Phytopathologia Mediterranea Firenze University Press www.fupress.com/pm The international journal of the Mediterranean Phytopathological Union Research Papers Citation: R.S. Brahmanage, M. Liu, D.N. Wanasinghe, M.C. Dayarathne, L. Mei, R. Jeewon, X. Li, K.D. Hyde (2020) Heterosporicola beijingense sp. nov. (Leptosphaeriaceae, Pleosporales) associated with Chenopodium quinoa leaf spots. Phytopathologia Mediterranea 59(2): 219-227. DOI: 10.14601/ Phyto-11113 Heterosporicola beijingense sp. nov. (Leptosphaeriaceae, Pleosporales) associated with Chenopodium quinoa leaf spots Rashika S. BRAHMANAGE1,2,3,#, Mei LIU1,#, Dhanushka N. WANASINGHE4, Monika C. DAYARATHNE2,7, Li MEI5, Rajesh JEEWON6, Xinghong LI1,*, Kevin D. HYDE2,4,8,* 1 Accepted: April 17, 2020 Published: August 31, 2020 Copyright: © 2020 R.S. Brahmanage, M. Liu, D.N. Wanasinghe, M.C. Dayarathne, L. Mei, R. Jeewon, X. Li, K.D. Hyde. This is an open access, peerreviewed article published by Firenze University Press (http://www.fupress. com/pm) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Editor: Vladimiro Guarnaccia, DiSAFA - University of Torino, Italy. Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, People’s Republic of China 2 Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, 57100, Thailand 3 School of Science, Mae Fah Luang University, Chiang Rai, 57100, Thailand 4 CAS key Laboratory for Plant Biodiversity and Biogeography of East Asia (KLPB), Kunming Institute of Botany, Chinese Academy of Science, Kunming 650201, Yunnan, China 5 Beijing Agricultural technology extension station, Beijing 100029, China 6 Department of Health Sciences, Faculty of Science, University of Mauritius, Reduit, Mauritius 7 Department of Plant Pathology, Agriculture College, Guizhou University, Guiyang, Guizhou Province, 550025, China 8 Innovative Institute of Plant Health, Zhongkai University of Agriculture and Engineering, Haizhu District, Guangzhou 510225, P.R. China *Corresponding authors: lixinghong1962@163.com, kdhyde3@gmail.com # Both authors contributed equally Summary. A coelomycetous fungus with hyaline, aseptate, oblong to ellipsoidal conidia was isolated from living Chenopodium quinoa leaves with leaf spots, in Beijing, China. Maximum likelihood and Bayesian analyses of a combined LSU, SSU, ITS and TEF sequence dataset confirmed its placement in Heterosporicola in Leptosphaeriaceae. The new taxon resembles other Heterosporicola species, but is phylogenetically distinct, and is introduced as a new species. Heterosporicola beijingense sp. nov. is compared with other Heterosporicola species, and comprehensive descriptions and micrographs are provided. Key words. DNA analyses, morpho-molecular taxonomy, pathogens. INTRODUCTION Quinoa (Chenopodium quinoa) is a dicotyledonous pseudocereal (VegaGalvez et al., 2010) which maintains high productivity in low fertility soils and under conditions of water shortage and high salinity (Tapia et al., 1997). Quinoa seeds are rich in nutrients, with high protein content including all Phytopathologia Mediterranea 59(2): 219-227, 2020 ISSN 0031-9465 (print) | ISSN 1593-2095 (online) | DOI: 10.14601/Phyto-11113 220 Rashika S. Brahmanage et alii nine essential amino acids with high concentrations of histidine, lysine and methionine. The seeds also lack gluten and contain high amounts of several minerals (including calcium, magnesium and iron), and healthpromoting compounds such as flavonoids (Dini et al., 1992; Wright et al., 2002). Quinoa was first introduced to Shanxi Province (China) in 2011 and transferred rapidly to other provinces, including Gansu, Jilin, Sichuan and Qinghai (Li et al., 2017). With the increase in quinoa crops, the number of pests and diseases of quinoa have also increased (Li et al., 2017). Among them, fungal diseases are responsible for significant production losses (Lee et al., 2019). Brown stalk rot, downy mildew, gray mould, leaf spot and root rot are the major fungal diseases that affect quinoa production in China and worldwide (Valencia-Chamorro, 2003; Testen et al., 2013; Li et al., 2017). Vilca (1972) described Ascochyta hyalospora from leaf spots of quinoa (Boerema et al., 