Endophytic Diaporthe Associated with Morinda officinalis in China

Diaporthe species are endophytes, pathogens, and saprobes with a wide host range worldwide. However, little is known about endophytic Diaporthe species associated with Morinda officinalis. In the present study, 48 endophytic Diaporthe isolates were obtained from cultivated M. officinalis in Deqing, Guangdong Province, China. The nuclear ribosomal internal transcribed spacer (ITS), partial sequences of translation elongation factor 1-α (tef1-α), partial calmodulin (cal), histone H3 (his), and Beta-tubulin (β-tubulin) gene regions were sequenced and employed to construct phylogenetic trees. Based on morphology and combined multigene phylogeny, 12 Diaporthe species were identified, including five new species of Diaporthe longiconidialis, D. megabiguttulata, D. morindendophytica, D. morindae, and D. zhaoqingensis. This is the first report of Diaporthe chongqingensis, D. guangxiensis, D. heliconiae, D. siamensis, D. unshiuensis, and D. xishuangbanica on M. officinalis. This study provides the first intensive study of endophytic Diaporthe species on M. officinalis in China. These results will improve the current knowledge of Diaporthe species associated with this traditional medicinal plant. Furthermore, results from this study will help to understand the potential pathogens and biocontrol agents from M. officinalis and to develop a disease management platform.


Introduction
Diaporthe Nitschke (1870, (syn. Phomopsis (Sacc.) Bubák), (Diaporthaceae) [1] includes important plant pathogens, endophytes, and saprobes [2]. Species identification in this genus is based on DNA-based phylogenetic approaches and incorporating sequences from type and voucher specimens to investigate species boundaries. In addition, the incorporation of morphological characters provided a more accurate identification [2]. Diaporthe is identified as a cryptic genus and thus morphology alone with phylogeny is challenging to delineate species [3]. Thus, some studies have shown the importance of incorporating additional approaches such as Genealogical Concordance Phylogenetic Species Recognition (GCPSR) and recombination analysis of incorrect species identification in this genus [3]. and Engineering (ZHKUCC). Herbarium materials (as dry cultures) were deposited at Zhongkai University of Agriculture and Engineering (ZHKU).

DNA Extraction and PCR Amplification
For DNA extraction, mycelia were scraped from about seven-day-old cultures on PDA. Total genomic DNA was extracted using the MagPure Plant DNA AS Kit (D6351-AS-06, Guangzhou Magen Biotechnology Co., Ltd., Guangzhou, China) using an Acid purification system (Auto-Pure 32A, Allsheng instruments Co., Ltd., Hangzhou, China). For preliminary species confirmation, the ITS gene region was amplified using the ITS1/ITS4 [22]. The sequences were aligned in GenBank by using the BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 1 July 2021). Then, four additional gene regions of tef-1α [22], cal [23], β-tubulin [24], and his [24,25] were amplified under each PCR conditions (Table 1) and sequenced. PCR amplicons were visualized on 1% agarose electrophoresis gel. The sequencing was performed by Tianyi Huiyuan Biotechnology Co., Ltd., Guangzhou, China. Initial sequence quality was checked using Bi-oEdit 7.25 [26]. The sequence data generated in this study have been deposited in Gen-Bank (Supplementary Table S1).

DNA Extraction and PCR Amplification
For DNA extraction, mycelia were scraped from about seven-day-old cultures on PDA. Total genomic DNA was extracted using the MagPure Plant DNA AS Kit (D6351-AS-06, Guangzhou Magen Biotechnology Co., Ltd., Guangzhou, China) using an Acid purification system (Auto-Pure 32A, Allsheng instruments Co., Ltd., Hangzhou, China). For preliminary species confirmation, the ITS gene region was amplified using the ITS1/ITS4 [22]. The sequences were aligned in GenBank by using the BLAST tool (https://blast.ncbi.nlm.nih. gov/Blast.cgi, accessed on 1 July 2021). Then, four additional gene regions of tef-1α [22], cal [23], β-tubulin [24], and his [24,25] were amplified under each PCR conditions (Table 1) and sequenced. PCR amplicons were visualized on 1% agarose electrophoresis gel. The sequencing was performed by Tianyi Huiyuan Biotechnology Co., Ltd., Guangzhou, China. Initial sequence quality was checked using BioEdit 7.25 [26]. The sequence data generated in this study have been deposited in GenBank (Supplementary Table S1).
The evolutionary models for each locus used in Bayesian analysis were selected using MrModeltest v. 2.3 [31]. The ML analyses were accomplished using RAxML-HPC2 on XSEDE (8.2.8) [32] in the CIPRES Science Gateway platform [33] using the GTR + I + G model of evolution with 1000 non-parametric bootstrap iterations. Bayesian analysis was performed in MrBayes v. 3.0b4 [30] for the portioned data set. The posterior probabilities (PPs) were determined by Markov chain Monte Carlo sampling (MCMC). Four simultaneous Markov chains were run for 20,000,000 generations, sampling the trees at every 100th generation. From the 200,000 trees obtained, the first 5000 representing the burn-in phase were discarded. The remaining 150,000 trees were used to calculate posterior probabilities (PPs) in a majority rule consensus tree.

