU066650.1 Phomopsis sp. 76CG/L 99 Phomopsis sp. REF-ISO 20 GU066617.1 Phomopsis sp. 27LD/T 97 Phomopsis sp. REF-ISO 21 KM362394.1 Diaporthe sp. C53-134 98 Diaporthe sp. REF-ISO 22 FJ799937.1 Diaporthe sp. SAB-2009a strain Q3310 18S 99 Diaporthe sp. REF-ISO 23 KP217184.1 Daldinia sp. P4.2 99 Daldinia sp. REF-ISO 24 JX559584.1 Guignardia mangiferae strain LGMF1163 99 Guignardia mangiferae REF-ISO 25 LM652417.1 Microascus cinereus 96 Microascus cinereus REF-ISO 26 LN482516.1 Aspergillus flavus 99 Aspergillus flavus REF-ISO 27 JQ763433.1 Aspergillus flavus 97 Aspergillus flavus REF-ISO 28 KF577897.1 Aspergillus flavus strain A0628 97 Aspergillus flavus REF-ISO 29 KF221065.1 Aspergillus flavus strain Bp5 97 Aspergillus flavus REF-ISO 30 JN709035.1 Aspergillus nomius strain SGE19 96 Aspergillus nomius REF-ISO 31 AB976023.1 Aspergillus fumigatus 97 Aspergillus fumigatus Table 2. Representative fungal endophytes isolated from med icinal plants in Albay, Phil ippines as identified through the sequencing of their ITS gene Reference Isolate Code Genbank Accession Number Most Closely Related Fungal Species based on ITS Sequence Homology % Homology Identity J.J.G. Guerrero et al. 43 Numerically, the upland area had the highest total endophytic count of 45 isolates among the sampling sites (Table 3). This, however, is not statistically signif icant compared to the coastal and lowland sampling areas. Medicinal plants from the upland, lowland, and coastal sampling sites constitute 37.5%, 35.8%, and 26.7% of the total isolates, respectively. No signif icant difference was detected in the number of fungal endophytes isolated from the same medicinal plants across different locations. Figure 1. Representative fungal endophytes in medicinal plants from Albay, Philippines grown on potato dextrose agar (PDA) for eight days: (A) Aspergillus fumigatus, (B) Aspergillus sp.1, (C) Aspergillus unguis, (D) Aspergillus sp.2, (E) Aspergillus sp. 3, (F) Colletotrichum gloeosporioides, (G) Phomopsis sp. , (H) Diaporthe sp. , and (I) Fusarium solani. Lowland 5 3 7 4 4 5 3 5 2 5 43 35.8 Coastal 5 1 2 2 4 3 5 3 3 4 32 26.7 Upland 3 7 4 7 2 2 4 6 3 7 45 37.5 Total 13 11 13 13 10 10 12 14 8 16 120 100 p-value = 0.2344 ns - no significant difference at 0.05 level of confidence CP M E M I A H C E CN M O O V M P BB Totalns %Location Total Count Table 3. Number of fungal endophytic isolates from med icinal plants in Albay, Phil ippines across d ifferent sampl ing locations Foliar Fungal Endophytes of Selected Medicinal Plants 44 The total number of unique species (Table 4), which refers to the number of unique individual fungus regardless of the number of times it was isolated from the hosts, is highest in the upland area and lowest in the coastal area. The obtained values for the total number of unique species do not signif icantly vary from each other. Most species of fungi had overlapping occurrence both in sampling sites and the host plant. Lowland 3 3 7 4 4 5 3 3 2 4 12 Coastal 4 1 2 2 3 3 4 3 3 3 10 Upland 2 5 3 5 2 1 4 4 2 7 14 Total 6 7 9 7 6 8 7 8 6 8 17 p-value = 0.3107 ns - no significant difference at 0.05 level of confidence CP M E M I A H CW CN M O O V M P TotalnsLocation Table 4. Number of unique species of fungal endophytes from med icinal plants in Albay, Phil ippines across d ifferent sampl ing sites BB Glomerella cingulata was the most frequent among the fungal endophytes, having been isolated from all the medicinal plants sampled, and in 70% of the sampling areas (Table 5). The total isolation rate is 10%, 2.33% of which is contributed by G. cingulata. Colletotrichum gloeosporioides had an isolation rate of 1.67%, while the genus Aspergillus collectively forms 1.32% of the isolation rate. Isolation rate represents the probability of encountering the same species from a pool of isolates. A higher isolation rate means that the species is grown more frequently