Endophytic Diaporthe Associated With Citrus grandis cv. Tomentosa in China

Diaporthe species are associated with Citrus as endophytes, pathogens, and saprobes worldwide. However, little is known about Diaporthe as endophytes in Citrus grandis in China. In this study, 24 endophytic Diaporthe isolates were obtained from cultivated C. grandis cv. “Tomentosa” in Huazhou, Guangdong Province in 2019. The nuclear ribosomal internal transcribed spacer (ITS), partial sequences of translation elongation factor 1-α (tef1), β-tubulin (tub2), and partial calmodulin (cal) gene regions were sequenced and employed to construct phylogenetic trees. Based on morphology and combined multigene phylogeny, eleven Diaporthe species were identified including two new species, Diaporthe endocitricola and D. guangdongensis. These are the first report of D. apiculata, D. aquatica, D. arecae, D. biconispora, D. limonicola, D. masirevicii, D. passifloricola, D. perseae, and D. sennae on C. grandis. This study provides the first intensive study of endophytic Diaporthe species on C. grandis cv. tomentosa in China. These results will improve the current knowledge of Diaporthe species associated with C. grandis. The results obtained in this study will also help to understand the potential pathogens and biocontrol agents and to develop a platform in disease management.

Diaporthe species are often reported as endophytes (Murali et al., 2006;Botella and Diez, 2011;Huang et al., 2015) and they may provide several advantages to the plants. They possibly contribute to resistance against pathogens and might also act as a secondary defence layer of the associated plant (Hyde and Soytong, 2008;Dini-Andreote, 2020). It is important to explore the relationship between natural products from the plant and its endophytes (Alvin et al., 2014) since this might reveal novel compounds with antimicrobial activities and medicinal properties. The objectives of the present study were to isolate and identify endophytic Diaporthe species associated with healthy C. grandis cv. "Tomentosa" trees collected in Huazhou, Guangdong, China. Detailed descriptions of novel species identified based on molecular phylogeny and morphology are provided.

Sampling and Isolation of Endophytic Fungi
Healthy C. grandis cv. "Tomentosa" leaves, twigs, and fruits were collected randomly from a Citrus orchard in Huazhou city, Guangdong Province, China in May 2019. Samples were placed in plastic zip-lock bags containing sterilized wet cotton to prevent drying, taken to the laboratory and isolations were made on the same day. The samples were initially washed with tap water and then with sterile water. The leaves were then cut into 3 mm × 3 mm segments, twigs into pieces 3 mm long, and the fruits into 3 cm 3 cubes. Each piece was surface sterilized by dipping sequentially into 75% ethanol for 40 s, 2.5% NaOCl (sodium hypochlorite) for 90 s, rinsed with three changes of sterile water, dried on sterilized filter paper and then placed on potato dextrose agar (PDA). Plates were incubated at 25 • C with 12 h dark and 12 h fluorescent light. In total, 80 tissue segments were obtained from leaves, twigs, and fruits. Fungi growing from the edges of the tissue were sub-cultured on fresh PDA plates. To obtain pure cultures, single spore isolation was carried out (Chomnunti et al., 2011).

