-
焦枯病是近年来树木或植物常见的一类叶部病害。其病因复杂,多由不适环境因素引起,如营养元素过量和钾元素缺乏、干旱胁迫、高温日晒等,造成植物水分生理失调,导致叶组织大面积产生枯斑,引发叶焦枯[1–2]。随着气候变暖和环境变化,病原菌导致植物叶焦枯病的种类增多,如由侵染性病原菌真菌拟茎点霉Phomopsis mollissim与其共生菌板栗蛇孢日规壳Ophiognomonia castaneae引起的板栗褐缘叶枯病[3–4];通过昆虫媒介传播的植物病原细菌木质部难养菌Xylella fastidiosa导致的叶焦枯病也日益频发。该细菌病原菌营养需求复杂、寄主范围很广,在多种重要的林木、观赏性植物及农业经济作物形成严重危害,已经成为全球性日益关注的新发流行病害[5–6]。
中国板栗(Castanea mollissima Bl.)属于壳斗科栗属的植物,原产于中国,有“干果之王”之称[7–8],河北板栗主要产于河北省北部的燕山山区,以颗粒饱满、香甜、皮薄、适于糖炒等特点著称于世,久负盛誉,是河北省出口农副产品中具有较大优势的土特产品之一,其产量、出口量和质量,均居全国第一位[8–9]。并且,板栗栽种面积占到全省有林地的10%,成为河北省重要的经济和生态树种[10]。近些年,青龙、迁西等板栗主产地相继发生一种新的叶部病害,板栗发病后,叶片上产生黄褐色的斑点,多个黄褐斑可渗透连片,以致使整个叶片上呈不规则的大面积焦枯状,引起果实重量减少,产量下降,造成栗农经济损失,该病害发病原因亟待明确,尽早进行有效的防控。
-
标本于2019年7月至9月,采于河北青龙县和迁西县(40.40° N, 118.95° E)5个病害发生严重的板栗果园,果园相距5~70 km。在栗园,观察症状的同时,放大镜镜检病症;在实验室,解剖镜镜检病组织表面病症,及显微镜镜检病组织真菌分生孢子器和子实体或细菌雾;实验室新鲜离体病叶组织湿培25℃,每天观察病症至第7天。
-
病原菌的分离和纯化采用组织分离法[11],将新鲜的病叶用清水冲洗干净,用75% 乙醇表面消毒,从其病健交界处用灭菌手术刀切取3~5 mm, 置于PDA培养基、MEA培养基和细菌蛋白胨培养基上25℃恒温培养,待菌丝或菌落长出后,进行转接纯化,并在PDA培养基上于25℃恒温培养,菌丝长满后,置于4℃冰箱中保存备用。每户叶片组织接种12个平板,每个PDA平板接3块组织。
-
分离菌株按形态初步归类后,用CTAB法提取和纯化菌株的基因组DNA[12],用通用引物ITS1和ITS4进行ITS rDNA基因片段的扩增,PCR产物经回收纯化后,由北京六合华大基因有限公司进行测序。所得序列经NCBI进行碱基Blast同源性分析,确定菌株分类地位。利用Mega X构建系统发育树,建树方法最大简约法(Maximum parsimony,MP),可靠性检验bootstrap法,初始值1000次,外群为Acervus globulosus HKAS 88987,系统发育树中本研究测得的序列号为MT373425-MT373459,Genbank中下载用于比对分析做系统发育树的序列见表1。
表 1 Genbank中下载用于比对分析做系统发育树的参考序列
Table 1. Sequences from Genbank for alignment and phylogenetic tree
序列来源
Source菌株号
Strain No.ITS序列号
ITS No.Alternaria alternata isolate Alt-C81 MN044802 Colletotrichum gloeosporioides IMI 356878 from type material NR_160754 Colletotrichum fructicola MFLU 090228 from type material NR_144783 Nigrospora oryzae isolate VARB205 KT966519 Ophiognomonia setacea CBS 128354 MH864895 Alternaria solani CBS 109158 KJ718239 Alternaria arborescens CBS 102605 NR_135927 Phomopsis sp. GJS 83-377 AF102999 Gnomoniopsis smithogilvyi CBS 130190 NR_166040 Coniothyrium pyrinum CBS 110.40 MH856053 Biscogniauxia sp. strain LPS-70 MF379340 Glomerella sp. isolate ERS038 KY413720 Leptostroma sp. strain Ecf-5 MK211262 Sporormiella sp. strain LTL532 MF663586 Dothiorella gregaria isolate hz-S8 FJ517548 Acervus globulosus HKAS 88987 NR_159565 -
依据1.3的结果,结合已报道致病性的菌株进行接种试验。室内离体接种选取健康无病的新鲜叶片,叶片采自温室1年健康苗的成熟叶片(停止伸展发育后20~30天),用清水冲洗干净,75% 乙醇消毒5 min,再用无菌水冲洗多次,置于事先灭菌的培养皿(直径150 mm,底部垫2~3层无菌滤纸,并加入适量无菌水保湿)中。因为发现在培养观察期间,分离菌株培养有产孢和不产孢,因此,接种体前者用分生孢子,后者用菌丝(菌饼)。将PDA平板上培养5~7天的分离物,无菌水冲洗孢子,制成106孢子·mL−1接种液;或用直径5 mm的打孔器在菌落边缘取菌饼。预实验发现无伤接种不成功,因此,用刺伤进行接种(用消毒昆虫针在健康叶片表面针刺后,将菌饼贴接于刺伤部位)。每处理接种5个叶片,每片叶接3个点,另以叶片相同部位接种无菌水处理为对照。接种封好后置于25℃培养箱黑暗培养。接种48 h后移去菌丝块,隔天观察发病情况并记录两周。