Morphological Characterisation
Agar plugs (5 mm diam.) were taken from the edges of actively growing cultures on PDA and transferred onto the middle of PDA plates and incubated at 25 • C equal hours of alternative dark and fluorescent light for over a month to induce sporulation [13]. Colony characters and pigmentation on PDA were recorded after 4-7 d, 15 d, and 30 d. Colony colours (upper and reverse) were described as in Rayner [37]. Cultures were examined periodically for the development of ascomata and conidiomata. Colony diameters were measured after 3-7 days. The shapes, sizes, and colours of at least 15 pycnidia were recorded with an Eclipse 80i photographic microscope (Nikon, Tokyo, Japan). Pycnidia were cut into 30 µm thin sections by a freezing sliding microtome (Bio-Key science and technology Co., LTD, LEICA CM1860, Weztlar, Germany) for photographing and measuring. Forty conidiophores, alpha conidia, and beta conidia were measured using NISElements BR 3.2, and mean sizes were calculated with their standard deviations (SDs).

Pairwise Homoplasy Index (PHI)
The PHI test was performed using SplitsTree4 v. 4.17.1, [38] to determine the recombination level within closely phylogenetically related species. The concatenated five-locus dataset (ITS + tef1-α + cal + his + β-tubulin) was used for the analyses. PHI test results (Fw) > 0.05 indicated no significant recombination within the dataset. The relationships between closely related taxa were visualized in split graphs with both the Log-Det transformation and splits decomposition options.

Isolation
In total, 48 endophytic Diaporthe strains were obtained (eight from roots and 40 from stems). All media used in this study were able to grow Diaporthe species. From 48 isolates, 9 isolates were obtained from LCA, 4 isolates were obtained from M + J medium, 10 isolates were obtained from MD, 6 isolates were obtained from CMA, 7 isolates from PDA, 8 were obtained from 2% PDA, and 4 isolates were obtained from P + J medium.

Phylogenetic Analyses
In the present study, we followed Norphanphoun et al. [39] for the taxonomic treatments of Diaporthe. As given in the methods, phylogenetic analyses were arranged in two steps. At first, we developed a genus tree including all species belonging to this genus as given in Guarnaccia et al. [9] and Yang et al. [27]. Then, following Norphanphoun et al. [39], species complexes belonging to isolates from this study were identified, and the final tree was developed. The final analyses were conducted using 271 Diaporthe strains (including types strains) with a combined ITS, tef1-α, cal, his, and β-tubulin sequence alignment. The phylogenetic tree was rooted in Diaporthella corylina (CBS 121124). The final maximum likelihood tree topology was similar to Bayesian analysis. The best scoring RAxML tree with a final likelihood value of −32,402.374230 is given in Figure 2. The matrix consisted of 1065 distinct alignment patterns, with 20.40% of undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.220036, C = 0.307463, G = 0.247268, T = 0.225234; substitution rates AC = 1.189728, AG = 3.654106, AT = 1.419097, CG = 0.937185, CT = 6.230555, GT = 1.000000; gamma distribution shape parameter α = 0.789923. For the Bayesian inference, GTR + I + G model was selected for ITS, TrN + I + G for tef1-α, TIM2 + I + G for cal, TIM1 + I + G for his, and TPM3uf + I + G for β-tubulin. The Bayesian analyses generated 200,000 trees (average standard deviation of split frequencies: 0.032560), from which 150,000 were sampled after 25% of the trees were discarded as burn-in. The alignment contained 1073 unique site patterns. In this resulted tree, isolates belonging to this study were clustered together with seven known Diaporthe species and five novel phylogenetic lineages. They belong to five  The best scoring RAxML tree obtained using the combined dataset of ITS, tef1-α, cal, his, and β-tubulin sequences. Diaporthella corylina (CBS 121124) was used to root the tree. Bootstrap support values equal to or greater than 60% in ML and BYPP equal to or greater than 0.95 are shown as ML/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, with T at the end of the strains numbers. The expected number of nucleotide substitutions per site is represented by the scale bar.

