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Research paper 研究论文 22 March 2021 40 3 473 486 Mycosystema ISSN1672 6472 CN11 5180 Q DOI 10 13346 j mycosystema 200333 基金项目 国家自然科学基金面上项目 31971658 31770015 31370063 国家自然科学基金国际 地区 合作与交流 项目 3155461143028 Supported by the National Natural Science Foundation of China 31971658 31770015 31370063 and NSFC Projects of International Cooperation and Exchanges 3155461143028 Corresponding author E mail yzhang Contributed equally to this work ORCID CHU Rui Tian 0000 0002 2081 7902 Received 2020 10 22 accepted 2020 11 23 Copyright 2021 Institute of Microbiology CAS All rights reserved jwxt Http journals Tel 86 10 64807521 菌物学报 473 Two novel species of Botryosphaeria causing stem canker of blueberries from China CHU Rui Tian 1 DOU Zhi Peng 2 3 HE Wei 3 ZHANG Ying 1 Institute of Microbiology School of Ecology and Nature Conservation Beijing Forestry University Beijing 100083 China Chinese Academy of Forestry CAF Beijing 100091 China Beijing Key Laboratory for Forest Pest Control Beijing Forestry University Beijing 100083 China Abstract Botryosphaeria spp are plant endophytes saprobes or parasites mostly associated with woody hosts In this paper two new species of Botryosphaeria causing stem canker of blueberries B dolichospermatii and B fujianensis are described and illustrated from southern China Phylogenetic analysis based on combined ITS LSU tef1 and TUB loci supports their separation from all other known species of Botryosphaeria Koch s postulate tests proved that both B dolichospermatii and B fujianensis were causal agents of stem canker of blueberries Key words Botryosphaeriaceae pathogen molecular phylogeny taxonomy 引用本文 褚睿天 豆志鹏 贺伟 张英 2021 引起蓝莓茎溃疡病的葡萄座腔菌属 Botryosphaeria 二新种 菌物学报 40 3 473 486 Chu RT Dou ZP He W Zhang Y 2021 Two novel species of Botryosphaeria causing stem canker of blueberries from China Mycosystema 40 3 473 486 CHU Rui Tian et al Two novel species of Botryosphaeria causing stem canker of blueberries from China Research paper 菌物学报 474 引起蓝莓茎溃疡病的葡萄座腔菌属 Botryosphaeria 二新种 褚睿天 1 豆志鹏 2 3 贺伟 3 张英 1 北京林业大学生态与自然保护区学院微生物研究所 北京 100083 中国林业科学研究院 北京 100091 北京林业大学北京市森林病虫害防治重点实验室 北京 100083 摘 要 在我国华南地区发现了两个引起蓝莓茎溃疡病的 Botryosphaeria 新种 即 B dolichospermatii 和 B fujianensis 本研究对这两个物种进行了详细的形态学描述 并制作了图版 基于 ITS LSU tef1 和 TUB 等 4 个基因片段的综合系统学分析 B dolichospermatii 和 B fujianensis 形成独立的分支 并与 Botryosphaeria 其他物种形成姐妹群 形态学特征也与系统发育关系较近的物种有明显区别 柯赫氏法则 检验表明 B dolichospermatii 和 B fujianensis 均为蓝莓枝干溃疡病的病原菌 关键词 葡萄座腔菌科 病原菌 分子系统发育 分类学 Botryosphaeriaceous fungi comprised plant endophytes saprobes and parasites mostly associated with woody hosts Phillips et al 2013 Ariyawansa et al 2016 Dissanayake et al 2016 Slippers et al 2017 Botryosphaeria Ces de Notaris 1863 Saccardo 1877 1882 Grossenbacher Putterill 1919 Trotter 1928 So far more than 200 epithets have been included in Index Fungorum http www indexfungorum org Aug 2018 The polyphyletic status of Botryosphaeria sensu lato has been well documented with species mostly encompassed in various genera of the Botryosphaeriaceae Slippers et al 2004 Crous et al 2006 Phillips et al 2008 2013 Dissanayake et al 2016 Based on the phylogenetic analysis of five loci SSU LSU ITS TUB and tef1 seven species was recognized within Botryosphaeria s str by Phillips et al 2013 namely B agaves Henn E J Butler B corticis Demaree Xu et al 2015a Ariyawansa et al 2016 Zhou et al 2016 2017 Li et al 2018 So far fifteen species have been included in Botryosphaeria s str Botryosphaeriacous fungi associated with blueberries have been widely documented Kong et al 2010 Yu et al 2012 2013a 2013b Xu et al 2015b Xu 2016 Wang et al 2016 Dou et al 2017 Three species of Botryosphaeria have been reported from blueberries Vaccinium spp namely B corticis B dothidea and B vaccinii