根据离体叶片接种结果,选取病原菌进行1年生盆栽苗接种实验,将菌丝块接种盆栽苗板栗叶片上,每种致病菌株接种3盆苗木,每苗木处理5个叶片,并用无菌水同时接种等数量的盆栽板栗苗作为对照,保鲜袋保湿24~48 h后去掉,定期观察发病情况。发病后取具有症状的组织再次进行病原菌分离。
-
病害在河北板栗园6月开始发病,病树整树叶片均出现症状,发病树木相对集中(图1a),果园树病率10%~50%,单株病叶率可至100%。该病害主要症状是,初始在叶片锐齿尖现褐色小点坏死(图1b),随着时间,顺沿叶缘至两侧脉之间的叶间组织呈现黄褐色至黑褐色病斑,病斑形状不规则,边缘不整齐,大小为2~3 cm,每叶片上能产生多个病斑(图1c),多个黄褐斑可渗透连片,以致使整个叶片上呈不规则的大面积焦枯状,均出现在叶脉间的叶组织上,病斑病健部有不规则宽度的亮黄色边缘线(图1d)。但病害不造成早期落叶。林间病叶未见病症。室内显微镜观察,也未见显著真菌分生孢子器和子实体。室内病组织保湿培养3天后,可镜检到链隔孢属Alternaria的分生孢子。栗园发病症状如图1。
-
栗园及室内在新鲜症状组织上均没有观察或镜检到病症,湿培3天后的病组织表面上可见有分散的较暗的细点,镜检确认是链隔孢属Alternaria的产孢梗及其分生孢子,极易被从病组织表面抹去。显微镜镜检也没有发现病健交界组织中的细菌菌溢现象。
病叶组织分离纯化共获得有效菌株102株,均为真菌。在菌落形态聚类的基础上,选取菌株扩增ITS。结果这些真菌归属于10科,12属,15种,丰度最高为Alternaria alternata(表2)。分离菌株的系统发育树见图2。分离菌株的主要培养物形态见图3。
表 2 河北板栗焦枯叶真菌菌株分离结果
Table 2. Fungi isolation from leaf scorch samples
分离菌号
Codes分离物鉴定
Isolates分离物数
Number of
isolates分离频率
Isolated
frequencies /%Bl21 Alternaria alternata 53 51.96 Bl36 Colletotrichum gloeosporioides 21 20.58 Bl30 Colletotrichum fructicola 7 6.86 Bl1 Nigrospora oryzae 3 2.94 Bl47 Ophiognomonia setacea 3 2.94 Bl12 Alternaria solani 2 1.96 Bl54 Alternaria arborescens 2 1.96 Bl62 Phomopsis sp. 2 1.96 Bl58 Gnomoniopsis smithogilvyi 2 1.96 Bl66 Coniothyrium pyrinum 2 1.96 Bl39 Biscogniauxia sp. 1 0.98 Bl5 Glomerella sp. 1 0.98 Bl53 Leptostroma sp. 1 0.98 Bl55 Sporormiella sp. 1 0.98 Bl61 Dothiorella gregaria 1 0.98 -
依据文献报道,重点挑选分离菌株中潜在的致病菌进行板栗离体叶片接种实验。选出8个菌:半壳霉属Leptostroma sp.,蛇孢日规壳Ophiognomonia setacea(Pers.) Sogonov,梨盾壳霉Coniothyrium pyrinum(Sacc.) J. Sheld.,稻黑孢菌Nigrospora oryzae (Berk. & Broome) Petch,拟茎点霉属Phomopsis sp.,胶孢炭疽菌Colletotrichum gloeosporioides (Penz.) Penz. & Sacc.,Gnomoniopsis smithogilvyi L.A. Shuttlew., E.C.Y. Liew & D.I. Guest,交链链格孢Alternaria alternata (Fr.) Keissl.。只有其中O. setacea和C. pyrinum两种菌接种的实验叶片出现了病斑,4~5天出现症状,一周后病斑基本稳定,不再变化,病斑位置局限于接种点附近(图4)。记号笔标出了接种部位,而其他几组菌种实验连续观察后均未出现明显病斑症状。另外我们还尝试混合了8种菌丝液,并混合Ophiognomonia setacea和Phomopsis sp.菌丝液进行板栗离体叶片接种实验,但并未出现明显症状。
在O. setacea和C. pyrinum离体叶片致病的基础上,进一步进行了两种菌株的1年生板栗盆栽苗致病实验。结果O. setacea和C. pyrinum两种菌均引起盆栽苗成熟叶片的病变,出现褐色坏死病斑。病斑接种后一周左右出现,大小不定,形状不规则,10~14天后病斑停止扩展趋于稳定(图5)。从发病部位可再次分离到这两种病原菌。
板栗叶焦枯病相关病菌分离及病因初探
Primary Study on Causes and Associated Pathogens for Chestnut Leaf Scorch
-
摘要:
目的 近年河北板栗主产区大面积严重发生板栗叶焦枯病,造成板栗减产和栗农经济损失,该病害发病原因亟待明确,尽早进行有效的防控。 方法 通过林间症状调查和病叶组织样本微生物分离、离体叶片及盆栽苗接种致病柯赫法则验证,结合果园管理措施和环境因素分析,对病因进行了探讨。 结果 该病栗园病树率可达10%~50%,单株树病叶率可至90%以上,病叶主要症状为叶缘及叶脉间组织干枯。林间、室内湿培及显微镜切片均未发现病组织存在病症或病原菌组织。组织分离纯化获得相关微生物菌株102株,均为真菌,归属10科12属15种,丰度最高为Alternaria alternata。致病性接种试验表明,其中的Ophiognomonia setacea和Coniothyrium pyrinum能引起接种组织局部褐变发病,但症状表现与林间叶焦枯病有差异。 结论 研究发现Ophiognomonia setacea和Coniothyrium pyrinum两种真菌具有致病性,可在中国板栗引发叶斑病,但症状不同于发生于栗园的板栗叶焦枯病。当前频发性树叶焦枯病类病害常见诱发因素,多与气候变暖、异常天气、环境污染以及生产和病虫害管理措施不当引起的品种生长不适应密切关联。河北大面积发生的板栗叶焦枯病病因有待综合非侵染因子进行深入研究和探明,以期为尽快采取有效防控措施奠定基础。 Abstract:Objective To investigate and analyze leaf scorch of Castanea mollissima occurred in the main chestnut producing areas in Hebei Province. Method Combined the investigation of the symptoms in the forest, the isolation of microbes from the symptomatic leaf tissue, the confirmation of Koch's law in vitro leaves and potted seedlings, with the management measures of orchard and the environmental factors, the causes of disease were analyzed and discussed. Result The disease rate of orchard trees was 20%–50%, the disease rate of individual tree leaves can be up to 100%, the main symptom of individual leaf was dry leaf margin and interveinal tissue. No pathogen was found in the forest symptomatic tissues and microscopic examination. A total of 102 strains were isolated and purified from symptomatic tissues, all of which were fungi, belonging to 10 families, 12 genera and 15 species with Alternaria alternata as the most abundant. The inoculation test showed that Ophiognomonia setacea and Coniothyrium pyrinum could induce the browning of leaf tissue of Chinese chestnut. However, the symptom was significantly different from that of chestnut forest leaf scorch. Conclusion Two pathogens, O. setacea and Coniothyrium Pyrinum, were newly found and verified to be pathogenic to Chinese chestnut leaves, causing chestnut tree leaf spots, which were different with the leaf scorch symptoms in chestnut observed. Currently, leaf scorches in trees are ordinarily connected with environmental and physical factors which influenced tree growth with global warming, extreme weather, pollution, inadequate silviculture and control pests/diseases managements. Therefore, integrating non-infection to investigate causes of the leaf scorch of Castanea mollissima occurred in Hebei Province is strongly proposed in order to effectively prevent and control the disease as soon as possible. -
表 1 Genbank中下载用于比对分析做系统发育树的参考序列
Table 1. Sequences from Genbank for alignment and phylogenetic tree
序列来源
Source菌株号
