Isolation and evaluation of endophytic fungi with antimicrobial ability from Phyllostachys edulis
Abstract
Endophytic fungi (30) isolates from bamboo branches were categorized into 12 genera, based on the blast analyses of ITS nrDNA sequence in GenBank and microscopic examination. The aim of this work was to investigate the antibacterial and antifungal activities of endophytic fungi. Inhibitory effects against clinical pathogens and phytopathogens have been screened for all the isolates preliminarily and strains tentatively identified as Cladosporium sphaerospermum (PE106), Simplicillium lanosoniveum (PE120), Curvularia sp. (PE127), Didymella sp. (PE128) and Penicillium cf. raistrickii (PE130) presented bioactivity against at least four tested pathogens using the agar diffusion method. Crude extracts of PE106, PE120, PE127 and PE130 displayed broad-spectrum activity against plant pathogenic fungi by mycelial radial growth test. All of the four isolates were found to have high bioactivity against the frequent plant pathogenic fungus Botryotinia fuckeliana, and two of the isolates (PE120 and PE130) also inhibited the growth of phytopathogen Thanatephorus cucumeris noteworthily. This study is the first report on the antimicrobial activity of endophytic fungi associated with branches of Ph. edulis.
Introduction
Endophytic fungi are common and diverse, and living asymptomatically within plant tissues or organs. The relationship between the endophytic fungi and their host plant is complicated (Saikkonen et al., 1998; Azevedo et al., 2000). They traditionally have been considered plant mutualists, and may provide some advantage to their hosts, including against array of biotic and abiotic stresses (Carroll, 1988; 1995; Wilson, 2000; Faeth and Fagan, 2002). Many endophytic fungi are capable of synthesizing bioactive compounds that can be used by the plant for defense against pathogenic fungi and bacteria, and producing a wide range of novel metabolites of pharmaceutical and agricultural importance. Since the paclitaxel (taxol®) in the endophytic fungus Taxomyces andreanae was firstly detected and extracted (Stierle et al., 1993; 1995), this group of microorganisms have obtained more and more attention. It is reported that many bioactive compounds, such as alkaloids, peptides, steroids, terpenoids, quinones, flavonoids, aliphatic compounds, and phenols, have been isolated from endophytic fungi in decades (Yu et al., 2010). Although there have been a broad-spectrum of biologically active metabolites from soil fungi, it is necessary to search for novel natural products from other fungi, especially interesting endophytic fungi, for resistant pathogenic strains and new diseases.
Bamboos, large perennial grass distributed widely from tropical and subtropical zones, belonging to the family Poaceae, are popularly known as the main food of the endangered giant panda (Ailuropoda melanoleuca). In Asian countries, different species and different organs of bamboos have been used for building material, handicraft article, food material and traditional medicine. For example, “Xian Zhu Li Kou Fu Ye†(as drug approved by SFDA, China) an extract of bamboo, is used as an active element to inhibit inflammation in the throat. Some biologically active components in bamboo and their potential health benefits have been widely studied (Lu et al., 2005; 2006; Keski-Saari et al., 2008; Panee et al., 2008; Mejia et al., 2009; Halvorson et al., 2011; Koide et al., 2011). Phyllostachys edulis (Carr.) H. De Lehaie (Bambusoideae, Poaceae) is mainly distributed in subtropic zones of South China. Because of high production, various purposes and wide distribution, it has long been considered the most important economic bamboo species in China.
In the last years, few studies have been conducted on the antipathogenic activity of the endophytic community of this plant (Umali et al., 1999). The aim of the present study was to explore the diversity of culturable endophytes from Ph. edulis, and to assess a promising source of bioactive compounds.
Materials and Methods
Collection of plant material and isolation of endophytic fungi
Branches of Ph. edulis collected from Yunle, Jingde County of the Anhui Province, in China. Twenty of the branches were chosen and each cut into 25 fragments less than 1 cm. The fragments were surface sterilized by being placed in 75% ethanol for 30 sec, 5% NaOCl for 10 min, and rinsing in sterile water. The small segments were cultured in Potato Dextrose Agar at 20°C without light. The dominant fungal isolates were classified by colony and hyphal characters. Cultures were deposited at China Forestry Culture Collection Center (CFCC).
Fungal culture and crude preparation
Endophytic fungi isolates were cultured in PDA solid media: PDA, containing (g/L): Potato 200 and dextrose 20; pH 6.0. The fresh mycelia of different endophytic fungi were grown on plates at 25°C for more than 7 d. 5 plugs (6 mm of diameter) of growing culture plus the adhering mycelium were subsequently added to 250 mL Erlenmeyer flasks containing 100 mL of PDA liquid media. All liquid cultures were kept at 25°C for 10 d with shaking (150 rpm).
The fermentation of each fungus was filtered to separate the mycelium from the filtrates. The mycelium was extracted with ethyl acetate (EtOAc) in order to obtain bioactive extracts (Hormazabal and Piontelli, 2009).
