林业科学  2018, Vol. 54 Issue (9): 80-88   PDF    
DOI: 10.11707/j.1001-7488.20180910
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文章信息

易润华, 陈玉娟, 韩静仪, 胡倩, 李慧琪, 吴海燕
Yi Runhua, Chen Yujuan, Han Jingyi, Hu Qian, Li Huiqi, Wu Haiyan
石碌含笑枯梢病菌的鉴定及生物学特性
Identification and Biological Characteristics of Diaporthe ueckerae Causing Dieback Disease on Michelia shiluensis
林业科学, 2018, 54(9): 80-88.
Scientia Silvae Sinicae, 2018, 54(9): 80-88.
DOI: 10.11707/j.1001-7488.20180910

文章历史

收稿日期:2017-02-14
修回日期:2018-02-16

作者相关文章

易润华
陈玉娟
韩静仪
胡倩
李慧琪
吴海燕

石碌含笑枯梢病菌的鉴定及生物学特性
易润华, 陈玉娟, 韩静仪, 胡倩, 李慧琪, 吴海燕     
广东海洋大学农学院生物技术系 湛江 524088
摘要:【目的】对广东省徐闻县2~3年生石碌含笑嫁接苗枯梢病进行病原菌的分离鉴定,分析其致病性及生物学特性,为该病的诊断和防控提供理论基础。【方法】将病枝表皮削除后,取病组织接种到PDA培养基中,25~30℃黑暗培养后,得到纯培养物,将其接种到健康寄主,测定其致病性。将病原菌的ITS、组蛋白HIS、延长因子TEF和微管蛋白TUB的基因扩增测序后,在GenBank进行序列比对,采用邻接法(NJ)和贝叶斯法(BI)进行多基因位点系统发育分析,确定病原菌与近缘种的关系。根据系统发育分析以及形态学特征对病原菌进行种类鉴定。将菌饼接种到培养基中,测定不同培养基、温度、pH、碳氮源和光照处理对病原菌的菌丝生长和形成分生孢子座的影响。【结果】从病组织中分离到的菌株可引起健康植株发病,其形态学特性与间座壳菌一致。GenBank比对结果显示,病原菌ITS序列与间座壳菌几个种的序列同源性高于99%,而HIS、TEF和TUB基因序列与Diaporthe ueckerae的序列同源性为100%。系统发育分析结果显示石碌含笑病原菌与Diaporthe ueckerae以自展支持率99%和后验概率1.00聚集在同一分枝。病原菌在不同培养基、温度、碳氮源条件下,菌丝生长速度和产生分生孢子座数量差异显著,在pH 4-8时菌丝生长和产分生孢子座差异不明显,不同光处理对病原菌生长影响差异不显著,但对产生分生孢子座影响差异显著,12 h光照+12 h黑暗处理与12 h光照+12 h黑暗+10 min紫外线处理不利于形成分生孢子座。【结论】石碌含笑枯梢病的病原菌是D.ueckerae Udayanga et Castlebury 2015,对环境因子适应性广,仅在低于15℃和高于35℃时不产生分生孢子座。
关键词:石碌含笑    枯梢病    Diaporthe ueckerae    生物学特性    
Identification and Biological Characteristics of Diaporthe ueckerae Causing Dieback Disease on Michelia shiluensis
Yi Runhua, Chen Yujuan, Han Jingyi, Hu Qian, Li Huiqi, Wu Haiyan    
Department of Biotechnology, Agricultural College, Guangdong Ocean University Zhanjiang 524088
Abstract: 【Objective】In 2014, dieback disease was observed on the 2-3-year-old grafted twigs of Michelia shiluensis in Xuwen County, Guangdong Province. To understand the pathogen causing the dieback, the isolate was collected and identified, the pathogenicity was tested according to the Koch's postulates, and the biological characteristics were determined. This study aims to provide basic theoretical knowledge for diagnosis and control of this disease.【Method】To get the pathogen, the infested tissues were cut from symptomatic twigs of which the barks were removed, placed on potato dextrose agar (PDA), and incubated at 25-30℃ in dark. The isolates were inoculated on the healthy hosts to test the pathogenicity according to the Koch's postulates. The sequences of internal transcribed spacer (ITS) rDNA, translation elongation factor (TEF) gene, β-tubulin gene and histone (HIS) gene were amplified, sequenced and blasted in GenBank. Based on the multi-locus (ITS, HIS, TEF, TUB) phylogenetic analyses with Neighbor joining (NJ) and Bayesian inference (BI) method, the phylogenetic relationship of the pathogen with Diaporthe spp. was analyzed. The pathogen was identified according to the morphological and molecular characteristics. The effects of culture medium, temperature, pH, carbon and nitrogen source as well as light treatment on mycelial growth and conidiomata formation were determined.【Result】The isolate from infected tissues caused twigs dieback on healthy hosts, and the morphological characters was the same as Diaporthe sp. The Blast result showed that the ITS sequences of the pathogen were above 99% homology with several species of Diaporthe, and the sequences of HIS, TEF and TUB gene were 100% homology with D. ueckerae. The pathogen was clustered in the same branch with D. ueckerae in a well-supported clade with Bootstrap support values above 99% and Bayesian posterior probability values 1.00. Under the condition of culture medium, temperature, pH, carbon and nitrogen source and light treatment, the mycelial growth and conidiomata formation had significant difference. The pH values ranged from 4 to 8 had insignificant effects on mycelial growth and conidiomata formation. Different light treatments had insignificant effect on the mycelial growth, but significant effect on conidiomata formation, thus it was not conducive to form the conidiomata under two light treatments, i.e. alternation of 12 hillumination and 12 h darkness, and alternation of 12 hillumination, 12 h darkness and 10 minultraviolet radiation.【Conclusion】The pathogen causing dieback on M. shiluensis has been identified as D. ueckerae Udayanga et Castlebury 2015, with strong adaptation to various environmental factors. The fungus could not form conidiomata below 15℃ or above 35℃.
Key words: Michelia shiluensis    dieback    Diaporthe ueckerae    biological characteristics    