Strain No.ITS序列号
ITS No.Alternaria alternata isolate Alt-C81 MN044802 Colletotrichum gloeosporioides IMI 356878 from type material NR_160754 Colletotrichum fructicola MFLU 090228 from type material NR_144783 Nigrospora oryzae isolate VARB205 KT966519 Ophiognomonia setacea CBS 128354 MH864895 Alternaria solani CBS 109158 KJ718239 Alternaria arborescens CBS 102605 NR_135927 Phomopsis sp. GJS 83-377 AF102999 Gnomoniopsis smithogilvyi CBS 130190 NR_166040 Coniothyrium pyrinum CBS 110.40 MH856053 Biscogniauxia sp. strain LPS-70 MF379340 Glomerella sp. isolate ERS038 KY413720 Leptostroma sp. strain Ecf-5 MK211262 Sporormiella sp. strain LTL532 MF663586 Dothiorella gregaria isolate hz-S8 FJ517548 Acervus globulosus HKAS 88987 NR_159565 表 2 河北板栗焦枯叶真菌菌株分离结果
Table 2. Fungi isolation from leaf scorch samples
分离菌号
Codes分离物鉴定
Isolates分离物数
Number of
isolates分离频率
Isolated
frequencies /%Bl21 Alternaria alternata 53 51.96 Bl36 Colletotrichum gloeosporioides 21 20.58 Bl30 Colletotrichum fructicola 7 6.86 Bl1 Nigrospora oryzae 3 2.94 Bl47 Ophiognomonia setacea 3 2.94 Bl12 Alternaria solani 2 1.96 Bl54 Alternaria arborescens 2 1.96 Bl62 Phomopsis sp. 2 1.96 Bl58 Gnomoniopsis smithogilvyi 2 1.96 Bl66 Coniothyrium pyrinum 2 1.96 Bl39 Biscogniauxia sp. 1 0.98 Bl5 Glomerella sp. 1 0.98 Bl53 Leptostroma sp. 1 0.98 Bl55 Sporormiella sp. 1 0.98 Bl61 Dothiorella gregaria 1 0.98 -
[1] 曹淑云. 板栗叶片焦枯症的发生与防治[J]. 烟台果树, 2005, (3):54-55. doi: 10.3969/j.issn.1005-9938.2005.01.043 [2] 周道明. 棉花红叶茎枯病及防治措施[J]. 农村科技, 2006,(12):34. [3] 姜淑霞, 仲伟元, 高 锋, 等. 板栗褐缘叶枯病发病规律及防治试验[J]. 中国森林病虫, 2010, 29(5):30-31. doi: 10.3969/j.issn.1671-0886.2010.05.010 [4] 龚 赛, 张秀停, 聂 阳, 等. 板栗褐缘叶枯病协同致病菌Ophiognomonia castaneae的生活史[J]. 菌物学报, 2016, 35(10):1178-1186. [5] Bucci E M. Xylella fastidiosa, a new plant pathogen that threatens global farming: Ecology, molecular biology, search for remedies[J]. Biochemical and Biophysical Research Communications, 2018, 502(2): 173-182. doi: 10.1016/j.bbrc.2018.05.073 [6] Sicard A, Zeilinger R A, Vanhove M, et al. Xylella fastidiosa: Insights into an emerging plant pathogen[J]. Annual Review of Phytopathology, 2018, 56: 181-202. doi: 10.1146/annurev-phyto-080417-045849 [7] 黄武刚, 程丽莉, 周志军, 等. 板栗野生居群遗传多样性研究[J]. 果树学报, 2010, 2(27):227-232. [8] 侯 明. 河北板栗资源开发设想[J]. 河北省科学院学报, 1993, (4):23-27. [9] 周定奎, 吕纪水. 开拓河北板栗市场[J]. 合作经济与科技, 2005, 13:10-11. doi: 10.3969/j.issn.1672-190X.2005.19.003 [10] 郗荣庭, 曲宪忠. 河北经济林[M]. 北京: 中国林业出版社, 2001.01. [11] 姬广海, 吴亚鹏, 张乃明, 等. 