【作者】 王璠；

【导师】 李国怀；

【作者基本信息】 华中农业大学 ， 果树学， 2012， 博士

【摘要】 葡萄座腔菌属(Botryosphaeria)真菌是许多单子叶、双子叶和裸子植物上常见的病原菌、内生菌和腐生菌。能引起许多多年生木本植物产生溃疡、枯萎、坏死和流胶等症状。桃(Prunus persica L.)流胶病过去多认为是逆境和管理粗放等引起的生理性病害,而葡萄座腔菌属真菌在我国作为桃流胶病病原菌并没有进行系统的研究。桃流胶病主要为害枝干,造成主干、主枝等部位树皮产生胶状溢出物,树胶初期无色透明,随时间推移逐渐变成红褐色至深褐色。湖北省是我国重要的桃产区,流胶病发生严重,本研究从湖北省桃流胶病病原学入手,在分生孢子形态学、培养特性、分子系统学和致病性研究的基础上,对桃流胶病菌进行鉴定。同时,设计PCR特异性引物用于病原菌快速检测；对不同桃品种的抗病性进行评价等。主要结果如下：1.我国湖北省桃流胶病菌为Fusicoccum aesculi Corda, Diplodia seriata De Not.和Lasiodiplodia theobromae (Pat.) GrifF&Maubl.。Botryosphaeria spp作为桃流胶病菌最早是20世纪70年代在美国Georgia州报道的。到目前为止,我国报道的引起桃流胶病的该属真菌只有Botryosphaeria dothidea (Moug.) Ces.&De Not.。湖北省是我国桃生产的重要产区,流胶病发生普遍。为了解该省桃流胶病的发生情况,在咸宁市、武汉市、孝感市、随州市、远安县(宜昌市)等地调查了14个果园的1914棵桃树,总发病株率99.2%。从武汉市、咸宁市、孝感市、远安县(宜昌市)、随州市、老河口市(襄阳市)、沙洋县(荆门市)、公安县(荆州市)、枣阳市共采集桃流胶病枝条170份,获得116个Botryosphaeria spp分离株。结合菌落培养特性、分生孢子形态学、致病性测定和rDNA内转录间隔区(rDNAITS, ITS1-5.8S-ITS2)、β微管蛋白(p-tubulin)和α延伸因子(EF1-α)的分子系统学将湖北省桃流胶病菌鉴定为：F. aesculi(有性阶段B. dothidea)、D. seriata(有性阶段B. obtusa (Schwein.) Shoemaker)和L. theobromae(有性阶段B. rhodina (Cooke) Arx),其中F. aesculi分离频率最高,分布最广泛；L. theobromae仅在沙洋县发现；D. seriata在公安县有分布。人工菌丝块接种当年生新梢和多年生枝条结果表明,这三种真菌对桃树都有致病力,能产生褐色病斑；L. theobromae致病力最强,且能引起多年生枝条产生大量胶液；D. seriata致病力最弱。2.通过分生孢子形态学、致病力测定、分子系统学和微卫星分析证明了我国桃流胶病菌F. aesculi的遗传多样性。形态学研究表明,我国桃流胶病菌F. aesculi主要有两种类型的分生孢子,一种是典型的纺锤形至椭圆形、无色透明的无隔Fusicoccum属分生孢子；另一种是纺锤形的一分隔分生孢子,并不是很常见。致病力试验中,18个F. aesculi分离株接种曙光油桃新梢5d时,病斑长度差异显著,最长达40.1mm(菌株XNHG-241),最短仅5.8mm (XNHG-62)。ITS、β-tubulin和EF1-α3个基因的分子系统学分析进一步证明了F. aesculi是我国最常见的桃流胶病菌,分布范围最广,存在种内分化现象。利用微卫星引物M13, T3B和(CTC)4RC获得了DNA指纹图谱,0-1矩阵在SAS8.1软件中Cluster(聚类)和建树(Tree)分析表明我国桃流胶病菌F. aesculi可以分为7类。第一类包括FH-11, MYA-6, HB-14, XG-52, WX-12, XX-25, SHYJ-1, CDA-20, CD-13, HB-11, YLA-6, WX-10, XX-1, WX11-12, SH-15, XX-23, FY-24, FY17, XX-44, JZ-7, GA-323, TA-44, JM-5, WH-1252等24个菌株；第二类包括CH-11, FYY-1, ZJG-42, FH-10, WX-3, JMA-1112等6个菌株；第三类包括菌株WH-1822；第四类包括菌株TA-1；第五类包括菌株JM-20；第六类包括菌株CSJ-10；第七类包括菌株SH-5。3.设计了桃流胶病F. aesculi的特异性引物。在β-tubulin基因序列区域设计了一个正向引物FaF,与非特异性反向引物Bt2b组成引物对,特异性的扩增桃流胶病菌F. aesculi基因组DNA,得到一条大小为322bp条带。参与检测的20个F. aesculi都能扩增出该条带,其它两种桃流胶病菌L. theobromae和D. seriata及桃树和其它植物上的其它属真菌显示为阴性。引物对FaF/Bt2b的灵敏度为浓度1pg/μl的基因组DNA。同时利用裂解法快速提取F. aesculi DNA用于PCR检测。结果表明,建立的桃流胶病菌分子检测方法可用于该病菌的分子检测,快速确定病原菌,及侵染源数量和病害流行的预测。4.结合田间调查和室内接种方法评价了不同桃品种对流胶病的抗病性。修订了桃流胶病病害分级标准,重新评价了7个桃园中的4个桃品种的发病严重程度。结果表明,同一果园病情指数比用旧标准调查的更低,如华中农业大学花果山中砂子早生、曙光油桃和早露蟠三个品种用旧标准调查的病情指数分别为84.0、94、82.8,而修定后调查的病情指数分别为52.1、61.7、43.4,更好地反映了田间发病情况；同一果园不同品种发病严重程度不同,感病性强弱结果为：曙光(油桃)>砂子早生(普通桃)>早露蟠(蟠桃)。同时,采用F. aesculi (菌株XNHG-241)室内人工接种了21个桃品种,所有品种均能发病；比较接种5d时病斑长度,发现曙光油桃最感病,早凤王最耐病。其中感病性曙光油桃>砂子早生>早露蟠,与田间调查结果一致。更多还原

【Abstract】 Species in the genus Botryosphaeria are found on a wide range of monocotyledonous, dicotyledonous and gymnospermous plants as cosmopolitan endophytes, pathogens and saprophytes. Although well-known as pathogens causing canker, blight, dieback and gummosis disease in numerous woody perennial hosts, Botryosphaeria spp. have been overlooked as important causal agents of peach(Prunus persica L.) tree gummosis in China. Peach tree gummosis was thought to be associated with physiological disorder caused by pruning, extensive management or stresses. Disease symptoms are always associated with lenticels, wounds, and include gum