New species and records of Diaporthe from Jiangxi Province, China

Abstract Diaporthe species have often been reported as important plant pathogens, saprobes and endophytes on a wide range of plant hosts. Although several Diaporthe species have been recorded, little is known about species able to infect forest trees in Jiangxi Province. Hence, extensive surveys were recently conducted in Jiangxi Province, China. A total of 24 isolates were identified and analysed using comparisons of DNA sequence data for the nuclear ribosomal internal transcribed spacer (ITS), calmodulin (cal), histone H3 (his3), partial translation elongation factor-1α (tef1) and β-tubulin (tub2) gene regions, as well as their morphological features. Results revealed five novel taxa, D. bauhiniae, D. ganzhouensis, D. schimae, D. verniciicola, D. xunwuensis spp. nov. and three known species, D. apiculatum, D. citri and D. multigutullata.

Species identification criteria in Diaporthe were originally based on host association, morphology and culture characteristics (Mostert et al. 2001;Santos and Phillips 2009;Udayanga et al. 2011), which led to the description of over 200 species (Hyde et al. 2020). Some species of Diaporthe were reported to colonise a single host plant, while other species were found to be associated with different host plants (Santos and Phillips 2009;Diogo et al. 2010;Santos et al. 2011;Gomes et al. 2013). In addition, considerable variability of the phenotypic characters was found to be present within a species (Rehner and Uecker 1994;Mostert et al. 2001;Santos et al. 2010;Udayanga et al. 2011). During the past decade, a polyphasic approach, based on multi-locus DNA data, morphology and ecology, has been employed for species boundaries in the genus Diaporthe (Crous et al. 2012;Huang et al. 2015;Gao et al. 2016Gao et al. , 2017Guarnaccia and Crous 2017;Guarnaccia et al. 2018;Yang et al. 2018Yang et al. , 2020. The classification of Diaporthe has been progressing and the basis for the species identification is a combination of morphological, cultural, phytopathological and phylogenetical analyses (Gomes et al. 2013;Udayanga et al. 2014Udayanga et al. , 2015Fan et al. 2015;Huang et al. 2015;Gao et al. 2016Gao et al. , 2017Guarnaccia and Crous 2017;Guarnaccia et al. 2018;Yang et al. 2018Yang et al. , 2020Manawasinghe et al. 2019).
In Jiangxi Province, China, some forest trees were observed to be infected with fungal pathogens that cause dieback and leaf spots. Cankered branches and leaves with typical Diaporthe fruiting bodies were also found in the area. However, we found that only limited research had been undertaken regarding the fungal pathogens isolated from forest trees in Jiangxi Province. Hence, the present study was conducted to identify Diaporthe species that cause dieback and leaf spots disease in the forest trees in Jiangxi Province through morphological and multi-locus phylogenetic analyses, based on modern taxonomic concepts.

Isolates
Fresh specimens of Diaporthe were isolated from the collected branches and leaves of six host plants during the collection trips conducted in Jiangxi Province (Table 1). A total of 24 isolates were established by removing a mucoid conidia mass from conidiomata, spreading the suspension on the surface of 1.8% potato dextrose agar (PDA) and incubating at 25 °C for up to 24 h. A single germinating conidium was plated on to fresh PDA plates. Specimens were deposited at the Museum of the Beijing Forestry University (BJFC). Axenic cultures were maintained at the China Forestry Culture Collection Centre (CFCC).

Morphological observation
Agar plugs (6 mm diam.) were taken from the edge of actively-growing cultures on PDA and transferred on to the centre of 9 cm diam. Petri dishes containing 2% tap water agar, supplemented with sterile pine needles (PNA; Smith et al. 1996) and potato dextrose agar (PDA) and incubated at 25 °C under a 12 h near-ultraviolet light/12 h dark cycle to induce sporulation, as described in recent studies (Gomes et al. 2013;Lombard et al. 2014). Colony characters and pigment production on PNA and PDA were noted in the 10-day culture. Colony features were rated according to the colour charts of Rayner (1970). Cultures were examined periodically for the development of conidiomata. The microscopic examination was based on the morphological features of conidiomata obtained from the fungal growth, mounted in clear lactic acid. At least 30 conidia were measured to calculate the mean size/length. Micro-morphological observations were done at 1000× magnification using a Leica compound microscope (DM 2500) with interference contrast (DIC) optics. Descriptions, nomenclature and illustrations of taxonomic novelties were deposited at MycoBank (www.MycoBank.org).