relative to the total number of isolates. Most isolates are from the phylum Ascomycota, although many are of the anamorphic form, such as Aspergillus spp. , Fusarium solani ( M a r t . ) S a c c . , a n d Co l l e t o t r i c h u m g l o e o s p o r i o i d e s . T h e b a s i d i o m y c e t e s Hormographiella aspergillata and Coprinopsis cinerea (Schaeff.) Redhead, Vilgalys & Moncalvo may be anamorph and teleomorph of the same species (Surmont et al. 2002). Should this be the case, in combination, they account for 1.16% of the isolation rate. Seven species of fungi were absent from the coastal areas, while f ive and three species were not found in the lowland and upland areas, respectively. Six species of fungi, namely Hormographiella aspergillata, Coprinopsis cinerea, Aspergillus unguis Weill & L. Gaudin, Aspergillus sp.1, Glomerella cingulata, Colletotrichum gloeosporioides, and Fusarium solani, were consistently isolated from all sampling sites. J.J.G. Guerrero et al. 45 Table 5. Fungal endophytes of med icinal plants in Albay, Phil ippines Hormographiella Basidiomycota 4 10.00 0.00 30.00 13.33 0.33 aspergillata Coprinopsis cinerea Basidiomycota 10 30.00 30.00 30.00 30.00 0.83 Aspergillus unguis Ascomycota 6 20.00 30.00 10.00 20.00 0.50 Aspergillus sp. 1 Ascomycota 3 10.00 10.00 10.00 10.00 0.25 Glomerella cingulata Ascomycota 28 50.00 70.00 90.00 70.00 2.33 Colletotrichum Ascomycota 20 70.00 10.00 50.00 43.33 1.67 gloeosporioides Phomopsis sp. Ascomycota 9 0.00 40.00 20.00 23.33 0.75 Diaporthe sp. Ascomycota 12 50.00 40.00 10.00 36.67 1.00 Daldinia sp. Ascomycota 5 20.00 0.00 30.00 16.67 0.42 Guignardia Ascomycota 1 10.00 0.00 0.00 3.33 0.08 mangiferae A.J. Roy Microascus Ascomycota 4 20.00 0.00 20.00 13.33 0.33 cinereus Curzi Aspergillus sp. 2 Ascomycota 1 10.00 0.00 0.00 3.33 0.08 Aspergillus sp. 3 Ascomycota 1 10.00 0.00 0.00 3.33 0.08 Aspergillus Ascomycota 3 0.00 10.00 20.00 10.00 0.25 flavus Link Aspergillus nomius Ascomycota 1 10.00 0.00 0.00 3.33 0.08 Kurtzman, B.W. Horn & Hesselt Aspergillus Ascomycota 1 0.00 10.00 0.00 3.33 0.08 fumigatus Fresenius Fusarium solani Ascomycota 11 30.00 30.00 40.00 33.33 0.92 Total 120 100.00 10.00 Upland Coastal Lowland Total Species P h y l u m Number of Isolates Isolation Rate (%) Total number of leaf discs plated = 1,200 Total number of [plant–site] = 30 DISCUSSION Med icinal plants as source of endophytes The fungi G. cingulata and C. gloeosporioides, the two most isolated species in this study, are common fungal endophytes of plants as they are also latent pathogens of many important fruits, such as mango, papaya, and avocado. In particular, for papaya, the C. gloeosporioides isolated in this study may be in its latency stage because no symptoms manifested on the leaf samples and symptoms of normal anthracnose disease often occur on the fruit. F. solani, likewise, was previously reported to be an endophyte of the medicinal plants Alpinia calcarata, Bixa orellana, Calophyllum inophyllum, Catharanthus roseus, and Aquilaria sinensis, all of which are plants known for their anticancer properties (Cui et al. 2011; Sunitha et al. 2013). Interestingly, F. solani isolated from A. sinensis exhibited antitumor properties, somehow mirroring the biological property of its host plant (Cui et al. 2011). Other endophytes isolated in this study form the f irst record of their isolation from specif ic medicinal plants. Foliar Fungal Endophytes of Selected Medicinal Plants 46 It is now well known that all plants are in a symbiotic relationship with at least one fungal endophyte (Arnold et al. 2000; Rodriguez and Redman 2008; Rodriguez et al. 2009), translating to fungal species diversity (Arnold and Lutzoni 2007). Rodriguez et al. (2009) grouped together endophytes that primarily reflect differences in evolutionary relatedness, taxonomy, plant hosts, and ecological functions. Because of the