• 中国中文核心期刊
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Volume 35 Issue 4
Jul.  2022
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Identification and Pathogenicity of Neofusicoccum occulatum the Agent Shoot Blight of Platycladus orientalis in Shandong Province, China

  • Corresponding author: WANG Qing-hai, wqhhai@126.com
  • Received Date: 2022-02-09
    Accepted Date: 2022-03-02
  • Objective The pathogens causing shoot blight of Platycladus orientalis (L.) Franco in the Jinan Forest Farm, Shandong province were identified to provide a basis for the prevention and control of the disease. Method The pathogens were isolated from the lesion borders of P. orientalis by tissue isolation, and the pure cultures were obtained by monosporic isolation. The isolated strains were identified by combining morphological and polygenic phylogenetic analysis (ITS, TEF-1α, TUB2). Result Colonies were initially white, flattened with tufts of white mycelium. After 14 days, mycelium turned to be dark gray-green and dark brown on the back. Conidia are hyaline, unicellular, fusiform, ellipsoid, and cymbiform, ovoid. Three representative isolates (CBL-02, CLB-03, and CBCB-02) were used for pathogenicity tests and phylogenetic analyses. All the isolates tested could cause similar symptoms on the P. orientalis. The multilocus phylogenetic tree was generated using the maximum parsimony method based on 3 genomic loci ITS, TEF-1α, and TUB2. In the maximum likelihood phylogenetic tree, all test isolates were clustered in N. occulatum clade with 89% bootstrap support. Based on the multi-gene phylogeny and morphology, all test isolates were identified as N. occulatum Sakalidis, M. L., Burgess, T. I. Conclusion It is clear that N. occulatum is the pathogenic bacteria causing shoot blight of P. orientalis, and P. orientalis in Jinan City, Shandong Province.
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  • [1] 李培琴, 明 洁, 张舒瑶, 等. 黄帝陵侧柏叶枯病的病原菌鉴定及防治药剂筛选[J]. 西北农林科技大学学报:自然科学版, 2021, 49(1):74-84.

    [2] 朱凤美. 松柏类之一种新病害“叶炎症”[J]. 中华农学会报, 1922, 33:21-29.

    [3] 戴雨生, 王学道, 林其瑞. 侧柏叶枯病病原菌研究[J]. 南京林业大学学报, 1992, 46:59-65.

    [4] 蒋金升, 田 毅, 张文玉, 等. 洋县汉王山林侧柏叶枯病初步调查[J]. 陕西林业科技, 1984, 3:11-13.

    [5] 赵怀谦. 园林病虫和防治[M]. 北京: 中国建筑工业出版社; 1978.

    [6]

    Sakalidis M L, Hardy G E S J, Burgess T I. Use of the Genealogical Sorting Index (GSI) to delineate species boundaries in the Neofusicoccum parvumNeofusicoccum ribis species complex[J]. Molecular Phylogenetics and Evolution, 2011, 60: 333-344. doi: 10.1016/j.ympev.2011.04.026
    [7]

    Scarlett K A, Shuttleworth L A, Collins D, et al. Botryosphaeriales associated with stem blight and dieback of blueberry (Vaccinium spp. ) in New South Wales and Western Australia[J]. Australasian Plant Pathology, 2019, 48: 45-57. doi: 10.1007/s13313-018-0584-6
    [8]

    Sakalidis M, Slippers B, Wingfield B D, et al. The challenge of understanding the origin, pathways and extent of fungal invasions: global populations of the Neofusicoccum parvumN. ribis species complex[J]. Diversity and Distributions, 2013, 19: 873-883. doi: 10.1111/ddi.12030
    [9]

    Ma X Y, Hyde K D, Phillips A J, et al. Three new host records of endophytic Neofusicoccum species reported from Dendrobium orchid[J]. Phytotaxa, 2021, 494: 193-207. doi: 10.11646/phytotaxa.494.2.2
    [10]

    Zlatkovic M. Botryosphaeriaceae species occurring on various woody hosts in Serbia [D]Novi-Sad: University of Novi Sad; 2016.
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Identification and Pathogenicity of Neofusicoccum occulatum the Agent Shoot Blight of Platycladus orientalis in Shandong Province, China

