Characterization of Diaporthe species on Camelliaoleifera in Hunan Province, with descriptions of two new species

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. At present, relatively little is known about the taxonomy and genetic diversity of Diaporthe on C.oleifera. Here, we conducted an extensive field survey in Hunan Province in China to identify and characterise Diaporthe species associated with tea-oil leaf spots. As a result, eleven isolates of Diaporthe were obtained from symptomatic C.oleifera leaves. These isolates were studied by applying a polyphasic approach including morphological and phylogenetic analyses of partial ITS, cal, his3, tef1 and tub2 gene regions. Two new Diaporthe species (D.camelliae-oleiferae and D.hunanensis) were proposed and described herein, and C.oleifera was revealed to be new host records of D.hubeiensis and D.sojae. This study indicated there is a potential of more undiscovered Diaporthe species from C.oleifera in China.


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 utilization 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 flavors, 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-tumor, lowering blood lipid, protecting liver and enhancing human immunity (Wang et al. 2007). Hunan Province leads the country in C. oleifera production with the average of 3.3~40,000 hm 2 to expand the cultivation area every year (Tan et al. 2018). By the end of 2017, the cultivation area of C. oleifera reached 1.4 million hm 2 , tea oil 290100 tons, and output value of 35 billion yuan (Tan et al. 2018). Thus, the development of C. oleifera industry is of great significance for the economic development of Hunan Province and the poverty alleviation of local farmers.
Diseases are a major constraint to C. oleifera production. Anthracnose disease caused by Colletotrichum species is one of the foremost diseases in southern China, which can infect leaves and fruits of C. oleifera, causing up to 40% fruit drop and up to 40% camellia seeds loss ). During July and August of 2020, 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. Diaporthe species are responsible for diseases on a wide range of plant hosts, including agricultural crops, forest trees and ornamentals, some of which can cause substantial yield losses (Santos et al. 2011;Gomes et al. 2013;Udayanga et al. 2015;Gao et al. 2016;Crous 2017, 2018;Yang et al. 2018Yang et al. , 2020Yang et al. , 2021. For instance, D. ampelina, the causal agent of Phomopsis cane and leaf spot, is known as a severe pathogen of grapevines (Hewitt and Pearson 1988), infecting all green tissues and causing yield reductions of up to 30% in temperate regions (Erincik et al. 2001). Diaporthe citri is another well-known pathogen exclusively found on Citrus spp. causing melanose, stem-end rot and gummosis in all the citrus production area except Europe (Mondal et al. 2007;Udayanga et al. 2014a; Crous 2017, 2018).
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. , 2017Guarnaccia and Crous 2017;Yang et al. 2018Yang et al. , 2020Yang et al. , 2021.
The classification of Diaporthe has been ongoing; 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 Hunan 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 Hunan 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. 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. 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).

Morphological and cultural characterization
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 done 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).

Phylogenetic analyses
The quality of the amplified nucleotide sequences was checked and combined using Se-qMan 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. , 2020Yang et al. , 2021. 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. 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 S28703 and S22703.
Culture characters. Culture incubated on PDA at 25 °C, originally flat with white fluffy aerial mycelium, becoming brown to black in the centre, with yellowish-cream conidial drops exuding from the ostioles after 20 days.
Culture characters. Culture incubated on PDA at 25 °C, originally flat with white fluffy aerial mycelium, becoming pale brown with age, with visible solitary conidiomata at maturity after 18 days.
Culture characters. Culture incubated on PNA at 25 °C, originally white, fluffy aerial mycelium, reverse yellowish pigmentation developing in centre, later becoming dark brown, with yellowish-cream drops exuding from the perithecia after 15 days.
Notes. Diaporthe sojae was first reported on pods and stems of soybean, and subsequently reported on a wide range of hosts (Dissanayake et al. 2015;Udayanga et al. 2015;Guo et al. 2020). It was also reported on some fruit trees in China, such as Vitis spp. (Dissanayake et al. 2015) and Citrus spp. (Huang et al. 2015). In the present, three isolates (HNZZ008, HNZZ010 and HNZZ022) are closely related to D. sojae in the combined phylogenetic tree (Fig. 1). The differences of nucleotides in the concatenated alignment (1/460 in ITS, 3/458 in cal, 1/320 in his3 and 3/433 in tub2) are minor. Compared with the description of the ex-type isolate FAU635, the isolate has wider asci (10-11.5 μm vs. 7-9 μm) . We therefore identify the isolates as belonging to D. sojae.

Discussion
In this study, an important oil-tea tree species, Camellia oleifera was investigated and Camellia leaf disease was found as a common disease in plantations in Hunan Province. Identification of our collections was conducted, based on isolates from symptomatic leaves of C. oleifera using five combined loci (ITS, cal, his3, tef1 and tub2), as well as morphological characters. It includes D. hubeiensis, D. sojae, as well as two new species named D. camelliae-oleiferae and D. hunanensis.
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 becoming the emerging Camellia leaf diseases in southern China (Gao et al. 2016;Yang et al. 2018;Zhou and Hou 2019). Understanding the diversity of Diaporthe species and the genetic variation within pathogen populations could help in developing sustainable disease management strategies.
As the species concept of Diaporthe has been improved a lot by using molecular data (Huang et al. 2015;Gao et al. 2016Gao et al. , 2017Guarnaccia and Crous 2017;Yang et al. 2018Yang et al. , 2020Yang et al. , 2021Manawasinghe et al. 2019;Guo et al. 2020), 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.