importance of medicinal plants, especially in the Philippine context, it becomes an ar tif icial plant group from where to isolate endophytic fungi. The medicinal benef its that may be derived from the plant may also theoretically be exhibited by the endophytes residing in them. Based on a survey by Mirandilla and Abalon (2013), the ten medicinal plants included in this study are the top ten medicinal plants used by households in the same sampling sites. Because of the importance of these medicinal plants to locals, their occurrence in the selected sampling areas is assured. Potential endophytes from these medicinal plants may also provide similar medicinal applications. The diversity of fungal endophytes cannot be oversimplif ied. Just as there are a diverse group of plants to act as hosts, a variety of fungal groups could also grow in nearly all parts of the plant. Aside from the stem and leaves, the roots have been noted to also harbor fungal endophytes which can form mutualistic associations functionally similar to mycorrhizal symbiosis (Jumpponen 2001). A wide host population for fungal endophytes has been observed from the salt-tolerant species of mangroves (Suryanarayanan et al. 1998; Kumaresan and Suryanarayanan 2001; A n a n d a a n d S r i d h a r 2 0 0 2 ) , t r o p i c a l f o r e s t s ( S u r y a n a r a y a n a n e t a l . 2 0 0 2 ; Suryanarayanan et al. 2003), medicinal plants (Huang et al. 2008), and important agricultural crops including rice (Naik et al. 2009) and wheat (Dingle and McGee 2003). Environmental factors also affect fungal endophyte diversity as seen in studies on altitudinal and grazing gradients (Granath et al. 2007), as well as precipitation (Herrera et al. 2011). Medicinal plants are important hosts to fungal endophytes because of their biological applications. Numerous studies have already explored medicinal plants and their associated fungal endophytes (Wiyakrutta et al. 2004; Li et al. 2005; Tejesvi et al. 2007), and almost all of these tackle their medicinal potentials. Whether the plants’ medicinal value is a product of the symbiosis with its endophyte or is independent of the relationship is relative. It is also possible that the plants’ medicinal value can be mirrored by the fungal endophyte, and thus, when isolated, the endophyte can also produce by itself the same active compounds in plants. The latter has already been shown to be true for many plants. For instance, Taxomyces andreanae Strobel, A. Stierle, D. Stierle & W.M. Hess produces taxol and taxane similar with its host, the Pacif ic yew (Stierle et al. 1993). J.J.G. Guerrero et al. 47 Considerations in sampl ing area Location may be a big factor in determining the number of species, as well as the uniqueness of the fungal community. Location can alter the microclimate experienced by the host plant, thereby directly affecting the presence or absence of plant species, and their distribution and habit. Although not statistically observed in this research, many other studies suggest that geographical location can determine the species composition of fungal endophytes among plants. For instance, Mishra et al. (2012) noted that some species of fungi occur mostly exclusively at a particular season and in locations where emissions of certain gases and particulates are present. Similar observations were noted by Göre and Bucak (2007) when the number of fungal species that were isolated in leaves differed between sampling sites and the dominant fungi were site-dependent. This was not observed in this study primarily because of the small number of samples and the proximity of sampling sites. Specif ic weather conditions that can support any correlation were not recorded. Sánchez-Márquez et al. (2008) suggested that increasing the number of plants or locations would most likely reveal new endophytic species. Endophytic relationship with plants Any