Curr Microbiol DOI 10.1007/s00284-014-0680-y
Diversity of Endophytic Fungi Associated with the Foliar Tissue of a Hemi-Parasitic Plant Macrosolen cochinchinensis Sheng-Liang Zhou • Shu-Zhen Yan Qi-Sha Liu • Shuang-Lin Chen
•
Received: 29 March 2014 / Accepted: 8 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Foliar fungal endophytes are an important plant-associated fungal group. However, little is known about these fungi in hemi-parasitic plants, a unique plant group which derive nutrients from living plants of its hosts by haustoria while are photosynthetic to some degree. In this paper, the endophytic fungi in the leaves of a species of hemi-parasitic plant, Macrosolen cochinchinensis, were studied by both culture-dependent and culture-independent methods. By culture-dependent method, a total of 511 isolates were recovered from 452 of 600 leaf fragments (colonization rate = 75.3 %) and were identified to be 51 taxa. Valsa sp. was the most abundant (relative abundance = 38.4 %), followed by Cladosporium sp. 1 (13.5 %), Ulocladium sp. (4.3 %), Phomopsis sp. 2 (3.7 %), Hendersonia sp. (3.5 %), and Diaporthe sp. 4 (3.5 %). The Shannon index (H0 ) of the isolated endophytic fungi was 2.628, indicating a moderate diversity. By culture-independent method, Aspergillus spp., Cladosporium sp., Mycosphaerella sp., Acremonium strictum, and Tremella sp. were detected. To our knowledge, the Tremella species have never been detected as endophytes so far. In
S.-L. Zhou S.-Z. Yan Q.-S. Liu S.-L. Chen (&) Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China e-mail: [email protected] S.-L. Zhou e-mail: [email protected] S.-L. Zhou Key Laboratory of Biotechnology for Medicinal Plants of Jiangsu Province, Jiangsu Normal University, Xuzhou 221116, China
addition, a cloned sequence was not similar with any current sequence in the Genbank, which may represent a novel species. Altogether, this study documented endophytic fungal assemble in the leaves of M. cochinchinensis which was worthy of our attention, and may expand our knowledge about endophytic fungi within the photosynthetic tissues of plants.
Introduction Endophytic fungi are one of plant-associated fungal groups which grow in healthy plant tissues for all or the most part of their life cycle without causing disease symptoms in the host plants [23]. They emerged around the Early Devonian (about 400 million years ago) according to molecular and fossil evidences [15, 30] and have been coevolving with their host since their existence [29]. Based on numerous investigations, endophytic fungi are believed to exist in various organs of almost all land plants. The communities of endophytic fungi residing in plant leaves are of special interest, as leaves are organs in which photosynthesis and transpiration take place, where endophytic fungi have to face more biochemical dynamics and environmental variations than woody tissues [1]. In turn, endophytic fungi have some effect on the physiological activities of foliar tissues. For example, Pinto et al. showed that endophytic Colletotrichum musae and Fusarium moniliforme impair the photosynthetic efficiency in maize and banana [24]. As foliar endophytic fungi may be an underlying aspect of plant biology and, as many studies suggested, a source of novel and/or active compounds [27], they have attracted increasing interest in recent years. In nature, many trees, shrubs, or herbs have a parasitic life mode, representing 4,400 species in approximately 19
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families of flowering plants [20]. Notably, about 80 % of parasitic plants are hemiparasites, which attach themselves to host plants by the haustoria to absorb nutrients from hosts. Interestingly, this group of plants contains chlorophyll when mature and hence their leaves are photosynthetic to some degree. In the past 30 years, a great deal of studies have been carried out on the diversity of endophytic fungi associated with foliar tissues of hundreds of species of plant [31], including all major lineages of land plants, such as moss, fern, lichen, gymnosperm, and angiosperm. However, rare attentions have been paid to endophytic fungi in the photosynthetic tissues of hemi-parasitic plants. Only recently, those of a hemiparasite Viscum album were studied by conventional culturing techniques [21]. In that study, only the favorably culturable fungal endophytes in the leaves of V. album were displayed due to the limitation of the culturing method. So, foliar fungal endophytes of hemi-parasitic plants are still poorly understood so far and further studies are needed for the unique plant group. Macrosolens cochinchinensis (Loranthaceae) is a perennial climbing woody hemi-parasitic shrub, and it is primarily distributed in tropical and subtropical regions and commonly occurs in southern China. To expand our knowledge about foliar endophytes of hemi-parasitic plants, the diversity of endophytic fungi in the leaves of M. cochinchinensis was studied by both a culture-based method using six kind of media side-by-side and a cultureindependent process that relies on nuclear ribosomal internal transcribed spacer (rDNA ITS) in this paper. The present study may also provide the foundation for understanding the relationship between the leaves of M. cochinchinensis and the endophytic fungi.
Materials and Methods Sampling Site and Procedure The sampling site was located at a tea-oil Camellia plantation in Qinzhou of Guangxi Zhuang Autonomous Region, southern China (20°540 –22°410 N, 107°270 –109°560 E). The mean annual temperature is 21.4–22.0 °C, and the average annual precipitation is 1,649.1–2,055.7 mm. Twenty plant individuals of M. cochinchinensis, with a minimum distance of 20 m from each other, were sampled in March, 2011, each of which invaded a separate Camellia oleifera tree of ca. 30 years old. Healthy leaves together with associated branches of the hemiparasite were collected from each selected individual, placed in plastic bags immediately, labeled and kept cold by ice. Samples were transported to the laboratory, stored at 4 °C and processed within 2 days.
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Culture-Dependent Method Leaves selected were removed from branches and cut into segments (5 9 5 mm). Thirty leaf segments of each individual plant were randomly selected for isolation of endophytic fungi. In total, 600 leaf segments (20 individuals 9 30 leaf segments) were used in this study. The segments were surface sterilized by consecutive immersion for 1 min in 75 % ethanol, 3 min in 3.25 % sodium hypochlorite, and 30 s in 75 % ethanol, then the fragments were surface dried with sterile paper towels. As some endophytic fungi might fail to grow on one medium while could develop on other [6], we employed six kinds of media to culture endophytic fungi side-by-side. Five surface-sterilized segments were evenly placed in each Petri dish (90 mm in diam) with the following media: Czapek agar, potato dextrose agar (PDA), corn meal agar, oat meal agar, malt extract agar, and PDA containing 10 % leave extract of M. cochinchinensis. Benzylpenicillin sodium (50 mg L-1) (North China Pharmaceutical Group Co., China) was added to all the six kinds of media above to suppress bacterial growth. The dishes were incubated at 22 °C, and examined periodically. When colonies developed, they were transferred to new PDA plates for purification. The pure strains were examined periodically, of which the sporulating ones were identified based on their morphological characteristics [5, 14]. The non-sporulating cultures were induced to sporulate according to the reported methods [25]. The strains which failed to sporulate after induction were designated as sterile mycelia and those were identified based on the internal transcribed spacer (ITS) region sequences of rDNA [18]. Total genomic DNA was extracted following the method described by Guo et al. [12]. The ITS region of each DNA sample was amplified using the universal primers ITS1/ITS4 [32], and sequenced at Sangon Biotech (Shanghai) Co., Ltd. All the sequences were initially aligned using the program package MEGA4.1. Then, each sequence was used as a query to search for similar sequences from GenBank using the FASTA and BLAST programs for identification. The colonization rate was calculated as the total number of fragments infected by one or more endophytic fungi divided by the total number of fragments incubated to measure the degree of endophytic fungal infection of the leaves. The relative abundance (RA) of each endophytic taxon was calculated as the number of isolates of a certain taxon divided by the total number of isolates obtained. Shannon’s diversity index (H’) was used to anlyze the diversity of endophytic fungi and calculated as follows: P H 0 ¼ ki¼1 Pi ln Pi, where k is the total number of fungal taxon, and Pi is the RA of taxon i. Species
Z. Sheng-Liang et al.: Diversity of Endophytic Fungi
accumulation curve was calculated with Estimate S Version 8.2.0.