    Corresponding author: WANG Qing-hai, wqhhai@126.com
  • 1. Jinan Forest Farm, Jinan, 250014, China
  • 2. Shandong Provincial Academy of Forestry, Jinan, 250014, China
  • 3. Jinan Academy of Landscape and Forestry, Jinan, 250103, China
  • 4. Yantai Academy of Agricultural Sciences, Yantai 265500, China

Abstract:  Objective The pathogens causing shoot blight of Platycladus orientalis (L.) Franco in the Jinan Forest Farm, Shandong province were identified to provide a basis for the prevention and control of the disease. Method The pathogens were isolated from the lesion borders of P. orientalis by tissue isolation, and the pure cultures were obtained by monosporic isolation. The isolated strains were identified by combining morphological and polygenic phylogenetic analysis (ITS, TEF-1α, TUB2). Result Colonies were initially white, flattened with tufts of white mycelium. After 14 days, mycelium turned to be dark gray-green and dark brown on the back. Conidia are hyaline, unicellular, fusiform, ellipsoid, and cymbiform, ovoid. Three representative isolates (CBL-02, CLB-03, and CBCB-02) were used for pathogenicity tests and phylogenetic analyses. All the isolates tested could cause similar symptoms on the P. orientalis. The multilocus phylogenetic tree was generated using the maximum parsimony method based on 3 genomic loci ITS, TEF-1α, and TUB2. In the maximum likelihood phylogenetic tree, all test isolates were clustered in N. occulatum clade with 89% bootstrap support. Based on the multi-gene phylogeny and morphology, all test isolates were identified as N. occulatum Sakalidis, M. L., Burgess, T. I. Conclusion It is clear that N. occulatum is the pathogenic bacteria causing shoot blight of P. orientalis, and P. orientalis in Jinan City, Shandong Province.

  • 侧柏(Platycladus orientalis (L.) Franco),属柏科(Cupressaceae)侧柏属(Platycladus)常绿乔木,耐干旱贫瘠,适应范围广,是我国荒山造林的先锋树种,也是重要的园林绿化树种,除新疆、青海外,全国均有分布。侧柏种植在防风固沙、调节生态气候等方面具有重要意义。侧柏是高龄树种,一些历史风景名胜地现存的古柏更是中华民族五千年历史文明的见证及珍贵历史文化遗存,具有极其重要的自然景观价值和历史价值。

    侧柏叶枯病是一种重要的侧柏叶部病害,在我国江苏、安徽、陕西等地大面积发生,在江苏盱眙县侧柏被害率达100%,病情指数高达50.0以上,危害严重时,侧柏干枯死亡,已成为我国侧柏最重要的病害之一[1]。目前的研究表明,Alternaria pruni McAlpine[2]A. alternata (Fr.) Keissler、Pestalotiopsis gracilis (Kleb.) Steyaert、Chloroscypha platycladus Y. S. Dai[3]、Monochaetia sp.[4]Keithia thujina Durand[5]均可以引起侧柏叶枯病。由于生长地理位置及生境差异,病原有所不同。2020年作者发现在济南市林场、千佛山风景区等侧柏林中,侧柏鳞叶出现枯黄脱落现象,病株率达90%以上,危害严重的侧柏整株枯死。济南市侧柏面积8.25万hm2,侧柏枝枯病主要发生于枝叶部,造成侧柏树势衰弱,可能诱发双条杉天牛、小蠹虫等次期性害虫的危害,造成侧柏林成片干枯,影响绿化效果,造成严重的生态和经济损失。为有效控制危害,明确病原种类是十分必要的。因此,本研究通过形态特征、多基因系统发育分析以及致病性测定,明确在济南市引起侧柏枝枯的致病因子,以期为该病害的发生、流行规律及其防治提供参考依据。

    • 感病的侧柏枝条及鳞叶,从山东省济南市佛慧山(36°37′37″ N, 117°2′44″ E)、仰山(36°36′26″ N, 117°1′43″ E)、蚂蚱鞍子山(36°37′21″ N, 117°44′46″ E)、廻龙山(36°37′25″ N, 117°4′26″ E)侧柏林采集。