PHI Analysis
In the phylogenetic analysis of Diaporthe species, our isolates developed five distinct clades within the genus with significant tree lengths and low statistical support. To confirm species, we conducted PHI analysis for these five clades. There was no evidence of significant genetic recombination (Fw > 0.05) between these novel species of Diaporthe and closely related species (Figure 3). These results confirmed that these taxa were significantly different from the existing species of Diaporthe. Figure 2. The best scoring RAxML tree obtained using the combined dataset of ITS, tef1-α, cal, his, and β-tubulin sequences. Diaporthella corylina (CBS 121124) was used to root the tree. Bootstrap support values equal to or greater than 60% in ML and BYPP equal to or greater than 0.95 are shown as ML/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, with T at the end of the strains numbers. The expected number of nucleotide substitutions per site is represented by the scale bar.

PHI Analysis
In the phylogenetic analysis of Diaporthe species, our isolates developed five distinct clades within the genus with significant tree lengths and low statistical support. To confirm species, we conducted PHI analysis for these five clades. There was no evidence of significant genetic recombination (Fw > 0.05) between these novel species of Diaporthe and closely related species (Figure 3). These results confirmed that these taxa were significantly different from the existing species of Diaporthe.    Pycnidia 130-1400 µm × 120-900 µm (x = 542 ± 415 µm × 388 ± 267 µm) oblate or subglobose, grey to black, single or multiple cavities, translucent to black conidial drops exuded from the ostioles. Pycnidia wall thick, exuding creamy to black conidial droplets from ostioles. Conidiophores hyaline, smooth, septate, densely aggregated, cylindrical, straight to sinuous, swelling at the base, tapering towards the apex. Conidiogenous cell hyaline, cylindrical, straight, inner wall buds produce sporulation in bottle form. Alpha conidia 5-10 µm × 2-3 µm (x = 7 ± 0.5 µm × 3 ± 0.2 µm) hyaline, aseptate, fusiform or ellipsoid, biguttulate or multi-guttulate. Beta and gamma conidia not observed.
Culture characteristics: Colonies on PDA reach 85 mm diam. After 5 days. White cotton flocculent aerial mycelium, while the perimeter edge sparse hyphae, then ochre brown mycelium with several black conidiomata for 15 days and a lot of darker black conidiomata at 30 days. Reverse white and become reddish-brown.
Habitat and host: Pear pyrifolia [10]. Known distribution: China [10]. Note: A single isolate (ZHKUCC 22-0043) obtained in this study clustered with D. chongqingensis (PSCG435) with 37% ML. Morphologically our isolates are similar to D. chongqingensis [10]. This species was introduced as a shoot canker pathogen on pear (Pear pyrifolia) in China [10]. This is the first report of
Culture characteristics: Colonies on PDA reach 85 mm diam. After 5 days. White cotton flocculent aerial mycelium, while the perimeter edge sparse hyphae, then ochre brown mycelium with several black conidiomata for 15 days and a lot of darker black conidiomata at 30 days. Reverse white and become reddish-brown.
Habitat and host: Pear pyrifolia [10]. Known distribution: China [10]. Note: A single isolate (ZHKUCC 22-0043) obtained in this study clustered with D. chongqingensis (PSCG435) with 37% ML. Morphologically our isolates are similar to D. chongqingensis [10]. This species was introduced as a shoot canker pathogen on pear (Pear pyrifolia) in China [10]. This is the first report of D. chongqingensis as an endophyte on the M. officinalis stem.
Culture characteristics: Colonies on PDA reach 85 mm diam. after 4 days. Cottony and radially with abundant aerial mycelium, sparse in the margin, white on surface, then turn to grey. Reverse white to pale yellow.
Habitat and host: Eucalyptus sp. [40]. Known distribution: Australia [40]. Note: In the multigene phylogenetic tree, two isolates (ZHKUCC 22-0044 and ZHKUCC 22-0045) from this study clustered together with the D. eucalyptorum (CBS 132525) with 85% ML and 1.00 BYPP values. Morphologically, the ZHKUCC 22-0044 isolate is similar to those in the original description of D. eucalyptorum [40]. The ZHKUCC 22-0044 strain developed both alpha and beta conidia while the D. eucalyptorum type strain (CBS 132525) develops alpha conidia only. Diaporthe eucalyptorum was introduced from diseased leaves of Eucalyptus sp. in Australia [40]. This is the first report of D.
Culture characteristics: Colonies on PDA reach 85 mm diam. after 4 days. Cottony and radially with abundant aerial mycelium, sparse in the margin, white on surface, then turn to grey. Reverse white to pale yellow.
Culture characteristics: Colonies on PDA reach 85 mm diam. after 25 days. White aerial mycelia and then turn to yellowish-brown. Reverse first white, then dark brownish-green.
Culture characteristics: Colonies on PDA reach 85 mm diam. after 25 days. White aerial mycelia and then turn to yellowish-brown. Reverse first white, then dark brownish-green.