Table 1 The community composition and shannon’s diversity index of endophytic fungi isolated from the leaves of Macrosolens cochinchinensis
Culture-Independent Process
Taxa of endophytes
The leaf samples of M. cochinchinensis were surface sterilized to remove organisms adhered to the surface of the leaves as described in Gao et al. [9]. Total DNA of the samples was extracted by a modified protocol of CTAB as in Guo et al. [13]. The ITS primers ITS1H/ITS4B, designed based on the universal primers ITS1/ITS4 [32], were used to amplify the ITS regions. Primer ITS1H, 50 -ACAC AAGCTTTCCGTAGGTGAACCTGCGG-30 , contains a HindIII restriction site (underlined); primer ITS4B, 50 -ACACGGATCCTCCTCCGCTTATTGATATGC-30 , contains a BamHI restriction site (underlined). The PCR products were analyzed on 2.0 % agarose gels. The objective bands were excised from the gels and purified with UltraSep Gel Extraction Kit (Omega Co.) following the manufacturer’s instructions. The purified PCR products and the pUC19 vector were digested by restriction endonucleases HindIII and BamHI. The digested PCR products and pUC19 vector were gel-purified as described above, ligated overnight at 16 °C, transformed into Escherichia coli DH5a, and recombinant clones were identified by bluewhite screening. Each recombinant plasmid DNA was extracted with SanPrep Column Plasmid Mini-Preps Kit (Sangon Biotech Co., China) and digested with HindIII and BamHI. The clones containing an insert of correct ITS size were sequenced at Sangon, and used for molecular identification. The clone library size was analyzed by coverage C and calculated as follows: C = 1 - n1/N, where n1 is the total number of operational taxonomic unit (OTU) which appears only once, and N is the total number of clones.
Result Endophytic Fungi Recovered by Culture-Dependent Method In total, 511 isolates were recovered from 452 of 600 leaf fragments used (colonization rate = 75.3 %), the remaining 148 fragments did not yield any endophytic fungus. The isolates were identified to be 51 taxa (Table 1), 17 of which were singletons (species that were observed only once). Identification of sterile mycelia is shown in Table 2. The Shannon diversity index (H0 ) of the culturable endophytes was 2.628. Valsa sp. was the most abundant (RA = 38.6 %), followed by Cladosporium sp. 1 (13.5 %), Ulocladium sp. (4.3 %), Phomopsis sp. 2 (3.7 %), Hendersonia sp. (3.5 %), and Diaporthe sp. 4 (3.5 %). the RA of
No. of isolates
Relative abundance (%)
Ascomycetes Acremoniella sp.
4
0.8
Aschersonia sp.
1
0.2
Ascomycota sp.1
8
1.6
Ascomycota sp.2
11
2.2
Ascomycota sp.3
8
1.6
Ascomycota sp.4
1
0.2
Ascomycota sp.5
1
0.2
Aspergillus sp.
2
0.4
Berkleasmium sp. Botryosphaeria dothidea
4 1
0.8 0.2
Botryosphaeria sp.1
13
2.5
Botryosphaeria sp.2
1
0.2
Catenularia sp.
2
0.4
Cephalosporium sp.
7
1.4
Chromosporium sp.1
4
0.8
Chromosporium sp.2
3
0.6
Cladosporium sp.1
69
13.5
Cladosporium sp.2
9
1.8
Corynespora sp.
3
0.6
Coryneum sp.
1
0.2
Curvularia sp.
3
0.6
Diaporthe sp.1
4
0.8
Diaporthe sp.2
4
0.8
Diaporthe sp.3 Diaporthe sp.4
13 18
2.5 3.5
Diaporthe sp.5
2
0.4
Diaporthe sp.6
1
0.2
Diaporthe sp.7
1
0.2
Diaporthe sp.8
1
0.2
Diaporthe sp.9
1
0.2
Fusarium sp.
1
0.2
Gilmaniella sp.
5
1.0
Glomerella sp.1
3
0.6
Glomerella sp.2
11
2.2
Glomerella sp.3
1
0.2
Helminthosporium sp.
1
0.2
Hendersonia sp.
18
3.5
Melanconium sp.
2
0.4
Neosartorya sp.
4
0.8
Pestalotiopsis sp. Phomopsis sp1
1 9
0.2 1.8
Phomopsis sp.2
19
3.7
Phomopsis sp.3
1
0.2
Sporidesmium sp.
2
0.4
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Z. Sheng-Liang et al.: Diversity of Endophytic Fungi Table 1 continued Taxa of endophytes
No. of isolates
Relative abundance (%)
Torula sp.1
1
0.2
Torula sp.2
2
0.4
22
4.3
197
38.6
1
0.2
Ulocladium sp. Valsa sp. Xylaria sp. Basidiomycetes Agaricomycetes sp.
3
0.6
Irpex sp.
6
1.2
511
100.0
Total Shannon’s diversity index (H0 )
2.628
the other species was between 2.5 % and 0.2 %. Species accumulation curve approximated an asymptote when singletons were removed; however, the curve continuously rose when singletons were included, indicating that further sampling would recover more rare endophyte taxa (Fig. 1). The overwhelming majority of the culturable endophytic fungi recovered were ascomycetes, which accounted for 98.2 % of the total isolates and were distributed in 3 classes (Sordariomycetes, Dothideomycetes, and Eurotiomycetes) except for Incertae sedis, the rest were basidiomycetes. As shown in Fig. 2, 59.1 % of isolates were assignable to Sordariomycetes, of which Diaporthales was dominant, accounting for 90.7 % of this group. The remaining Sordariomycetes isolates belonged to Hypocreales, Chaetosphaeriales, Xylariales, and Incertae sedis, accounting for 3.0, 0.7, 0.7, and 5.0 %, respectively. There were 28.6 % of isolates in Dothidemycetes. Of the Dothidemycetes group, more than half (53.4 %) were in Capnodiales, 36.3 and 10.3 %, respectively, were in Pleosporales and Botryosphaeriales. A considerable fraction of isolates (9.4 %) were Incertae sedis of ascomycetes, and only a small portion were in Eurotiomycetes (1.2 %), all belonging to Eurotiales. In addition, 1.8 % of isolates were in Agaricomycetes of Basidiomycota. Endophytic Fungi Detected by Culture-Independent Method Three major fragment sizes of PCR products were obtained from DNA samples extracted directly from the leaf of M. cochinchinensis (Fig. 3). Of which, the putative ITS sequences (500–700 bp) were purified and cloned. The recombinant clones were identified by blue-white screening and most white transformants were shown to carry an insert fragment similar to the expected size of the objective
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ITS sequences (Fig. 4). A total of 80 such recombinant clones were isolated in this study. The result of DNA sequencing and sequence alignments of 80 recombinant clones revealed 16 different insert sequences, no chimera was found. The coverage C of the library size was 86.3 %. The abundance, length and the most homologous sequences of the cloned sequences identified in this study are summarized in Table 3. Among the 16 cloned sequences, 14 had relatively high sequence similarities with species of their closely identified phylogenetic relative belonging to Ascomycota (C98 %), one sequence, J2A19, was related to Basidiomycota at ITS similarity of 89 %, J2G90 was not similar to any current sequence in the database. The clone sequences were further identified to be species of Aspergillus (70 clones), Cladosporium (4 clones), Mycosphaerella (2 clones), Acremonium (2 clones), and Tremella (1 clone) except J2G90, which may represent a novel species.
Discussion In this paper, endophytic fungal community associated with the leaves of M. cochinchinensis were studied by both a culture-dependent method and a culture-independent process. The conventional method for studying the diversity of endophytic fungi is culture-dependent method. The main limitation of the method is that unculturable species and some slow-growing or weak-competitive species may not be isolated. To make up the shortcomings of the culturing method, culture-independent methods were developed in recent years [3]. However, studies showed that endophytic fungi recoverd by culture-dependent method were often different from those detected by culture-independent methods, and some commonly isolated strains were never found by unculture methods [3, 13]. In the present study, the endophyte communities detected by the two methods were completely different. The most cloned sequences were closely relative to species of Aspergillus according to the culture-independent method. Although one Aspergillus species was also isolated by culturing method, the ITS sequence of which differed significantly from the cloned ones (around 22.5 % of sequence divergence), indicating that different Aspergillus species were detected by the two methods. Similarly, the species of Cladosporium detected by the two methods were not the same according to the sequence comparison. The reasons that cause the difference between endophytic fungal assembles detected by culturing and unculturing methods have been discussed in previous studies [13]. Generally, the Shannon index (H0 ) is usually between 1.5 and 3.5, the lowest is 1.5 and the highest is 3.5 [10]. The H0 of the endophytic fungi isolated from the leaves of
Z. Sheng-Liang et al.: Diversity of Endophytic Fungi Table 2 Molecular identification of sterile mycelia recovered from the leaves of Macrosolens cochinchinensis based on ITS sequences Identification
Closestly matched species in GenBank (Accection number)
Classification of matched speciesa
Cover (%)
KF675752
Aschersonia sp.