Agar diffusion assay
The endophytic fungi were screened using the agar diffusion method, as a rapid and qualitative selection of the bioactive microorganisms. Endophytic fungi were cultivated on the PDA solid media at 25°C over 7 d. The agars (6 mm of diameter) of growing culture plus the adhering mycelium were subsequently added to LB and PDA solid media, supplemented with 0.5% olive oil previously spread with bacteria (Staphylococcus aureus, Bacillus subtilis, Listeria monocytogenes, Salmonella bacteria), yeasts (Rhodotorula rubra, Saccharomyces cerevisiae, Candida albicans), and filamentous fungi (Curvularia eragrostidis, Pleospora herbarum, Arthrinium sacchari, Arthrinium phaeospermum and Phoma herbarum). These cultures of bacteria and fungi were also deposited at CFCC. The mycelia of filamentous fungi were fragmented with pestle and mortar. Plates were incubated at 37°C for 24 hours for the bacteria and 28°C for 2-7 d for the fungi. The inhibition zones around the agars were measured, to confirm the antimicrobial activity (de Siqueira et al., 2011).
Mycelial radial growth test
The antifungal activities of fungal extracts were tested in a number of pathogenic fungi: Botryotinia fuckeliana, Alternaria alternate, Thanatephorus cucumeris, Gibberella avenacea, and Colletotrichum lagenarium. These cultures fungi were all deposited at CFCC. The bioactive extracts were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/mL. From this solution, 0.3 ml samples were then poured on sterile Petri dishes containing 15 mL PDA to make 200 μg/mL concentration. To check the growth inhibition whether is due to DMSO, the negative control was prepared using 0.3 mL DMSO. PDA solid media were poured in three plates. Plant pathogenic fungi were inoculated in the centre of plates and the diameters of the inhibition zones were measured until the negative control plates full of mycelium. The percent of growth inhibition of extract was calculated as the following formula:
PGI = ((ND-ED)/ND)*100%
Where, ND = diameter of plant pathogenic fungi in plate with negative control; ED = diameter of plant pathogenic fungi in plate with extracts from mycelium of endophytic fungi. All the experiments were repeated three times. PGI for the each replicate was calculated and analysis of variance of the PGI was conducted by SSPS 18.0.
Results
All of fungal isolates were obtained from the branches of Phyllostachys edulis. Among them, over 150 distinct isolates were categorized into different morphotypes on the basis of colony distinction and hyphal characters. The nr ITS DNA sequence analyses were conducted to confirm the identification of these endophytic fungi. 30 representatives were identified belonging to 12 genera, namely Cladosporium, Shiraia, Colletotrichum/Glomerella, Microdochium, Arthrinium, Penicillium, Aureobasidium, Simplicillium, Phoma, Curvularia and Didymella (Table I). For molecular identification of Colletotrichum species, the sequences of ex-types were used (Hyde et al., 2009). Due to lacking of ex-type or ex-epitype of other species, the relevant sequences published in journals were also cited. The list of their respective accession number were obtained by GenBank as well as the accession number of ITS sequences from cultural endophytic fungi for the present study and the analyzed and categorized information about 30 strains involved in the study are given in Table I.
Table I:Culturable endophytic fungi isolated from Phyllostachys edulis branches
| Strain (PE) |
%Isolation frquency | Numbers of isolates | ITS (No.)a | Most closely related species | %Similarity | Reference ITS (No.)b |
|---|---|---|---|---|---|---|
| 101 | 2.7 | 4 | JX875918 | Cladosporium oxysporum | 99 | Hyde et al., 2009 EF136374 |
| 102 | 1.3 | 2 | JX875919 | Shiraia sp. Slf14 | 99 | Zhu et al., 2010 GQ355934 |
| 103 | 3.3 | 5 | JX875920 | Cladosporium sp. P31E1 | 99 | Loro et al., 2012 JN207316 |