石碌含笑(Michelia shiluensis)是木兰科(Magnoliaceae)含笑属(Michelia)的常绿高大乔木,主要分布海南省昌江、太平、霸王岭、东方和保亭等地(陈焕镛, 1963; 中国科学院中国植物志编辑委员会, 1996),属于我国重点保护野生植物。石碌含笑树形优美,叶泽光亮,花优雅、芳香,且适应性强,在我国广东、云南、广西和湖南等省作为盆景以及城市公园、街道、花圃的绿化和观赏树种被广泛种植。

木兰科植物受多种病害危害,主要有褐根病又称南方根腐病(Phellinus noxius)(Ann et al., 2002)、白绢病(Sclerotium rolfsii)(Hsiao et al., 2006)、根腐病(Pythium splendens)(Fu et al., 2005)、白粉病(Oidium sp.)(Holcomb, 1999)、溃疡病(Fusarium decemcellulare)(Wang et al., 2015)、炭疽病(Colletotrichum fioriniae)(Shivas et al., 2009)、拟茎点霉属叶斑病(Phomopsis micheliae)(Sankaran et al., 1987; Mitra et al., 2014)、轮枝枯萎病(Verticillium dahliae)和疫霉根腐病(Phytophthora cinnamomi)以及多种叶斑病(Knox et al., 2012)。2014年,在广东省徐闻县农场繁育石碌含笑种苗时发现2~4年生的嫁接苗很容易枝梢枯死,发病率为30%~40%,严重影响种苗的繁育。为确定引起该病的原因,从采集的病枝样品分离到间座壳菌(Diaporthe sp.),通过致病性测定确定其为病原菌,根据形态学和分子特征对其进行鉴定,并研究病原菌的生物学特性,以期为该病的防治提供理论依据。

1 材料与方法 1.1 病原菌分离

从广东省徐闻县农场2~3年生的石碌含笑嫁接苗采集发病枝梢,在无菌条件用刀片削去树皮,将树枝切成0.2 cm长的方块,接入到含20 μg·mL-1青霉素的PDA培养基,置25~30 ℃黑暗培养4~5天,待长出菌丝后,挑取菌丝尖端转接到PDA平板,纯化后保存备用。