1977). The first symptoms are light spots of indefinite area on the quinoa leaves (Boerema et al., 1977; Alandia et al., 1979; Li et al., 2017), and with time pycnidia can be observed, and the leaves become dry and fall (Li et al., 2017). Leaf spots have become a rapidly increasing fungal disease in the cultivation of quinoa in China (Wang et al., 2014). Heterosporicola (as Heterospora) was initially considered as a section of Phoma by Boerema (1977). De Gruyter et al. (2013) raised Heterospora to generic rank to accommodate two species (H. chenopodii and H. dimorphospora) of Phoma sect. Heterospora that clustered in Leptosphaeriaceae. The current name Heterosporicola was proposed to accommodate Heterospora by Wijayawardene et al. (2018) as the remaining species of Phoma sect. Heterospora clustered in the family Didymellaceae (Aveskamp et al., 2010). The sexual morph of Heterospora is presently undetermined (De Gruyter et al., 2013 Ariyawansa et al., 2015; Wijayawardene et al., 2017; Hyde et al., 2018). The present study introduces a novel potential plant pathogenic Heterosporicola species, associated with quinoa leaf spots occurring in Mentougou and Yanging districts (Beijing) in China. A multigene-based phylogram is also presented to infer phylogenetic relationships of this species. MATERIALS AND METHODS Sample collections, examination and isolation Symptomatic quinoa leaves were collected from six fields in Beijing Mentougou and Yanging districts in August and July 2018. Leaf samples placed in Zip-lock plastic bags were brought to the laboratory and incubated at room temperature (25°C). An attempt was made to obtain axenic cultures following the single spore isolation method (Chomnunti et al., 2014) onto potato dextrose agar (PDA) and malt extract agar (MEA). A tissue isolation method was also used in attempts to isolate fungi from diseased leaves. Small leaf pieces (0.5 × 0.5 cm) were surface sterilized (Schulz et al., 1993) to eliminate epiphytic fungi, and were then incubated on PDA or MEA. Three replicates from each sample were maintained. Digital images of fruiting structures were captured with a Canon 450D digital camera fitted to a Nikon ECLIPSE 80i compound microscope. Squash mount preparations were made from conidiomata near symptomatic leaf areas. Measurements of fungus structures were made using the Tarosoft (R) Image Frame Work program, and the images used for figures were processed with Adobe Photoshop CS3 Extended v. 10.0 (Adobe®). Herbarium specimens of the new species were deposited in the Mae Fah Luang University Herbarium (MFLU) and the Beijing Academy of Agricultural and Forestry Sciences (JZB), China. Faces of fungi and Index Fungorum numbers were registered according to Jayasiri et al. (2015) and Index Fungorum (2020). The new species was established following the guidelines of Jeewon and Hyde (2016). DNA extraction, PCR amplifications and sequencing A DNA extraction kit (E.Z.N.A.® Forensic DNA kit, D3591-01, Omega Bio-Tek) was used to extract DNA from fresh fruiting bodies from fungal isolates, following the manufacturer’s instructions (as conidia did not germinate on any of the media used). Extracted DNA was used for PCR reactions with the following ingredients: each amplification reaction contained 0.125 μL of 5 units μL-1 Ex-Taq DNA polymerase (TaKaRa), 2.5 μL of 10 × PCR buffer, 2 μL of 2 mM MgCl2, 2.5 μL of 2 mM dNTPs, 1 μL of 0.2–1.0 μM primer, <500 ng DNA template, and was adjusted with double-distilled water to a total volume of 25 μL. PCR amplification and sequencing was performed of the ITS gene region using the primer pair ITS5 and ITS4 (Carbone and Kohn, 1999). The LSU, SSU and TEF gene regions were amplified and sequenced, respectively, using the primer pairs LR0R/ LR5 (Vilgalys and Hester, 1990), NS1/NS4 (White et al., 1990) and EF1-983F/EF1-2218R (Rehner and Buckley, 2005). The amplification profiles for all four gene regions were as follows: an initial denaturing step for 2 min at 94°C, followed by 35 amplification cycles of denaturation at 94°C for 60 s, annealing at 55°C for 60 s and exten- 221 Heterosporicola beijingense sp. nov. sion at 72°C for 90 s, and a final