| 104 | 2.7 | 4 | JX875921 | Cladosporium cladosporioides strain D10 | 99 | Bukovska et al., 2010 GU566222 |
| 105 | 1.3 | 2 | JX875922 | Cladosporium cladosporioides strain LPSC1088 | 99 | Llorente et al., 2012 JF949719 |
| 106 | 0.7 | 1 | JX875923 | Cladosporium sphaerospermum isolate wb311 | 99 | Buzina et al., 2003 AF455481 |
| 107 | 1.3 | 2 | JX875924 | Cladosporium sp. 7306 | 99 | Yarden et al., 2007 EF120415 |
| 108 | 1.3 | 2 | JX875925 | Cladosporium colombiae strain CBS 274.80B | 99 | Schubert et al., 2009 FJ936159 |
| 109 | 1.3 | 2 | JX875926 | Colletotrichum dematium | 97 | Hyde et al., 2002 AJ301954 |
| 110 | 1.3 | 2 | JX875927 | Microdochium sp. 5/97-31 | 99 | Ernst et al., 2011 AM502258 |
| 111 | 0.7 | 1 | JX875928 | Arthrinium sacchari isolate A09 | 99 | Gorfer et al., 2011 HQ115646 |
| 112 | 0.7 | 1 | JX875929 | Shiraia sp. JP185 | 95 | Morakotkarn et al., 2006 AB354994 |
| 113 | 0.7 | 1 | JX875930 | Shiraia sp. JP232 | 97 | Morakotkarn et al., 2006 AB255303 |
| 114 | 1.3 | 2 | JX875931 | Penicillium citrinum strain P-1637 | 99 | Alborch et al., 2012 JQ316514 |
| 115 | 3.3 | 5 | JX875932 | Colletotrichum sp. JP9 | 98 | Morakotkarn et al., 2006 AB255243 |
| 116 | 0.7 | 1 | JX875933 | Shiraia sp. slf14 | 99 | Zhu et al., 2010 GQ355934 |
| 117 | 0.7 | 1 | JX875934 | Aureobasidium pullulans strain ZD-3D | 98 | Zhang et al., 2011 JF422784 |
| 118 | 0.7 | 1 | JX875935 | Colletotrichum sp. JP9 | 99 | Morakotkarn et al., 2006 AB255243 |
| 119 | 0.7 | 1 | JX875936 | Cladosporium cladosporioides isolate wb146 | 99 | Buzina et al., 2003 AF455535 |
| 120 | 0.7 | 1 | JX875937 | Simplicillium lanosoniveum strain CBS 962.72 | 97 | Zare et al., 2008 EF641862 |
| 121 | 0.7 | 1 | JX875938 | Shiraia sp. slf14 | 99 | Zhu et al., 2010 GQ355934 |
| 122 | 0.7 | 1 | JX875939 | Arthrinium sacchari strain FBC.045 | 100 | Shrestha et al., 2011 EF076711 |
| 123 | 0.7 | 1 | JX875940 | Phoma sp. P48E2 | 99 | Loro et al., 2012 JN207349 |
| 124 | 0.7 | 1 | JX875941 | Colletotrichum sp. JP9 | 99 | Morakotkarn et al., 2006 AB255243 |
| 125 | 0.7 | 1 | JX875942 | Penicillium sclerotiorum isolate M9 | 99 | Yuan et al., 2011 HM595498 |
| 126 | 0.7 | 1 | JX875943 | Shiraia sp. slf14 | 99 | Zhu et al., 2010 GQ355934 |
| 127 | 0.7 | 1 | JX875944 | Curvularia sp. M5 | 99 | Akita et al., 2011 HM371207 |
| 128 | 0.6 | 1 | JX875945 | Didymella exitialis strain CBS 446.82 | 95 | Simon et al., 2000 EU167564 |
| 129 | 0.6 | 1 | JX875946 | Colletotrichum sp. JP9 | 98 | Morakotkarn et al., 2006 AB255243 |
| 130 | 0.6 | 1 | JX875947 | Penicillium raistrickii strain FRR 1044 | 99 | Haugland et al., 2004 AY373927 |
| aITS nrDNA sequences of cultural endophytic fungi were deposited at GenBank; bMatches of ITS nrDNA sequences published in journals were also from GenBank (Cont.) | ||||||
A total of 30 fungal endophytes were isolated to evaluate their antimicrobial activities against S. aureus, B. subtilis, L. monocytogenes, S. bacteria, C. albicans, R. rubra, S. cerevisiae, C. eragrostidis, Pleospora herbarum, A. sacchari, A. phaeospermum and Phoma herbarum by diffusion agar assay (Table II and III). The preliminary screening revealed that, of 30 isolates, five endophytic fungi (PE106, PE120, PE127, PE128 and PE130) inhibited the growth of at least two or more human pathogenic bacteria, which showed higher activity than others significantly (Table II). These isolates were also all active against human pathogenic fungi C. albicans, but PE127 and PE128 had less effect. There were two strains, PE130 and PE120, both having strongest activity against S. aureus, B. subtilis and C. albicans. In the antibacterial assay, PE130 displayed the widest spectrum of anti-activity, and also inhibited C. albicans significantly. PE120 exhibited more antifungal activity against R. rubra, less bacterial activity against L. monocytogenes and S. bacteria.