formation on trunks, scaffold limbs, and braches, and necrotic lesions. The work presented in this thesis was initiated by the identification of Botryosphaeria spp. causing the disease in Hubei Province, which is one of the major peach producing areas in China, by analysis of conidial morphology, cultural characteristics, and nucleotide sequences of three genomic regions of the internal transcribed spacer region (ITS, ITS1-5.8S-ITS2), a partial sequence of β-tubulin gene, and the translation elongation factor1-α gene (EF1-α). And then, identification, pathogenicity and virulence of Botryosphaeria spp. in China, specific primer for PCR detection, and the fungal gummosis susceptibility of peach cultivars were also studied in this paper. The results were summarized as follows:1. Botryosphaeria spp. have been identified as causal agents of peach tree gummosis in Georgia, USA since1970s. To date, B. dothidea (Moug.) Ces.&De Not. has been reported as the only species associated with cankers on P. persica in China. Hubei Province is one of the most important peach producing areas in China. Fungal gummosis on trunks and branches has become a growing threat to the peach industry in this province and throughout Southern China. In order to determine the occurrence of gummosis and Botryosphaeria species in Hubei Province, a field survey was conducted of1,914trees in14peach orchards throughout the province. Gummosis was observed in1,899of1,914trees examined. A total of170samples of branches with gummosis were collected from orchards in Wuhan City, Xianning City, Xiaogan City, Yuan’an County (Yichang City), Suizhou City, Laohekou City, Shayang County (Jingmen City), Gong’an County (Jingzhou City), Zaoyang City, covering an area of300km by260km.. A total of116isolates of Botryosphaeria spp. were obtained. Culture characteristics, conidial morphology, pathogenicity, along with phylogenetics analysis of ITS, β-tubulin and EFl-a showed that at least three Botryosphaeria species occur on peach trees in Hubei Province, including B. dothidea (anamorph Fusicoccum aesculi Corda), B. rhodina (Berk.&M. A. Curtis)(anamorph Lasiodiplodia theobromae (Pat.) Griffon&Maubl.) and B. obtusa (Schwein.)(Diplodia seriata De Not.). Fusicoccum aesculi was isolated in all regions, but L. theobromae was found only in Shayang County in Jingmen City, and D. seriata only in Gong’an in Jingzhou City. Via artificial inoculation using mycelia on wounded twigs or branches, these three species were all found to be pathogenic, causing dark lesions and, sometimes, with gum exudation from diseased parts. Isolates of L. theobromae were the most virulent and caused most copious gum, and D. seriata had less gum than the other two species.2. The genetic diversity of F. aesculi isolated from different regions in China was studied based on conidial morphology, pathogenicity. phylogenetics and microsatellites analysis. The results showed that:in culture, isolates of F. aesculi had two types of conidia, namely fusiform, ellipsoid or ovoid, hyaline, aseptate conidia typical of the genus Fusicoccum, and fusiform, one septate conidia that are not typically observed. Five days after inoculation, vascular discoloration developed on peach