1.2 致病性测定

取健康石碌含笑枝条(长30 cm)用70%(V/V)酒精消毒放入瓷盆,接种经活化的病原菌菌饼(Φ=0.8 cm),保湿,25~30 ℃培养,以不带菌琼脂块做对照。将病原菌接种到经高压蒸汽灭菌的石碌含笑枝条(长3 cm),培养15天后接种在田间健康枝条上,保湿,定期检查发病情况,以不带菌枝条做对照。

1.3 病原菌鉴定

形态学特性:参考Baumgartner等(2013)Gao等(2016)Gomes等(2013)Udayanga等(2015)方法,观察病原菌菌落形态、产孢体结构、孢子形态和大小,用Nikon DXM 1200F数码显微成像系统拍照。

分子鉴定:将病原菌接种到PDA培养基25 ℃黑暗培养3天,用牙签挑取少量菌丝进行菌落PCR(Lu et al., 2012)。扩增ITS、β-微管蛋白(β-tubulin, TUB)和延长因子(translation elongation factor, TEF)基因序列。ITS引物为ITS 1(TCC GTA GGT GAA CCT GCG G, )和ITS 4(TCC TCC GCT TAT TGA TAT)(White et al., 1990);TEF引物为EF1-728F(CAT CGA GAA GTT CGA GAA GG)(Carbone et al., 1999)和EF2-R(GGA RGT ACC AGT SAT CAT GTT)(Glass et al., 1995),TUB引物为2Fd(GTB CAC CTY CAR ACC GCY CAR TG)和4Rd(CCR GAY TGR CCR AAR ACR AAG TTG TC)(Groenewald et al., 2013)。

采用TaKaRa公司的MightyAmp DNA Polymerase Ver2试剂盒,PCR扩增体系60 μL,上下游引物浓度分别为0.3 μmol·L-1,Amp酶1.2 U, dNTP浓度为400 μ μmol·L-1。PCR扩增程序:98 ℃预变性5 min;98 ℃变性10 s;56 ℃退火15 s;72 ℃延伸30 s,36个循环后72 ℃延伸10 min。PCR产物由生工生物工程(上海)股份有限公司测序。

所得序列在GenBank进行BLAST,比较病原菌与近缘种序列的同源性,并选取序列经MEGA 6.0软件(Tamura et al., 2013)比对,选用邻接法(neighbor-joining,NJ)构建系统发育树,通过内部分枝检验(Interior-branch test),重复抽样1 000次对进化树分枝的支持率进行评估。贝叶斯法(Bayesian inference,BI)构建系统发育树时,比对序列根据AIC准则(Akaike information criterion),应用jModelTest V2.1.4(Santorum et al., 2014)软件选择ΔAICc=0的核苷酸替换模型为最优模型(TrN+I+G),用MrBayes3.2程序(Ronquist et al., 2003)重建系统发育树。分析程序参考童依婷等(2016)易润华等(2017),用马尔科夫链蒙特卡罗法(Markov Chain Monte Carlo method,MCMC)计算后验概率(posterior probability)(Mossel et al., 2005)评价系统发育树的可信度。

1.4 生物学特性

供试菌株在PDA培养基25 ℃培养5天后,用打孔器(Φ=5 mm)取菌饼分别接种到装有培养基的培养皿中央进行培养,根据实验目的,选择相应的培养基和培养条件,分别测定培养基、碳氮源、温度、pH和光照对病原菌生长以及分生孢子座形成的影响。除研究温度对病原菌生长以及形成分生孢子座的影响外,其他培养条件均为25~28 ℃黑暗。每个处理3次重复。

测试培养基及配方:1)马铃薯葡萄糖琼脂培养基(PDA);2)马铃薯蔗糖琼脂培养基(PSA);3)察氏培养基(Czapek);4)燕麦琼脂培养基(OMA);5)玉米粉蔗糖琼脂培养基(MSA);6)蛋白胨酵母膏葡萄糖琼脂培养基(PYGA);7)豆芽汁蔗糖培养基(SSA)和8)胡萝卜蔗糖琼脂培养基(CSA)。其中1)—6)参考童依婷等(2016)易润华等(2017)。7)和8)分别将20.0 g胡萝卜和豆芽煮为汁液后,加入蔗糖30.0 g和琼脂20.0 g,定容至1 000 mL。

