J Phytopathol

ORIGINAL ARTICLE Isolation and Molecular Identification of gloeosporioides Causing Brown Spot Disease of Camellia oleifera in Hainan of China Min Xu, Rui He, Yun Peng, Can-Bin Zeng, Yang Liu, Guang-Hua Tang and Hua Tang

Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources, College of Agriculture, Hainan University, No. 58 Renmin Avenue, Haikou, Hainan 570228, China

Keywords Abstract internal transcribed spacer sequences, morphological investigation, pathogen, tea-oil Colletotrichum gloeosporioides is an important pathogen that causes wide- tree spread brown spot disease on the leaves of the tea-oil tree (Camellia olei- fera) in China. This study was designed to isolate, identify and Correspondence characterize this fungal pathogen, based on morphology, molecular char- H. Tang, Hainan Key Laboratory for acteristics and pathogenicity. One pathogenic , named CCG4, was Sustainable Utilization of Tropical isolated from wild-type Camellia oleifera of Hainan Province. Colonies were Bioresources, College of Agriculture, Hainan – University, Haikou, China. regular circular in shape with 50 60 mm diameter after 5 days of incuba- E-mail: [email protected] tion at 28°C on potato dextrose agar (PDA) medium, and woolly with a small amount of jacinth pigment; the colony colour changed from white Received: November 22, 2016; accepted: to black during later stages of infection. The mycelium produced was February 21, 2017. branched and septate. Conidia were cylindrical-truncate, oblong-obtuse to doliform, colourless with 1–2 hyaline oil globules and 4.5– doi: 10.1111/jph.12571 5.3 lm 9 7.7–17.5 lm. The sporodochia were cushion-shaped. The pathogen was identified as Colletotrichum gloeosporioides on the basis of morphological characteristics and internal transcribed spacer sequence, which showed 100% query cover and 99% similarity to the sequence Colletotrichum gloeosporioides JN887341.1, from a pathogenic fungus known to cause brown spot disease of Camellia oleifera.

in inducing antioxidant enzymes in vitro and in vivo, Introduction as well as protecting against oxidative injury to liver The tea-oil tree (Camellia oleifera) is an important tissues and gastrointestinal mucosa (Cheng et al. source of natural and edible oil, which can be 2015). Camellia oleifera shell is an important by-pro- obtained from its seeds, in tropical and subtropical duct of woody edible oil production, which can be regions of Asia, especially south-west China (Chen used to produce saponin, xylose, tanin, xylitol, fur- et al. 2010). It is regarded as one of the world’s four fural, potassic salt and medium (Zhu et al. 2013). Fur- most important woody edible oil crops, along with oil thermore, tea saponin and the residue of camellia oil palm, olive and coconut, and has a very high level of are good industrial raw materials that can be widely comprehensive utilization. Camellia oil is rich in used in many fields including cosmetics, papermak- unsaturated fatty acids, which can reach 90% of the ing, printing and dyeing, and chemical fibre produc- total fatty acids, such as oleic acid and linoleic acid tion (Chen et al. 2010; Zhu et al. 2013). Therefore, with fine colour and flavour (Feas et al. 2013). It is Camellia oleifera not only is of high value for nutrition also known as the ‘eastern olive oil’, with high nutri- and health, but also is of key economic value. tional value and uses in hygiene applications (Zong Several species of the genus Camellia grow in China, et al. 2015). It is reported that Camellia oleifera oil has such as Camellia oleifera, Camellia meiocarpa, Camellia potential hepatoprotective and anti-ulcer bioactivities vietnamensis, Camellia semiserrata and Camellia yuh- (Feas et al. 2013). The oil also plays an important role sienensis. Camellia oleifera has been proven to be very

380 J Phytopathol 165 (2017) 380–386 Ó 2017 Blackwell Verlag GmbH M. Xu et al. Pathogen for brown spot disease of Camellia oleifera

