Endophytic Fungi from Lycium chinense Mill and Characterization of Two New Korean Records of Colletotrichum

Chinese boxthorn or matrimony vine (Lycium chinense Mill) is found primarily in southeastern Europe and Asia, including Korea. The dried ripe fruits are commonly used as oriental medicinal purposes. Endophytic fungi were isolated from surface sterilized tissues and fruits of the medicinal plant in 2013 to identify the new or unreported species in Korea. Among 14 isolates, 10 morphospecies were selected for molecular identification with the internal transcribed spacer (ITS) gene. Phylogenetic analysis revealed that all isolates belonged to Ascomycota including the genera Acremonium, Colletotrichum, Cochliobolus, Fusarium, Hypocrea and Nemania. Two Colletotrichum species were identified at the species level, using three genes including internal transcribed spacer (ITS), glycerol-3-phosphate dehydrogenase (GAPDH) and Actin (ACT) for PCR and molecular data analysis along with morphological observations. The fungal isolates, CNU122031 and CNU122032 were identified as Colletotrichum fructicola and C. brevisporum, respectively. Morphological observations also well supported the molecular identification. C. brevisporum is represented unrecorded species in Korea and C. fructicola is the first record from the host plant.


Results
A total of 10 endophytic fungal morphospecies obtained from L. chinense in Korea were selected from 14 isolates for identification (Table 1). Endophytic fungi were identified by analysis of the ITS region of the rDNA gene. Six distinctive fungal taxa were detected at a >90% sequence similarity threshold (Table 1)  The sequence of CNU122031 completely matched with C. fructicola C1263.3 when retrieved from GenBank with maximum bootstrap value shown in the phylogenetic tree. The isolate CNU122032 showed 99% sequence similarity with two similar isolates C. brevisporum LC0600 and Glomerella magna L2.5 with 100% bootstrap support. Therefore, we used Actin and GAPDH primers to confirm the identification, and sequence similarity with C. brevisporum was 99%-100%. The isolate CNU122033, CNU122034, CNU122035, CNU122036 and CNU122037 showed 99%-100% sequence similarity with Acremonium sp. r116, Cochliobolus lunatus Cs-1C, Fusarium cf. equiseti AM-48, Colletotrichum sp. ITCC 2041 and Hypocrea citrina GJS 91-61, and a high bootstrap value.

Figure 1.
Neighbor-joining phylogenetic tree showing the placement of the representative endophytic isolates based on the sequences of the ITS region. The Kimura two-parameter model is used for pairwise distance measurement. The tree is rooted with Rhizopus microsporus (EU798703). Only bootstrap values >50% (1000 replications) are shown in at the internal nodes.

Molecular Phylogeny
A molecular phylogenetic analysis was generated using the multilocus molecular dataset ( Figure 2). The combined dataset of ITS, ACT and GAPDH comprised 24 sequences and produced 54 most parsinimonious trees (TL = 86, CI = 0.791 and RI = 0.933). One of the most parsimonious trees is shown in Figure 2.  The isolates CNU122031 and CNU122032 formed a monophyletic clade with reliable reference relatives from GenBank isolates. The phylogenetic tree constructed from the combined dataset of ITS, GAPDH, and ACT gene sequences clearly showed that the isolates CNU122031 and CNU122032 are C. fructicola and C. brevisporum, respectively with high bootstrap support ( Figure 2).

Morphological Characterization
Taxonomic descriptions and micromorphographs of the morphological structures for the two species (C. fructicola CNU122031 and C. brevisporum CNU122032) are shown in details below.

CNU122031-Colletotrichum fructicola
Fast growing colonies on PDA were white at the starting of the growth period. Over time, colonies became gray to dark gray at the center ( Figure 3A,B). The reverse color was grayish to blackish with a white halo. Aerial mycelia were whitish, dense, cottony, and without visible conidial masses. Single-celled conidia were observed after the sporulation period. The cylindrical conidia had obtuse to rounded ends and the size ranged from 8.7-29.5 μm long and 2.8-5.9 μm wide ( Figure 3I,J). The shape of appresoria varied among species. Appresoria were commonly ovoid, sometimes clavate. The sizes of the appresoria were 10.5-14.5 × 6-11 μm ( Figure 3C-H).