Aschersonia sp. (KC771478.1)
Clavicipitaceae, Hypocreales, Sordariomycetes
94
99
KF159990
Ascomycota sp.1
Pichia guilliermondii (HM037942.1)
Pichiaceae, Saccharomycetales, Saccharomycetes
95
99
Debaryomyces hansenii (DQ534408.1)
Incertae sedis, Saccharomycetales, Saccharomycetes
95
99
Ustilaginoidea virens (JF271122.1)
Incertae sedis, Hypocreales, Sordariomycetes
95
99
Phomopsis sp. (HQ832826.1)
Diaporthaceae, Diaporthales, Sordariomycetes
93
98
Diaporthe sp. (JF441199.1)
Diaporthaceae, Diaporthales,Sordariomycetes
93
99
Alternaria mali (EU732731.1)
Pleosporaceae, Pleosporales, Dothideomycetes
94
98
Fusarium oxysporum (JN400690.1)
Nectriaceae, Hypocreales, Sordariomycetes
96
99
Cyttaria darwinii (EU107253.1)
Cyttariaceae, Cyttariales, Leotiomycetes
96
99
Botryosphaeria dothidea (GQ855797.1)
Botryosphaeriaceae, Botryosphaeriales, Dothideomycetes
96
99
Phomopsis sp. (HQ832826.1)
Diaporthaceae, Diaporthales, Sordariomycetes
95
99
Diaporthe sp. (DQ145729.1)
Diaporthaceae, Diaporthales, Sordariomycetes
95
99
Alternaria mali (EU732731.1)
Pleosporaceae, Pleosporales, Dothideomycetes
96
98
Fusarium lateritium (KF159980.1)
Nectriaceae, Hypocreales, Sordariomycetes
96
99
Aspergillus clavatus (KC215115.1)
Trichocomaceae, Eurotiales, Eurotiomycetes
96
97
Aschersonia sp. (AY225332.1)
Clavicipitaceae, Hypocreales, Sordariomycetes
96
97
Accession number
Indent (%)
Ascomycetes
KF159992
KF159996
KF675756
KF675759
Ascomycota sp.2
Ascomycota sp.3
Ascomycota sp.4
Ascomycota sp.5
KF495994
Aspergillus
Aspergillus niger (JN672584.1)
Trichocomaceae, Eurotiales, Eurotiomycetes
95
99
KF675755
Botryosphaeria dothidea
Botryosphaeria dothidea (KC492490.1)
Botryosphaeriaceae, Botryosphaeriales, Dothideomycetes
97
99
KF160000
Botryosphaeria sp.1
Botryosphaeria dothidea (KC492490.1)
Botryosphaeriaceae, Botryosphaeriales,Dothideomycetes
94
99
KF675753
Botryosphaeria sp.2
Botryosphaeria sp. (JQ772020.1)
Botryosphaeriaceae, Botryosphaeriales, Dothideomycetes
73
97
KF159973
Cladosporium sp.1
Cladosporium sp. (JX675049.1)
Davidiellaceae, Capnodiales, Dothideomycetes
98
99
KF159974
Cladosporium sp.2
Cladosporium sphaerospermum (HM595522.1)
Davidiellaceae, Capnodiales, Dothideomycetes
97
99
KF159976
Diaporthe sp.1
Diaporthe eucalyptorum (JX069862.1)
Diaporthaceae, Diaporthales, Sordariomycetes
95
99
KF159977
Diaporthe sp.2
Diaporthe eucalyptorum (JX069862.1)
Diaporthaceae, Diaporthales, Sordariomycetes
95
99
KF159984
Diaporthe sp.3
Diaporthe eucalyptorum (JX069862.1)
Diaporthaceae, Diaporthales, Sordariomycetes
94
98
KF159993
Diaporthe sp.4
Diaporthe perseae (KC343173.1)
Diaporthaceae, Diaporthales, Sordariomycetes
97
98
KF675745
Diaporthe sp.5
Diaporthe sp. (JF773672.1)
Diaporthaceae, Diaporthales, Sordariomycetes
96
99
KF675746
Diaporthe sp.6
Diaporthe sp. (KF159984.1)
Diaporthaceae, Diaporthales, Sordariomycetes
96
99
KF675747
Diaporthe sp.7
Diaporthe sp. (KF160008.1)
Diaporthaceae, Diaporthales, Sordariomycetes
99
99 100
KF675748
Diaporthe sp.8
Diaporthe sp. (KF160008.1)
Diaporthaceae, Diaporthales, Sordariomycetes
96
KF675750
Diaporthe sp.9
Diaporthe hongkongensis (KC343119.1)
Diaporthaceae, Diaporthales, Sordariomycetes
95
99
KF675751
Fusarium sp.
Fusarium oxysporum (JN400719.1)
Nectriaceae, Hypocreales, Sordariomycetes
97
99
KF159983
Glomerella sp.1
Glomerella sp. (GQ352479.1)
Glomerellaceae, Incertae sedis, Sordariomycetes
97
99
KF159986
Glomerella sp.2
Glomerella cingulata (EU520087.1)
Glomerellaceae, Incertae sedis, Sordariomycetes
97
99
KF675757
Glomerella sp.3
Glomerella sp. (KF159997.1)
Glomerellaceae, Incertae sedis, Sordariomycetes
98
99
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Z. Sheng-Liang et al.: Diversity of Endophytic Fungi Table 2 continued Accession number
Identification
Classification of matched speciesa
Closestly matched species in GenBank (Accection number)
Cover (%)
Indent (%)
KF159988
Neosartorya sp.
Neosartorya hiratsukae (HE578066.1)
Trichocomaceae, Eurotiales, Eurotiomycetes
94
99
KF675754
Pestalotiopsis sp.
Pestalotiopsis microspora (HQ889705.1)
Amphisphaeriaceae, Xylariales, Sordariomycetes
96
99
KF159989
Phomopsis sp1
Phomopsis sp. (GU066685.1)
Diaporthaceae, Diaporthales, Sordariomycetes
95
99
KF159991
Phomopsis sp.2
Phomopsis sp. (GU066617.1)
Diaporthaceae, Diaporthales, Sordariomycetes
96
99
KF675749
Phomopsis sp.3
Phomopsis sp. (JQ809670.1)
Diaporthaceae, Diaporthales, Sordariomycetes
96
98
KF675758
Xylaria sp.
Xylaria sp. (AB449098.1)
Xylariaceae, Xylariales, Sordariomycetes
97
99
Bjerkandera adusta (JN198491.1)
Meruliaceae, Polyporales, Agaricomycetes Basidiomycota
96
99
Trichaptum abietinum (FJ768676.1)
Incertae sedis, Hymenochaetales, Agaricomycetes Basidiomycota,
94
100
Thanatephorus cucumeris (FJ467377.1)
Ceratobasidiaceae, Cantharellales, Agaricomycetes, Basidiomycota
95
99
Ganoderma lobatum (JQ520165.1)
Ganodermataceae, Polyporales, Agaricomycetes, Basidiomycota
94
99
Irpex lacteus (HQ670693.1)
Meruliaceae, Polyporales, Agaricomycetes, Basidiomycota
97
99
Basidiomycetes KF159979
KF159981
Irpex sp.
Fungal taxonomy applied follows Index Fungorum (http://www.indexfungorum.org/Names/Names.asp)
Numbet of species of endophytic fungi
a
Agaricomycetes sp.