Material examined: China, Guangdong Province, Zhaoqing, isolated from a healthy stem of M. officinalis. June 2020, W. Guo, dried culture, ZHKU 22-0034, and living culture (ZHKUCC 22-0046) Habitat and host: Vitis vinifera [12]. Known distribution: China [12]. Note: In the phylogenetic analyses, a single isolate (ZHKUCC 22-0046) from this study clustered together with the D. guangxiensis strain (JZB320094), 76% ML and 1.0 BYPP values. Morphologically, the present isolate is similar to those in the original description of D. guangxiensis [12]. Diaporthe guangxiensis was introduced from diseased V. vinifera trunks [12]. The type strain (JZB320094) of D. guangxiensis developed both alpha and beta conidia while the ZHKUCC 22-0046 strain develop beta conidia only. This is the first report of D. guangxiensis as an endophyte on M. officinalis.
Culture characteristics: Colonies on PDA reach 85 mm diam. after 15 days. Aerial mycelium abundant, cottony, white, dense in the center, sparse near the margin. White on surface side, then yellowish-brown; reverse white to brown.
Known distribution: China [41]. Note: In the multigene phylogenetic analysis, two isolates (ZHKUCC 22-0047 and ZHKUCC 22-0048) from the present study clustered together with the D. heliconiae (SAUCC194.77) with 100% ML and 1.00 BYPP values. Morphologically, the present isolate is similar to those in the original description of D. heliconiae [41]. Diaporthe heliconiae was introduced as a pathogen on H. metallica petiole [41]. This is the first report of D.
Culture characteristics: Colonies on PDA reach 85 mm diam. after 15 days. Aerial mycelium abundant, cottony, white, dense in the center, sparse near the margin. White on surface side, then yellowish-brown; reverse white to brown.
Culture characteristics: Colonies on PDA reach 85 mm diam. after 5 days. White cotton with some polygon mycelium as stars in the center, then with purple pigmentation. Fruiting body occurred from about 15 days, grey to dark black with orange surroundings. Reverse white with some purple pigmentation.
Habitat and host: Healthy stem and root of M. officinalis. Known distribution: China (Zhaoqing, Guangdong Province). Note: In the polygenic analysis, eight isolates from the present study clustered together with the Diaporthe biconispora and Diaporthe pometiae ex-type strain with 100% ML and 1.00 BYPP values. Morphologically, Alpha conidia of strain ZHKUCC 22-0058 (x = 8 ± 1 × 3 ± 0.3 µm) were similar to those in the original description of D. biconispora [13]. The colony morphology of our isolates was different to those in the original description of D. biconispora, the surface of the colony is white and light yellow, the center of the back is black, and the sides are light yellow [13]. Alpha conidia of D. pometiae (x = 6.7 × 3.1 µm) were smaller than the ZHKUCC 22-0058 strain. The colony morphology of our isolate was different to those in the original description of D. pometiae. The surface of the colony is white, and the back is white to light grey [42]. Diaporthe biconispora has 3% nucleotide Note: In the polygenic analysis, eight isolates from the present study clustered together with the Diaporthe biconispora and Diaporthe pometiae ex-type strain with 100% ML and 1.00 BYPP values. Morphologically, Alpha conidia of strain ZHKUCC 22-0058 (x = 8 ± 1 × 3 ± 0.3 µm) were similar to those in the original description of D. biconispora [13]. The colony morphology of our isolates was different to those in the original description of D. biconispora, the surface of the colony is white and light yellow, the center of the back is black, and the sides are light yellow [13]. Alpha conidia of D. pometiae (x = 6.7 × 3.1 µm) were smaller than the ZHKUCC 22-0058 strain. The colony morphology of our isolate was different to those in the original description of D. pometiae. The surface of the colony is white, and the back is white to light grey [42]. Diaporthe biconispora has 3% nucleotide differences in ITS (497 nucleotides), 3% differences in tub2 (353 nucleotides), 4% differences and 4% gaps in tef1-α (355 nucleotides). Diaporthe pometiae has 5% differences and 3% gaps in ITS (541 nucleotides), 6% differences, and 1% gaps in tub2 (471 nucleotides). In addition, there is no evidence of significant genetic recombination (Fw = 0.300) in the PHI analysis. Considering both morphological and molecular data, these isolates were identified as a new species.
Culture characteristics: Colonies on PDA reach 85 mm diam. after 5 days. White cotton flocculent aerial mycelium in the center, surrounding sparse hyphae, and then become yellowish brown, margin lobate. Reverse white and then purple grey due to pigment formation.
Culture characteristics: Colonies on PDA reach 85 mm diam. after 9 days. Surface white and turning to grey with ageing, reverse white to grey with dark grey at the centre. Habitat and host: Carya illinoinensis, Citrus sp., Citrus unshiu, Fortunella margarita, Glycine max and Vitis vinifera [45].
Culture characteristics: Colonies on PDA reach 85 mm diam. after 9 days. Surface white and turning to grey with ageing, reverse white to grey with dark grey at the centre.