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from colonies grown on cellophane-covered PDA, using a CTAB (cetyltrimethylammonium bromide) method (Doyle and Doyle 1990). DNA was estimated by electrophoresis in 1% agarose gel and the yield was measured using the NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA), following the user manual (Desjardins et al. 2009). The PCR amplifications were performed in the DNA Engine Peltier Thermal Cycler (PTC-200; Bio-Rad Laboratories, Hercules, CA, USA). The primer set ITS1/ITS4 (White et al. 1990) was used to amplify the ITS region. The primer pair CAL228F/CAL737R (Carbone and Kohn 1999) was used to amplify the calmodulin gene (cal) and the primer pair CYLH4F (Crous et al. 2004) and H3-1b (Glass and Donaldson 1995) were used to amplify part of the histone H3 (his3) gene. Newly sequenced material is indicated in bold type. NA, not applicable.
The primer pair EF1-728F/EF1-986R (Carbone and Kohn 1999) was used to amplify a partial fragment of the translation elongation factor 1-α gene (tef1). The primer sets T1 (O'Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) were used to amplify the beta-tubulin gene (tub2); the additional combination of Bt2a/ Bt2b (Glass and Donaldson 1995) was used in case of amplification failure of the T1/ Bt2b primer pair. The PCR amplifications of the genomic DNA with the phylogenetic markers were done using the same primer pairs and conditions as in Yang et al. (2018). The PCR products were assayed via electrophoresis in 2% agarose gels, while the DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyser with a Big-Dye Terminater Kit v.3.1 (Inv-itrogen, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Phylogenetic analyses
The quality of the amplified nucleotide sequences was checked and combined using SeqMan v.7.1.0 and reference sequences were retrieved from the National Center for Biotechnology Information (NCBI), based on recent publications on the genus Diaporthe Yang et al. 2018Yang et al. , 2020. Sequences were aligned using MAFFT v. 6 (Katoh and Toh 2010) and corrected manually using Bioedit 7.0.9.0 (Hall 1999). The best-fit nucleotide substitution models for each gene were selected using jModelTest v. 2.1.7 (Darriba et al. 2012) under the Akaike Information Criterion. The phylogenetic analyses of the combined gene regions were performed using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. ML was conducted using PhyML v. 3.0 (Guindon et al. 2010), with 1000 bootstrap replicates while BI was performed using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.0 (Ronquist et al. 2003). Two MCMC chains, started from random trees for 1,000,000 generations and trees, were sampled every 100 th generation, resulting in a total of 10,000 trees. The first 25% of trees were discarded as burn-in of each analysis. Branches with significant Bayesian Posterior Probabilities (BPP) were estimated in the remaining 7500 trees. Phylogenetic trees were viewed with FigTree v.1.3.1 (Rambaut and Drummond 2010) and processed by Adobe Illustrator CS5. Sequence alignment and phylogenetic trees were deposited in TreeBASE (submission ID: S25213). The nucleotide sequence data of the new taxa were deposited in GenBank (Table 1).
Culture characters. Colony originally flat with white fluffy aerial mycelium, becoming yellowish to pale green mycelium with age, marginal area irregular, conidiomata absent.    Notes. Diaporthe apiculatum was originally described as an endophyte from healthy leaves of Camellia sinensis in Jiangxi Province, China (Gao et al. 2015). In the present study, three isolates (CFCC 53068, CFCC 53069 and CFCC 53070) from symptomatic branches of Rhus chinensis were found congruent with D. apiculatum, based on DNA sequence and morphological data (Fig. 1). The clade was, therefore, confirmed to be D. apiculatum and was found to be both an endophyte and a pathogen. Diagnosis. Distinguished from the phylogenetically closely-related species D. psoraleae-pinnatae in alpha and beta conidia.
Culture characters. Colony at first white, becoming wine-red in the centre with age. Aerial mycelium white, dense, fluffy, conidiomata absent.
Culture characters. Colony originally flat with white fluffy aerial mycelium, becoming greyish mycelium with age, with yellowish-cream conidial drops exuding from the ostioles. Notes. Diaporthe citri is a widely distributed species in citrus-growing regions. In the present study, four isolates (CFCC 53079, CFCC 53080, CFCC 53081 and CFCC 53082) from symptomatic leaves of Citrus sinensis were congruent with D. citri, based on DNA sequence and morphological data (Fig. 1). The clade was, therefore, confirmed to be D. citri. Diagnosis. Distinguished from the phylogenetically closely-related species D. vawdreyi in having longer conidiophores and wider alpha conidia.
Notes. Diaporthe multiguttulata was originally described as an endophyte from a healthy branch of Citrus grandis in Fujian Province, China (Huang et al. 2015). In the present study, three isolates (CFCC 53095, CFCC 53096 and CFCC 53097) from symptomatic branches of Citrus maxima were congruent with D. multigutullata, based on DNA sequence data and confirmed from the morphological analysis (Fig. 1). The clade, therefore, was verified as D. multigutullata which could exist both as an endophyte and a pathogen. Diagnosis. Distinguished from the phylogenetically closely-related species D. sennae in having larger alpha conidia and longer beta conidia.
Culture characters. Colony entirely white, with fluffy aerial mycelium, concentric zonation, margin fimbricate, reverse slightly yellowish. Notes. Diaporthe schimae occurs in an independent clade (Fig. 1) and was revealed to be phylogenetically distinct from D. sennae. Diaporhe schimae can be distinguished with D. sennae by 41 nucleotides in concatenated alignment, in which three were distinct in the ITS region, 20 in the tef1-α region and 18 in the tub2 region. Diaporthe schimae differs morphologically from D. sennae in having larger alpha conidia and longer beta conidia (8-8.5 × 2.5-3 vs. 5.5-6.3 × 1.5-1.7 μm in alpha conidia; 27.5-38.5 vs. 18.4-20 μm in beta conidia) (Yang et al. 2017a). Diagnosis. Distinguished from the phylogenetically closely-related species D. rostrata in having smaller alpha conidia; and from D. juglandicola in having wider alpha conidia.

