Article An Emerging Pathogen from Rotted Chestnut in China: Gnomoniopsis daii sp. nov. Ning Jiang and Chengming Tian * The Key Laboratory for Silviculture and Conservation of Ministry of Education, Beijing Forestry University, Beijing 100083, China; [email protected] * Correspondence: [email protected] Received: 23 October 2019; Accepted: 11 November 2019; Published: 13 November 2019 Abstract: Nut quality is fundamental to the economic viability of the Chinese sweet chestnut industry, but fruit rot disease significantly reduces this quality. In this study, we investigated chestnut rot in Anhui and Hubei provinces in China. Typical brown rot symptoms were observed, affecting nuts from different plantations. Isolates were obtained from symptomatic tissues of rotted fruits that were identified based on morphological comparison and phylogenetic analyses of partial internal transcribed spacer (ITS), and tef1 and tub2 gene sequences. The inoculation results showed that the tested fungal species is pathogenic to chestnut fruits. Hence, a new and severe pathogen that causes Chinese sweet chestnut brown rot, Gnomoniopsis daii sp. nov., is introduced herein. Keywords: Castanea mollissima; fruit disease; plant pathology; taxonomy 1. Introduction China has the largest chestnut industry in the world, producing more than 2 106 tons of chestnuts × annually since 2013 [1]. The Chinese sweet chestnut (Castanea mollissima Bl.) is widely cultivated in most provinces in China, providing gluten-free, low fat, and cholesterol-free crop nuts for human consumption [2]. Chestnut orchards and stands are also important to the economy as sources of timber [3]. Traditionally, several pathogens were considered the causal agents of chestnut rot in China, including Alternaria Nees, Botryosphaeria Ces. & De Not., Colletotrichum Corda, Diaporthe Nitschke, Fusarium Link, and Penicillium Link species [4–7]. However, no detailed studies on these pathogens have been conducted in China in the past decade. European sweet chestnut (Castanea sativa Mill.), known as one of the four major chestnut species in the world, has been well studied in nut rot by several phytopathologists and taxonomists [8–15]. Several important fungal species, Cryphonectria parasitica M.E. Barr, Gnomoniopsis smithogilvyi L.A. Shuttlew., E.C.Y. Liew & D.I. Guest (syn. G. castaneae Tamietti), Phytophthora cinnamomi Rands, and Sirococcus castanea J.B. Mey., Senn-Irlet & T.N. Sieber, have been reported on Castanea sativa from Australia and Europe [16–20]. The genus Gnomoniopsis Berl. (Gnomoniaceae G. Winter, Diaporthales Nannf.) was first described as a subgenus within Gnomonia Ces. & De Not. for species having ascospores that develop additional septa [21]. However, the development of additional septa was thought to be an occasional occurrence; Gnomoniopsis was subsequently proposed as a synonym of Gnomonia [22]. Sogonov et al. reevaluated concepts of the leaf-inhabiting genera in Gnomoniaceae based on the DNA sequence data of these genera, and restricted the genus Gnomoniopsis to G. chamaemori Berl. (type), G. comari Sogonov, G. fructicola Sogonov, G. macounii Sogonov, G. paraclavulata Sogonov, G. racemula Sogonov, and G. toementillae Sogonov [21]. Subsequently, nine additional species were added to this genus [23]. Gnomoniopsis castaneae and G. smithogilvyi were described independently from Europe and Australia, but Shuttleworth Forests 2019, 10, 1016; doi:10.3390/f10111016 www.mdpi.com/journal/forests Forests 2019, 10, 1016 2 of 10 Forests 2019, 10, x FOR PEER REVIEW 2 of 10 et al. proved that both names refer to a single species based on a comparative morphological analysis and five-markerbut Shuttleworth phylogenetic et al. proved analysis that [both18]. names refer to a single species based on a comparative Duringmorphological the surveys analysis of and chestnut five-marker rot conducted phylogenetic in analysis Anhui and[18]. Hubei provinces in China, typical brown rotDuring symptoms the surveys were of observed chestnut (Figurerot conducted1). Our in Anhui aim in and this Hubei study provinces was to identifyin China, pathogenstypical brown rot symptoms were observed (Figure 1). Our aim in this study was to identify pathogens associated with chestnut brown rot in China. We conducted pathogenicity tests on healthy nuts to associated with chestnut brown rot in China. We conducted pathogenicity tests on healthy nuts to assessassess their their pathogenicity. pathogenicity. FigureFigure 1. Symptoms 1. Symptoms of of chestnut chestnut brown brown rot: rot: (aa,b,b)) diseased diseased nuts nuts and and (c) ( discoloredc) discolored kernels. kernels. 