Table II: Antimicrobial activity of fungal isolates from Phyllostachys edulis against
| Isolate No. (PE) |
Staphylococcus aureus | Bacillus subtilis | Listeria monocytogenes | Salmonella bacteria | Candida albicans | Rhodotorula rubra | Saccharomyces cerevisiae |
|---|---|---|---|---|---|---|---|
| 101 | - | - | - | - | - | - | - |
| 102 | - | - | - | - | - | - | - |
| 103 | - | - | - | - | - | - | - |
| 104 | - | - | - | - | - | - | - |
| 105 | - | - | - | - | - | - | - |
| 106 | + | + | - | - | ++ | - | - |
| 107 | - | - | - | - | - | - | - |
| 108 | - | - | - | - | - | - | - |
| 109 | - | - | - | - | - | - | - |
| 110 | - | - | - | - | - | - | - |
| 111 | - | - | - | - | - | - | - |
| 112 | - | - | - | - | - | - | - |
| 113 | - | - | - | - | - | - | - |
| 114 | - | - | - | - | - | - | - |
| 115 | - | - | - | - | - | - | - |
| 116 | - | - | - | - | - | - | - |
| 117 | - | - | - | - | - | - | - |
| 118 | - | - | - | - | - | - | - |
| 119 | - | - | - | - | - | - | - |
| 120 | +++ | +++ | + | - | +++ | ++ | - |
| 121 | - | - | - | - | - | - | - |
| 122 | - | - | - | - | - | - | - |
| 123 | - | - | - | - | - | - | - |
| 124 | - | - | - | - | - | - | - |
| 125 | - | - | - | - | - | - | - |
| 126 | - | - | - | - | - | - | - |
| 127 | - | + | + | + | + | - | - |
| 128 | + | + | - | - | + | + | - |
| 129 | - | - | - | - | - | - | - |
| 130 | +++ | +++ | ++ | ++ | +++ | + | - |
| - : No activity (<10 mm); +: Activity (10-15 mm); ++: Good activity (15-20 mm); +++: Very good activity (>20 mm) | |||||||
To test anti-plant pathogenic fungi activity of the endophytic fungi, five bambusicolous pathogenic fungi (C. eragrostidis, Pleospora herbarum, A. sacchari, A. phaeospermum and Phoma herbarum) were selected for further research (Table III). None of the cultivable isolates were found effective against C. eragrostidis and Phoma herbarum, and five strains (PE106, PE120, PE127, PE128, PE130) had bioactivity against Pleospora herbarum. PE120 and PE130 were found most effective against two pathogenic fungi A. sacchari and A. phaeospermum, and PE106 presented the highest activity against Pleospora herbarum.
Table III: Antimicrobial activity of fungal isolates from Phyllostachys edulis against plant pathogens
| Isolate No. (PE) |
Curvularia eragrostidis | Pleospora herbarum | Arthrinium sacchari | Arthrinium phaeospermum | Phoma herbarum |
|---|---|---|---|---|---|
| 101 | - | - | - | - | - |
| 102 | - | - | - | - | - |
| 103 | - | - | - | - | - |
| 104 | - | - | - | - | - |
| 105 | - | - | - | - | - |
| 106 | - | +++ | ++ | ++ | - |
| 107 | - | - | - | - | - |
| 108 | - | - | - | - | - |
| 109 | - | - | - | - | - |
| 110 | - | - | - | - | - |
| 111 | - | - | - | - | - |
| 112 | - | - | - | - | - |
| 113 | - | - | - | - | - |
| 114 | - | - | - | - | - |
| 115 | - | - | - | - | - |
| 116 | - | - | - | - | - |
| 117 | - | - | - | - | - |
| 118 | - | - | - | - | - |
| 119 | - | - | - | - | - |
| 120 | - | ++ | +++ | +++ | - |
| 121 | - | - | - | - | - |
| 122 | - | - | - | - | - |
| 123 | - | - | - | - | - |
| 124 | - | - | - | - | - |
| 125 | - | - | - | - | - |
| 126 | - | - | - | - | - |
| 127 | - | + | + | + | - |
| 128 | - | + | - | - | - |
| 129 | - | - | - | - | - |
| 130 | - | ++ | +++ | +++ | - |
| -: No activity (<8 mm); +: Mild activity (8-10 mm); ++: Good activity (12-15 mm); +++: Very good activity (>15 mm) | |||||
Of 30 isolates, five strains (PE106, PE120, PE127, PE128 and PE130) presented bioactivity against at least four tested pathogenic microorganisms, and they were selected to continuation of searching bioactive compounds from fermentation broth. But unfortunately, strain PE128 could only grow on the solid media and it was not suitable for the growth of PE128 in submerged fermentation, so the bioactivities of extracts were also not calculated. Active extracts from mycelium of the other four endophytic fungi (PE106, PE120, PE127 and PE130) were submitted to the percent of growth inhibition (PGI), which is an indication of the efficacy of antifungal activity, and PGI of ethyl acetate extracts were tested at 200 μg/mL against pathogens. From the calculation of PGI, ethyl acetate extracts of four strains had the best effect against typical plant pathogenic fungi B. fuckeliana (Table IV), and they also displayed the same board-spectrum of bioactivity against T. cucumeris, but extracts of PE120 and PE130 inhibited the growth of the pathogen more weakly. Especially extracts of PE106 had low activity against T. cucumeris. In the mycelial radial growth test, no endophytic fungi exhibit antifungal activity against A. alternate and G. avenacea significantly.