    • 采用组织分离法。先用70%乙醇将样品表面消毒,然后在侧柏细枝、鳞叶病健交界处利用无菌解剖刀取5 mm2的组织块,依次用3%次氯酸钠浸泡1 min,无菌水冲洗3次。将处理好的组织块置于PDA平板上,每个平皿放置5块,25 ℃培养3 d。将生长的菌落纯化转接至新的PDA平板上,25 ℃下培养,采取孢子稀释分离法获取单菌落。分离获得的菌株均保存于山东省林业科学研究院森林保护研究所。

    • 分离菌株致病性测定采用室内离体接种法及室外活体接种法。室内离体接种法,选择健康的侧柏细枝条,自来水冲洗干净,晾干后,用70%酒精表面消毒,备用。将分离获得的菌株在PDA培养基上培养5 d,在菌落边缘用打孔器制作菌饼($\phi$ = 5 mm),转移至低营养琼脂培养基(SNA)平板上,菌饼菌丝贴在平板上,在菌饼周围放置2根无菌松针,紫外灯照射条件下培养10 d诱导产孢。将孢子配置成106孢子·mL−1孢子悬浮液,备用。先用无菌的挑针($\phi $ = 0.5 mm)轻微刺伤鳞叶,在伤口处滴200 μL孢子悬浮液,以无菌水为对照。将处理后的枝条置于含有少量无菌水的无菌烧杯中,用保鲜膜封闭,置于25 ℃恒温培养箱中培养7 d,观察。每组试验各做5次平行处理,重复2次。

      室外活体接种,选用健康无病生长一致的播种2年生盆栽侧柏苗进行活体接种。鳞叶用挑针刺伤,干基部用无菌手术刀做“T”伤口,在伤口处滴200 μL 孢子悬浮液,并放上无菌水浸湿的脱脂棉,用剪好的封口膜包扎,以接种无菌水为对照。为了防止水分蒸发过快,每一株侧柏用黑色塑料袋包裹,2 d后除去黑色塑料袋,每天进行观察;每组试验各做5次平行处理,重复2次。

    • 将分离获得的单孢菌株在PDA培养基平板活化培养5 d,在菌落边缘利用打孔器制作菌饼($\phi $ = 5 mm),将菌饼置于PDA、MEA平板中央,每个平板置1块菌饼,25 ℃、12 h照射/12 h黑暗交替培养,观察记录菌落特征。

    • DNA的提取采用改进的CTAB法。扩增的目的基因分别为核糖体转录间隔区序列(ITS)、翻译延伸因子−1α(TEF1-α)、β-微管蛋白(TUB2)。引物序列及扩增反应条件参数见表1。扩增反应体系为25 μL,其中2 μL DNA 模板、8.5 μL ddH2O、12.5 μL Taq 酶 mix、各1 μL上下游引物。PCR扩增产物由上海派森诺生物科技有限公司进行双向测序。

      基因
      Gene
      引物
      Primer
      序列
      Sequence
      预变性
      Initial denaturation
      变性
      Denaturation
      退火
      Annealing
      延伸
      Extension
      最后延伸
      Final extension
      °Cmin°Cs°Cs°Cmin°Cmin
      ITSITS1TCCGTAGGTGAACCTGCGG95594305830721.5727
      ITS4TCCTCCGCTTATTGATATGC
      TEF1-αEF1-728FCATCGAGAAGTTCGAGAAGG94594305230721.5727
      EF-986RTACTTGAAGGAACCCTTACC
      TUB2BT-2aGGTAACCAAATCGGTGCTGCTTTC9459445553072457210
      BT-2bACCCTCAGTGTAGTGACCCTTGGC