Distinguishing Characters
C. fructicola CNU122031 was distinctly separated from the closely related species C. siamense by colony color, conidial size, and shape. C. siamense produced a pinkish colony color and the reverse was yellowish to pinkish whereas, the present isolate produced gray to dark gray colony color, and completely matched with the reference species, C. fructicola strains [18]. The size of the colony of present isolate was shorter than that of C. siamense. The CNU122031 isolate produced cylindrical, obtuse conidia but C. siamense produced fusiform conidia, indicating a clear difference between the species ( Table 2). The fungus, C. fructicola is a newly isolated species from L. chinense.

CNU122032-Colletotrichum bresvisporum
The colony on PDA was white at first, became grayish over time. The mycelium was in small tufts. The reverse was dark blackish at the center and the edge was white to grayish with a white halo ( Figure 4A,B). Aerial mycelia were white in color and dense. Conidial masses were not observed. Conidia produced were single celled. Conidia were cylindrical with rounded ends, smooth-walled, hyaline, and ranged in size from 12.2-24.2 × 2.6-6 μm ( Figure 4F,G). Appresoria were 10-16.8 × 5-9.4 μm in slide cultures, irregular in shape, sometimes ovoid, often becoming complex over time ( Figure 4C-E).

Distinguishing Characters
The isolate was identified as C. brevisporum by molecular data analysis. The morphological characteristics supported the molecular identification.
The isolate was closely related to C. cliviae, but differed in conidial shape and length. Conidia of the present isolate were cylindrical with distinctly round ends, whereas the conidia of C. cliviae were cylindrical, straight, or curved, and obtuse at the ends. The present isolate did not produce any curved conidia, and not obtuse at the ends ( Table 2). The morphological description of the present isolate completely matched that of C. brevisporum described by Noireung et al. 2012 [13]. This is the first fungal record of C. brevisporum isolated in Korea.

Discussion
Endophytic fungal distributions vary with plant-associated habitats which may affect microbial communities that colonize roots, leaves, stems, branches, fruits, pods, and leaves [20]. Previous investigations revealed that endophytes were isolated from different plant tissues or organs. In this study, we isolated and characterized endophytic fungi from leaves and fruits of the L. chinense Mill plant and identified two unreported fungi isolated from the L. chinense Mill plant.
Endophytic fungal communities associated with various kinds of plants from tropical, sub-tropical, temperate, and Arctic ecosystems were investigated previously and fungal diversity was high [21]. From the Chinese boxthorn plant we isolated 14 fungi. Among them, 10 morphospecies were selected and sequenced with ITS gene. Six different genera were observed (Acremonium, Colletotrichum, Cochliobolus, Fusarium, Hypocrea and Nemania) and included common endophytic fungi from different regions around the world. The distribution of dominant species agreed with previous report [6,7,[22][23][24][25]. The Dominant fungi described here are commonly associated with disease symptoms in several crop plants. Species of Acremonium, Colletotrichum, Cochliobolus, Fusarium, Hypocrea, and Nemania cause severe disease in many cultivated and non-cultivated plants. The anthracnose of chili pepper caused by C. acutatum is an example and Fusarium oxysporum causes disease in the same plant and many other cultivated and non-cultivated plants, and both were found in endophytic association with leaves, roots, pods, and many other plants. They were found in association with leaves and stems of medicinal plant Tylophora indica in India [24], and with the orchid species, Dendrobium loddigesii in China [6]. We isolated fungi associated with a host plant that did not show any disease symptoms. It is possible that they are latent pathogens.
A number of endophytic fungi consist of sterile mycelia or non-sporulating fungi and consequently cannot be identified by morphological characters. Previously, molecular techniques have been employed successfully for the identification of different endophytic fungal community [6,21,26]. In this study, we also used the molecular strategy to identify fungi specifically utilizing of ITS rDNA gene sequence and phylogenetic analysis.

Sampling
Chinese boxthorn (Lycium chinense Mill) plants were selected because of their medicinal applications in Korea. Medicinal plants may live in association with a number of new or unreported microorganisms, especially fungal species. Sampling sites of this study were Cheongyang farmer's field, South Chungcheong province, Republic of Korea ( Figure 5). Living symptomless leaf and fruit samples were collected and stored in sterile polyethylene bags.