symbiotic relationship with the plant can be fluid, and therefore, fungal endophytes cannot be clear-cut mutualists all the time. Species such as F. solani, C. gloeosporioides, and G. cingulata are known pathogens of plants, and yet, are considered endophytes because of their location in the plant. Endophytism, by itself, should only be regarded as the location of the fungus and does not fully depict the fungus’ interaction with the host. According to Faeth (2002), the predominant defensive mutualism perspective is a product of a long list of research involving agronomic grass cultivars and may not be the case among native grasses. The preference to look at plant-fungal endophyte interactions in the light of defensive mutualism may be because of the low genetic diversity and altered growing environment of domesticated grasses, and this concept should not be generalized. This defensive mutualism is rare even in introduced and domesticated grasses. Models suggest that host and fungus genotypes, as well as the environmental conditions, affect the direction of interactions (Faeth and Fagan 2002). Faeth and Sullivan (2003) also challenged defensive mutualism by showing that mutualistic asexual endophytes of native grasses are usually parasitic because of the negative effects on plant reproduction (number and mass of seeds and germination rate). Moreover, Sieber (2007) mentioned that evidence for such is mostly circumstantial but agreed that plants would probably not survive many environmental stresses Foliar Fungal Endophytes of Selected Medicinal Plants 48 without the symbiosis with fungal endophytes. To prove unequivocally the positive impact of endophytes to its host, an endophyte-free control must be developed which, according to Sieber (2007), is a major problem. In addition, fungi can switch from quiescence to pathogenicity when factors are favorable for the fungi and unfavorable for the host. This switch is genetic, as observed in the genus Colletotrichum which causes anthracnose in cucurbits (Freeman and Rodriguez 1993). T h e s w i t c h l a t e r o n l e a d s t o t h e m a i n t e n a n c e o f c o m p a t i b i l i t y b e t w e e n Colletotrichum spp. and cucurbits, similar to that of a pathogen, as well as the survival of the host in what is known to be a balanced plant-endophyte status (Kogel et al. 2006). Colletotrichum spp. can switch from being pathogens to mutualists based on the host genotype, and thus, its ability to be an endophyte is not strictly conf ined (Rodriguez et al. 2005). Potential biological appl ications of fungal endophytes G. cingulata and C. gloeosporioides, two of the most isolated endophytes in this study, are known to have anticancer properties (Gangadevi et al. 2008), while Phomopsis spp. are known to produce terpenoids and isoflavonoids acting as antimicrobials (Redko et al. 2006; Nithya and Muthumary 2010). There is no literature stating the biological applications of Hormographiella aspergillata which is known to cause disease in humans (Surmont et al. 2002). H. aspergillata isolated from this study was not well characterized for this reason. F. solani, although known to be a plant pathogen, is also known to produce Taxol, an anticancer agent (Kusari et al. 2009). Because biological properties often differ due to location and host, the applications of the isolates from this research may be the subject of future investigations. CONCLUSION AND RECOMMENDATIONS Medicinal plants are good sources of fungal endophytes from which metabolites can be obtained with wide spectrum of applications. From this research, fungal endophytes from pre-selected medicinal plants were isolated and identif ied. A comparison of their occurrence across sampling sites and host plants displayed no signif icant difference. It is yet to be established whether a general mirroring of medicinal value can be observed from the