Table IV: Antifungal activity of ethyl acetate extracts of the mycelium of endophytic fungi from Phyllostachys edulis branches calculated by mycelial radial growth test
| Isolate No (PE) | The percent of growth inhibition %a | ||||
|---|---|---|---|---|---|
| Botryotinia fuckeliana | Alternaria alternata | Thanatephorus cucumeris | Gibberella avenacea | Colletotrichum lagenarium | |
| 106 | 67.4 ± 5.58h | 4.0 ± 0.6b | 20.6 ± 6.7de | 5.4 ± 4.3bc | 26.8 ± 0.6ef |
| 120 | 81.0 ± 2.4i | 26.2 ± 3.3ef | 68.0 ± 5.5h | 15.0 ± 2.7cd | 41.5 ± 5.1g |
| 127 | 80.7 ± 0.8i | 12.9 ± 4.6bcd | 75.9 ± 4.9hi | 28.1 ± 7.9ef | 14.2 ± 2.9bcd |
| 128 | NE | NE | NE | NE | NE |
| 130 | 74.4 ± 4.0hi | 4.0 ± 0.7b | 45.8 ± 4.7g | 33.3 ± 3.3f | 13.9 ± 4.6bcd |
| aValue are average of three replicates ± standard deviation. Statistical analysis of the data was performed with SSPS 18.0 using Student-Newman-Keuls test for determining significant difference (α = 0.05). bcno activity, delow activity, fgmoderate activity, hihigh activity | |||||
With bioactivity against B. fuckeliana, PGI of extracts from PE120 and PE127 were higher than PE106 significantly under the same condition. Ethyl acetate extracts of PE120 were found to exhibit most bioactive against A. alternate and C. lagenarium. PE127 and PE130 showed higher activity against G. avenacea than the other strains significantly, and the most prominent activity against T. cucumeris was shown from extracts of PE120 and PE127.
Discussion
The present study is the first to investigate the diversity and antimicrobial activity of endophytic fungi from Ph. edulis. The results have shown that the fungal isolates were diverse both in morphology and molecular data. All of the cultivable isolates were affiliated with 12 genera by ITS data. The fungal community is common on terrestrial and submerged bamboos pathogen, saprophyte or endophytes (Hyde et al., 2001; Cai et al., 2003; Morakotkarn et al., 2007). The present results showed that the fungi isolated from bamboo branches are extremely frequent, and some have already been reported as pathogens or endophytes on bamboos. Strain PE120 was obviously close to Shiraia sp., which was proved to be different from S. bambusicola and other Shiraia-like fungi (Morakotkarn et al., 2008). The Shiraia sp. was common pathogenic and endophytic fungus of bamboos, but had not yet been reported on Ph. edulis (Li et al., 2009). The fruit-body of S. bambusicola Henn., has been used as an orally taken folk medicine in China over hundred years (Liu, 1978; Li et al., 2009), for its antitumor activity and antiangiogenesis (Mazzini et al., 2001; Tong et al., 2004; Chen et al., 2005). Hypocrellins, as dominant compounds, were extracted from S. bambusicola (Wan and Chen, 1981), and have attracted a great deal of attention because of their light-induced antifungal, antiviral and antitumor activity, especially against the human immunodeficiency virus (HIV) (Kocisova et al., 1999; Wang et al., 1999; Mirossay et al., 2000; Xu et al., 2001; Yang et al., 2001; Ali and Olivo, 2002; Deininger et al., 2002; Zhou et al., 2003; Chin et al., 2004). However, its efficient antimicrobial activity was only against clinical pathogens and there have been few reports of the antimicrobial activity against plant pathogens (Ma et al., 2004; Su et al., 2009). In the present study, PE120 exhibited broad-spectrum and effective antimicrobial activity, especially showed high bioactivity against human pathogens (S. aureus, B. subtilis, C. albicans and R. rubra) and phytopathogens (A. Sacchari, A. Phaeospermum, B. fuckeliana and T. cucumeris) in antagonistic test, which would be used as biocontrol agent after further studies.
As newly bambusicolous fungus, strain PE130 was closely related to Penicillium spp., isolated from plants as endophytes. It has proved Penicillium spp. to be a good source for the production of bioactive compounds, where Penicillium striatisporum, Penicillium canescens and Penicillium janczewskii all have antifungal activity against typical pathogens (Nicoletti et al., 2007; Ma et al., 2008). In our study, PE130 was also found active against the most of the test pathogens, and displayed the best inhibitory halos against S. aureus, B. subtilis, L. monocytogenes, S. bacteria, C. albicans, A. Phaeospermum and G. avenacea respectively. This isolates may also be useful source of effective antifungal agents to improve clinical study and plant defense.