60 50
I II
40 30 20 10 0
0
5
10
15
20
25
Number of trees
Fig. 1 Species accumulation curve of endophytic fungi isolated from the leaves of Macrosolens cochinchinensis, I, singletons were included. II, singletons were removed
M. cochinchinensis was 2.628 in our results, indicating the diversity was moderate. Among these endophytic fungi, the Sordariomycetes and Dothideomycetes accounted for more than 87.7 % of isolates (Fig. 2). The result was consistent with previous reports, in which the Sordariomycetes and Dothideomycetes represented the majority of foliar endophyte species [1]. Totally, culture-dependent and cultureindependent methods revealed that the endophytic fungi detected were distributed mainly in 3 classes of ascomycetes and 2 classes of basidiomycetes, indicating a broad spectrum of endophytic fungi within the leaves of M. cochinchinensis. Furthermore, the composition structure of endophytic fungi
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in this study was also in agreement with those in nonparasitic plants, in which the overwhelming majority of endophytic fungi recovered were ascomycetes, occasionally a small portion of basidiomycetes or zygomycetes. The endophyte communities are generally dominated by one or more species, which are supposed to be closely related to their hosts [11]. Among the culturable endophytic fungi in the present study, Valsa sp. was the most abundant (RA = 38.4 %). Valsa species have been rarely detected as endophytic fungi so far. To our knowledge, only one endophytic species of Valsa was reported as dominant endophyte from the Populus tremula in Spain [26]. Valsa is a widespread genus of fungi within the family Valsaceae (Diaporthales, Sordariomycetes). Generally, Valsa species were found as saprophytic or pathogenic fungi. Some species of Valsa may invade in various deciduous fruit trees or broad-leaved trees, causing serious lesion of the hosts. It has long been recognized that a fungal species may act as endophyte in one host while pathogen in other plants [1]. Some fungal pathogens, e.g., species of Apiognomonia, Ophiovalsa, Pezicula or Phomopsis, have been reported as dominant endophytic fungi in different trees [29]. Our results showed for the first time that the endophyte community in the leaves of a hemi-parasitic plant might also dominated by the fungus that is considered a pathogen. However, the relationship between endophytic Valsa sp. and M. cochinchinensis needs further studies. Many studies showed that endophytic fungi in the leaves of woody plants spread via spores (horizontal transmission)
Z. Sheng-Liang et al.: Diversity of Endophytic Fungi
Fig. 2 The community composition of endophytic fungi isolated from the leaves of Macrosolens cochinchinensis
Fig. 3 Agarose gel electrophoresis profiles of PCR products amplified from DNA extracted directly from leaves of Macrosolens cochinchinensis. Lanes 1 and 2, PCR products from two independent DNA samples as templates. The arrow shows the objective bands of ITS sequences (500–700 bp). Lane M, DNA size markers
[2, 8], which may flow with air movement, attach to plant leaves, then germinate and invade into the foliar tissues. In our study, the species of Cladosporium, Phomopsis and Diaporthe were isolated with relatively high frequency. Cladosporium spores are wind-dispersed, and the endophytic fungi in this genus are known to be not host specificity, which can be found in various hosts [22]. The host range of endophytic Phomopsis are also very wide, they may inhabit in various plant hosts which are taxonomically irrelevant [29]. Similarly, Diaporthe (the teleomorph of Phomopsis) has also been reported as one of the most frequently encountered genera of endophytic fungi in
Fig. 4 Agarose gel electrophoresis profiles of 7 digested recombinant plasmid DNA samples. The arrow shows the size of objective bands of ITS sequence. Right lane, DNA size markers
several plant hosts [7, 19]. In addition, species of Glomerella, Fusarium, Pestalotiopsis, Xylaria, and Botryosphaeria detected in this study with low frequency are also known to be ubiquitous genera as endophytic fungi [4, 28]. It seems that the non-host-specificity endophytic species mentioned above invade into the leaves of M. cochinchinensis via horizontal transmission. However, all of the fungal species mentioned above did not sporulate on the culture media despite of various methods of induction, their transmission mechanism remain unknown. Among the endophyte community detected in the present study, Aspergillus and Cladosporium were detected by both culture-dependent and culture-independent methods.
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Z. Sheng-Liang et al.: Diversity of Endophytic Fungi Table 3 The abundance, length and the most homologous sequences of the ITS of endophytic fungi detected from the leaves of Macrosolen cochinchinensis by cultureindependent method
ITS clone sequences
No. of clones
Length of the sequence (bp)
GenBank accession numbers
Closely identified phylogenetic relative (Genebank accession number)
Query coverage (%)
J2C68
7
572
JX123325
Aspergillus ustus strain UWFP 546 (AY213637.1)
100
99
J2E39
1
569
JX123326
Aspergillus ustus strain UWFP 546 (AY213637.1)
100
99
J2A35
54
568
JX123331
Aspergillus sp. MS-2011F42(HE608807.1)
100
100
J2G54
1
567
JX123337
Aspergillus sp. MS-2011F42(HE608807.1)
100
99
J2G20
1
567
JX123341
Aspergillus sp. MS-2011F42(HE608807.1)
100
99
J2G48
1
570
JX123347
Aspergillus sp. MS-2011F42(HE608807.1)
100
98
J2E32
1
566
JX123353
Aspergillus versicolor strain CanR-30 (JF817276.1)
99
99
J2F149
4
569
JX123354
Aspergillus versicolor isolate UOA/HCPF8640 (FJ878625.1)
100
100
J2A19
1
500
JX123327
Tremella globispora strain CBS 6972 (AF444432.1)
100
89
J2A26
1
551
JX123328
Uncultured Cladosporium clone OS_2d_H09(JF449702.1)
100
100
J2A28
2
553
JX123329
Cladosporium sp. MS-2011-F14 (HE608784.1)
100
99
J2G25
1
551
JX123330
Cladosporium sp. MS-2011-F14 (HE608784.1)
100
98
J2A37
2
574
JX123349
Acremonium strictum genogroup III strain(AY138846.1)
100
100
J2E4
1
542
JX123351
Mycosphaerella sp. Ponipodef 06 (HQ731642.1)
100
99
J2E25
1
542
JX123352
Mycosphaerella sp. Ponipodef 06 (HQ731642.1)
100
99
J2G90
1
559
JX123356
–
–
Endophytic Aspergillus spp. were widely reported to produce novel metabolites and bioactive procucts, such as antimicrobial, anticancer, antioxidant, anti-phytopathogenic, and phytohormonal activity [17]. Similarly, endophytic Cladosporium spp. were also known as a source of new natural products and bioactive metabolites, including antifungal, antitumor, antiviral, antimitotic, and cytostatic activities [33]. In addition, Acremonium strictum detected in this study was reported to mediate an antagonism toward herbivorous insects [16]. The results indicated that the leaves of M. cochinchinensis, and reasoningly other hemiparasitic plants, might represent an untapped reservoir of endophytic fungi with novel and/or active metabolites and potential interest for biological control. Furthermore, by culture-independent methods, a species of the genera Tremella was detected and to our knowledge, the Tremella species have never been reported as endophytic fungi so
123
Max Ident (%)
–
far. Another clone, J2G90, may represent a novel species. The results clearly suggested that the special habitat might be a source of novel endophytic fungi. Overall, this study documented endophytic fungal assemble in the leaves of M. cochinchinensis and may expand our knowledge about endophytic fungi within the photosynthetic tissues of hemi-parasitic plants. Our results showed a broad spectrum of endophytic fungi within the leaves of M. cochinchinensis, which were worthy of our attention. This study may also provide the foundation for understanding the relationship between the leaves of M. cochinchinensis and the endophytic fungi. Acknowledgments We are deeply grateful to Prof. Guo LiangDong, Dr. Sun Xiang, and Dr. Sun Dong-Xu for their valuable suggestions to the improvement of our manuscript. Special thanks are due to Dr. Wang Mei-Xia and Dr. Dai Qun for their kindly helps in the
Z. Sheng-Liang et al.: Diversity of Endophytic Fungi experiments. This study was supported by the fund of National Natural Science Foundation of China (Project no. 31300067). 18.