碳氮源:以等质量的葡萄糖(单糖)、果糖(单糖)、麦芽糖(双糖)、乳糖(双糖)、可溶性淀粉(多糖)、甘露醇(醇糖)和山梨醇(醇糖)为碳源替换察氏培养基中的蔗糖(双糖);以等质量的甘氨酸、尿素、酵母膏、硝酸铵、氯化铵和胰蛋白胨为氮源替换察氏培养基中的硝酸钠。

pH:以PDA为培养基,pH分别调至4、5、6、7、8、9和10。

温度:以PDA为培养基,接种病原菌后分别置于5、10、15、20、25、30和35 ℃黑暗培养。

光照:以PDA为培养基,接种后分别置于24 h黑暗、24 h光照、12 h黑暗+12 h光照、24 h黑暗+10 min紫外光、24 h光照+10 min紫外光、12 h黑暗+12 h光照+10 min紫外光。

2 结果与分析 2.1 病害症状

石碌含笑枯梢病主要危害2~4年生的嫁接苗枝梢,发病初期在枝条形成圆形病斑,病斑中央灰白色,边缘漆黑色,病斑扩展缓慢。病原菌侵入后,沿韧皮部与木质部交界处在枝梢内扩展蔓延,导致树皮缓慢脱绿、皱缩,当韧皮部破坏后,造成叶片萎蔫,枝梢枯死(图 1A)。受危害枝条表面由绿变黑,上下裂开(图 1B),流胶,油漆状(图 1C),树皮内部组织崩解,仅留下表层。

图 1 石碌含笑枯梢病病症状 Figure 1 Symptoms of dieback on Michelia shiluensis A:枝梢枯死Die back;B:枯枝Dead wood;C:溃疡与流胶Canker and gummosis.
2.2 病原菌的鉴定 2.2.1 形态学特征

从10条病枝的病组织中分离得到46个菌株,菌落形态特征一致。随机选取了菌株SLHX3、SLHX6、SLHX11和SLHX14接种到健康枝条,室内接种10天后,在枝条上形成水渍状病斑,田间接种1个月后出现枝梢枯死。从人工接种的发病组织中可重新分离到与接种菌相同形态的菌株,对照不发病,证明所分离的菌株为石碌含笑枯梢病的病原菌。

在PDA上菌落2周可长满直径9 cm培养皿(图 2A)。菌落正面边缘整齐,白色,中心为淡黄色,反面中心为黑色,气生菌丝较发达,无色透明。在25 ℃黑暗培养2周后形成分生孢子器,球形或不规则,多室或分散,有孔口,黑色。分泌物从分生孢子器孔口流出,初浅黄色,后变黑(图 2BC)。

图 2 A:枝梢枯死Die back;B:枯枝Dead wood;C:溃疡与流胶Canker and gummosis. Figure 2 Morphological characteristics of pathogen causing dieback on M. shiluensis A: PDA培养基25 ℃黑暗培养20天的菌落Colony on PDA at 25 ℃ in the dark for 20 d;B, C:分生孢子座及分泌物Conidiomata with conidial droplets exuded from ostioles;D-H:分生孢子Conidia(E: α型孢子α-spore;G,H:β型孢子β-spore).标尺Bar: D. 20 μm; E-H. 10 μm.

分生孢子梗无色透明,无分枝,产孢细胞圆柱形,顶端尖细。在PDA培养基中形成α和β 2种孢子(图 2D—H)。α型孢子无色、单胞,纺锤形至椭圆形,含1~2个油球(图 2E),(4.3~8.8)μm ×(1.6~3.7)μm[(6.8±0.9)μm×(2.8±0.5)μm](n>50);β型孢子无色、单胞,线形,一端弯曲呈钩状(图 2 G、H),(16.4~37.7)μm ×(0.7~1.1)μm[(25.1±4.3) μm×(0.9±0.2)μm](n>50)。

