﻿Two new species of Diaporthe (Diaporthaceae, Diaporthales) associated with Camelliaoleifera leaf spot disease in Hainan Province, China

﻿Abstract Tea-oil tree (Camelliaoleifera Abel.) is an important edible oil woody plant with a planting area over 3,800,000 hectares in southern China. Species of Diaporthe inhabit a wide range of plant hosts as plant pathogens, endophytes and saprobes. Here, we conducted an extensive field survey in Hainan Province to identify and characterise Diaporthe species associated with tea-oil leaf spots. As a result, eight isolates of Diaporthe were obtained from symptomatic C.oleifera leaves. These isolates were studied, based on morphological and phylogenetic analyses of partial ITS, cal, his3, tef1 and tub2 gene regions. Two new Diaporthe species (D.hainanensis and D.pseudofoliicola) were proposed and described herein.


Introduction
Tea-oil tree, Camellia oleifera Abel., is a unique woody edible oil species in China, mainly distributed in the Qinling-Huaihe River area.It has a long history of cultivation and utilisation for more than 2300 years since ancient China (Zhuang 2008).Camellia oil, obtained from C. oleifera seeds, is rich in unsaturated fatty acids and unique flavours and has become a rising high-quality edible vegetable oil in China.The edible of tea-oil is also conducive to preventing cardiovascular sclerosis, anti-tumour, lowering blood lipid, protecting liver and enhancing human immunity (Wang et al. 2007).According to the Three-year Action Plan for Accelerating the Development of oil tea Industry, Hainan Province is listed as a key development area of oil tea and the total area of oil tea planting in the Province is planned to reach 16,667 hm 2 by 2025.The development of C. oleifera industry is of great significance for the economic development of Hainan Province and the poverty alleviation of local farmers.
The expanding cultivation of C. oleifera over the last several decades has attracted increasing attention from plant pathologists to infectious diseases on this crop.Therein, diseases caused by Diaporthe species have become the emerging Camellia leaf diseases in southern China (Zhou and Hou 2019;Yang et al. 2021).During July and August of 2022, new leaf spots were detected on tea-oil tree with irregular, brownish-grey lesions, often associated with leaf margins.Infected leaves cultured on medium had dark pycnidia producing ellipsoid guttulate conidia, similar to that of Diaporthe species (Yang et al. 2021).The asexual morph is characterised by ostiolate conidiomata, with cylindrical phialides producing three types (alpha, beta and gamma conidia) of hyaline, aseptate conidia (Udayanga et al. 2011;Gomes et al. 2013).
Species identification criteria in Diaporthe has mainly relied on host association, morphology and culture characteristics (Mostert et al. 2001;Santos and Phillips 2009;Udayanga et al. 2011), which resulted in the description of over 200 species.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;Udayanga et al. 2011).During the past decade, a polyphasic approach, based on multi-locus DNA data, morphological, phytopathological and phylogenetical analyses, has been employed for species boundaries in the genus Diaporthe (Huang et al. 2015;Gao et al. 2016Gao et al. , 2017;;Guarnaccia andCrous 2017, 2018;Guarnaccia et al. 2018;Yang et al. 2018Yang et al. , 2020Yang et al. , 2021;;Cao et al. 2022;Bai et al. 2023;Zhu et al. 2023).
The classification of Diaporthe has been on-going; however, little is known about species able to infect C. oleifera.Thus, the objective of the present study was to identify the prevalence of Diaporthe spp.associated with tea-oil tree leaf spot in the major plantations in Hainan Province, based on morphological and phylogenetic features.

Fungal isolation
Leaves of C. oleifera with typical symptoms of leaf spots were collected from the main tea-oil camellia production fields in Hainan Province.Small sections (3 × 3 mm) were cut from the margins of infected tissues and surface-sterilised in 75% ethanol for 30 s, then sterilised in 5% sodium hypochlorite for 1 min, followed by three rinses with sterilised water and finally dried on sterilised filter paper (Yang et al. 2021).The sections were then plated on to PDA plates and incubated at 25 °C.Fungal growth was examined daily for up to 7 d.Isolates were then transferred aseptically to fresh PDA and purified by single-spore culturing (Fan et al. 2015).All fungal isolates were placed on PDA slants and stored at 4 °C.Specimens and axenic cultures are maintained in the Central South University of Forestry and Technology (CSUFT) in Changsha, Hunan Province.

Morphological and cultural characterisation
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 after 10 d.Colony colours were rated according to Rayner (1970).Cultures were examined periodically for the development of ascomata and conidiomata.The morphological characteristics were examined by mounting fungal structures in clear lactic acid and 30 measurements at 1000× magnification were determined for each isolate using a Leica compound microscope (DM 2500) with interference contrast (DIC) optics.Descriptions, nomenclature and illustrations of taxonomic novelties are deposited in MycoBank (Crous et al. 2004a).

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 quality was measured using the NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA), following the user manual (Desjardins et al. 2009).PCR amplifications were performed in a 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 primers CYLH4F (Crous et al. 2004b) and H3-1b (Glass and Donaldson 1995) were used to amplify part of the histone H3 (his3) gene.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 set T1 (O'Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) was 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 undertaken using the same primer pairs and conditions as in Yang et al. (2018).PCR amplification products were assayed via electrophoresis in 2% agarose gels.DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyzer with a BigDye Terminater Kit v.3.1 (Invitrogen, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).
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 and Huelsenbeck 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.The nucleotide sequence data of the new taxa were deposited in GenBank (Table 1).The multilocus sequence alignments were deposited in TreeBASE (www.treebase.org)as accession S30780.

Phylogenetic analyses
The five-gene sequence dataset (ITS, cal, his3, tef1 and tub2) was analysed to infer the interspecific relationships within Diaporthe.The dataset consisted of 259 sequences including the outgroup taxon, Diaporthella corylina (CBS 121124).A total of 2909 characters including gaps (528 for ITS, 608 for cal, 563 for his3, 646 for tef1 and 564 for tub2) were included in the phylogenetic analysis.The best nucleotide substitution model for ITS, his3 and tub2 was TrN+I+G, while HKY+I+G was selected for both cal and tef1.The topologies resulting from ML and BI analyses of the concatenated dataset were congruent (Fig. 1).According to the phylogenetic tree, D. hainanensis and D. pseudofoliicola are new to science, based on the distinct and well-supported molecular phylogenetic placement with their closest described relatives.Phylogenetical- Etymology.In reference to the Hainan Province, from where the fungus was first collected.
Culture characters.Culture incubated on PDA at 25 °C, originally flat with white fluffy aerial mycelium, becoming pale brown due to pigment formation, with yellowish-cream conidial drops exuding from the ostioles after 20 days.
As the species concept of Diaporthe has been greatly improved by using molecular data (Huang et al. 2015;Gao et al. 2016Gao et al. , 2017;;Guarnaccia and Crous 2017;Guarnaccia et al. 2018;Yang et al. 2018Yang et al. , 2020Yang et al. , 2021;;Manawasinghe et al. 2019;Guo et al. 2020;Jiang et al. 2021;Cao et al. 2022;Bai et al. 2023;Zhu et al. 2023), many new species have been discovered and reported in recent years.In this study, the Diaporthe isolates from C. oleifera were identified, based on sequence analysis and morphological characteristics.Future studies should focus on pathogenicity, epidemiology and fungicide sensitivity of the important plant fungal pathogen to develop effective management of C. oleifera disease and on the pathogenic molecular mechanism.

Table 1 .
Isolates and GenBank accession numbers used in the phylogenetic analyses of Diaporthe.