DNA Extraction and PCR Amplification
Mycelia were scraped from 7 days old pure cultures growing on PDA and total genomic DNA was extracted using the CTAB method (Sun et al., 2009). The ITS region was amplified and sequenced with primers ITS1/ITS4 (White et al., 1990). BLAST searches in GenBank with the ITS sequences provided genus level identifications. Once the BLAST results confirmed the isolates as Diaporthe species, additional three gene regions, namely translation elongation factor-1α (tef1), β-tubulin (tub2), and calmodulin (cal) were amplified and sequenced. The protocols for PCR amplification were followed as given in Udayanga et al. (2012) and Manawasinghe et al. (2019). The primer pairs and their respective amplification conditions are given in Table 1. Positive PCR amplicons were observed on 1% agarose electrophoresis gel. Sequencing (forward direction, both directions when necessary) was done by Tianyi Huiyuan Biotechnology Co., Ltd., Guangzhou, China. Initial sequence quality was checked with BioEdit 7.25 (Hall, 2006). All sequence data generated in this study were submitted to GenBank (Supplementary Table 1).
For the MP analysis, ambiguous regions in the alignment were excluded and gaps were treated as missing data. Tree stability was evaluated with 1,000 bootstrap replications. Zerolength branches were collapsed, and all parsimonious trees were saved. Tree parameters; tree-length (TL), consistency index (CI), retention index (RI), relative consistency index (RC), and homoplasy index (HI) were calculated. Kishino-Hasegawa tests (KHT) were conducted to evaluate differences between the trees inferred under different optimality criteria (Kishino and Hasegawa, 1989). MrModeltest v. 2.3 (Nylander, 2004) was used to determine the evolutionary models for each locus to be used in Bayesian and maximum likelihood analyses. The maximum likelihood analyses were conducted using RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis, 2014) in the CIPRES Science Gateway platform (Miller et al., 2010). The GTR + I + G evolutionary model was employed with 1,000 non-parametric bootstrapping iterations. Bayesian analysis was performed in MrBayes v. 3.0b4 (Ronquist and Huelsenbeck, 2003). Six simultaneous markov chains were run for 10 6 generations, sampling the trees at every 200th generation. From the 5,000 trees obtained, the first 2,000 representing the burn-in phase were discarded. The remaining 3,000 trees were used to calculate posterior probabilities (BYPPs) in a majority rule consensus tree. The final sequence alignment generated in this study was submitted to TreeBASE ID 26384 2 . Taxonomic novelties were submitted to Index Fungorum 3 and 1 http://www.ebi.ac.uk/Tools/msa/mafft/ 2 http://purl.org/phylo/treebase/phylows/study/TB2:S26384?x-access-code= e34e24939299e00766a5bf45970dabb0&format=html 3 www.indexfungorum.org Faces of Fungi database (Jayasiri et al., 2015). Newly generated sequences were deposited in GenBank.

Morphological Characterization
Agar plugs (5mm diam) were taken from actively growing cultures on PDA and transferred onto PDA, malt extract agar (MEA) (Crous et al., 2009) and pine needle agar (PNA: 2% WA with three sterilized pine needles) (Smith et al., 1996) plates and incubated at 25 • C with 12 hours of alternating darkness and fluorescent light per day for over a month to induce sporulation (Gomes et al., 2013;Huang et al., 2015). Colony characters and pigmentation on MEA and PDA were recorded after 7, 15, and 30 days. Colony color (upper and reverse) was described by referring to the color charts of Rayner (1970). Colony diameters were measured after 3-7 days. Digital images of morphological structures (shape, size, and color) were recorded with an Eclipse 80i photographic microscope (Nikon, Japan). Conidial length and width were measured for 40 conidia per isolate using NIS-Elements BR 3.2, and the mean values were calculated with their standard deviations (SDs).
Culture Characteristics: Colonies on PDA reach 75 mm diam. after 5 days at 25 • C. White fluffy aerial mycelium, margin filiform. Later turning to brownish yellow, pigmentation developing from the center. Reverse initially white and then turning brownish-yellow from the center, some became brownish-green.
Note: A single isolate in the present study clustered together with the Diaporthe apiculata ex-type strain (CGMCC 3.17533) with 71% maximum likelihood bootstrap value and 0.89 Bayesian posterior probabilities. Colony morphology and conidial dimensions of the present isolate (ZHKUCC 20-0001) were similar to those in the original description of D. apiculata (Gao et al., 2016). Diaporthe apiculata was introduced by Gao et al. (2016) for a species associated with healthy leaves of Camellia. This is the first report of D. apiculata on C. grandis cv. "Tomentosa" (Farr and Rossman, 2020).  (Figure 3).