Arx Barr 1972 Phillips et al 2006 Wright Yu et al 2012 Xu et al 2015b In the course of an ongoing survey of blueberry diseases in China two new taxa with general characteristics of Botryosphaeria were collected Molecular characteristics as well as the phylogeny based on combined ITS LSU tef1 and TUB nuDNA sequences support their new status within Botryosphaeria 1 MATERIALS AND METHODS 1 1 Sampling isolation and morphology Cankered stems of blueberries were collected from Fujian Province China in 26 February 2018 About 20cm cankered branches were cut for sampling The wood segments were first surface sterilized Pavlic et al 2004 cut about 0 5cm 0 5cm 0 2cm from the junction of disease and health and then incubated on malt extract agar MEA Isolates grown on MEA were kept at ambient temperatures about 28 C in the dark Fungal isolates were deposited in Beijing Forestry University BJFU with duplicates in the China General Microbiological Culture Collection Center CGMCC Herbarium specimens were deposited in Herbarium Mycologicum Academiae Sinicae HMAS To induce sporulation colonies resembling Botryosphaeriaceae spp were selected and transferred to synthetic nutrient poor agar SNA with sterilized pine needles placed onto the medium Plates were incubated at 28 C under continuous near UV light for two weeks Released conidia and squash mounts of pycnidia formed on the pine needles were mounted in water on microscope slides and examined microscopically Measurements and digital photographs were made using a Nikon Coolpix 995 digital camera connected to a trinocular Leitz Orthoplan microscope and processed with Adobe Photoshop Elements 10 software Measurements of conidia paraphyses and conidiogenous cells were made from water mounts 1 2 DNA extraction and PCR amplification DNA was extracted from mycelium grown on MEA plates with CTAB plant genome DNA fast extraction kit Aidlab Biotechnologies Co Ltd Beijing China The internal transcribed spacer of rDNA ITS was amplified and sequenced with primers ITS1 and ITS4 White et al 1990 The translation elongation factor 1 tef1 was amplified and sequenced with primers EF1 688F and EF1 1251R Alves et al 2008 The tubulin gene TUB was amplified and sequenced with primers Bt2a and Bt2b Glass Posada Huelsenbeck B Inoculated by Botryosphaeria dolichospermatii C Inoculated by B fujianensis photographing Pathogenicity was determined by the length of the necrotic lesion The fungi were re isolated by cutting small pieces of wood from the edges of the lesions cultivated in MEA at 28 C morphologically studied and sequenced again for Koch s postulates 2 RESULTS 2 1 Phylogeny analyses Phylogenetic analysis of the combined ITS LSU tef1 and TUB sequence dataset comprising 2 268bp revealed 293 parsimony informative characters The outgroup taxon was Neofusicoccum luteum and N parvum The heuristic search with random addition of taxa 500 replicates generated 5 000 most parsimonious trees of 511 steps CI 0 783 RI 0 894 RC 0 699 HI 0 217 The phylogenetic tree resulting from the Bayesian analysis using the general time reversible model of DNA evolution Rodr guez et al 1990 including estimation of invariable sites and assuming a discrete gamma distribution with six rate categories GTR G had a topology identical to the MP tree presented In both analyses MP and Bayesian the clade of Botryosphaeria received a high level of support 100 for MP and 1 00 for BI The subclade of B dolichospermatii and B fujianensis sibling to other species of Botryosphaeria while B dolichospermatii closely related to B sinensia B auasmontanum B minutispermatia B dothidea and B wangensis and B fujianensis to B fabicerciana and B fusispora Fig 2 2 2 Taxonomy Botryosphaeria dolichospermatii Z P Dou W He Y Zhang sp nov Fig 3 MycoBank