      Table 1.  Primers used in this study, with sequence and PCR conditions

      将获得的ITS、TEF1-αTUB2序列提交NCBI 数据库,并利用Blast进行比对。选取38株Neofusicoccum sp.菌株作为参考菌株,以Botryosphaeria dothidea (CBS 115476)、Dothiorella iberica (CBS 115041)和D. sarmentorum (IMI63581b) 作为外围菌株(表2)。利用 Fasta alignment joiner 软件(https://users-birc.au.dk/~palle/php/fabox/alignment_joiner.php#)通过首尾相连的方法,将 ITS、TEF1-αTUB2基因串联。利用MEGA 7.0 软件,采用最大似然法(Maximum Likelihood)构建多基因系统发育树,循环1 000次,并以自展支持率(Bootstrap)校对检测。

      种类
      Species
      菌株编号
      Cultrue accession
      寄主
      Host
      国家
      Country
      基因编号
      GenBank accessions
      ITSTEF-1αTUB2
      Neofusicoccum algerienseCBS137504Vitis viniferaAlgeriaKJ657702KJ657715KX505915
      N. algerienseCAA322Eucalyptus globulus-KX505906KX505894KX505916
      N. andinumCBS117453Eucalyptus sp.VenezyelaAY693976AY693977KX464923
      N. arbutiCBS116131Arbutus menziesiiUSAAY819720KF531792KF531793
      N. australeCMW6837Acacia sp.AustraliaAY339262AY339270AY339254
      N. batangarumCBS124924Terminalia catappaAfricaFJ900607FJ900653FJ900634
      N. brasilienseCMM1338Mangifera indicaBrazilJX513630JX513610KC794031
      N. buxiCBS113714B.sempervirensFranceKX464164KX464677KX464954
      N. cordaticolaCBS123634Syzygium cordatumSouth AfricaEU821898EU821868EU821838
      N. corticosaeCBS120081--DQ923533KX464682KX464958
      N. cryptoaustraleCMW23785Eucalyptus sp.South AfricaFJ752742FJ752713FJ752756
      N. eucalvpticolaCBS115766Eucalyptus rossiiAustraliaAY615143AY615135AY615127
      N. eucalypticolaCBS115679Eucalyptus rossiiAustraliaAY615141AY615133AY615125
      N. hellenicumCERC1947Pistachia veraGreeceKP217053KP217061KP217069
      N. illiciiCGMCC3.18311Illicium verumChinaKY350150KY817756KY350156
      N. kwambonambienseCBS123639Syzygium cordatumSouth AfricaEU821900EU821870EU821840
      N. lumnitzeraeCMW41469--KP860881KP860724KP860801
      N. luteumCBS110299Vitis viniferaPortugalAY259091AY573217DQ458848
      N. macroclavatumWAC12444Eucalyptus globulusAustraliaDQ093196DQ093217DQ093206
      N. mangiferaeCBS118531Mangifera indicaAustraliaAY615185DQ093221AY615172
      N. mangroviorumCMW41365--KP860859KP860702KP860779
      N. mediterraneumCBS121718Eucalyptus sp.GreeceGU251176GU251308GU251836
      N. occulatumCBS128008Eucalyptus grandis hybridAustraliaEU301030EU339509EU339472
      MUCC286Eucalyptus pellitaAustraliaEU736947EU339511EU339474
      CBL-02*Platycladus orientalisChinaOM283294OM315202OM403995
      CBL-03*Platycladus orientalisChinaOM283295OM315203OM691696
      CBCB-02*Platycladus orientalisChinaOM283296OM315204OM403996
      N. parvumCMW9081Actinidia deliciosaNew ZealandAY236943AY236888AY236917
      N. parvumCBS110301Vitis viniferaPortugalAY259098AY573221EU673095
      N. pennatisporumMUCC510Vitis vinifera-EF591925EF591976EF591959
      N. pistaciaeCBS595.76Pistachia veraGreeceKX464163KX464676KX464953
      N. pistaciarumCBS113083Pistachia veraUSAKX464186KX464712KX464998
      N. protearumCBS114176-South AfricaAF452539KX464720KX465006
      N. ribisCBS115475Ribes sp.USAAY236935AY236877AY236906
      N. sinenseCGMCC3.18315-ChinaKY350148KY817755KY350154
      N. stellenboschianaCBS282.70--KX464225KX464758KX465051
      N. terminaliaeCMW26679--GQ471802GQ471780KX465052
      N. umdonicolaCBS123645Syzygium cordatumSouth AfricaEU821904EU821874EU821844
      N. ursorumCMW24480Eucalyptus sp.South AfricaFJ752746FJ752709KX465056
      N.viticlavatumCBS112878Vitis viniferaSouth AfricaAY343381AY343342KX465058
      N. vitifusiformeCBS110887Vitis viniferaSouth AfricaAY343383AY343343KX465061
      Dothiorella ibericaCBS115041Quercus ilexSpainAY573202AY573222EU673096
      D. sarmentorumIMI63581bUlmus sp.United KingdomAY573212AY573235EU673102
      Botryosphaeria dothideaCBS115476Prunus sp.SwitzerlandAY236949AY236898AY236927
      *表示的是研究菌株。
      *=Strains collected in the present study.