extension step of 72°C for 10 min (Brahmanage et al., 2019). Purification and sequencing of PCR products were carried out using the above-mentioned PCR primers at Bio-med Biotech Company (Beijing, China). Sequences were checked for ambiguity, assembled and deposited in GenBank. Phylogenetic analysis Sequence data were compared by BLAST searches in the GenBank database at the National Centre for Biotechnology Information (NCBI) (https://www.ncbi.nlm. nih.gov/nucleotide/). Initial BLAST similarity indices showed that the isolates were very similar to Heterosporicola. Heterosporicola strains were compared with other related sequences of Leptosphaeriaceae, following procedures of Dayarathne et al. (2015) and Tennakoon et al. (2017) (Table 1). Sequences were aligned with MAFFT v. 7.0 (Kuraku et al., 2013), combined using Bioedit 7 (Hall, 1999) and refined manually. Phylogenetic trees were generated using maximum likelihood (ML) and Bayesian inference (BI). The ML trees were generated with RAxML-HPC2 on XSEDE (v. 8.2.8) (Stamatakis, 2014) in the CIPRES Science Gateway platform (Miller et al., 2010), using the GTR+I+G model of evolution. Bayesian analyses were performed for both individual and combined datasets using MrBayes v. 3.0b4 (Ronquist and Huelsenbeck, 2003). Nucleotide substitution models were determined with MrModeltest v. 2.2 (Nylander, 2004). A dirichlet state frequency was predicted for all four data partitions and GTR+I+G was the best model. The heating parameter was set to 0.2 and trees were saved every 1,000 generations (Ronquist et al., 2012). Posterior probabilities (PP) (Rannala et al., 1998; Zhaxybayeva and Gogarten, 2002) were defined by the Bayesian Markov Chain Monte Carlo (BMCMC) sampling method in MrBayes v. 3.0b4 (Huelsenbeck and Ronquist, 2001). The resulting trees were viewed with FigTree v.1.4.0 (Rambaut, 2009) and the final layout was done using Microsoft PowerPoint (2016). RESULTS AND DISCUSSION Symptoms Numerous yellowish brown to reddish brown circular spots with lighter surrounding tissues were observed on affected quinoa leaves at the initial stage of disease development. Whirls of black conidiomata and shot holes on the leaves also were observed at later stages (Figure 1). Phylogenetic analysis The combined LSU, SSU, ITS, and TEF sequence dataset belonging to Leptosphaeriaceae, with Phoma herbarum (CBS 615.75) as the outgroup taxon, comprised 36 taxa with 2,436 nucleotide characters. RAxML analysis of the combined dataset yielded a best tree (Figure 2) with a final ML optimization likelihood value of -8815.833844. The matrix had 438 distinct alignment patterns, with 20.53% undetermined characters or gaps. Estimated base frequencies were; A = 0.244796, C = 0.219925, G = 0.271915, T = 0.263364. Substitution rates were AC = 1.726279, AG = 2.859320, AT = 2.066497, CG = 0.464869, CT = 6.764103, and GT = 1.000000. The gamma distribution shape parameter α = 0.171569. Phylogenetic trees obtained from ML and BI were similar in topology. Phylogenetic results indicated that isolates of Heterosporicola beijingense clustered together in a subclade with strong support (100% ML, 1.00 PP), and closely related to H. chenopodii and H. dimorphospora (Figure 2). Taxonomy Heterosporicola beijingense Brahmanage & K.D. Hyde, sp. nov. Figure 2 Index Fungorum: IF 557214, Facesoffungi number: FoF 07325 Etymology: Name refers to the geographical region Beijing, China, where the species was first found. Holotype: JZB3400001 Saprobic or pathogenic on leaves of quinoa (Chenopodium quinoa). Leaf spots on quinoa leaves irregular, necrotic, with conidiomata arranged in several whorls. Sexual morph: undetermined. Asexual morph: Coelomycetous. Conidiomata 200–600 μm wide, pycnidia immersed to semi-immersed, globose to subglobose, black and each with an inconspicuous ostiole. Conidiomatal wall 15–60 μm wide, composed of 3–5 layers of cells of textura angularis, pale yellowish brown. Conidiogenous cells 4–8 × 4–6 μm (x̄ = 6 × 5 μm, n = 20), phialidic, subglobose to short conical. Microconidia 3.8–4.4 × 1.4–2.1 μm (x̄ = 4.2 × 1.8 μm, n = 30) hyaline, aseptate, oblong to ellipsoidal with two to many