在PDA培养基上生长速度为(7.1±0.5)mm·d-1。察氏培养基上菌落形成同心环圈,边缘白色,中心灰白色,反面相同,边缘呈花瓣状,菌丝疏松呈绒毛状,生长速度为(6.9±1.0) mm·d-1。在蛋白胨酵母膏葡萄糖琼脂培养基上,菌落正面呈灰绿色,反面为黄绿色,边缘呈锯齿状,菌落厚实呈棉絮状,生长速度为(5.4±0.6) mm·d-1。玉米粉蔗糖琼脂培养基上菌落稀薄,正反两面皆为乳白色,菌丝稀疏边缘呈放射状,生长速度为(10.7±1.8) mm·d-1,菌丝致死温度51 ℃。

2.2.2 基因序列同源性比较

病原菌ITS、HIS、TEF和TUB基因序列与GenBank的间座壳菌Diaporthe sp.(无性型拟茎点霉Phomopsis sp.)基因序列同源性高于90%。其中,ITS序列(登录号:KY565424、KP677503、KY565425和KY565426)与GenBank中的Diaporthe longicolla菌株A0633、A0643和A0649的序列(KF494833、KF577901和KF577903),D. passifloricola菌株CPC 27480的序列(KX228292)、D. phaseolorum菌株D006的序列(KU377474)、3个内生真菌菌株STRI:ICBG-Panama: TK1037、TK1304和TK356的序列(KF435932、KF436125和KF435178),Phomopsis phaseoli(有性型D. phaseoli)菌株INBio:154B的序列(EU236702)、P. longicolla(有性型D. longicolla)的序列(EU236702)以及Phomopsis sp.14个菌株的序列(如JN153055和KM229696等)同源性为99%。

TUB基因序列(登录号:KY569387、KY569386、KY569385和KY569384)与GenBank中的D. absenteum(D. miriciae)菌株LC3429和D. passifloricola菌株CPC 27480的序列(KP293477和KX228387)同源性为100%。TEF基因序列(登录号:KY569391、KY569390、KY569389和KY569388)与D. absenteum(D. miriciae)菌株LC3564和LC3429的序列(KP267986和KP267971)同源性达100%。HIS基因序列(登录号:KY569395、KY569394、KY569393和KY569392)与D. absenteum(D. miriciae)菌株LC3564和LC3429序列(KP293559和KP293547)同源性为100%。

2.2.3 系统发育分析

根据BLAST结果选择间座壳属(Diaporthe)的65个种、Diaporthella corylina(外群)以及石碌含笑枯梢病菌的ITS、TEF、HIS和TUB序列比对后建立数据矩阵,采用邻接法和贝叶斯法得到2棵系统发育树,石碌含笑枯梢病菌均与D. ueckeraeD. miriciae以高支持率聚集在同一分枝(自展支持率及后验概率分别为99%和1.0)(图 3)。

图 3 基于ITS、TEF、HIS及TUB基因序列构建的系统发育树 Figure 3 Phylogenetic tree reconstructed by neighbor-joining (NJ) and Bayesian inference (BI) using ITS, TEF and TUB sequence dataset 菌株编号上标T表示该菌株为模式菌株The strains with ‘T’ superscript were type strains. ▲:石碌含笑枯梢病菌菌株The isolates of pathogen causing dieback on Michelia shiluensis.分枝上的数字分别代表后验概率和自举值,“-”表示后验概率小于50。The numbers above the branches indicate posterior probability values or bootstrap percentages resulting from different analyses in the order: BI /NJ. The values lower than 50 are given as“-”for BI。

Gao等(2016)对间座壳属的真菌进行系统发育分析时将D. miriciae R.G. Shivas, S.M. Thomps & Y. P.Tan 2015作为一个异名并入D. ueckerae Udayanga et Castlebury 2015,因此根据形态学、基因序列比对以及系统发育分析,将石碌含笑枯梢病菌鉴定为D. ueckerae Udayanga et Castlebury 2015 (Thompson et al., 2015; Udayanga et al, 2015; Gao et al, 2016)。