sampled medicinal plants and their associated endophytes. The investigation of the biological activities of the isolates is recommended. J.J.G. Guerrero et al. 49 ACKNOWLEDGMENTS The researchers extend their gratitude to Bicol University for the funding of the research, Engr. Alein Navares and Mr. Francis Edward Lacanlale for the statistical analysis, Mr. Erwin Bañares for the assistance during the sampling, and to Ms. Eliza Ann A. Arena and Ms. Mae Breech R. Bernal for the photographs, isolation, and maintenance of cultures in the laboratory. REFERENCES Abe R, Ohtani K. 2013. An ethnobotanical study of medicinal plants and traditional therapies on Batan Island, the Philippines. Journal of Ethnopharmacology. 145(2):554- 565. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology. 215:403-410. Ananda K, Sridhar KR. 2002. Diversity of endophytic fungi in the roots of mangrove species on the west coast of India. Canadian Journal of Microbiology. 48(10):871-878. Arnold AE, Lutzoni F. 2007. Diversity and host range of foliar fungal endophytes: Are tropical leaves biodiversity hotspots? Ecology. 88(3):541-549. A r n o l d A E , M a y n a r d Z , G i l b e r t G S , Co l ey P D, Ku r s a r TA . 2 0 0 0 . A r e t r o p i c a l f u n g a l endophytes hyperdiverse?. Ecology Letters. 3(4):267-274. Carroll G. 1988. Fungal endophytes in stems and leaves: from latent pathogen to mutualistic symbiont. Ecology. 69(1):2-9. Cui JL, Guo SX, Xiao PG. 2011. Antitumor and antimicrobial activities of endophytic fungi from medicinal parts of Aquilaria sinensis. Journal of Zhejiang University Science B. 12(5):385-392. Dingle J, Mcgee PA . 2003. Some endophytic fungi reduce the density of pustules of Puccinia recond ita f. sp. tritici in wheat . Mycological Research. 107(3):310-316. Faeth SH. 2002. Are endophytic fungi defensive plant mutualists? Oikos. 98(1):25-36. Faeth SH, Fagan WF. 2002. Fungal endophytes: common host plant symbionts but uncommon mutualists. Integrative and Comparative Biology. 42(2):360-368. Faeth SH, Sullivan TJ. 2003. Mutualistic asexual endophytes in a native grass are usually parasitic. The American Naturalist. 161(2):310-325. Freeman S, Rodriguez RJ. 1993. Genetic conversion of a fungal plant pathogen to a nonpathogenic, endophytic mutualist. Science. 260(5104):75-79. Foliar Fungal Endophytes of Selected Medicinal Plants 50 Gangadevi V, Muthumary J. 2008. Isolation of Colletotrichum gloeosporioides, a novel endophytic Taxol-producing fungus from Justicia gendarussa. Mycological Balcanica. 5:1-4. Göre ME, Bucak C. 2007. Geographical and seasonal influences on the distribution of fungal endophytes in Laurus nobilis. Forest Pathology. 37(4):281-288. Granath G, V icari M, Bazely DR, Ball JP, Puentes A , Rakocevic T. 2007. Variation in the a b u n d a n c e o f f u n g a l e n d o p h y t e s i n f e s c u e g r a s s e s a l o n g a l t i t u d i n a l a n d g r a z i n g gradients. Ecography. 30(3):422-430. Herrera J, Poudel R, Nebel KA, Collins SL. 2011. Precipitation increases the abundance of some groups of rootassociated fungal endophytes in a semiarid grassland. Ecosphere. 2(4):1-14. Huang WY, Cai YZ, Hyde KD, Corke H, Sun M. 2008. Biodiversity of endophytic fungi associated with 29 traditional Chinese medicinal plants. Fungal Diversity. 33:61-75. Jumpponen ARI. 2001. Dark septate endophytes-are they mycorrhizal? Mycorrhiza. 11(4):207-211. Kogel KH, Franken P, Hückelhoven R. 2006. Endophyte or parasite–what decides? Current Opinion in Plant Biology. 9(4):358-363. Kumar S, Stecher G, Tamura K. 2015. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0. Molecular Biology and Evolution. 33(7):1870-1874. Kumaresan V, Suryanarayanan TS. 2001. Occurrence and distribution of endophytic fungi in a mangrove community. Mycological Research. 105(11):1388-1391. Kusari S, Zühlke S, Spiteller M. 2009. An endophytic fungus from Camptotheca acuminata that produces camptothecin and analogues. Journal of Natural Products. 