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Fig. 1 Brown spot symptoms caused by Col- letotrichum gloeosporioides observed in the field. (A) Brown spots on the surface of leaves; (B) infected leaves. Bar, 1 cm. [Colour figure can be viewed at wileyonlinelibrary.com] adaptable to environmental conditions and disease surface-sterilized with 70% ethanol for 3–5 s, placed pressures, with a distribution mainly in East Asia, in 0.1% corrosive sublimate for 3 min and then South Asia and South-East Asia. However, China and rinsed three times in sterilized water. The tissue was Vietnam represent the major growing regions of then cut into 2- to 3-mm squares with sterilized Camellia oleifera. tweezers and scissors in sterilized water to make At present, the known fungal disease agents of sure that every tissue sample came from the junc- Camellia oleifera are Colletotrichum gloeosporioides, tion between the healthy and diseased leaves. The Myrothecium camellia, Pestalotiopsis microspora, Fusarium tissue was pushed into PDA plates at a 45-degree proliferatum, Meliola camellia and Cortium scutellare angle. Culture dishes were sealed with Parafilm and (Guangtao Song 2012; Ronglin Kuang et al. 2012; placed upside down in a constant temperature incu- Shaofeng Peng et al. 2015). Among these, Col- bator for 3–5 days at 28°C. letotrichum gloeosporioides causes one of the most Once strains had germinated in PDA, hyphae were important and devastating diseases (Guangtao Song picked up from the edge of the Petri dish, transferred 2012). Typical symptoms of this disease are brown to new PDA, sealed with Parafilm and placed in the spots on fruits, branches and leaves. This results in constant temperature incubator for 3–5 days at 28°C dropping of buds, petals and fruits, often causing to obtain purified strains. Purified strains were death of plants, which has caused enormous eco- transferred to new PDA medium and incubated for nomic loss to Camellia oleifera plantations (Guangtao 3–5 days at 28°C, and the characteristics of the colo- Song 2012; Aixian Jin et al. 2009). nies were recorded. Strains were also grown on steril- Disease caused by Colletotrichum gloeosporioides ized coverslips, which were inserted into the medium occurs in most Camellia oleifera plantations in Hainan at a 45-degree angle at 28°C for 3–7 days. Mycelium Province, with an incidence of 10–30%, even reach- was stained with 10.0 g/l Alcian blue 8GX dye (1.0 g ing 80% in some plantations (Hui Zhu et al. 2015). of Alcian blue 8GX dissolved in 3% acetic acid to The objectives of this study were to determine the 100 ml, stored at 4°C for 1–2 years) for 25–30 min to occurrence of this disease and to isolate, identify and visualize acidic mucins and mucopolysaccharides characterize the fungal pathogens involved using a (Kameyama et al. 2015). Mycelia were then rinsed combination of molecular and morphological three times with sterile water to remove excess dye, characteristics. covered by cover slips and observed under an inverted microscope. Materials and Methods Observation of histopathological staining Morphological identification and characterization, and Alcian blue 8GX was successfully used in whole-leaf mycelium staining staining to visualize intercellular hyphae and investi- Diseased samples of wild-type Camellia oleifera from gate septal development in fungi. Materials were fixed Hainan Province were collected for pathogen isola- in 1 : 1 (w/v) 95% alcohol and glacial acetic acid for tion (Fig. 1). Newly infected leaves were collected as 24 h. Infected leaves were then transferred into satu- separation materials. General tissue separation (Yi rated chloral aqueous solution for 24 h and stained in et al. 2015) was the most suitable method for 10.0 g/l Alcian blue 8GX dye solution for 20–30 min isolating the fungus. The diseased leaves were cut before washing three times with sterilized water. with scissors into 5 mm 9 5 mm tissue blocks, Based on the conventional method of tissue