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Diversity of Endophytic Fungi Associated with the Foliar Tissue of a Hemi-Parasitic Plant Macrosolen cochinchinensis Sheng-Liang Zhou • Shu-Zhen Yan Qi-Sha Liu • Shuang-Lin Chen
•
Received: 29 March 2014 / Accepted: 8 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Foliar fungal endophytes are an important plant-associated fungal group. However, little is known about these fungi in hemi-parasitic plants, a unique plant group which derive nutrients from living plants of its hosts by haustoria while are photosynthetic to some degree. In this paper, the endophytic fungi in the leaves of a species of hemi-parasitic plant, Macrosolen cochinchinensis, were studied by both culture-dependent and culture-independent methods. By culture-dependent method, a total of 511 isolates were recovered from 452 of 600 leaf fragments (colonization rate = 75.3 %) and were identified to be 51 taxa. Valsa sp. was the most abundant (relative abundance = 38.4 %), followed by Cladosporium sp. 1 (13.5 %), Ulocladium sp. (4.3 %), Phomopsis sp. 2 (3.7 %), Hendersonia sp. (3.5 %), and Diaporthe sp. 4 (3.5 %). The Shannon index (H0 ) of the isolated endophytic fungi was 2.628, indicating a moderate diversity. By culture-independent method, Aspergillus spp., Cladosporium sp., Mycosphaerella sp., Acremonium strictum, and Tremella sp. were detected. To our knowledge, the Tremella species have never been detected as endophytes so far. In
S.-L. Zhou S.-Z. Yan Q.-S. Liu S.-L. Chen (&) Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China e-mail: [email protected] S.-L. Zhou e-mail: [email protected] S.-L. Zhou Key Laboratory of Biotechnology for Medicinal Plants of Jiangsu Province, Jiangsu Normal University, Xuzhou 221116, China
addition, a cloned sequence was not similar with any current sequence in the Genbank, which may represent a novel species. Altogether, this study documented endophytic fungal assemble in the leaves of M. cochinchinensis which was worthy of our attention, and may expand our knowledge about endophytic fungi within the photosynthetic tissues of plants.
Introduction Endophytic fungi are one of plant-associated fungal groups which grow in healthy plant tissues for all or the most part of their life cycle without causing disease symptoms in the host plants [23]. They emerged around the Early Devonian (about 400 million years ago) according to molecular and fossil evidences [15, 30] and have been coevolving with their host since their existence [29]. Based on numerous investigations, endophytic fungi are believed to exist in various organs of almost all land plants. The communities of endophytic fungi residing in plant leaves are of special interest, as leaves are organs in which photosynthesis and transpiration take place, where endophytic fungi have to face more biochemical dynamics and environmental variations than woody tissues [1]. In turn, endophytic fungi have some effect on the physiological activities of foliar tissues. For example, Pinto et al. showed that endophytic Colletotrichum musae and Fusarium moniliforme impair the photosynthetic efficiency in maize and banana [24]. As foliar endophytic fungi may be an underlying aspect of plant biology and, as many studies suggested, a source of novel and/or active compounds [27], they have attracted increasing interest in recent years. In nature, many trees, shrubs, or herbs have a parasitic life mode, representing 4,400 species in approximately 19
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families of flowering plants [20]. Notably, about 80 % of parasitic plants are hemiparasites, which attach themselves to host plants by the haustoria to absorb nutrients from hosts. Interestingly, this group of plants contains chlorophyll when mature and hence their leaves are photosynthetic to some degree. In the past 30 years, a great deal of studies have been carried out on the diversity of endophytic fungi associated with foliar tissues of hundreds of species of plant [31], including all major lineages of land plants, such as moss, fern, lichen, gymnosperm, and angiosperm. However, rare attentions have been paid to endophytic fungi in the photosynthetic tissues of hemi-parasitic plants. Only recently, those of a hemiparasite Viscum album were studied by conventional culturing techniques [21]. In that study, only the favorably culturable fungal endophytes in the leaves of V. album were displayed due to the limitation of the culturing method. So, foliar fungal endophytes of hemi-parasitic plants are still poorly understood so far and further studies are needed for the unique plant group. Macrosolens cochinchinensis (Loranthaceae) is a perennial climbing woody hemi-parasitic shrub, and it is primarily distributed in tropical and subtropical regions and commonly occurs in southern China. To expand our knowledge about foliar endophytes of hemi-parasitic plants, the diversity of endophytic fungi in the leaves of M. cochinchinensis was studied by both a culture-based method using six kind of media side-by-side and a cultureindependent process that relies on nuclear ribosomal internal transcribed spacer (rDNA ITS) in this paper. The present study may also provide the foundation for understanding the relationship between the leaves of M. cochinchinensis and the endophytic fungi.
Materials and Methods Sampling Site and Procedure The sampling site was located at a tea-oil Camellia plantation in Qinzhou of Guangxi Zhuang Autonomous Region, southern China (20°540 –22°410 N, 107°270 –109°560 E). The mean annual temperature is 21.4–22.0 °C, and the average annual precipitation is 1,649.1–2,055.7 mm. Twenty plant individuals of M. cochinchinensis, with a minimum distance of 20 m from each other, were sampled in March, 2011, each of which invaded a separate Camellia oleifera tree of ca. 30 years old. Healthy leaves together with associated branches of the hemiparasite were collected from each selected individual, placed in plastic bags immediately, labeled and kept cold by ice. Samples were transported to the laboratory, stored at 4 °C and processed within 2 days.
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Culture-Dependent Method Leaves selected were removed from branches and cut into segments (5 9 5 mm). Thirty leaf segments of each individual plant were randomly selected for isolation of endophytic fungi. In total, 600 leaf segments (20 individuals 9 30 leaf segments) were used in this study. The segments were surface sterilized by consecutive immersion for 1 min in 75 % ethanol, 3 min in 3.25 % sodium hypochlorite, and 30 s in 75 % ethanol, then the fragments were surface dried with sterile paper towels. As some endophytic fungi might fail to grow on one medium while could develop on other [6], we employed six kinds of media to culture endophytic fungi side-by-side. Five surface-sterilized segments were evenly placed in each Petri dish (90 mm in diam) with the following media: Czapek agar, potato dextrose agar (PDA), corn meal agar, oat meal agar, malt extract agar, and PDA containing 10 % leave extract of M. cochinchinensis. Benzylpenicillin sodium (50 mg L-1) (North China Pharmaceutical Group Co., China) was added to all the six kinds of media above to suppress bacterial growth. The dishes were incubated at 22 °C, and examined periodically. When colonies developed, they were transferred to new PDA plates for purification. The pure strains were examined periodically, of which the sporulating ones were identified based on their morphological characteristics [5, 14]. The non-sporulating cultures were induced to sporulate according to the reported methods [25]. The strains which failed to sporulate after induction were designated as sterile mycelia and those were identified based on the internal transcribed spacer (ITS) region sequences of rDNA [18]. Total genomic DNA was extracted following the method described by Guo et al. [12]. The ITS region of each DNA sample was amplified using the universal primers ITS1/ITS4 [32], and sequenced at Sangon Biotech (Shanghai) Co., Ltd. All the sequences were initially aligned using the program package MEGA4.1. Then, each sequence was used as a query to search for similar sequences from GenBank using the FASTA and BLAST programs for identification. The colonization rate was calculated as the total number of fragments infected by one or more endophytic fungi divided by the total number of fragments incubated to measure the degree of endophytic fungal infection of the leaves. The relative abundance (RA) of each endophytic taxon was calculated as the number of isolates of a certain taxon divided by the total number of isolates obtained. Shannon’s diversity index (H’) was used to anlyze the diversity of endophytic fungi and calculated as follows: P H 0 ¼ ki¼1 Pi ln Pi, where k is the total number of fungal taxon, and Pi is the RA of taxon i. Species
Z. Sheng-Liang et al.: Diversity of Endophytic Fungi
accumulation curve was calculated with Estimate S Version 8.2.0.