Discussion
Endophytic fungi live inside the healthy host tissues [48,49]. They are a common and diverse group of fungi [12,49]. Diaporthe species are ubiquitous endophytes on numerous hosts. Skaltsas et al. [50] isolated endophytic Diaporthe from Hevea brasiliensis, H. guianensis, and Micandra spp. in Cameroon, Mexico. Rhoden [51] isolated 97 strains from Trichilia elegans (Meliaceae) in Brazil, while the Diaporthe were the most frequently isolated genus. This reflects the species richness of Diaporthe species as endophytes in many hosts. In the present study, 48 endophytic Diaporthe isolates were obtained from M. officinalis in China from stems and roots. Based on the multigene phylogeny, all 48 isolates from this study were grouped in 12 distinct clades within the Diaporthe phylogenetic tree. Among them, seven new host records were identified: Diaporthe chongqingensis, D. guangxiensis, D. heliconiae, D. siamensis, D. unshiuensis, and D. xishuangbanica. The remaining five species were identified as novel species: Diaporthe longiconidialis, D. megabiguttulata, D. morindendophytica, D. morindae, and D. zhaoqingensis. Our study is the first comprehensive study on endophytic fungi associated with M. officinalis in China.
During the last few years, many Diaporthe species were introduced. For example, in 2020, 34 new species were introduced, and in 2021, 37 new species were introduced (Index Fungorum 2022, accessed on 15 June 2022). Almost all these species were introduced based on molecular phylogeny following the phylogenetic species concept [13,27,46]. However, Diaporthe species do not have enough morphologies to distinguish; thus, the phylogenetic tree with five loci became the key tool to define the species. This resulted in over 200 species which are genetically closer and define Diaporthe as cryptic species. What is missing here is that there are no discussions on the different species or different genotypes of the existing species. In the phylogenetic analysis of the present study, D. morindae, a new species from our study, was clustered together with D. hubeiensis, D. tectonae, and D. tulliensis. These four species develop a distinct clade from other Diaporthe species in the tree. Within this clade, these species develop independent lineages, yet their morphologies are overlapping. However, it is quite interesting as this clustering might be based on the geography and host in which, D. morindae and D. hubeiensis introduced from China are associated with Citrus (this study) and Vitis [12]. Diaporthe tectonae was introduced from m Tectona grandis from Thailand [52]. In contrast, D. tulliensis was introduced from the rotted stem end of the fruit of Theobroma cacao in Australia [53].
Additional analyses such as recombination tests and phylogenetic incompatibility are employed, but the species delineation enigma of Diaporthe is unresolved. Therefore, in the present study, we followed polyphasic taxonomic approaches which included phylogeny, morphology, and recombination analysis to identify the species. For existing species, species definition was based on phylogeny and morphology. Furthermore, we emphasise that taxon sampling is critical to establishing a primary tree to void the possible false introduction of species. However, it is necessary to discuss "what is the species" in Diaporthe.
Medicinal plants have a high economical and important role in human cultures worldwide. Morinda officinalis is a famous traditional medicinal plant, which has various biological effects such as anti-inflammatory activities [53], antiosteoporotic [54], antifatigue [55], anti-rheumatoid arthritis [56], and anti-oxidant [57]. It has been mentioned that endophytes live inside the host might affect the phenotype of the host in many ways, including providing resistance to pathogens [58,59], promoting seed germination or/and plant growth [60], herbivores [61], weed control [62,63], resistance to abiotic stresses, or even in litter decomposition [64]. In addition, some studies have revealed that secondary metabolites produced by endophytic fungi could be a novel source of medicinal compounds [64]. Though two new metabolites from M. officinalis endophytic Alternaria sp. A744 [63] and two polyketide compounds from the M. officinalis endophytic Trichoderma spirale A725 [65] were found. However, little is known about the endophytic fungi of M. officinalis. Therefore, further studies are necessary to explore the relationship between medicinal properties and a plant's endophytic biota.