Discussion
The current study described eight Diaporthe species from 24 strains, based on a large set of freshly-collected specimens. It includes five new species and three known species, which were sampled from six host genera distributed in Jiangxi Province of China (Table 1). In this study, 142 reference sequences (including outgroup) were selected, based on BLAST searches of NCBIs GenBank nucleotide database and included in the phylogenetic analyses (Table 1). Phylogenetic analyses, based on five combined loci (ITS, cal, his3, tef1 and tub2), as well as morphological characters, revealed the diversity of Diaporthe species in Jiangxi Province, mainly focusing on diebacks from major ecological or economic forest trees. The identification and characterisation of novel taxa and new host records indicate the high potential of Diaporthe to evolve rapidly. In the present study, five species were first reported in China as pathogens. Amongst these species, D. bauhiniae was characterised by having longer alpha conidia (9-13 × 2-2.5 μm). Diaporthe ganzhouensis and D. xunwuensis were isolated from unknown dead wood, but D. ganzhouensis can be distinguish from D. xunwuenesis in having beta conidia and was supported by analysis of the sequence data. Diaporthe schimae was identified as the most widespread species from isolates collected in Jiangxi Province. Diaporthe verniciicola have conidiomata with single necks erumpent through the host bark. Furthermore, two new host records were described, D. apiculatum from Rhus chinensis and D. multiguttulata from Citrus maxima.
Recent plant pathological studies have revealed that several Diaporthe species cause disease, particularly to important plant hosts on a wide range of economically-significant agricultural crops, such as blueberries, citrus, grapes, oaks, sunflowers, soybeans, tea plants, tropical fruits, vegetables and various trees (van Rensburg et al. 2006;Santos and Phillips 2009;Santos et al. 2011;Thompson et al. 2011;Grasso et al. 2012;Lombard et al. 2014;Huang et al. 2015;Udayanga et al. 2015;Gao et al. 2016;Guarnaccia et al. 2018;Yang et al. 2020). For example, research conducted by Huang et al. (2015) revealed seven endophytic Diaporthe species on Citrus; Gao et al. (2016) demonstrated that Diaporthe isolates associated with Camellia spp. could be assigned to seven species and two species complexes; Guarnaccia et al. (2018) explored the occurrence, diversity and pathogenicity of Diaporthe species associated with Vitis vinifera and revealed four new Diaporthe species; Yang et al. (2018) provided the first molecular phylogenetic framework of Diaporthe diversity associated with dieback diseases in China. Following the adoption of DNA sequence-based methods, Diaporthe taxonomy is actively changing, with numerous species being described each year.
The present study is the first evaluation of Diaporthe species, associated with dieback diseases in Jiangxi Province using the combined morphology and molecular data and provided useful information for evaluating the pathogenicity of various species. Multiple strains from different locations should also be subjected to multi-locus phylogenetic analysis to determine intraspecific variation and redefine species boundaries. The descriptions and molecular data of Diaporthe species, provided in this study, represent a resource for plant pathologists, plant quarantine officials and taxonomists for identification of Diaporthe.