J Phytopathol 165 (2017) 380–386 Ó 2017 Blackwell Verlag GmbH 381 Pathogen for brown spot disease of Camellia oleifera M. Xu et al. sectioned, the infected tissues were observed under predenaturation at 94°C for 5 min; then denaturation an optical microscope. at 94°C for 1 min, annealing at 54°C for 30 s, exten- sion at 72°C for 30 s, for 30 cycles; extension at 72°C for 10 min; hold at 16°C. The PCR products were Pathogenicity test verified by staining with GoldView on 1.0% agarose Pathogenicity tests were conducted by an inoculation electrophoresis gels and purified using a BioTeKe method using older leaves and tender new leaves of purification kit according to the manufacturer’s Camellia oleifera from Ganzhou, Jiangxi Province, and instructions. DNA sequencing of the PCR products Hainan Academy of Agricultural Sciences (HAAS), was conducted by Sangon Company (Shanghai, Hainan Province. Firstly, leaves were surface-steri- China). lized in 75% ethanol for 30 s and rinsed three times In order to investigate the evolutionary relation- with distilled water. After air-drying, leaves were ships between different members of the Colletotrichum inoculated with fungal isolate CCG4. Leaves were gloeosporioides species complex, phylogenetic analysis wounded in the middle using a sterilized dissecting was performed using MEGA6.0 (Tamura et al. 2013) needle. Inoculations were conducted by placing an by applying the neighbour-joining method (www.me 8-mm agar plug from the edge of an actively growing gasoftware.net). Bootstrap values were obtained from 5-day-old culture onto the leaf. Petioles were then 1000 bootstrap replicates. The type strain Col- wrapped with absorbent paper soaking in distilled letotrichum aeschynomenes ICMP 17673 is a representa- water. The leaves were divided into two groups: the tive of the genus Colletotrichum species complex that negative control group and the infected experimental causes anthracnose of pepper and coffee. To compare group. Each group contained 20 older and 20 tender the fungal sequences obtained in this study, 19 leaves from HAAS and 20 older and 20 tender leaves sequences from different species and isolates of the from Ganzhou. The negative control leaves were inoc- genus Colletotrichum available in the NCBI database ulated with 8-mm-diameter plugs of non-colonized were included in the analysis. Glomerella sp. PDA medium, while the leaves in the infected group KC110794 was used as the out-group. were inoculated with 8-mm-diameter plugs of colo- nized PDA medium. Inoculated leaves were immedi- Results ately placed in plastic containers, arranged in a completely randomized design, with distilled water to Morphological identification and characterization maintain a humid environment, and incubated at 28°C under artificial light (12/12-h light/dark cycles). Brown spot disease was observed on tea-oil tree leaves The mean number of lesions on the inoculated leaves in the field, as shown in Fig. 1, and it affected the pro- was recorded after 5 days. duction of Camellia oleifera oil. The pathogen was iso- lated, and investigated by light microscopy (Fig. 2). Regular circular colonies with 50–60 mm diameter, a Molecular identification and phylogenetic analysis woolly appearance and a small amount of jacinth pig- Total genomic DNA of the pathogen was extracted by ment in the centre were generated after 5 days of the modified protocol of Xu et al. (1994). Total geno- incubation on PDA medium at 28°C; the colonies mic DNA was extracted from fresh mycelium changed from white to black at later stages of growth (500 mg) scraped from the margin of a colony on a (Fig. 2A, B). The mycelium was branched and septate. PDA plate incubated at 28°C for 5–7 days and verified The conidia were cylindrical-truncate, oblong-obtuse by staining with GoldView (Aidlab, Beijing, China) on to doliform, colourless with 1–2 hyaline oil globules 1.0% agarose electrophoresis gels. The concentration in every conidium and 4.5–5.3 lm 9 7.7–17.5 lm. and purity of DNA were assessed using an ultraviolet The sporodochia were cushion-shaped (Fig. 2C, D). spectrophotometer. The internal transcribed spacer (ITS) was amplified using primer pair ITS1/ITS4; ITS1: Observation of histopathological staining TCCGTAGGTGAACCTGCGC; ITS4: TCCTCCGCTTATT GATATGC (White et al. 1990). PCRs were performed The following macroscopic features were clearly seen in a total volume of 50 ll. The PCR mixtures on the surface of the diseased leaves (Fig. 3A): scalene contained 29 Taq PCR MasterMix (Aidlab) 25 ll, triangle or close globose appressoria; cylindrical coni- ITS1 3.0 ll (10 lmol/l), ITS4 3.0 ll (10 lmol/l), DNA dia; branched, septate hyphae; and complicated germ template 2.0 ll (100–500 ng) and double-distilled tubes. In addition, the hyphae were inserted into stom- water 17 ll. PCR conditions were as follows: atal cells, which control the transport of substances in