Table 1 The community composition and shannon’s diversity index of endophytic fungi isolated from the leaves of Macrosolens cochinchinensis
Culture-Independent Process
Taxa of endophytes
The leaf samples of M. cochinchinensis were surface sterilized to remove organisms adhered to the surface of the leaves as described in Gao et al. [9]. Total DNA of the samples was extracted by a modified protocol of CTAB as in Guo et al. [13]. The ITS primers ITS1H/ITS4B, designed based on the universal primers ITS1/ITS4 [32], were used to amplify the ITS regions. Primer ITS1H, 50 -ACAC AAGCTTTCCGTAGGTGAACCTGCGG-30 , contains a HindIII restriction site (underlined); primer ITS4B, 50 -ACACGGATCCTCCTCCGCTTATTGATATGC-30 , contains a BamHI restriction site (underlined). The PCR products were analyzed on 2.0 % agarose gels. The objective bands were excised from the gels and purified with UltraSep Gel Extraction Kit (Omega Co.) following the manufacturer’s instructions. The purified PCR products and the pUC19 vector were digested by restriction endonucleases HindIII and BamHI. The digested PCR products and pUC19 vector were gel-purified as described above, ligated overnight at 16 °C, transformed into Escherichia coli DH5a, and recombinant clones were identified by bluewhite screening. Each recombinant plasmid DNA was extracted with SanPrep Column Plasmid Mini-Preps Kit (Sangon Biotech Co., China) and digested with HindIII and BamHI. The clones containing an insert of correct ITS size were sequenced at Sangon, and used for molecular identification. The clone library size was analyzed by coverage C and calculated as follows: C = 1 - n1/N, where n1 is the total number of operational taxonomic unit (OTU) which appears only once, and N is the total number of clones.
Result Endophytic Fungi Recovered by Culture-Dependent Method In total, 511 isolates were recovered from 452 of 600 leaf fragments used (colonization rate = 75.3 %), the remaining 148 fragments did not yield any endophytic fungus. The isolates were identified to be 51 taxa (Table 1), 17 of which were singletons (species that were observed only once). Identification of sterile mycelia is shown in Table 2. The Shannon diversity index (H0 ) of the culturable endophytes was 2.628. Valsa sp. was the most abundant (RA = 38.6 %), followed by Cladosporium sp. 1 (13.5 %), Ulocladium sp. (4.3 %), Phomopsis sp. 2 (3.7 %), Hendersonia sp. (3.5 %), and Diaporthe sp. 4 (3.5 %). the RA of
No. of isolates
Relative abundance (%)
Ascomycetes Acremoniella sp.
4
0.8
Aschersonia sp.
1
0.2
Ascomycota sp.1
8
1.6
Ascomycota sp.2
11
2.2
Ascomycota sp.3
8
1.6
Ascomycota sp.4
1
0.2
Ascomycota sp.5
1
0.2
Aspergillus sp.
2
0.4
Berkleasmium sp. Botryosphaeria dothidea
4 1
0.8 0.2
Botryosphaeria sp.1
13
2.5
Botryosphaeria sp.2
1
0.2
Catenularia sp.
2
0.4
Cephalosporium sp.
7
1.4
Chromosporium sp.1
4
0.8
Chromosporium sp.2
3
0.6
Cladosporium sp.1
69
13.5
Cladosporium sp.2
9
1.8
Corynespora sp.
3
0.6
Coryneum sp.
1
0.2
Curvularia sp.
3
0.6
Diaporthe sp.1
4
0.8
Diaporthe sp.2
4
0.8
Diaporthe sp.3 Diaporthe sp.4
13 18
2.5 3.5
Diaporthe sp.5
2
0.4
Diaporthe sp.6
1
0.2
Diaporthe sp.7
1
0.2
Diaporthe sp.8
1
0.2
Diaporthe sp.9
1
0.2
Fusarium sp.
1
0.2
Gilmaniella sp.
5
1.0
Glomerella sp.1
3
0.6
Glomerella sp.2
11
2.2
Glomerella sp.3
1
0.2
Helminthosporium sp.
1
0.2
Hendersonia sp.
18
3.5
Melanconium sp.
2
0.4
Neosartorya sp.
4
0.8
Pestalotiopsis sp. Phomopsis sp1
1 9
0.2 1.8
Phomopsis sp.2
19
3.7
Phomopsis sp.3
1
0.2
Sporidesmium sp.
2
0.4
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Z. Sheng-Liang et al.: Diversity of Endophytic Fungi Table 1 continued Taxa of endophytes
No. of isolates
Relative abundance (%)
Torula sp.1
1
0.2
Torula sp.2
2
0.4
22
4.3
197
38.6
1
0.2
Ulocladium sp. Valsa sp. Xylaria sp. Basidiomycetes Agaricomycetes sp.
3
0.6
Irpex sp.
6
1.2
511
100.0
Total Shannon’s diversity index (H0 )
2.628
the other species was between 2.5 % and 0.2 %. Species accumulation curve approximated an asymptote when singletons were removed; however, the curve continuously rose when singletons were included, indicating that further sampling would recover more rare endophyte taxa (Fig. 1). The overwhelming majority of the culturable endophytic fungi recovered were ascomycetes, which accounted for 98.2 % of the total isolates and were distributed in 3 classes (Sordariomycetes, Dothideomycetes, and Eurotiomycetes) except for Incertae sedis, the rest were basidiomycetes. As shown in Fig. 2, 59.1 % of isolates were assignable to Sordariomycetes, of which Diaporthales was dominant, accounting for 90.7 % of this group. The remaining Sordariomycetes isolates belonged to Hypocreales, Chaetosphaeriales, Xylariales, and Incertae sedis, accounting for 3.0, 0.7, 0.7, and 5.0 %, respectively. There were 28.6 % of isolates in Dothidemycetes. Of the Dothidemycetes group, more than half (53.4 %) were in Capnodiales, 36.3 and 10.3 %, respectively, were in Pleosporales and Botryosphaeriales. A considerable fraction of isolates (9.4 %) were Incertae sedis of ascomycetes, and only a small portion were in Eurotiomycetes (1.2 %), all belonging to Eurotiales. In addition, 1.8 % of isolates were in Agaricomycetes of Basidiomycota. Endophytic Fungi Detected by Culture-Independent Method Three major fragment sizes of PCR products were obtained from DNA samples extracted directly from the leaf of M. cochinchinensis (Fig. 3). Of which, the putative ITS sequences (500–700 bp) were purified and cloned. The recombinant clones were identified by blue-white screening and most white transformants were shown to carry an insert fragment similar to the expected size of the objective
123
ITS sequences (Fig. 4). A total of 80 such recombinant clones were isolated in this study. The result of DNA sequencing and sequence alignments of 80 recombinant clones revealed 16 different insert sequences, no chimera was found. The coverage C of the library size was 86.3 %. The abundance, length and the most homologous sequences of the cloned sequences identified in this study are summarized in Table 3. Among the 16 cloned sequences, 14 had relatively high sequence similarities with species of their closely identified phylogenetic relative belonging to Ascomycota (C98 %), one sequence, J2A19, was related to Basidiomycota at ITS similarity of 89 %, J2G90 was not similar to any current sequence in the database. The clone sequences were further identified to be species of Aspergillus (70 clones), Cladosporium (4 clones), Mycosphaerella (2 clones), Acremonium (2 clones), and Tremella (1 clone) except J2G90, which may represent a novel species.
Discussion In this paper, endophytic fungal community associated with the leaves of M. cochinchinensis were studied by both a culture-dependent method and a culture-independent process. The conventional method for studying the diversity of endophytic fungi is culture-dependent method. The main limitation of the method is that unculturable species and some slow-growing or weak-competitive species may not be isolated. To make up the shortcomings of the culturing method, culture-independent methods were developed in recent years [3]. However, studies showed that endophytic fungi recoverd by culture-dependent method were often different from those detected by culture-independent methods, and some commonly isolated strains were never found by unculture methods [3, 13]. In the present study, the endophyte communities detected by the two methods were completely different. The most cloned sequences were closely relative to species of Aspergillus according to the culture-independent method. Although one Aspergillus species was also isolated by culturing method, the ITS sequence of which differed significantly from the cloned ones (around 22.5 % of sequence divergence), indicating that different Aspergillus species were detected by the two methods. Similarly, the species of Cladosporium detected by the two methods were not the same according to the sequence comparison. The reasons that cause the difference between endophytic fungal assembles detected by culturing and unculturing methods have been discussed in previous studies [13]. Generally, the Shannon index (H0 ) is usually between 1.5 and 3.5, the lowest is 1.5 and the highest is 3.5 [10]. The H0 of the endophytic fungi isolated from the leaves of
Z. Sheng-Liang et al.: Diversity of Endophytic Fungi Table 2 Molecular identification of sterile mycelia recovered from the leaves of Macrosolens cochinchinensis based on ITS sequences Identification
Closestly matched species in GenBank (Accection number)
Classification of matched speciesa
Cover (%)
KF675752
Aschersonia sp.