芦荟根腐病病原菌的鉴定[J]. 植物病理学报, 2007, 37(2):207-209. doi: 10.3321/j.issn:0412-0914.2007.02.015 [12] Chang S, Puryear J, Cairney J. A simple and efficient method for isolating RNA from pine trees[J]. Plant Molecular Biology Reporter, 1993, 11(2): 113-116. doi: 10.1007/BF02670468 [13] 姜淑霞, 刘传忠, 王庆华, 等. 板栗新病害褐缘叶枯病及病原鉴定[J]. 林业科学, 2011, 47(5):177-180. doi: 10.11707/j.1001-7488.20110127 [14] 陆家云. 植物病原真菌学[M]. 北京: 中国农业出版杜, 2001, 461-462. [15] 谢宝多. 板栗病害[M]. 北京: 中国林业出版杜, 1998, 31-37. [16] 戚佩坤, 姜子德, 向梅梅. 中国真菌志-第三十四卷, 拟茎点霉属[M]. 北京: 科学出版杜, 2007, 2-74. [17] 贺 伟, 叶建仁. 森林病理学[M]. 北京: 中国林业出版社, 2017, 295-313. [18] 于秋香. 介绍几个燕山板栗优良品种[J]. 河北果树, 2016, (6):28-29. [19] Gong S, Zhang X, Jiang S, et al. A new species of Ophiognomonia from Northern China inhabiting the lesions of chestnut leaves infected with Diaporthe eres[J]. Mycological Progress, 2017, 16(1): 83-91. doi: 10.1007/s11557-016-1255-z [20] Walker D M, Castlebury L A, Rossman A Y, et al. Phylogeny and taxonomy of Ophiognomonia (Gnomoniaceae, Diaporthales), including twenty-five new species in this highly diverse genus[J]. Fungal Diversity, 2012, 57(1): 85-147. doi: 10.1007/s13225-012-0200-y [21] Sogonov M V, Castlebury L A, Rossman A Y, et al. Leaf-inhabiting genera of the Gnomoniaceae, Diaporthales[J]. Studies in Mycology, 2008, 62: 1-77. doi: 10.3114/sim.2008.62.01 [22] Stoykov D Y. New records of Ophiognomonia (Gnomoniaceae, Diaporthales) from Bulgaria, Greece and Turkey[J]. Phytologia Balcanica: International Journal of Balkan Flora and Vegetation, 2016, 22(3): 297-301. [23] Ahmed N, Ahmed T. Fruits related problems and their management in Rajouri district of Jammu and Kashmir[J]. Journal of the Humanities and Social Sciences, 2013, 12(2): 65-75. [24] Fazli M A, Razdan V K. A new record of Coniothyrium pyrinum on almond trees[J]. Indian Phytopathology, 1991, 44(3). [25] Pasche S, Calmin G, Auderset G, et al. Gnomoniopsis smithogilvyi causes chestnut canker symptoms inCastanea sativashoots in Switzerland[J]. Fungal Genetics and Biology, 2016, 87: 9-21. doi: 10.1016/j.fgb.2016.01.002 [26] Visentin I, Gentile S, Valentino D, et al. Gnomoniopsis castanea sp. nov. (Gnomoniaceae, Diaporthales) as the causal agent of nut rot in sweet chestnut[J]. Journal of Plant Pathology, 2012: 411-419. [27] Shuttleworth L A, Walker D M, Guest D I. The chestnut pathogen Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales) and its synonyms[J]. Mycotaxon, 2016, 130(4): 929-940. doi: 10.5248/130.929