guttules. Macroconidia not observed. Material examined: CHINA, Beijing, Mentougou, on living leaves of Chenopodium quinoa (Amaranthaceae), July 2018, Rashika S. Brahmanage, LC41 (JZB3400001, holotype), ibid., LC43 (JZB3400002), ibid., Yanging district, on living leaves of Chenopodium quinoa (Amaranthaceae), July 2018, Rashika S. Brahmanage, LC44 222 Rashika S. Brahmanage et alii Table 1. Taxa used in this study and their GenBank accession numbers for SSU, LSU, ITS and TEF DNA sequence data. Type strains are indicated with T and newly generated sequences are in bold. GenBank accessions Taxa Strain number italicaT Alloleptosphaeria Alternariaster bidentisT Alternariaster helianthiT Alternariaster centaureaediffusaeT Alternariaster trigonosporusT Heterosporicola chenopodiiT Heterosporicola chenopodii Heterosporicola dimorphospora Heterosporicola dimorphospora Heterosporicola beijingense Heterosporicola beijingense Heterosporicola beijingense Heterosporicola beijingense Leptosphaeria slovacica Leptosphaeria doliolumT Leptosphaeria doliolum Leptosphaeria ebuliT Leptosphaeria conoidea Neoleptosphaeria rubefaciensT Neoleptosphaeria jonesiiT Paraleptosphaeria macrospora Paraleptosphaeria nitschkeiT Paraleptosphaeria rubiT Paraphoma radicina Plenodomus pimpinellae Plenodomus guttulatus Plenodomus salviae Pseudoleptosphaeria etheridgeiT Sphaerellopsis macroconidiale Sphaerellopsis hakeae Sphaerellopsis paraphysata Subplenodomus valerianae Subplenodomus violicolaT Subplenodomus galicolaT ITS SSU LSU TEF MFLUCC 14-0934 CBS 134021 CBS 327.69 KT454722a NR159551 KC609335 KC584627 KT454714 KC609341 KC584369 - MFLUCC 14-0992 KT454724 KT454731 KT454716 - MFLU 15-2237 CBS 448.68 CBS 115.96 CBS 345.78 CBS 165.78 JZB3400001 JZB3400002 JZB3400003 JZB3400004 CBS 389.80 CBS 505.75 MFLUCC 15-1875 MFLUCC 14-0828 CBS 616.75 CBS 223.77 MFLUCC 16-1442 CBS 114198 CBS 306.51 MFLUCC 14-0211 CBS 111.79 CBS 101637 MFLUCC 151876 MFLUCC 130219 CBS 125980 CBS 658.78 CPC 29566 CPC 21841 CBS 630.68 CBS 306.68 MFLU 15-1863 NR159558 FJ427023 JF740227 JF740203 JF740204 MN733734 MN733735 MN733736 MN733737 JF740247 JF740205 KT454727 NR155323 MH860957 JF740243 NR152375 JF740238 JF740239 KT454726 NR156556 JF740240 KT454721 KT454725 NR111620 KP170659 NR155859 NR137956 JF740251 FJ427083 NR154454 EU754088 JF740098 MN733738 MN733739 MN733740 MN733741 JF740101 NG062778 KP753954 JF740099 NG063625 KT454733 EU754092 KT454729 KT454732 KY554199 GU238231 - KY674858 EU754187 EU754188 GU238069 JF740281 MN737597 MN737598 MN737599 MN737600 JF740315 GU301827 KT454734 KP744488 MH872726 JF740312 KY211870 JF740305 JF740308 KT454718 EU754191 JF740309 KT454713 KT454717 MH875320 KP170727 KY173555 GU238150 GU238156 KY554199 GU349077 MN786372 MN786373 MN786374 MN786375 GU349069 KY211872 KF253130 KP170684 - CBS: Centraalbureau voor Schimmelcultures, Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; JZB: Beijing Academy of Agricultural and Forestry Sciences; MFLU: Mae Fah Luang University Herbarium; MFLUCC: Mae Fah Luang University Culture Collection a Sequence data from Dayarathne et al. (2015) and Tennakoon et al. (2017). (JZB3400003), ibid., LC45 (JZB3400004). Living cultures are not available. Notes: Heterosporicola beijingense isolated from Chenopodium quinoa resembles H. chenopodii and H. dimorphospora (Van der Aa and van Kesteren, 1979, de Gruyter et al., 2013). To support establishment of the new taxon as per the guidelines of Jeewon and Hyde (2016), we examined the nucleotide differences within the ITS and TEF regions. ITS base pair differences between H. beijingense and H. chenopodii were 6.5% (36 out of 550bp), and 6.9% (38 out of 550 bp) between H. beijinense and H. dimorphospora. The TEF base pair difference between H. beijingense and H. chenopodii was 2.3% (23 out of 895bp), but there were no TEF data generated from H. dimorphospora for comparison. Heterosporicola beijingense sp. nov. 223 Figure 1. Chenopodium quinoa leaf spots. a–b, Diseased plants in the field. c, Closeup of a diseased plant. d–e, Closeup of a diseased leaf (e, upper surface, d, lower surface). Scale bars: = 1 cm. 224 Rashika S. Brahmanage et alii Figure 3. Heterosporicola beijingense on Quinoa