Aschersonia sp. (KC771478.1)
Clavicipitaceae, Hypocreales, Sordariomycetes
94
99
KF159990
Ascomycota sp.1
Pichia guilliermondii (HM037942.1)
Pichiaceae, Saccharomycetales, Saccharomycetes
95
99
Debaryomyces hansenii (DQ534408.1)
Incertae sedis, Saccharomycetales, Saccharomycetes
95
99
Ustilaginoidea virens (JF271122.1)
Incertae sedis, Hypocreales, Sordariomycetes
95
99
Phomopsis sp. (HQ832826.1)
Diaporthaceae, Diaporthales, Sordariomycetes
93
98
Diaporthe sp. (JF441199.1)
Diaporthaceae, Diaporthales,Sordariomycetes
93
99
Alternaria mali (EU732731.1)
Pleosporaceae, Pleosporales, Dothideomycetes
94
98
Fusarium oxysporum (JN400690.1)
Nectriaceae, Hypocreales, Sordariomycetes
96
99
Cyttaria darwinii (EU107253.1)
Cyttariaceae, Cyttariales, Leotiomycetes
96
99
Botryosphaeria dothidea (GQ855797.1)
Botryosphaeriaceae, Botryosphaeriales, Dothideomycetes
96
99
Phomopsis sp. (HQ832826.1)
Diaporthaceae, Diaporthales, Sordariomycetes
95
99
Diaporthe sp. (DQ145729.1)
Diaporthaceae, Diaporthales, Sordariomycetes
95
99
Alternaria mali (EU732731.1)
Pleosporaceae, Pleosporales, Dothideomycetes
96
98
Fusarium lateritium (KF159980.1)
Nectriaceae, Hypocreales, Sordariomycetes
96
99
Aspergillus clavatus (KC215115.1)
Trichocomaceae, Eurotiales, Eurotiomycetes
96
97
Aschersonia sp. (AY225332.1)
Clavicipitaceae, Hypocreales, Sordariomycetes
96
97
Accession number
Indent (%)
Ascomycetes
KF159992
KF159996
KF675756
KF675759
Ascomycota sp.2
Ascomycota sp.3
Ascomycota sp.4
Ascomycota sp.5
KF495994
Aspergillus
Aspergillus niger (JN672584.1)
Trichocomaceae, Eurotiales, Eurotiomycetes
95
99
KF675755
Botryosphaeria dothidea
Botryosphaeria dothidea (KC492490.1)
Botryosphaeriaceae, Botryosphaeriales, Dothideomycetes
97
99
KF160000
Botryosphaeria sp.1
Botryosphaeria dothidea (KC492490.1)
Botryosphaeriaceae, Botryosphaeriales,Dothideomycetes
94
99
KF675753
Botryosphaeria sp.2
Botryosphaeria sp. (JQ772020.1)
Botryosphaeriaceae, Botryosphaeriales, Dothideomycetes
73
97
KF159973
Cladosporium sp.1
Cladosporium sp. (JX675049.1)
Davidiellaceae, Capnodiales, Dothideomycetes
98
99
KF159974
Cladosporium sp.2
Cladosporium sphaerospermum (HM595522.1)
Davidiellaceae, Capnodiales, Dothideomycetes
97
99
KF159976
Diaporthe sp.1
Diaporthe eucalyptorum (JX069862.1)
Diaporthaceae, Diaporthales, Sordariomycetes
95
99
KF159977
Diaporthe sp.2
Diaporthe eucalyptorum (JX069862.1)
Diaporthaceae, Diaporthales, Sordariomycetes
95
99
KF159984
Diaporthe sp.3
Diaporthe eucalyptorum (JX069862.1)
Diaporthaceae, Diaporthales, Sordariomycetes
94
98
KF159993
Diaporthe sp.4
Diaporthe perseae (KC343173.1)
Diaporthaceae, Diaporthales, Sordariomycetes
97
98
KF675745
Diaporthe sp.5
Diaporthe sp. (JF773672.1)
Diaporthaceae, Diaporthales, Sordariomycetes
96
99
KF675746
Diaporthe sp.6
Diaporthe sp. (KF159984.1)
Diaporthaceae, Diaporthales, Sordariomycetes
96
99
KF675747
Diaporthe sp.7
Diaporthe sp. (KF160008.1)
Diaporthaceae, Diaporthales, Sordariomycetes
99
99 100
KF675748
Diaporthe sp.8
Diaporthe sp. (KF160008.1)
Diaporthaceae, Diaporthales, Sordariomycetes
96
KF675750
Diaporthe sp.9
Diaporthe hongkongensis (KC343119.1)
Diaporthaceae, Diaporthales, Sordariomycetes
95
99
KF675751
Fusarium sp.
Fusarium oxysporum (JN400719.1)
Nectriaceae, Hypocreales, Sordariomycetes
97
99
KF159983
Glomerella sp.1
Glomerella sp. (GQ352479.1)
Glomerellaceae, Incertae sedis, Sordariomycetes
97
99
KF159986
Glomerella sp.2
Glomerella cingulata (EU520087.1)
Glomerellaceae, Incertae sedis, Sordariomycetes
97
99
KF675757
Glomerella sp.3
Glomerella sp. (KF159997.1)
Glomerellaceae, Incertae sedis, Sordariomycetes
98
99
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Z. Sheng-Liang et al.: Diversity of Endophytic Fungi Table 2 continued Accession number
Identification
Classification of matched speciesa
Closestly matched species in GenBank (Accection number)
Cover (%)
Indent (%)
KF159988
Neosartorya sp.
Neosartorya hiratsukae (HE578066.1)
Trichocomaceae, Eurotiales, Eurotiomycetes
94
99
KF675754
Pestalotiopsis sp.
Pestalotiopsis microspora (HQ889705.1)
Amphisphaeriaceae, Xylariales, Sordariomycetes
96
99
KF159989
Phomopsis sp1
Phomopsis sp. (GU066685.1)
Diaporthaceae, Diaporthales, Sordariomycetes
95
99
KF159991
Phomopsis sp.2
Phomopsis sp. (GU066617.1)
Diaporthaceae, Diaporthales, Sordariomycetes
96
99
KF675749
Phomopsis sp.3
Phomopsis sp. (JQ809670.1)
Diaporthaceae, Diaporthales, Sordariomycetes
96
98
KF675758
Xylaria sp.
Xylaria sp. (AB449098.1)
Xylariaceae, Xylariales, Sordariomycetes
97
99
Bjerkandera adusta (JN198491.1)
Meruliaceae, Polyporales, Agaricomycetes Basidiomycota
96
99
Trichaptum abietinum (FJ768676.1)
Incertae sedis, Hymenochaetales, Agaricomycetes Basidiomycota,
94
100
Thanatephorus cucumeris (FJ467377.1)
Ceratobasidiaceae, Cantharellales, Agaricomycetes, Basidiomycota
95
99
Ganoderma lobatum (JQ520165.1)
Ganodermataceae, Polyporales, Agaricomycetes, Basidiomycota
94
99
Irpex lacteus (HQ670693.1)
Meruliaceae, Polyporales, Agaricomycetes, Basidiomycota
97
99
Basidiomycetes KF159979
KF159981
Irpex sp.
Fungal taxonomy applied follows Index Fungorum (http://www.indexfungorum.org/Names/Names.asp)
Numbet of species of endophytic fungi
a
Agaricomycetes sp.