72:2-7. Li H, Qing C, Zhang Y, Zhao Z. 2005. Screening for endophytic fungi with antitumour and antifungal activities from Chinese medicinal plants. World Journal of Microbiology and Biotechnology. 21(8-9):1515-1519. Melo FM, Fiore MF, Moraes LA, Silva-Stenico ME, Scramin S, Teixeira MD, Melo IS. 2009. Antifungal compound produced by the cassava endophyte Bacillus pumilus MAIIIM4A. Scientia Agricola. 66(5):583-592. Mirandilla MTA , Abalon ED. 2013. Assessment of the common medicinal plants planted and used in various households in Albay, Philippines. R&D e-journal. 1:1-10. M i s h r a A , G o n d S K , Ku m a r A , S h a r m a V K , Ve r m a S K , K h a r w a r R N , S i e b e r T N . 2 0 1 2 . Season and tissue type affect fungal endophyte communities of the Indian medicinal plant Tinospora cord ifolia more strongly than geographic location. Microbial Ecology. 64(2):388-398. J.J.G. Guerrero et al. 51 Naik BS, Shashikala J, Krishnamurthy YL. 2009. Study on the diversity of endophytic c o m m u n i t i e s f r o m r i c e ( O r y z a s a t i v a L . ) a n d t h e i r a n t a g o n i s t i c a c t i v i t i e s i n v i t r o. Microbiological Research. 164(3):290-296. Nithya K, Muthumary J. 2010. Secondary Metabolite from Phomopsis sp. Isolated from Plumeria acutifolia. Recent Research in Science and Technology. 2:99-103. Penecilla GL, Magno CP. 2011. Antibacterial activity of extracts of twelve common m e d i c i n a l p l a n t s f r o m t h e P h i l i p p i n e s . J o u r n a l o f M e d i c i n a l P l a n t s Re s e a r c h . 5(16):3975-3981. Pe te r o s N P, U y M M . 2 0 1 0 . A n t i ox i d a n t a n d cyto tox i c a c t i v i t i e s a n d p h y to c h e m i c a l screening of four Philippine medicinal plants. Journal of Medicinal Plants Research. 4(5):407-414. R: A language and environment for statistical computing [internet]. 2018. R Foundation for Statistical Computing, Vienna, Austria; [cited 2018 Feb 21]. Available online at https://www. R-project.org/. Radu S, Kqueen CY. 2002. Preliminary screening of endophytic fungi from medicinal plants in Malaysia for antimicrobial and antitumor activity. The Malaysian Journal of Medical Sciences. 9(2):23. Rajendran L, Akila R, Kar thikeyan G, Raguchander T, Samiyappan R. 2015. Defense r e l a t e d e n z y m e i n d u c t i o n i n c o c o n u t b y e n d o p h y t i c b a c t e r i a ( E P C 5 ) . A c t a Phytopathologica et Entomologica Hungarica. 50(1):29-43. Redko F, Clavin M, Weber D, Anke T, Mar tino V. 2006. Search for active metabolites of Erythrina crista-galli and its endophyte Phomopsis sp. Molecular Medicine and Medicinal Chemistry. 10:24-26. Rodriguez RJ, Redman RS, Henson JM. 2005. Symbiotic lifestyle expression by fungal endophytes and the adaptation of plants to stress: unraveling the complexities of intimacy. Mycology Series. 23:683. Rodriguez R, Redman R. 2008. More than 400 million years of evolution and some plants still can’t make it on their own: plant stress tolerance via fungal symbiosis. Journal of Experimental Botany. 59(5):1109-1114. Rodriguez RJ, White Jr JF, Arnold AE, Redman ARA . 2009. Fungal endophytes: Diversity and functional roles. New Phytologist. 182(2):314-330. Saikkonen K, Faeth SH, Helander M, Sullivan TJ. 1998. Fungal endophytes: A continuum of interactions with host plants. Annual Review of Ecology and Systematics. 29(1):319- 343. Sánchez-Márquez M, Bills GF, Zabalgogeazcoa I. 2008. Diversity and structure of the fungal endophytic assemblages from two sympatric coastal grasses. Fungal Diversity. 33:87-100. Foliar Fungal Endophytes of Selected Medicinal Plants 52 Schoch CL, Seifer t KA , Huhndorf S, Robert V, Spouge JL, Levesque CA, Miller AN. 2012. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences. 