2. Materials2. Materials and and Methods Methods 2.1. Sample2.1. Sample Collection Collection and and Isolation Isolation AnhuiAnhui and Hubeiand Hubei provinces provinces are twoare two important important chestnut chestnut production production bases bases in in China. China. Samples Samples were randomlywere randomly collected incollected local storehouses in local storehouses from di fromfferent different chestnut chestnut plantations plantations after after harvest, harvest, then then packed packed in paper bags, and posted to the laboratory for further study. Rotted chestnuts were surface- in paper bags, and posted to the laboratory for further study. Rotted chestnuts were surface-sterilized sterilized for 1 min in 75% ethanol, 3 min in 1.25% sodium hypochlorite, and 1 min in 75% ethanol, for 1 minthen inrinsed 75% for ethanol, 2 min in 3 sterile min in water 1.25% and sodium blottedhypochlorite, on dry sterile filter and paper. 1 min Infected in 75% ethanol,nut tissues then were rinsed for 2 mincut into in sterile small pieces water (0.2 and cm blotted × 0.2 cm) on dryusing sterile a ster filterile scalpel paper. and Infected transferred nut onto tissues the weresurface cut of into malt small piecesextract (0.2 cm agar0.2 (MEA; cm) using30 g malt a sterile extract, scalpel 5 g peptone, and transferred 15 g agar/L; onto Aobox the surface Company of malt Limited, extract Beijing, agar (MEA; × 30 g maltChina). extract, After 5inoculation, g peptone, agar 15 g plates agar/L; were Aobox left at Company 25 °C in the Limited, dark for Beijing, 2 days. China). Then, single After inoculation,hyphal agar platesstrands were were left transferred at 25 ◦C into thefresh dark medium for 2 days.plates Then,under singlea dissecting hyphal stereomicroscope strands were transferred with a sterile to fresh mediumneedle. plates Specimen under aof dissectingthe new species stereomicroscope was deposited with in the a sterile Museum needle. of Beijing Specimen Forestry of theUniversity, new species was depositedBeijing, China in the (BJFC). Museum The ex-type of Beijing culture Forestry was maintained University, in Beijing, the China China Forestry (BJFC). Culture The ex-typeCollection culture was maintainedCenter, Beijing, in the China China (CFCC). Forestry Culture Collection Center, Beijing, China (CFCC). 2.2. DNA2.2. DNA Extraction Extraction and and Phylogenetic Phylogenetic Analysis Analysis Genomic DNA was extracted from 15-day-old mycelium grown on MEA using the CTAB Genomic DNA was extracted from 15-day-old mycelium grown on MEA using the CTAB (cetyltrimethylammonium bromide) method [24]. DNA sequences were generated for the internal (cetyltrimethylammoniumtranscribed spacer (ITS) regions bromide) including method the [5.8S24]. gene DNA of the sequences ribosomal were RNA generated operon ampli forfi theed with internal transcribedprimers spacerITS1/ITS4 (ITS) [25], regions the translation including elongation the 5.8S genefactor of1a the (tef1 ribosomal) amplified RNA with operonprimers amplifiedEF1- with728F/EF1-1567R primers ITS1/ ITS4[26], and [25 ],the the b-tubulin translation gene 2 elongation(tub2) amplifi factored with 1a primers (tef1) T1 amplified/Bt2b [27]. with The PCR primers EF1-728Fconditions/EF1-1567R were: [initial26], and denaturation the b-tubulin step geneof 5 min 2 (tub2 at 94) amplified°C, followed with by primers35 cycles T1of /30Bt2b s at [ 2794]. °C, The 50 PCR conditionss at 48 were:°C (ITS) initial or 54denaturation °C (tef1) or 52 step°C (tub2 of 5), minand 1 at min 94 ◦atC, 72 followed °C, and a byfinal 35 elongation cycles of 30step s atof 947 min◦C, 50 s at 72 °C. The PCR amplification products were scored visually by electrophoresis in 2% agarose gel. at 48 ◦C (ITS) or 54 ◦C(tef1) or 52 ◦C(tub2), and 1 min at 72 ◦C, and a final elongation step of 7 min The DNA sequencing was performed using an ABI Prism 3730xl DNA Analyzer (ABI, Foster City, at 72 ◦C. The PCR amplification products were scored visually by electrophoresis in 2% agarose gel. CA, USA) with Big-Dye Terminator kit v.3.1 (Invitrogen, Beijing, China) at Shanghai Invitrogen The DNA sequencing was performed using an ABI Prism 3730xl DNA Analyzer (ABI, Foster City, CA, Biological Technology Co. Ltd. (Beijing, China). USA) with Big-Dye Terminator kit v.3.1 (Invitrogen, Beijing, China) at Shanghai Invitrogen Biological Sequences of the three individual loci (ITS, tef1 and tub2) were aligned and edited manually Technologyusing MEGA6 Co. Ltd. (Table (Beijing, 1). Maximum China). likelihood (ML) analysis was used for phylogenetic inferences of Sequencesthe concatenated of the alignments. three individual ML analysis loci (ITS, wastef1 implementedand tub2) on were the alignedCIPRES andScience edited Gateway manually portal using MEGA6using (Table RAxML-HPC1). Maximum BlackBox likelihood v. 8.2.10 based (ML) on analysis single ITS was and used combined for phylogenetic sequences