382 J Phytopathol 165 (2017) 380–386 Ó 2017 Blackwell Verlag GmbH M. Xu et al. Pathogen for brown spot disease of Camellia oleifera

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Fig. 2 Morphological characteristics of Col- letotrichum gloeosporioides CCG4 from leaves of Camellia oleifera. (A, B) Colony appearance on PDA; (C) branched, septate hyphae with conidia (red arrow); (D) sporodochia (red arrow). (C, D) Stained with Alcian blue 8GX. Bars, 10 lm. [Colour figure can be viewed at m m wileyonlinelibrary.com] 10 µ 10 µ

A B

Fig. 3 Observation of histopathological stain- ing of Camellia oleifera with Alcian blue 8GX. (A) Conidia (black arrow) and branched, sep- tate hyphae on the surface of infected leaves; (B) appressorium and germ tube (black arrow) into a stomatal cell. Bars, 10 lm. [Colour figure 10 µm 10 µm can be viewed at wileyonlinelibrary.com] and out of cells and are related to resistance to diseases Leaves from HAAS showed greater resistance than (Fig. 3B). leaves from Ganzhou, Jiangxi Province, to Col- letotrichum gloeosporioides (Fig. 4B). Statistical analysis showed that the rate of infection of leaves from Ganz- Pathogenicity tests hou, Jiangxi Province, was 50%, while it was only The pathogenicity test resulted in the appearance of 25% in leaves from HAAS. dark-grey disease spot symptoms on the leaves of the infected experimental group. The negative control Molecular identification and phylogenetic analysis group was not infected (Fig. 4A). Initial symptoms were observed as grey spots, which developed on the From the amplification of the ITS region, an approxi- leaves after 3 days of inoculation. At later stages, the mately 545-bp PCR product was obtained for the fun- spot tissues of the leaves became black and rotten. gal isolate, and the isolate was identified as

J Phytopathol 165 (2017) 380–386 Ó 2017 Blackwell Verlag GmbH 383 Pathogen for brown spot disease of Camellia oleifera M. Xu et al.

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1 cm abcd

B

Fig. 4 Pathogenicity tests. (A) Control group. (B) Group infected by Colletotrichum gloeospo- rioides CCG4. (a, b) Older and tender leaves, respectively, from HAAS, Hainan Province. (c, d) Older and tender leaves, respectively, from Ganzhou, Jiangxi Province. Bars, 1 cm. [Colour figure can be viewed at wileyonlinelibrary.- 1 cm abcdcom]

7 colletotrichum aeschynomenes, Aeschynomene virginica, ICMP17673 Type strain 4 Colletotrichum gloeosporioides, Boehmeriae Sawada, GQ120490 26 Colletotrichum gloeosporioides, Cymbidium ensifolium, KC010541 6 23 Colletotrichum gloeosporioides, Walnut, GU597322 colletotrichum fruticola, unknown plant, JN943082 34 colletotrichum siamense, Coffea canephora, HE655488 colletotrichum fragariae, unknown plant, AB087221 , , JN887341 9 27 Colletotrichum gloeosporioides Citrus reticulata 68 Colletotrichum gloeosporioides, Huperzia serrata, KP900224 68 colletotrichum gloeosporioides, Mango, JN697582 Colletotrichum gloeosporioides, Blueberry, KC122769 CCG4 Camellia oleifera 57 7 47 Colletotrichum gloeosporioides, Blueberry, JX878503 15 Colletotrichum gloeosporioides, Capscicum annuum, HQ264182 22 Colletotrichum gloeosporioides, unknown plant, KM357285 43 Colletotrichum gloeosporioides, Vitex negundo, KF923863 Colletotrichum gloeosporioides, unknown plant, JF710554 32 colletotrichum tropicale, Trichilia tuberculata, GU994329 Colletotrichum gloeosporioides, Maytenus hookeri Loes, FJ224105 Glomerella sp.KC110794 Out-group