Isolation of Endophytic Fungi
Samples were cleaned under running tap water to remove debris, then air dried and processed within 5 h of collection. Tissues were cut into small (1 cm length and 0.5 cm width) pieces. Then surface sterilized by immersing in 95% ethanol for 1 min, sodium hypochlorite (4% chlorine) for 4 min and 95% ethanol for 30 s, and then samples were washed in sterile water three times to remove the surface sterilization agents. Samples were allowed to dry on paper towel in a laminar air flow chamber. A total of 120 segments were selected and plated (100 segments from leaf samples and 20 segments from fruit samples). Ten segments per petri dish were placed horizontally in potato dextrose agar (PDA, Difco, Franklin Lakes, NJ, USA) and rose bengal chloramphenicol agar (DRBC, Difco, Franklin Lakes, NJ, USA) supplemented with the antibiotic streptomycin sulfate (0.4 mg/mL, SIGMA-ALDRICH, Munich, Germany) to stop bacterial growth. After incubation at 25 °C for 5, 10, and 25 days, individual hyphal tips of the developing fungal colonies were collected and placed onto PDA media and incubated for 5-10 days, and checked for culture purity. Eventually, pure cultures were transferred to PDA slant tubes and eppendorf tubes with 20% glycerol stock solution. Strain numbers were assigned for selected isolates and deposited in the Fungal Herbarium of the Chungnam National University, Daejeon, Korea. Cultures of the isolates were also deposited in the Environmental Microbiology Lab. (EML) Herbarium, Chonnam National University, Gwangju, Korea.
PCR amplification was carried out [28] in an i-Cycler (BIO-RAD, Hercules, CA, USA) for 30 cycles of 94 °C for 1 min denaturing, 55 °C for 1 min annealing and 72 °C for 1.30 min extension. Initial denaturing at 94 °C was extended to 5 min and the final extension was for 10 min at 72 °C. For the specific identification of the Colletotrichum species, two additional genes were used for PCR amplification: Actin (Forward ACT-512F & reverse ACT-783R) and Glyceraldehyde-3-phosphate dehydrogenase (Forward GDF & reverse GDR) ( Table 3). The PCR conditions for these genes are showing in Table 3. All PCR products were purified using the Wizard PCR prep kit (Promega, Madison, WI, USA). Purified double-stranded PCR fragments were directly sequenced with BigDye terminator cycle sequencing kits (Applied Biosystems, Foster City, CA, USA) following the manufacturer instructions. The gel electrophoresis and data collection were performed on an ABI prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

Phylogenetic Analysis
The rDNA ITS, Actin, and GAPDH gene sequences were compared by BLAST search with sequence available in the GenBank database. Sequences generated from materials in this study and retrieved from GenBank (Table 4) were initially aligned using CLUSTAL X [31], and the alignment was refined manually using PHYDIT program version 3.2 [32]. A neighbor-joining tree for the ITS sequences was constructed with Kimura's 2-parameter distance model [33] using the PHYLIP 3.57c package [34]. Maximum parsimony trees were constructed for the combined datasets of ITS, Actin, and GAPDH gene sequences using MEGA5 program. Bootstrap analysis using 1000 replications was performed to assess the relative stability of the branches.

Morphological Observation
The isolates CNU122031 and CNU122032 were used for morphological description. Colony characteristics (color, size, and texture) were determined after 7 days at 25 °C in the dark grown on PDA plates. Conidial morphology was examined in standard conditions [13]. Plates were maintained in a chamber without humidity control at 25 °C and held under a fluorescent light/dark cycle of 12/12 h. Conidia were mounted in lactophenol picric acid solution (SIGMA-ALDRICH, Munich, Germany) and measured using a light microscope (OLYMPUS BX50, Tokyo, Japan) and an Artray Artcam 300MI digital camera system (Artray Co., Ltd., Tokyo, Japan). Randomly selected conidia were counted for the morphological description. A slide culture technique [35] was used for the production of appresoria and their size and shape were examined.

Conclusions
One of the main objectives of the present study was to find unreported fungal species in Korea. The ITS sequence-based identification showed that CNU122031 and CNU122032 were consistent with C. fructicola and C. brevisporum, respectively, both of which were previously unrecorded from Chinese borxthorn plant and C. brevisporum is new record in Korea. Further study was needed to clarify the identification. A multi-locus molecular study was carried out with Actin and GAPDH gene sequences. The combined dataset produced a clear molecular identification for these two fungi. Each fungus was isolated as endophytic fungi previously. C. brevisporum was reported as an endophytic new fungus from Thailand. The distinguishing characteristics of the present fungi and their reference species and related species was described. We conclude that Chinese boxthorn in Korea is the host species of unrecorded fungi and plays a potentially important role in understanding microbial diversity in Korea.