2.3 生物学特性 2.3.1 培养基

病原菌在测试的8种培养基中生长速度及形成子座的数量差异显著(P<0.05)(图 4)。其中,在SSA、CSA、MSA、PSA、PDA和OMA中,病原菌生长差异不明显,培养7天后,菌落直径分别为7.19、7.72、6.78、6.82、6.96和7.06 cm,在CSA培养基中生长最快;在Czapek和PYGA培养基中生长较慢,分别为3.98和4.46 cm。病原菌在PDA培养基上生长时形成分生孢子座数量最多,而在MSA、OMA和PYGA培养基上则较少。

图 4 培养基对石碌含笑枯梢病菌的生长及形成子座的影响 Figure 4 Effect of culture media on mycelial growth and conidiomata formation of the pathogen SSA:豆芽汁蔗糖培养基Bean-sprout sugar agar;CSA:胡萝卜蔗糖琼脂培养基Carrot sugar agar;MSA:玉米粉蔗糖琼脂培养基Maize sugar agar;PSA:马铃薯蔗糖琼脂培养基Potato sugar agar;PDA:马铃薯葡萄糖琼脂培养基Potato dextrose agar;OMA:燕麦琼脂培养基;Czapek:察氏培养基Czapek medium;PYGA:蛋白胨酵母膏葡萄糖琼脂培养基Peptone yeast extract glucose agar.相同图柱上标有不同字母表示经Duncan氏新复极差法检验差异显著(P<0.05)The different letters above the same column diagram mean significant differences by Duncan’s new multiple range test (P < 0.05).
2.3.2 碳氮源

碳氮源对病原菌生长和形成分生孢子座影响差异显著(表 1)。以淀粉为碳源时,病原菌生长最快,培养7天后,菌落直径为6.14 cm,以甘露醇为碳源形成分生孢子座数量最多;在无碳源培养基中生长速度最慢且不形成分生孢子座。

表 1 碳氮源对石碌含笑枯梢病菌的生长及形成分生孢子座的影响 Tab.1 Effect of carbon and nitrogen sources on mycelial growth and conidiomata formation of the pathogen

以胰蛋白胨为氮源时,病原菌生长最快,其次为牛肉膏,培养7天后,菌落直径分别为7.48和5.16 cm,而以甘氨酸为氮源时生长最慢,但产生分生孢子座最多。

2.3.3 温度和pH

病原菌在温度5~35 ℃条件下均可生长,最适生长温度范围为25~30 ℃,在5 ℃和35 ℃时生长缓慢,25 ℃最有利于生长,菌落直径为7.15 cm。在25 ℃和30 ℃,病原菌的生长速度和形成分生孢子座的数量差异不显著,但在30 ℃生长最快,形成分生孢子座数量最多(表 2),在5、10、15和35 ℃均不形成分生孢子座。

表 2 温度和pH值对石碌含笑枯梢病菌的生长及形成子座的影响 Tab.2 Effect of temperatures and pH values on mycelial growth and conidiomata formation of the pathogen

病原菌在pH4—10时均能生长,pH4—8时生长差异不显著,pH4—10时形成分生孢子座数量差异显著(图 2)。pH为5时生长最快,菌落直径为8.28 cm、pH10时生长最慢。

2.3.4 光照

光照对病原菌生长影响差异不显著,对分生孢子座的形成影响差异显著(图 5)。在不同的光照条件下,病原菌生长速度由快到慢依次为:12 h黑暗+12 h光照+10 min紫外光>24 h光照+10 min紫外光>12 h黑暗+12 h光照>24 h黑暗>24 h黑暗+10 min紫外光>24 h光照,24 h光照最不利于病原菌生长。在24 h光照、24 h黑暗、24 h光照+10 min紫外光和24 h黑暗+10 min紫外光4种光照处理对病原菌形成分生孢子座的数量影响差异不显著(图 5)。12 h黑暗+12 h光照+10 min紫外光和12 h黑暗+12 h光照2种光照处理形成分生孢子座的数量比其他4种光照明显减少。