One endophytic species might occur in different tissues in the same host [13]. In the present study, endophytes Diaporthe were successfully isolated from M. officinalis's roots and stems. Diaporthe evcalyptorum, D. longiconidialis, D. morindae and D. xishuangbanica were isolated from roots and stems of M. officinalis, while other species were isolated from only one tissue type. However, we were not able to isolate any Diaporthe species from leaves. Gond et al. [66] obtained only two endophytic Diaporthe strains from leaves of Aegle marmelos Correae (Rutaceae). Dong et al. [14] identified two endophytic Diaporthe species from Citrus grandis cv. Tomentosa while 22 strains were from fruits or twigs. Huang et al. [13] did not find any endophytes Diaporthe from Citrus leaves. These variations might be a result of differences in the tissue organization structure and the different nutrition content of each tissue type [13,67]. However, the exact underlying reasons and mechanisms for these variations are not known. At the same time, further studies are needed to compare the variations in endophytic colonization according to different seasons or different stages of maturity of the plant.
Optimization of cultural conditions is the most common and simple method to obtain different fungal diversity. Zhou et al. [68] found that the addition of vitamins in the media significantly increased the diversities of isolated fungi, and the increment reached 207% and 81% at the generic level at both 4 • C and 25 • C, respectively, conditions. In the present study, D. morindendophytica was isolated from three media (MD, CMA, and P++J medium), while two species of them including D. longiconidialis, and D. morindae were isolated from all the media tested (PDA, 2% PDA, LCA, M + J, MD and P + J medium). In addition, these four species were isolated from both roots and stems. Therefore, these species might be the dominant endophytic Diaporthe species in M. officinalis. Even though Diaporthe species can grow in different media, D. biguttulata, D. chongqingensis, D. heliconiae, D. heterophyllae, D. guangxiensis, and D. megabiguttulata were only isolated in one medium. Thus, optimizing and employing several media to isolate endophytes will enhance the diversity of endophytes obtained from a particular host.
Several endophytes such as Colletotrichum and Botryosphaeriaceae have been considered latent pathogens or opportunistic pathogens [69,70]. Diaporthe is also known to be opportunistic pathogens. Huang et al. [13] observed that some Diaporthe species associated with Citrus in China may act as opportunistic pathogens. Dong et al. [14] found Diaporthe limonicola as endophyte in C. grandis cv. Tomentosa, while it has also been reported as pathogenic on Citrus sp. In China [13] and as a dieback pathogen of lemon trees in Europe [7]. In the present study, all species isolated are known pathogenic species from a different host [8,[10][11][12][13][41][42][43]. Diaporthe unshiuensis was also reported as an endophyte of Citrus in China [13]. It was reported as a dieback disease associated with Carya illinoinensisin [71], peach [72], and grapevine [12] in China and as Citrus disease in Europe [7]. Therefore, further studies are necessary to understand the pathogenicity of these endophytic strains and the exact role of endophytic fungal taxa in the medicinal properties of these host plants.
In conclusion, in the present study, 12 endophytic species Diaporthe species were isolated from a traditional medicinal plant in China. To delineate these isolated taxa employing polyphasic approaches are necessary. Either morphology alone or phylogeny is difficult to delineate species in Diaporthe. Fungal growth media might have a significant effect on the number of different species isolated. Moreover, species occurrence is varied based on the plant tissue type as well. All previously known species obtained in this study have been reported as pathogens on various hosts in China and other countries. Therefore, future studies are necessary to understand the pathogenicity of these species on M. officinalis. The present study will be a baseline to understand the endophytic fungal diversity of Chinese Traditional Medicinal plants and thus to understand the effects of endophytes on medical properties.