FIGURE 1 | Continued
Frontiers in Microbiology | www.frontiersin.org FIGURE 1 | The best scoring RAxML tree obtained using the combined dataset of ITS, tef1, tub2, and cal sequences. Diaporthella corylina (CBS 121124) was used to root the tree. Bootstrap support values equal to or greater than 50% in ML and MP and BYPP equal or greater than 0.95 are shown as ML/MP/BYPP above the respective node. The isolates belonging to the current study are given in blue for known species, and novel taxa are shown in red. Ex-type strains are bold. Expected number of nucleotide substitutions per site is represented by the scale bar.
Culture Characteristics: Colonies on PDA reach 85 mm diam. after 5 days at 25 • C. Aerial mycelium, white, undulate margin, forming concentric rings of pycnidia. Reverse white, and then turning to brown-yellow from the center.
Habitat and host: Freshwater fungus (Hu et al., 2012). Known distribution: China (Hu et al., 2012). Note: Isolate ZHKUCC 20-0002 obtained in this study clusters together with the ex-type isolate of Diaporthe aquatica (IFRDCC3051) with 66% maximum likelihood bootstrap and 0.95 Bayesian posterior probability values. Alpha and beta conidia of this strain have a similar length to Diaporthe aquatica (Hu et al., 2012). So far, this species has been reported only as a freshwater fungus from China (Hu et al., 2012). This is the first report of D. aquatica on C. grandis cv. "Tomentosa" (Farr and Rossman, 2020).  (Figure 4).
Culture Characteristics: After 5 days at 25 • C colonies reach 85 mm diam. on PDA. White radial, margin undulate. Reverse white, becoming tawny then dark brown from the center.

Diaporthe biconispora
Culture Characteristics: Cultures reach 75 mm diam. on PDA at 25 • C after 5 days. White to light yellow, aerial mycelium, circular, entire margin. Reverse light yellow, black pigmentation at the center.
Etymology-In reference to its endophytic nature in Citrus grandis.
Culture characteristics: Cultures reach 85 mm diam., after 5 days on PDA at 25 • C. White with radial hyphal growth at the rim, circular form with entire margin, with some irregular conidiomata after 20 days. Reverse white becoming yellowbrown, with zonations.
Culture Characteristics: Cultures reach 85 mm diam. after 3 days on PDA at 25 • C. White turn to yellowish-white with time, circular, entire margin. Reverse white and turn to reddish-brown from the center with time.
Culture Characteristics: At 25 • C colonies reach 85 mm diam. on PDA after 5 days of inoculation. White, circular, entire margin, becoming cream to smoke-gray. Reverse white turning brown and dark gray, dark brown scattered pigmentation. Colonies on MEA at first white, then cream and become yellowish, flat, and dense. Reverse pale brown with conidiomata appearing as brownish dots that become black solitary or aggregated conidiomata at maturity.
Note: Two isolates obtained in this study clustered together with Diaporthe limonicola (CBS H-23126) with 68% maximum likelihood, 52% maximum parsimony bootstrap, and 1.0 Bayesian posterior probability values. Morphologically both strains produced similarly shaped and similar sized conidia to the original description of D. limonicola (Guarnaccia and Crous, 2017). Diaporthe limonicola was introduced by Guarnaccia and Crous (2017) as a species associated with serious trunk and branch cankers of C. limon. This is the first report of D. limonicola on C. grandis cv. "Tomentosa" (Farr and Rossman, 2020).
Note: Three isolates obtained in the present study clustered together with Diaporthe masirevicii (BRIP 57892a) with 94% maximum likelihood, 92% maximum parsimony bootstrap, and 1.0 Bayesian posterior probability values. Morphologically these strains produce beta and gamma conidia with similar lengths of those of the Diaporthe masirevicii type description (Thompson et al., 2015). Diaporthe masirevicii was introduced by Thompson et al. (2015) as a species associated with cankers or dead trees of Chrysanthemoides monilifera subsp. Rotundata, Glycine max, Helianthus annuus, and Zea mays. To our knowledge, this is the first report of D. masirevicii on C. grandis cv. "Tomentosa" (Farr and Rossman, 2020).  (Figure 10).
Culture Characteristics: Colonies on PDA reach 85 mm diam. on PDA at 25 • C after 7 days. White, fluffy, aerial mycelium, and filiform margins. Reverse white, and later become yellow-brown to ochreous from the center.
Note: In the combined multigene phylogenetic analysis of ITS, tef1, tub2, and cal, eight strains isolated in this study developed a well-supported clade with the D. passifloricola (CPC 27480) with 96% ML, 96% MP bootstrap and 1.0 BYPP values.
In comparison between D. passifloricola and strains in the present study, they share morphologically similar characters as given in Crous et al. (2016). However, isolates obtained in this study have faster growth rate on PDA and radial margins. In addition, comparisons of gene regions between isolates from this study and D. passifloricola type, there is a 1.4% nucleotide difference in ITS with 555 nucleotides. In protein-coding regions, 0.55% differences in tub2 (547 nucleotides). This is the first record of D. passifloricola on C. grandis cv. "Tomentosa" (Farr and Rossman, 2020).
Culture Characteristics: Colonies on PDA at 25 • C reach 85 mm diam. after 4 days. White and later turns pale white with patches of sienna, filamentous, entire margin. Reverse white, and with age produce umber color patches turning into sienna.
Note: A single isolate from the present study clustered together with the Diaporthe perseae (CBS 151.73) with 73% maximum likelihood, 72% maximum parsimony bootstrap, and 1.0 bayesian posterior probability values. Morphologically this strain produces a similar length of both alpha and beta conidia to the Diaporthe perseae (Gomes et al., 2013). Diaporthe perseae was introduced by Gomes et al. (2013) as a species associated with branches of dying Persea gratissima. This is the first report of D. perseae on C. grandis (Farr and Rossman, 2020).
Culture Characteristics: After 5 days on PDA, colonies reach 85 mm diam. at 25 • C. White, radial hyphal growth, becoming gray-white, develop pycnidia after 7 days. Reverse white, then pale brown from the center and forming concentric rings of pycnidia.
Habitat and host: Senna bicapsularis (Yang et al., 2017a). Known distribution: China (Yang et al., 2017a). Note: The ZHKUCC 20-0011 isolate obtained in this study clustered together with the D. sennae (CFCC 51636) with 95% maximum likelihood, 92% maximum parsimony bootstrap, and 1.0 Bayesian posterior probability values. Morphologically the isolate in this study produces a similar size of alpha and beta conidia to the D. sennae type description (Yang et al., 2017a).
Gamma conidia were also observed in strain ZHKUCC 20-0011 of this study after 7 days on PDA. Diaporthe sennae was introduced by Yang et al. (2017a) as a species associated with dieback of Senna bicapsularis. This is the first record of D. sennae on C. grandis cv. "Tomentosa" (Farr and Rossman, 2020).