MB826937 Holotype CHINA Fujian Province Nanping Jianyang District Huilong from cankered branch of Vaccinium uliginosum 26 February 2018 L Zhao HMAS 255410 ex type living culture CGMCC 3 19096 CHU Rui Tian et al Two novel species of Botryosphaeria causing stem canker of blueberries from China Research paper 菌物学报 480 Fig 2 Maximum parsimony tree generated from sequence analysis of the combined ITS tef1 TUB and LSU dataset of Botryosphaeria species Designated out group taxon is Neofusicoccum luteum and N parvum Bootstrap support values for maximum parsimony MP greater than 60 are shown above at the nodes Bayesian bootstrap BP posterior probability scores above 0 80 are shown under the branches MP value less than 60 or BP value less than 0 80 The species characterized in this study are in red and the ex type strains are in boldface 研究论文 22 March 2021 40 3 473 486 Mycosystema ISSN1672 6472 CN11 5180 Q 菌物学报 481 Fig 3 Botryosphaeria dolichospermatii From holotype 3 19096 A Colony growing on MEA after 5d B Conidiomata formed on pine needles in culture C D Conidiogenous cells E Spermatia F Conidia with mucous apical sheath arrowheads G Germinating conidia connected with tubes arrowheads H Conidia Scale bars A 1cm B 500 m C H 10 m Diagnosis Botryosphaeria dolichospermatii is characterized by its conidia with mucilaginous sheath and obvious curved spermatia Etymology GK comp dolichospermatii referring to the relatively longer spermatia produced in culture Description Sexual morph unknown Conidiomata pycnidial stromatic produced on pine needles on SNA within 2 3wk solitary immersed to semi immersed iron grey to black covered with dense mycelium mostly uniloculate 140 315 m diam thick walled globose without papilla Paraphyses not observed Conidiogenous cells holoblastic discrete hyaline smooth thin walled cylindrical to ampulliform producing a single conidium at the tip 8 9 15 2 3 5 m av 11 5 4 1 m n 30 Conidia fusiform aseptate straight hyaline thin walled smooth apex slightly sharp base subtruncate to bluntly rounded sometimes with a mucous apical sheath mostly one or two septa formed before germination sometimes different conidia connected with tubes when germinating 21 22 30 32 5 8 m av of 50 conidia 24 8 6 5 m L W ratio 3 85 range from 3 14 to 4 8 Spermatia hyaline aseptate smooth oblong to cylindrical straight to obvious curved most rounded ends up to 9 4 m Culture characteristics Colonies on MEA at 28 C in darkness oliveaceous becoming grey with reverse black Mycelial mat moderately dense margin irregular Colonies reaching 53 9mm on MEA after 3d in the dark at 28 C Additional specimens examined CHINA Fujian Province Nanping city Jianyang district CHU Rui Tian et al Two novel species of Botryosphaeria causing stem canker of blueberries from China Research paper 菌物学报 482 Huilong town from cankered branch of Vaccinium uliginosum 26 February 2018 L Zhao paratype HMAS 255411 living culture CGMCC 3 19097 paratype HMAS 255412 living culture CGMCC 3 19098 Note Phylogenetically Botryosphaeria dolichospermatii is distinct from but closely related to B sinensia B auasmontanum B minutispermatia B dothidea and B wangensis Morphologically the condia of B dolichospermatii 21 22 30 32 5 8 m are larger than those of B auasmontanum 8 8 5 11 5 13 2 5 3 4 5 m and B minutispermatia 8 14 3 4 m Slippers et al 2014 Ariyawansa et al 2016 The larger and sometimes curved spermatia of B dolichospermatii up to 9 4 m are distinguishable from those of B dothidea 3 6 1 5 2 m B sinensia 4 7 2 3 m and B wangensis 3 5 4 5 1 1 5 m Phillips et al 2013 Zhou et al 2017 Li et al 2018 Furthermore the apical sheath presenting on some conidia of B dolichospermatii also differs from that of other five species Botryosphaeria fujianensis Z P Dou W He Y