      Table 2.  Strains and sequences of the genus Neofusicoccum used in this study

    2.   结果与分析
    • 危害叶片,多由叶片前端或中部枯黄、变褐枯死。危害细枝,呈段斑状变褐(图1a, b)。在枯死的鳞叶和细枝上产生黑色的颗粒物(图1d)。树冠内部和下部发生严重。严重时,叶片大量脱落,枝条枯死,在主干或枝干上萌发出一丛丛的小枝叶,形成所谓“树胡子”。连续数年受害,最终全株枯死(图1c)。

      Figure 1.  Shoot blight disease symptom on Platycladus orientalis; (a)~(c) symptom in the field; (d) fruiting bodies produced on leaves and shoots (e) longitudinal section of pycnidium Bars=20 μm

    • 从4个地点共采集样品20份,分离获得菌株87株。根据菌落形态特征分为3种类型,其中链格孢属真菌27株(31.0%),细菌菌株4株(4.6%),其余为1种真菌共56株(64.4%),菌落形态特征一致,分生孢子形态与侧柏鳞叶和枝条上子实体分生孢子形态相似(图1e)。

      选择代表性菌株CBL-02、CBL-03和CBCB-02菌株室内离体接种7 d后,在鳞叶、枝条接种部位,均可以引起相同症状,致使侧柏鳞叶变色,形成黑褐色病斑(图2a),枝条接种点黑褐色(图2d)。显微观察,在鳞叶、枝条接种部位均有黑色子实体产生(图2b~d)。而空白对照未出现相应症状(图2f)。室外活体接种30 d后,在接种部位侧柏鳞叶变黄、枯死(图2e)。接种60 d,枝条、鳞叶干枯脱落,侧柏整株死亡(图2g),与田间观察症状一致。从接种发病部位重新分离,获得的菌株与接种菌株形态特征一致。表明,分离菌株为侧柏枝枯病的病原菌。

      Figure 2.  Pathogenicity test; (a)~(c) symptoms on Platycladus orientalis leaves inoculated with isolate CBCB-02 for 7 d; (d) symptoms on inoculation sites of P. orientalis shoot; (e) symptoms on seedlings (2-years) inoculated with isolate CBCB-02 for 30 d; (f) inoculated with sterile distilled water; (g) symptoms on seedlings (2-years) inoculated with isolate CBCB-02 for 60 d, L: control, R: treated

    • 在PDA培养基上,初期白色,稀疏,向四周扩散,培养5 d后,菌落达到平板边缘($\phi $ = 70 mm),菌丝开始呈现白色絮状,菌丝致密,中期颜色逐渐变成灰色(图3a),后期呈现深灰绿色,背面黑褐色,边缘完整(图3b)。在MEA培养基上,初期白色,絮状向四周扩散生长,后渐变为灰白色。培养3 d,菌落达到平板边缘($ \phi $ = 70 mm),菌落边缘完整(图3d),后期灰色、灰褐色,背面深灰绿色(图3e)。没有观察到有性特征。分生孢子器于7 d内产生于松针上,单生或聚生,外面包被白色菌丝,暗褐色至黑色(图3c)。分生孢子单孢透明、梭形、椭圆形、卵球形,顶端钝圆,基部平截,有时两端变窄。无隔膜,表面光滑,大小为20.6 ± 2.4 μm × 7.6 ± 1.2 μm(L/W = 2.7,n = 50)(图3f)。