图 5 光照对石碌含笑枯梢病菌的生长及形成子座的影响 Figure 5 Effect of light treatments on mycelial growth and conidiomata formation of pathogen A: 24 h光照24 h light;B: 24 h黑暗24 h darkness;C: 12 h黑暗+12 h光照Alternation of 12 h illumination and 12 h darkness;D: 24 h光照+10 min紫外光Alternation of 24 h darkness and 10 min ultraviolet radiation;E: 24 h黑暗+10 min紫外光Alternation of 24 h darkness and 10 min ultraviolet radiation;F: 12 h黑暗+12 h光照+10 min紫外光Alternation of 12 h illumination, 12 h darkness and 10 min ultraviolet radiation。相同图柱上标有不同字母表示经Duncan氏新复极差法检验差异显著(P<0.05)。The different letters above the same column diagram mean significant differences by Duncan’s new multiple range test (P < 0.05).
3 讨论

拟茎点霉Phomopsis (Sacc.) Bubák (有性型:间座壳菌Diaporthe Nitschke)是一种世界性、营寄生、腐生和内生生活方式的真菌,寄主范围广,在亚洲,寄主植物约800多种,20多种木兰科植物受Diaporthe americanaD. arctiiD. binoculataD. pterocarpiD. delitescensD. eresPhomopsis liriodendriP. magnoliae(D. magnoliae)、P. magnoliicolaP. magnolinaP. micheliaeP. micheliicolaP. viridarii等多种真菌危害(Farr et al., 2017)。枝梢枯死病是一种常见的植物病害,可由多种病原菌如松壳色二孢菌(Diplodia pinea)(黄敬林等, 2005)、可可球二孢菌(Botryodiplodia theobromae)(岑炳沾等, 1994)、竹喙球菌(Ceratosphaeria phyllostachydis)(邱子林等, 1991)、Neofusicoccum australe(Auger et al., 2013)和葡萄生拟茎点霉菌(Phomopsis viticola)(Úrbez-Torres et al., 2013)等引起。

据报道,D. ueckerae在油茶(Camellia sinensis)(Gao et al., 2016)、香瓜(Cucumis melo)(Udayanga et al., 2015)、大豆(Glycine max)(Thompson et al., 2015; Udayanga et al., 2015)、向日葵(Helianthus annuus)、皱果荠(Rapistrum rugosum)和绿豆(Vigna radiata)(Thompson et al., 2015)等寄主植物上营腐生或内生生活,也可在人体脓疮上生活(Gomes et al., 2013; Udayanga et al., 2015),本研究证实其可侵染石碌含笑导致枝梢枯死。

病原菌在8种测试的培养基中,CSA培养基最适合菌丝生长,在PDA培养基中形成分生孢子座数量最多;25~30 ℃最有利于病原菌的生长和形成分生孢子座;病原菌对pH的适应范围较广,pH 4—8范围菌丝生长差异不明显,形成分生孢子座的数量差异不显著;不同光照处理对病原菌菌丝生长差异不显著,对形成分生孢子座影响差异显著,12 h黑暗+12 h光照+10 min紫外光处理和12 h黑暗+12 h光照处理后产生分生孢子座的数量明显低于其他处理,说明光暗交替不利于病原菌形成产孢体。在广东徐闻,石碌含笑枯梢病主要危害在2~3年生嫁接苗,主要在5—6月最容易发病,可持续发病到11月,这与病原菌在25~30 ℃最有利于病原菌的生长和形成分生孢子座的特点相吻合。有关该菌的生物学特性未见文献报道。

石碌含笑枯梢病菌可在病枝存活很长时间,修剪病枝是控制该病在嫁接苗发生发展的一项重要措施,苯菌灵(benomyl)和烯菌灵(imazalil)等杀菌剂对拟茎点霉菌(有性型:间座壳菌)有抑制作用(Otta et al., 1980; Kliejunas, 1988),但对石碌含笑枯梢病防治的效果需要进一步研究。

4 结论

本研究将从石碌含笑发病的枝条分离得到的菌株接种到健康的寄主,可引起枝梢枯死,确定分离到的菌为致病菌,根据形态学与分子特征将其鉴定为D. ueckerae Udayanga et Castlebury 2015。生物学特性研究结果表明,培养基、温度、pH、碳氮源和光照对病原菌菌丝生长和形成分生孢子座影响差异明显,研究结果可为石碌含笑枯梢病的防控提供理论基础。

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