Strain PE106 is a strain of Cladosporium sp. belonging to Capnodiales, which had high similarity to Cladosporium sphaerospermum, is a kind of relatively common Hyphomycetes, and is widespread mostly as saprophyte of different substrata including soil, grain, fruits, and grass litter (Braun et al., 2003; Park et al., 2008). Despite the pathogenic nature of the fungus, Cladosporium sp. has also been reported to display antiviral, antifungal and antitumor activity (Seto et al., 2005; Wang et al., 2007; Hamayun et al., 2009; Zhang et al., 2009; de Medeiros et al., 2011). The present strain PE106, isolated from Ph. edulis branches for the first time, had the best effect on the test fungi Pleospora herbarum, and was also found bioactive against C. albicans and B. fuckeliana significantly. Further investigations are required to make use of it as a biocontrol agent.
Strain PE127 was similar to Curvularia sp., which was first isolated from Ph. edulis. As a plant pathogenic fungus, the distribution of Curvularia sp. was widespread among plants, including some bamboo species but without Ph. edulis (Mohanan, 2002). Another interesting aspect of Curvularia sp. was to produce several bioactive compounds, which included Benzopyrans (Teles et al., 2005), cell cycle inhibitor (Honda et al., 2001), laccase and cellobiase (Banerjee, 1990; Banerjee and Vohra, 1991), Multiplolides A and B (Boonphong et al., 2001), phenylacetic acid derivatives and 4-epiradicinol (Varma et al., 2006), curvulapyrone, curvulalide and curvulalic acid (Trisuwan et al., 2011). Our observations suggest that fungus PE127 have broad-spectrum but mild activity against human pathogens (B. subtilis, L. monocytogenes, S. bacteria and C. albicans), and high activity against only two plant pathogens (B. fuckeliana and T. cucumeris). Further study is in progress, to identify new and useful bioactive agents.
Isolate PE128 from genus of Didymella, exhibited different strength of biological activities. PE128 was found antibacterial against S. aureus and B. subtilis by agar diffusion array, and antifungal against C. albicans, R. rubra and Pleospora herbarum. But with the normal growth on PDA solid media, there was no suited submerged fermentation method for PE128. The extracts of fermentation broth were not evaluated by mycelial radial growth test, and the data of PGI were not calculated. Future research should optimize the submerged fermentation media of PE128 and evaluate the bioactive compounds of mycelium.
Acknowledgement
We are grateful to Dr. Roland Kirschner for criticizing the manuscript and giving valuable suggestions.
References
Ali SM, Olivo M. Efficacy of hypocrellin pharmacokinetics in phototherapy. Int J Oncol. 2002; 21: 1229-37.
Azevedo JL, Maccheroni Jr W, Pereira JO, De Arajo WL. Endophytic microorganisms: A review on insect control and recent advances on tropical plants. Electron J Biotechn. 2000; 3: 15-16.
Banerjee U. Production of β-glucosidase (cellobiase) by Curvularia sp. Lett Appl Microbiol. 1990; 10: 197-99.
Banerjee U, Vohra R. Production of laccase by Curvularia sp. Folia Microbiol. 1991; 36: 343-46.
Boonphong S, Kittakoop P, Isaka M, Pittayakhajonwut D, Tanticharoen M, Thebtaranonth Y. Multiplolides A and B, new antifungal 10-membered lactones from Xylaria multiplex. J Nat Prod. 2001; 64: 965-67.
Braun U, Crous PW, Dugan F, Groenewald JZE, Sybren De Hoog G. Phylogeny and taxonomy of Cladosporium-like hyphomycetes, including Davidiella gen. nov., the teleomorph of Cladosporium s. str. Mycol Prog. 2003; 2: 3-18.
Cai L, Zhang K, McKenzie EHC, Hyde KD. Freshwater fungi from bamboo and wood submerged in the Liput River in the Philippines. Fungal Divers. 2003; 13: 1-12.
Carroll G. Fungal endophytes in stems and leaves: From latent pathogen to mutualistic symbiont. Ecology 1988; 2-9.
Carroll G. Forest endophytes: Pattern and process. Can J Bot. 1995; 73: 1316-24.
Chin W, Lau W, Cheng C, Olivo M. Evaluation of Hypocrellin B in a human bladder tumor model in experimental photodynamic therapy: Biodistribution, light dose and drug-light interval effects. Int J Oncol. 2004; 25: 623-29.
de Medeiros LS, Murgu M, de Souza AQL, Rodrigues-Fo E. Antimicrobial depsides produced by Cladosporium uredinicola, an endophytic fungus isolated from Psidium guajava fruits. Helv Chim Acta. 2011; 94: 1077-84.
de Siqueira VM, Conti R, de Araujo JM, Souza-Motta CM. Endophytic fungi from the medicinal plant Lippia sidoides Cham. and their antimicrobial activity. Symbiosis 2011; 53: 89-95.
Deininger MH, Weinschenk T, Morgalla MH, Meyermann R, Schluesener HJ. Release of regulators of angiogenesis following Hypocrellin-A and -B photodynamic therapy of human brain tumor cells. Biochem Biophys Res Commun. 2002; 298: 520-30.
Faeth SH, Fagan WF. Fungal endophytes: Common host plant symbionts but uncommon mutualists. Integr Comp Biol. 2002; 42: 360-68.