leaves (JZB3400001 holotype). a–c, Pycnidia on leaf surface. d–e, Conidial contacts. f, Conidia. Scale bars: a = 100 μm, b = 500 μm, c = 200 μm, d-f = 10 μm. Figure 2. Maximum Likelihood tree generated by RAxML based on combined LSU, SSU, ITS and TEF sequence data from taxa of Leptosphaeriaceae. Bootstrap support values for ML ≥65% and Bayesian posterior probabilities >0.95 are given above each branch. Newly generated strains are in blue bold and ex-type sequences are in bold black. DISCUSSION Heterosporicola species have been reported as pathogens on Chenopodium species (Boerema, 1997; De Gruyter et al., 2013 Alves et al., 2013). Among two previously reported Heterosporicola species, H. dimorphospora is a parasite on species of Chenopodium in Northand South-America (van der Aa and van Kesteren, 1979). In some parts of South America, this fungus causes eye-shaped stem lesions on Chenopodium quinoa (van der Aa and van Kesteren, 1979). Heterosporicola chenopodii (= Phoma variospora) is a very common pathogen on species of Chenopodium in Europe. On account of the septate macroconidia in vivo, H. chenopodii is sometimes confused with Ascochyta caulina. Heterosporicola is closely related to Subplenodomus. No sexual morph is known for Heterosporicola (De Gruyter et al., 2013). The new species reported here, H. beijingense, produces “bird eye”-like yellowish brown to reddish brown spots in characteristic circular arrangements on living Chenopodium quinoa leaves. However, Heterosporicola chenopodii produces pale yellowish brown or whitish leaf spots with narrow purplish-brown borders mostly on C. album, while H. dimorphospora formed pale brown leaf spots or eye-shaped lesions on stems especially on C. quinoa (Boerema et al., 1997). Morphological differences between H. beijingense, H. chenopodii and H. dimorphospora are described in Table 2, and it is clear that these three species differ from one another in conidiomata, conidiogenous cell and conidium dimensions. The other two Heterosporicola species produce two types of conidia (macro- and micro-conidia). However, we did not observe macroconidia in H. beijingense. We could not obtain axenic cultures for this species on PDA and MEA or oatmeal agar (OA), either by single spore isolation or tissue isolation methods. However, H. chenopodii and H. dimorphospora are known from cul- 225 Heterosporicola beijingense sp. nov. Table 2. Morphological comparison of Heterosporicola species. Size (μm) Species name H. chenopodii H. dimorphospora H. beijingense Conidiomata width Conidiomatal Conidiogenous wall cells 100–550 wide 6–14 4–10 80–200 seldom up to 300 wide 10–25 3–8 200–600 wide 15–60 4–8 × 4–6 tures on different media including PDA and OA (Boerema et al., 1997). Heterosporicola beijingense may have specific growth requirements for macroconidium production. This study focused on identifying fungal species associated with leaf spots on quinoa, and confirmation of their identity. Data were not collected to estimate disease severity, incidence and pathogenicity of Heterosporicola beijingense. Pathogenicity experiments, disease severity and incidence evaluations with appropriate field trials are recommended to confirm the pathogenicity of this species. ACKNOWLEDGEMENTS This study was funded by grants from the Thailand Research Fund (project No. TRG5880152) and the Mushroom Research Foundation. Rashika Brahmanage thanks Prof. Alan Phillips, Dr Kasun Thambugala, Dr Ruvishika Jayawardena and Ms Pranami Abeywickrama for their helpful comments and advice. Mei Liu thanks the Beijing Agriculture Innovation Consortium (BAIC07-2020) and the project of Improvement and Demonstration of Simplified and High-efficiency Cultivation Techniques of quinoa (Item Number: 20180311). D.N. Wanasinghe thanks the CAS President’s International Fellowship Initiative (PIFI) for funding his postdoctoral research (number 2019PC0008), the National Science Foundation of China and the Chinese Academy of Sciences gave financial support (Grant numbers 41761144055, 41771063 and Y4ZK111B01). Rajesh Jeewon thanks the University of Mauritius for support. 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