Fig. 5 Phylogenetic analysis. Molecular phylogenetic analysis of isolate CCG4 with 19 species of the genera Colletotrichum and Glomerella obtained from GenBank. The tree was constructed by the neighbour-joining method based on ITS sequences. Bootstrap values after 1000 replicates are expressed as percentages at branching points. [Colour figure can be viewed at wileyonlinelibrary.com]

384 J Phytopathol 165 (2017) 380–386 Ó 2017 Blackwell Verlag GmbH M. Xu et al. Pathogen for brown spot disease of Camellia oleifera

Colletotrichum gloeosporioides based on BLAST analysis isolate formed a clade with members of the genus Col- of the ITS sequence against the NCBI database. The letotrichum Corda; however, it could not be identified isolate was named CCG4. The sequence had 100% to the species level. Colletotrichum aeschynomenes ICMP query cover and 99% similarity to that of Col- 17673 was a type strain of Colletotrichum sp, while letotrichum gloeosporioides (GenBank JN887341.1). Glomerella sp. KC110794 was used as an out-group. For phylogenetic analysis, a total of 19 sequences representing members of the genus Colletotrichum Discussion Corda, including a type strain and an out-group strain, obtained from GenBank, and the CCG4 Camellia oleifera has been cultivated in China for more sequence were used to construct the phylogenetic than 2000 years, primarily for the edible oil extracted tree. Information concerning sequences used for phy- from seeds, and is mainly distributed throughout 14 logenetic analysis is given in Table 1. Nucleotide provinces in regions south of the Yangtze River (Gao sequences were analysed using MEGA6.0 (Tamura et al. 2015). With the development of Camellia oleifera et al. 2013) for neighbour-joining tree analysis. Boot- production and the expansion of cultivated areas, dis- strap values were obtained from 1000 bootstrap repli- eases of Camellia oleifera are becoming more and more cates. The phylogenetic tree (Fig. 5) revealed that the prominent. Members of the genus Colletotrichum Corda cause one of the most important diseases of Table 1 Information concerning sequences used for phylogenetic anal- Camellia oleifera in China. They infect and cause ysis anthracnose disease in at least 470 different host spe- cies and are considered the major causal agent of GenBank Length postharvest disease in fruits such as citrus, apple, accession no. (bp) Organism Host olive, mango, banana and (Reboledo et al. 2015). Disease resistance varies among different GQ120490 576 Colletotrichum Ramie gloeosporioides varieties of Camellia, with Camellia sasanqua having KC010541 583 Colletotrichum Cymbidium the best resistance while the common Camellia oleifera gloeosporioides ensifolium is the most susceptible. GU597322 584 Colletotrichum Walnut Morphological identification and characterization gloeosporioides and the BLAST analysis of the ITS sequence against JN943082 549 Colletotrichum Unknown the NCBI database revealed that the isolate we fruticola obtained in this study could be identified as Col- HE655488 488 Colletotrichum Coffea siamense canephora letotrichum gloeosporioides. However, it could not be AB087221 490 Colletotrichum Unknown identified to the species level on the basis of neigh- fragariae bour-joining tree analysis of ITS sequences of patho- JN887341 545 Colletotrichum Citrus reticulata gens in the genus Colletotrichum Corda. According to gloeosporioides our study, the closely related species could not be sep- KP900224 538 Colletotrichum Huperzia arated by neighbour-joining analysis, in which the gloeosporioides serrata support was low at some key evolutionary tree nodes. JN697582 557 Colletotrichum Mango gloeosporioides Previous studies that utilized morphology or single KC122769 549 Colletotrichum Blueberry genes also failed to resolve the phylogenetic relation- gloeosporioides ships among these species (Chen et al. 2015). There- JX878503 549 Colletotrichum Blueberry fore, in future work, it will be necessary to identify gloeosporioides species based on multiple conserved gene sequences, HQ264182 537 Colletotrichum Capsicum including beta-tubulin, 28S ribosomal DNA, ITS gloeosporioides annuum region sequences and similar. KM357285 525 Colletotrichum Plant gloeosporioides Colletotrichum gloeosporioides has been reported to KF923863 548 Colletotrichum Vitex negundo have wide geographical and host ranges in China, and gloeosporioides has been reported on mango (Hong et al. 2016), citrus JF710554 542 Colletotrichum Unknown fruit (Reboledo et al. 2015) and human skin (Lin gloesporioides et al. 2015). So far, however, there is no effective GU994329 553 Colletotrichum Trichilia method to prevent disease caused by Colletotrichum tropicale tuberculata in plantations. Cur- FJ224105 524 Colletotrichum Maytenus gloeosporioides Camellia oleifera gloeosporioides hookeri Loes rently, chemical methods are often used; however, the drawback of this approach is environmental