Zhang sp nov Fig 4 MycoBank MB826938 Holotype CHINA Fujian Province Nanping Jianyang District Huilong from cankered branch of Vaccinium uliginosum 26 February 2018 L Zhao HMAS 255413 ex type living culture CGMCC 3 19099 Diagnosis Botryosphaeria fujianensis is characterized by its larger conidia dimension colonies with a circular area lacking aerial mycelium in the middle and different tef1 sequence Fig 4 Botryosphaeria fujianensis From holotype 3 19099 A Colony growing on MEA after 5d B Conidiomata formed on pine needles in culture C Conidiogenous cells and paraphyses D Young conidia E Spermatia F Connected conidia with tubes arrowheads G Aged conidia Scale bars A 1cm B 500 m C G 10 m 研究论文 22 March 2021 40 3 473 486 Mycosystema ISSN1672 6472 CN11 5180 Q 菌物学报 483 Etymology in reference to the location where the species was first described Description Sexual morph unknown Conidiomata pycnidial stromatic produced on pine needles on SNA within 1 2wk solitary semi immersed to superficial iron grey to black covered with dense mycelium uniloculate 140 330 m diam thick walled globose without papilla Paraphyses hyaline septate cylindrical sometimes tapering towards the apices arising between the conidiophores and conidiogenous cells up to 62 4 m Conidiophores when present hyaline cylindrical sometimes branches smooth 0 1 septate Conidiogenous cells holoblastic discrete hyaline smooth thin walled cylindrical to ampulliform producing a single conidium at the tip 8 19 2 4 m av 13 1 3 1 m n 30 Conidia fusiform aseptate mostly straight hyaline thin walled smooth with granular contents widest in the middle both ends rounded base sometimes truncate mostly forming one to three septa before germination 20 21 29 31 7 9 m av of 30 conidia 24 7 8 m L W ratio 3 08 range from 2 63 to 3 63 aged conidia hyaline fusiform and becoming 0 3 septate more shorter and slender sometimes two aged conidia connected with tube 19 24 5 7 m av of 30 conidia 21 3 5 6 m L W ratio 3 87 range from 3 14 to 4 8 Spermatia hyaline aseptate smooth rod shaped straight most rounded ends 4 8 2 m Culture characteristics Colonies on MEA at 28 C in darkness oliveaceous becoming grey with reverse black Abundant aerial mycelium reaching to the lid of Petri dishes margin irregular with a circular black area lacking aerial mycelium in the middle Colonies reaching 71 3mm on MEA after 3d in the dark at 28 C Additional specimens examined CHINA Fujian Province Nanping city Jianyang district Huilong town from cankered branch of Vaccinium uliginosum 26 February 2018 L Zhao paratype BJFU180226 3 living culture BJFUCC180226 3 paratype BJFU180226 4 living culture BJFUCC180226 4 Note Phylogenetically Botryosphaeria fujianensis is closely related but distinct from B fabicerciana and B fusispora Morphologically conidiomata of B fujianensis 140 330 m are larger than those of B fusispora 140 180 m while smaller than those of B fabicerciana 346 470 m Liu et al 2012 Phillips et al 2013 The colonies of B fujianensis with a circular area lacking aerial mycelium in the middle differ from those of B fabicerciana and B fusispora 2 3 Pathogenicity tests Four isolates were inoculated on the blueberry stems After two weeks of inoculation a few black necrotic lesions were observed on the surface of branches while these were not seen in negative controls Fig 1 Koch s postulates were fulfilled by successful re isolation of pathogen from all the necrotic stems and the morphological characteristics and DNA sequences of re isolated pathogen was consistent with those of pathogen isolated from original diseased blueberry stems CHU Rui Tian et al Two novel species of Botryosphaeria causing stem canker of blueberries from China Research paper 菌物学报
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