Gardes M, Bruns TD. ITS primers with enhanced specificity for basidiomycetes: Application to the identification of mycorrhizae and rusts. Mol Ecol. 1993; 2: 113-18.
Halvorson JJ, Cassida KA, Turner KE, Belesky DP. Nutritive value of bamboo as browse for livestock. Renew Agric Food Syst. 2011; 26: 161-70.
Hamayun M, Khan S, Ahmad N, Tang DS, Kang SM, Na CI, Sohn EY, Hwang YH, Shin DH, Lee BH, Kim JG, Lee IJ. Cladosporium sphaerospermum as a new plant growth-promoting endophyte from the roots of Glycine max (L.) Merr. World J Microb Biot. 2009; 25: 627-32.
Honda Y, Ueki M, Okada G, Onose R, Usami R, Horikoshi K, Osada H. Isolation, and biological properties of a new cell cycle inhibitor, curvularia, isolated from Curvularia sp. RK97-F166. J Antibiot. 2001; 54: 10-16.
Hormazabal E, Piontelli E. Endophytic fungi from Chilean native gymnosperms: Antimicrobial activity against human and phytopathogenic fungi. World J Microb Biot. 2009; 25: 813-19.
Hyde K, Ho W, McKenzie E, Dalisay T. Saprobic fungi on bamboo culms. Fungal Divers. 2001; 7: 35-48.
Keski-Saari S, Ossipov V, Julkunen-Tiitto R, Jia JB, Danell K, Veteli T, Zhang GQ, Xiong YW, Niemela P. Phenolics from the culms of five bamboo species in the Tangjiahe and Wolong Giant Panda Reserves, Sichuan, China. Biochem Syst and Ecol. 2008; 36: 758-65.
Kocisova E, Jancura D, Sanchez-Cortes S, Miskovsky P, Chinsky L, Garcia-Ramos JV. Interaction of antiviral and antitumor photoactive drug hypocrellin A with human serum albumin. J Biomol Struct Dyn. 1999; 17: 111-20.
Koide CL, Collier AC, Berry MJ, Panee J. The effect of bamboo extract on hepatic biotransforming enzymes: Findings from an obese-diabetic mouse model. J Ethnopharmacol. 2011; 133: 37-45.
Li XM, Gao J, Yue YD, Hou CL. Studies on systematics, biology and bioactive substance of Shiraia bambusicola. Forest Res. 2009; 22: 279-84.
Liu B. In: Chinese Medical Fungi. Liu B. Taiyuan, China, Shanxi People Press, 2001.
Lu B, Wu X, Tie X, Zhang Y. Toxicology and safety of antioxidant of bamboo leaves. Part 1: Acute and subchronic toxicity studies on antioxidant of bamboo leaves. Food Chem Toxicol. 2005; 43: 783-92.
Lu B, Wu X, Shi J, Dong Y, Zhang Y. Toxicology and safety of antioxidant of bamboo leaves. Part 2: Developmental toxicity test in rats with antioxidant of bamboo leaves. Food Chem Toxicol. 2006; 44: 1739-43.
Ma G, Khan SI, Jacob MR, Tekwani BL, Li Z, Pasco DS, Walker LA, Khan IA. Antimicrobial and antileishmanial activities of hypocrellins A and B. Antimicrob Agents Chemother. 2004; 48: 4450-52.
Ma Y, Chang Z, Zhao J, Zhou M. Antifungal activity of Penicillium striatisporum Pst10 and its biocontrol effect on Phytophthora root rot of chilli pepper. Biol Control. 2008; 44: 24-31.
Mazzini S, Merlini L, Mondelli R, Scaglioni L. Conformation and tautomerism of hypocrellins: Revised structure of shiraia chrome A. J Chem Soc Perkin Trans. 2001; 2: 409-16.
Mejia AI, Gallardo C, Vallejo JJ, Ramirez G, Arboleda C, Durango ES, Jaramillo FA, Cadavid E. Plants of the genus bambusa: Importance and application in the pharmaceutical, cosmetic and food indusry. Vitae-Columbia. 2009; 16: 396-405.
Mirossay A, Mojzis J, Tothova J, Hajikova M, Lackova A, Mirossay L. Hypocrellin and hypericin-induced phototoxicity of HL-60 cells: Apoptosis or necrosis? Phytomedicine 2000; 7: 471-76. Publishers, 2002.
Morakotkarn D, Kawasaki H, Seki T. Molecular diversity of bamboo associated fungi isolated from Japan. Fems Microbiol Lett. 2007; 266: 10-19.
Morakotkarn D, Kawasaki H, Tanaka K, Okane I, Seki T. Taxonomic characterization of Shiraia-like fungi isolated from bamboos in Japan. Mycoscience 2008; 49: 258-65.
Nicoletti R, Lopez-Gresa MP, Manzo E, Carella A, Ciavatta ML. Production and fungitoxic activity of Sch 642305, a secondary metabolite of Penicillium canescens. Mycopathologia 2007; 163: 295-301.