J Phytopathol 165 (2017) 380–386 Ó 2017 Blackwell Verlag GmbH 385 Pathogen for brown spot disease of Camellia oleifera M. Xu et al. pollution and pesticide residues. Therefore, it is impor- profiles of the related defense genes in postharvest tant to understand the molecular mechanisms of the mango fruit against Colletotrichum gloeosporioides. response of Camellia oleifera to Colletotrichum gloeospori- Gene 576:275–283. oides so that it may be possible to suppress the disease Jin A, Zhou G, Li H. (2009) Process, problem and prospect using the resistance of the plants themselves. of oil Camelliae anthracnose (Colletotrichum gloeospori- – In conclusion, brown spot of Camellia oleifera occurs oides) research. For Pest Dis 28:27 31 (in Chinese). in most plantations in China. To prevent and reduce Kameyama A, Dong W, Matsuno YK. (2015) Succinyla- the disease occurrence, Camellia oleifera plantations tion-Alcian Blue Staining of Mucins on Polyvinylidene – should be well managed, particularly in the aspects of Difluoride Membranes. Methods Mol Biol 1314:325 331. hygiene and pruning practice. Morphological identifi- Kuang R, Sun S, Wang J, Huang Y. (2012) Research pro- cation of Colletotrichum gloeosporioides was supported gress on Camellia oleifera diseases and control. Biol Disas- by sequences of ITS regions that showed 99% similar- ter Sci 35:435–438 (in Chinese). ity with a Colletotrichum gloeosporioides sequence in Lin LY, Yang CC, Wan JY, Chang TC, Lee JY. (2015) Cuta- GenBank (JN887341.1). The study provides a theoret- neous Infection Caused by Plant Pathogen Colletotrichum ical basis for future research into the prevention of gloeosporioides. JAMA Dermatol 151:1383–1384. the disease and breeding of resistance. It also provides Peng S, Jia L, Jinxiu Y, Chen Y, Ma L, Peng Y. (2015) a basis for our better understanding of molecular Resistance of 21 Camellia germplasms to Colletotrichum adaptation mechanisms in Colletotrichum gloeosporioides gloeosporioides and Myrothecium camelliae. J Cent South infections of Camellia oleifera. Univ For Technol 35:20–24 (in Chinese). Reboledo G, Del Campo R, Alvarez A, Montesano M, Mara H, Ponce De Leon I. (2015) Physcomitrella patens acti- Acknowledgements vates defense responses against the pathogen Col- This work was supported by the following grants: letotrichum gloeosporioides. Int J Mol Sci 16:22280–22298. 31360364 (National Natural Science Foundation of Song GT. (2012) The study on detection and biological China, NSFC), lhxm-2012-2 (Joint Support Program control to the main pathogens of Camellia Oleifera. from Tropical Crop Breeding Engineering Center of Changsha, Hunan, China, Central South University of Ministry of Education and National Crop Science Key Forestry and Technology, PhD Thesis (in Chinese). 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