109(16):6241- 6246. Schulz B, Boyle C, Draeger S, Römmer t AK, Krohn K. 2002. Endophytic fungi: A source of novel biologically active secondary metabolites. Mycological Research. 106(9):996- 1004. Sieber TN. 2007. Endophytic fungi in forest trees: are they mutualists? Fungal Biology Reviews. 21(2):75-89. Stierle A , Strobel G, Stierle D. 1993. Taxol and taxane production by Taxomyces and reanae, an endophytic fungus of Pacif ic yew. Science. 260(5105):214-216. S u n i t h a V H , N i r m a l a D e v i D , S r i n i v a s C . 2 0 1 3 . E x t r a c e l l u l a r e n z y m a t i c a c t i v i t y o f endophytic fungal strains isolated from medicinal plants. World Journal of Agricultural Sciences. 9(1):1-9. Surmont I, Aelst FV, Verbanck J, Hoog GD. 2002. A pulmonary infection caused by Coprinus cinereus (Hormographiella aspergillata) diagnosed after a neutropenic episode. Medical Mycology. 40(2):217-219. Suryanarayanan TS, Kumaresan V, Johnson JA . 1998. Foliar fungal endophytes from two species of the mangrove Rhizophora. Canadian Journal of Microbiology. 44(10):1003- 1006. Suryanarayanan TS, Murali TS, Venkatesan G. 2002. Occurrence and distribution of fungal endophytes in tropical forests across a rainfall gradient. Canadian Journal of Botany. 80(8):818-826. Suryanarayanan TS, Venkatesan G, Murali TS. 2003. Endophytic fungal communities in leaves of tropical forest trees: diversity and distribution patterns. Current Science. 85(4):489-492. Ta n t i a d o RG . 2 0 1 2 . S u r v ey o n e t h n o p h a r m a co l o g y o f m e d i c i n a l p l a n t s i n I l o i l o, Philippines. International Journal of Bio-Science and Bio-Technology. 4(4):11-26. Tejesvi MV, Kini KR, Prakash HS, Subbiah V, Shetty HS. 2007. Genetic diversity and antifungal activity of species of Pestalotiopsis isolated as endophytes from medicinal plants. Fungal Diversity. 24(3):37-54. To r r e s J M O, D e l a C r u z T E E . 2 0 1 5 . A n t i b a c t e r i a l a c t i v i t i e s o f f u n g a l e n d o p h y t e s associated with the Philippine endemic tree, Canarium ovatum. Mycosphere. 6:266- 273. Vieira WA , Michereff SJ, de Morais MA, Hyde KD, Câmara MP. 2014. Endophytic species of Co l l e t o t r i c h u m a s s o c i a ted w i t h m a n g o i n n o r t h e a s te r n B r a z i l . F u n g a l D i ve r s i t y. 67(1):181-202. J.J.G. Guerrero et al. 53 Villaseñor IM, Lamadrid MRA. 2006. Comparative anti-hyperglycemic potentials of medicinal plants. Journal of Ethnopharmacology. 104(1-2):129-131. Watanabe T. 2010. Pictorial atlas of soil and seed fungi: Morphologies of cultured fungi and key to species. Florida: CRC Press. Wiyakrutta S, Sriubolmas N, Panphut W, Thongon N, Danwisetkanjana K, Ruangrungsi N, Meevootisom V. 2004. Endophytic fungi with anti-microbial, anti-cancer and anti- malarial activities isolated from Thai medicinal plants. World Journal of Microbiology and Biotechnology. 20(3):265-272. _____________ Jonathan Jaime G. Guerrero is a faculty of the Department of Biology, College of Science, Bicol University. His research interest includes mycology, environmental science, and biodiversity conservation. He is currently f inishing his master’s degree in Plant Pathology at the University of the Philippines Los Baños and Kasetsart University – Bangkok. Mheljor A. General is a B.S. Biology graduate of Bicol University, currently aff iliated with the Environmental Management Bureau of the Department of Environment and Natural Resources. He was a research assistant at the Bicol University College of Science. He is currently f inishing his M.S. Biology degree at Bicol University as a CHED scholar. Jazzlyn T. Imperial is an M.S. Biology degree holder from the University of the Philippines Diliman, majoring in Genetics. Prof. Imperial is currently aff iliated with Bicol University as a full-time professor handling genetics subjects for B.S. Biology students. Her work includes bioactive compounds of fungal endophytes.