Panee J, Liu W, Lin Y, Gilman C, Berry MJ. A novel function of bamboo extract in relieving lipotoxicity. Phytother Res. 2008; 22: 675-80.
Park YS, Kim KC, Lee JH, Cho SM, Choi YS, Kim YC. Cladosporium sp. is the major causal agent in the microbial complex associated with the skin sooty dapple disease of the asian pear in Korea. Plant Pathol J. 2008; 24: 118-24.
Saikkonen K, Faeth S, Helander M, Sullivan T. Fungal endophytes: A continuum of interactions with host plants. Annu Rev Ecol Syst. 1998; 319-43.
Seto Y, Kogami Y, Shimanuki T, Takahashi K, Matsuura H, Yoshihara T. Production of phleichrome by Cladosporium phlei as stimulated by diketopiperadines of Epichloe typhina. Biosci Biotech Bioch. 2005; 69: 1515-19.
Stierle A, Strobel G, Stierle D. Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific yew. Science 1993; 260: 214-16.
Stierle A, Strobel G, Stierle D, Grothaus P, Bignami G. The search a taxol-producing microorganism among the endophytic fungi of the pacitic yew, taxus-brevifolia. J Nat Prod. 1995; 58: 1315-24.
Su YJ, Yin XY, Rao SQ, Cai YJ, Reuhs B, Yang YJ. Natural colourant from Shiraia bambusicola: Stability and antimicrobial activity of hypocrellin extract. Int J Food Sci Tech. 2009; 44: 2531-37.
Teles HL, Silva GH, Castro-Gamboa I, Bolzani VS, Pereira JO, Costa-Neto CM, Haddad R, Eberlin MN, Young MCM, Araújo ÂR. Benzopyrans from Curvularia sp., an endophytic fungus associated with Ocotea corymbosa (Lauraceae). Phytochemistry. 2005; 66: 2363-67.
Trisuwan K, Rukachaisirikul V, Phongpaichit S, Preedanon S, Sakayaroj J. Modiolide and pyrone derivatives from the sea fan-derived fungus Curvularia sp PSU-F22. Arch Pharm Res. 2011; 34: 709-14.
Umali T, Quimio T and Hyde KD. Endophytic fungi in leaves of Bambusa tuldoides. Fungal Sci. 1999; 14: 11-18.
Varma GB, Fatope MO, Marwah RG, Deadman ME, Al-Rawahi FK. Production of phenylacetic acid derivatives and 4-epiradicinol in culture by Curvularia lunata. Phytochemistry. 2006; 67: 1925-30.
Wan X, Chen Y. Hypocrellin A, a new drug for photochemotherapy. Kexue Tongbao. 1981; 26: 1040-42.
Wang FW, Jiao RH, Cheng AB, Tan SH, Song YC. Antimicrobial potentials of endophytic fungi residing in Quercus variabilis and brefeldin A obtained from Cladosporium sp. World J Microb Biot. 2007; 23: 79-83.
Wang ZJ, He YY, Huang CG, Huang JS, Huang YC, An JY, Gu Y, Jiang LJ. Pharmacokinetics, tissue distribution and photodynamic therapy efficacy of liposomal-delivered hypocrellin A, a potential photosensitizer for tumor therapy. Photochem Photobiol. 1999; 70: 773-80.
White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR protocols: A guide to methods and applications. Innis MA, Gelfand DH, Sninsky JJ, White TJ, 1st ed. San Diego, USA, Academic Press, 1990, pp 315-22.
Wilson D. Ecology of woody plant endophytes. In: Microbial endophytes. Bacon CW, White JF Jr, eds., 1st ed. New York, USA, CRC Press, 2001, pp 389-420.
Xu S, Chen S, Zhang M, Shen T, Liu Z, Zhao Y, Wu Y. Cyclohexylamino-demethoxy-hypocrellin B and photodynamic therapy decreases human cancer in vitro. Anticancer Drug Des. 2001; 16: 271-77.
Yang H, Wu T, Zhang M, Zhang Z. A novel photosensitizer, 2-butylamino-2-demethoxy-hypocrellin B (2-BA-2-DMHB) - its photodynamic effects on HeLa cells: Efficacy and apoptosis. Biochim Biophys Acta. 2001; 1540: 22-31.
Yu H, Zhang L, Li L, Zheng C, Guo L, Li W, Sun P, Qin L. Recent developments and future prospects of antimicrobial metabolites produced by endophytes. Microbiol Res. 2010; 165: 437-49.
Zhang P, Zhou PP, Yu LJ. An endophytic taxol-producing fungus from taxus media, Cladosporium cladosporioides MD2. Curr Microbiol. 2009; 59: 227-32.
Zhou Z, Yang H, Zhang Z. Role of calcium in phototoxicity of 2-butylamino-2-demethoxy-hypocrellin A to human gastric cancer MGC-803 cells. Biochim Biophys Acta. 2003; 1593: 191-200.