DISCUSSION
In the present study, Diaporthe species were isolated as endophytes from C. grandis cv. "Tomentosa" in China with 24 isolates from fruits, leaves, and twigs. Based on the multigene phylogeny, all 24 isolates from this study were grouped in 11 distinct clades within the Diaporthe phylogenetic tree. Among them, two species (D. arecae and D. biconispora) are already known to be associated with C. grandis. Nine new host records were identified namely: D. apiculata, D. aquatica, D. limonicola, D. masirevicii, D. passifloricola, D. perseae, and D. sennae. The remaining two species were identified as novel and introduced here as D. endocitricola, and D. guangdongensis. This study is the first comprehensive analysis of endophytic fungi associated with C. grandis in China.
The host species in this study is C. grandis cv. "Tomentosa, " which is commonly known as "huajuhong" in China is a famous traditional Chinese medicinal plant, which has been used to alleviate cough and phlegm for several hundred years (Jiang et al., 2014). It has been proved that the endophytic fungi associated with medicinal plants have the ability to act as biological control agents (Carvalho et al., 2012) and some have anticancer activities (Carvalho et al., 2012). A few studies have revealed that secondary metabolites produced by endophytic fungi could be novel sources of medicinal compounds (Strobel et al., 2001;Kusari et al., 2012). Therefore, further studies are necessary to understand the relationship between medicinal properties and a plant's endophytic biota.
In the present study, endophytes were isolated from fruits, leaves, and twigs. Diaporthe masirevicii and D. passifloricola were isolated from fruits and twigs of C. grandis, while the other species were isolated from only one tissue type. A lower degree of colonization was observed on leaves (the only two species isolated were; Diaporthe perseae and Diaporthe biconispora). Similar to this study, Gond et al. (2007) and Huang et al. (2015) observed a lower number of endophytic species in leaves of Citrus spp. One endophytic species might occur in different tissues in the same host (Huang et al., 2015). However, the endophytic colonization in different tissues of the same plant might also vary (Taylor et al., 1999;Huang et al., 2015). For example, greater numbers of endophytic fungi were isolated from flowers and seeds than that from vegetative organs like stems and leaves (Braun et al., 2003). It has also been observed that greater numbers of endophytes could be isolated from veinal tissues than from interveinal tissues (Taylor et al., 1999). These variations might be a result of differences in the tissue organizational structure and the different nutrition content of each tissue type (Rana et al., 1997;Huang et al., 2015). However, the exact underlying reasons and mechanisms for these variations are not known. It is thus clear that further studies are needed to compare the variations in endophytic colonization according to different seasons or different stages of maturity of the plant.
Diaporthe biconispora was previously reported as an endophyte in branches of Citrus sinensis in China (Huang et al., 2015). In the present study, this species was isolated from leaves. Thus, D. biconispora may be a common endophytic species in Chinese Citrus plants. Diaporthe limonicola has been reported as pathogenic on Citrus sp. (Huang et al., 2015;Guarnaccia and Crous, 2017) and was first reported as a dieback pathogen of lemon trees in Europe. This species causes serious cankers on Citrus limon, C. aurantiifolia, C. reticulata, and C. sinensis (Guarnaccia and Crous, 2017). Identification of previously known pathogenic species as endophytes might reveal the opportunistic pathogenic nature of the Diaporthe species (Manawasinghe et al., 2019). Moreover, this is also important to develop quarantine measures to prevent the introduction of these species into new localities. Two of the species isolated in this study have been reported as pathogens on several other hosts. Diaporthe masirevicii has been reported causing peanut stem and peg dieback in Australia (Thompson et al., 2018), Gloriosa superba leaf blight in India (Naveen et al., 2018) and Physalis peruviana fruit rot in Brazil (Pazdiora et al., 2018). Diaporthe perseae was reported causing stem-end rot of mango (Lim et al., 2019). Therefore, further studies are necessary to understand the pathogenicity of these endophytic strains and the factors that determine their pathogenicity on Citrus.
In addition to previously known species from Citrus, in the present study, we identified nine novel host records and two new species. Novel species and host records are an indication of the ability of Diaporthe to evolve rapidly (Manawasinghe et al., 2019). Furthermore, this reflects the high species diversity of Diaporthe associated with a single host. Several studies have revealed the high species richness of Diaporthe species as endophytes on different hosts (Skaltsas et al., 2011;Rhoden et al., 2012). When there are diverse species associated with a single host, there is the potential of emerging new pathogens on Citrus. This could be a result of the species developing into taxa with greater virulence, or divergence of existing species into a novel species via long term exposure to natural and human-mediated factors. One possible phenomenon is the application of fungicides for other known phytopathogens while non-target fungal species become pathogenic a few years later. Thus, it might be challenging to control current disease while eliminating new disease occurrences. To overcome this, further studies are necessary to understand the interaction of endophytes with phytopathogenic genera and their effect on the Citrus microbiome.