60 50
I II
40 30 20 10 0
0
5
10
15
20
25
Number of trees
Fig. 1 Species accumulation curve of endophytic fungi isolated from the leaves of Macrosolens cochinchinensis, I, singletons were included. II, singletons were removed
M. cochinchinensis was 2.628 in our results, indicating the diversity was moderate. Among these endophytic fungi, the Sordariomycetes and Dothideomycetes accounted for more than 87.7 % of isolates (Fig. 2). The result was consistent with previous reports, in which the Sordariomycetes and Dothideomycetes represented the majority of foliar endophyte species [1]. Totally, culture-dependent and cultureindependent methods revealed that the endophytic fungi detected were distributed mainly in 3 classes of ascomycetes and 2 classes of basidiomycetes, indicating a broad spectrum of endophytic fungi within the leaves of M. cochinchinensis. Furthermore, the composition structure of endophytic fungi
123
in this study was also in agreement with those in nonparasitic plants, in which the overwhelming majority of endophytic fungi recovered were ascomycetes, occasionally a small portion of basidiomycetes or zygomycetes. The endophyte communities are generally dominated by one or more species, which are supposed to be closely related to their hosts [11]. Among the culturable endophytic fungi in the present study, Valsa sp. was the most abundant (RA = 38.4 %). Valsa species have been rarely detected as endophytic fungi so far. To our knowledge, only one endophytic species of Valsa was reported as dominant endophyte from the Populus tremula in Spain [26]. Valsa is a widespread genus of fungi within the family Valsaceae (Diaporthales, Sordariomycetes). Generally, Valsa species were found as saprophytic or pathogenic fungi. Some species of Valsa may invade in various deciduous fruit trees or broad-leaved trees, causing serious lesion of the hosts. It has long been recognized that a fungal species may act as endophyte in one host while pathogen in other plants [1]. Some fungal pathogens, e.g., species of Apiognomonia, Ophiovalsa, Pezicula or Phomopsis, have been reported as dominant endophytic fungi in different trees [29]. Our results showed for the first time that the endophyte community in the leaves of a hemi-parasitic plant might also dominated by the fungus that is considered a pathogen. However, the relationship between endophytic Valsa sp. and M. cochinchinensis needs further studies. Many studies showed that endophytic fungi in the leaves of woody plants spread via spores (horizontal transmission)
Z. Sheng-Liang et al.: Diversity of Endophytic Fungi
Fig. 2 The community composition of endophytic fungi isolated from the leaves of Macrosolens cochinchinensis
Fig. 3 Agarose gel electrophoresis profiles of PCR products amplified from DNA extracted directly from leaves of Macrosolens cochinchinensis. Lanes 1 and 2, PCR products from two independent DNA samples as templates. The arrow shows the objective bands of ITS sequences (500–700 bp). Lane M, DNA size markers
[2, 8], which may flow with air movement, attach to plant leaves, then germinate and invade into the foliar tissues. In our study, the species of Cladosporium, Phomopsis and Diaporthe were isolated with relatively high frequency. Cladosporium spores are wind-dispersed, and the endophytic fungi in this genus are known to be not host specificity, which can be found in various hosts [22]. The host range of endophytic Phomopsis are also very wide, they may inhabit in various plant hosts which are taxonomically irrelevant [29]. Similarly, Diaporthe (the teleomorph of Phomopsis) has also been reported as one of the most frequently encountered genera of endophytic fungi in
Fig. 4 Agarose gel electrophoresis profiles of 7 digested recombinant plasmid DNA samples. The arrow shows the size of objective bands of ITS sequence. Right lane, DNA size markers
several plant hosts [7, 19]. In addition, species of Glomerella, Fusarium, Pestalotiopsis, Xylaria, and Botryosphaeria detected in this study with low frequency are also known to be ubiquitous genera as endophytic fungi [4, 28]. It seems that the non-host-specificity endophytic species mentioned above invade into the leaves of M. cochinchinensis via horizontal transmission. However, all of the fungal species mentioned above did not sporulate on the culture media despite of various methods of induction, their transmission mechanism remain unknown. Among the endophyte community detected in the present study, Aspergillus and Cladosporium were detected by both culture-dependent and culture-independent methods.
123
Z. Sheng-Liang et al.: Diversity of Endophytic Fungi Table 3 The abundance, length and the most homologous sequences of the ITS of endophytic fungi detected from the leaves of Macrosolen cochinchinensis by cultureindependent method
ITS clone sequences
No. of clones
Length of the sequence (bp)
GenBank accession numbers
Closely identified phylogenetic relative (Genebank accession number)
Query coverage (%)
J2C68
7
572
JX123325
Aspergillus ustus strain UWFP 546 (AY213637.1)
100
99
J2E39
1
569
JX123326
Aspergillus ustus strain UWFP 546 (AY213637.1)
100
99
J2A35
54
568
JX123331
Aspergillus sp. MS-2011F42(HE608807.1)
100
100
J2G54
1
567
JX123337
Aspergillus sp. MS-2011F42(HE608807.1)
100
99
J2G20
1
567
JX123341
Aspergillus sp. MS-2011F42(HE608807.1)
100
99
J2G48
1
570
JX123347
Aspergillus sp. MS-2011F42(HE608807.1)
100
98
J2E32
1
566
JX123353
Aspergillus versicolor strain CanR-30 (JF817276.1)
99
99
J2F149
4
569
JX123354
Aspergillus versicolor isolate UOA/HCPF8640 (FJ878625.1)
100
100
J2A19
1
500
JX123327
Tremella globispora strain CBS 6972 (AF444432.1)
100
89
J2A26
1
551
JX123328
Uncultured Cladosporium clone OS_2d_H09(JF449702.1)
100
100
J2A28
2
553
JX123329
Cladosporium sp. MS-2011-F14 (HE608784.1)
100
99
J2G25
1
551
JX123330
Cladosporium sp. MS-2011-F14 (HE608784.1)
100
98
J2A37
2
574
JX123349
Acremonium strictum genogroup III strain(AY138846.1)
100
100
J2E4
1
542
JX123351
Mycosphaerella sp. Ponipodef 06 (HQ731642.1)
100
99
J2E25
1
542
JX123352
Mycosphaerella sp. Ponipodef 06 (HQ731642.1)
100
99
J2G90
1
559
JX123356
–
–
Endophytic Aspergillus spp. were widely reported to produce novel metabolites and bioactive procucts, such as antimicrobial, anticancer, antioxidant, anti-phytopathogenic, and phytohormonal activity [17]. Similarly, endophytic Cladosporium spp. were also known as a source of new natural products and bioactive metabolites, including antifungal, antitumor, antiviral, antimitotic, and cytostatic activities [33]. In addition, Acremonium strictum detected in this study was reported to mediate an antagonism toward herbivorous insects [16]. The results indicated that the leaves of M. cochinchinensis, and reasoningly other hemiparasitic plants, might represent an untapped reservoir of endophytic fungi with novel and/or active metabolites and potential interest for biological control. Furthermore, by culture-independent methods, a species of the genera Tremella was detected and to our knowledge, the Tremella species have never been reported as endophytic fungi so
123
Max Ident (%)
–
far. Another clone, J2G90, may represent a novel species. The results clearly suggested that the special habitat might be a source of novel endophytic fungi. Overall, this study documented endophytic fungal assemble in the leaves of M. cochinchinensis and may expand our knowledge about endophytic fungi within the photosynthetic tissues of hemi-parasitic plants. Our results showed a broad spectrum of endophytic fungi within the leaves of M. cochinchinensis, which were worthy of our attention. This study may also provide the foundation for understanding the relationship between the leaves of M. cochinchinensis and the endophytic fungi. Acknowledgments We are deeply grateful to Prof. Guo LiangDong, Dr. Sun Xiang, and Dr. Sun Dong-Xu for their valuable suggestions to the improvement of our manuscript. Special thanks are due to Dr. Wang Mei-Xia and Dr. Dai Qun for their kindly helps in the
Z. Sheng-Liang et al.: Diversity of Endophytic Fungi experiments. This study was supported by the fund of National Natural Science Foundation of China (Project no. 31300067). 18.
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