      Figure 3.  Neofusicoccum occulatum (a)~(b) cultural character (cultured on PDA for 7 d, 14 d), (c) conidiomata formed on pine needles in culture, (d)~(e) cultural character (cultured on MEA for 3 d, 7 d), (f) conidia, Bars=50 μm

    • CBL-02、CBL-03、CBCB-02菌株与38株Neofusicoccum spp.参考菌株进行比对,以Botryosphaeria dothidea CBS115476、Dothiorella iberica CBS115041、D. sarmentorum IMI63581B为外围菌株,基于 ITS、TEF1-αTUB2基因序列数据,运用最大似然法(Maximum Likelihood)构建进化树。CBL-02,CBL-03和CBCB-02菌株与Neofusicoccum occulatum聚在一个进化分枝上,自展支持率为 89%(图4)。结合分离菌株形态特征、培养性状以及多基因系统发育分析,本试验获得的菌株为N. occulatum Sakalidis, M. L., Burgess, T. I.。

      Figure 4.  Phylogenetic tree of all isolates tested with allied taxa calculated with sequence data of concatenated ITS, TEF-1α and TUB2 using maximum likelihood method (1,000 bootstrap replicates; bootstrap values indicated at nodes. Botryosphaeria dothidea, Dothiorella sarmentorum and D. iberica represent the out group. The scale bar indicates the number of expected changes per site.

    3.   讨论
    • N. occulatum是Sakalidis 2011年[6]首次在澳大利亚的桉树(Eucalyptus spp.),瓦勒迈杉(Wollemia nobilis W. G. Jones, K. D. Hill & J. M. Allen)上分离获得。已有研究表明,N. occulatum已在非洲(乌干达)、大洋洲(澳大利亚、美国夏威夷)、南美洲(乌拉圭)、亚洲(泰国)等5个国家有分布,可以危害南洋杉(Araucaria cunninghamii Sweet),邓恩桉(Eucalyptus dunnii Maiden),大桉(E. grandis W. Mill ex Maiden),巨桉杂交种(E. grandis hybrid),粗皮桉(E. pellita F. V. Muell.),桉树(Eucalyptus sp.),尾叶桉和赤桉杂交种(Eucalyptus urophylla S. T. Blake x E. camaldulensis Dehnh.),瓦勒迈杉(W. nobilis),银桦(Grevillea sp.),Blepharocalyx salicifolius (Kunth) O. Berg,蓝莓(Vaccinium spp.),葡萄(Vitis vinifera L.),束花石斛(Dendrobium chrysanthum Wall. ex Lindl.)等寄主植物[7-9]。本研究结果表明N. occulatum已在我国有分布,这是国内有关N. occulatum的首次报道。目前,对N. occulatum的研究仅局限分类研究,其生物学、生态学、流行学、种群遗传结构和有效的防治措施尚需进一步系统研究。

      据已有的文献报道,新壳梭孢属真菌(Neofusicoccum sp.)可以危害美国扁柏(Chamaecyparis lawsoniana (A. Murray bis) Parlatore)、日本扁柏(Ch. Obtuse (Siebold et Zuccarini) Enelicher)、日本花柏(Ch. pisifera (Siebold et Zuccarini) Enelicher)、柏木(Cupressus funebris Endl.)、地中海柏木(Cu. sempervirens L.)、欧洲刺柏(Juniperus communis L.)、北美香柏(Thuja occidentalis L.)、北美乔柏(T. plicata Donn ex D. Don)、罗汉柏(Thujopsis dolabrata (Thunberg ex L. f.) Sieb. et Zucc.)、巨杉(Sequoiadendron giganteum (Lindl.) J. Buchholz)[10],尚未见危害侧柏的报道,是侧柏上发生的一种新病害。

    4.   结论
    • 本研究通过组织分离、单孢纯化,致病性测定,表明获得的单孢菌株CBL-02,CBL-03和CBCB-02均可以引起侧柏枝枯病。通过形态学以及多基因系统发育分析,证实分离获得菌株为N. occulatum

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