CONCLUSION
In the present study, eleven endophytic Diaporthe species were isolated and identified from C. grandis cv. "Tomentosa" in China. Two new species D. endocitricola and D. guangdongensis were introduced. This study reveals the existence of several previously known pathogenic Diaporthe species as endophytes. Thus, it reflects the opportunistic nature of Diaporthe species as phytopathogens. However, further studies are necessary to understand the pathogenic potential of these endophytic taxa on C. grandis or other Citrus species in China. These results will open a discussion on interactions between fungal species on a particular host as endophytes, pathogens and potential biocontrol agents. In addition, these results will provide a platform to develop antimicrobial compounds, and to understand the contribution of endophytes to the medicinal values of the plant.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

AUTHOR CONTRIBUTIONS
ZD and ML conceived the research and planned the basic research. YS provided the materials. YS, YH, and ML conducted the experiments. ZD, ML, and IM prepared the manuscript. ZD, ML, IM, and AP analyzed the data. AP, KH, AD, and MX revised the manuscript. All authors read and approved the final manuscript.

ACKNOWLEDGMENTS
We would like to thank Shaun Pennycook for the guidance for new species names. ZD would like to thank the Department of Science and Technology, Guangdong Province for funding the Key Realm R&D Program of Guangdong Province (2018B020205003). KH thanks the Thailand Research grant entitled Impact of climate change on fungal diversity and biogeography in the Greater Mekong Subregion (grant no: RDG6130001) for supporting this study. AP acknowledges the support from UIDB/04046/2020 and UIDP/04046/2020 Centre grants from FCT, Portugal (to BioISI).