twigs inoculated with each of the isolates of F. aesculi. There was significant difference in length of vascular discoloration among the18isolates ranging from5.8mm to40.1mm. Phylogenetic analysis combining three gene sequences of ITS, P-tubulin and EF1-α revealed the three Botryosphaeria species distributed in China, and differentiation existed in the isolates of F. aesculi. In the meantime, DNA fingerprinting produced by microsatellite primer M13, T3B, or (CTC)4RC were applied to analyze the difference among the isolates of F. aesculi. Seven groups were divided by Single Linkage in Cluster progress and Tree progress in SAS8.1software. Type one comprised of24isolates; type two comprised of6isolates. The other five types comprised of isolate WH-1822, TA-1, JM-20, CSJ-10and SH-5, respectively. The four aspects of conidial morphology, pathogenicity, phylogeny and microsatellite analysis confirmed the genetic diversity among isolates of F. aesculi in China.3. Fusicoccum aesculi is the most common pathogen of Botryosphaeria spp. associated with peach tree gummosis in China. In this study, a polymerase chain reaction (PCR)-based assay was developed for the specific detection of the fungal pathogen F. aesculi from peach trees. Variation among β-tubulin gene sequences of Botryosphaeria spp. was exploited to design primer pairs. Forward primer FaF, when used with the nonspecific reverse primer Bt2b, amplified a322bp DNA fragment for all20tested isolates of F. aseculi collected from peach trees. Specificity was also confirmed using those of closely related fungi including L. theobromae and D. seriata, and the other three fungal species isolated from peach or other hosts. The PCR assay using this pair of primers was sensitive enough to detect1pg/μl of genomic DNA of F. aesculi. A rapid DNA extraction procedure was applied to detect and identify F. aesculi from peach. These results indicate that this new primer is potentially useful for studies in areas or hosts in which the pathogen may be present, and prediction of inoculum production and disease development.4. Surveys in the field and experiments in doors were conducted to evaluate the peach cultivar susceptibility. A new grading standard for disease incidence was developed to score the severity of peach trees in the field. Seven peach orchards were surveyed, revealing that less severity of the same orchards compared with previous results, which was more effective and reliable for evaluation. Peach tree fungal gummosis, incited by Botryosphaeria spp., significantly depresses growth and yield on susceptible peach cultivars, on which little is known about the relative susceptibility in China. As F. aesculi is the most common species in peach trees,21cultivars were tested for susceptibility via artificial inoculation with F. aesculi isolate XNHG-241, which was identified as strong virulence in previous studies. Peach twigs inoculated with F. aesculi were placed in the plastic boxes in25℃for5days. Disease severity was evaluated by the length of vascular discoloration. Significant variation in disease susceptibility was detected across cultivars. Of the21cultivars tested, none of them was immune, and ’Shuguang’ was the most susceptible and ’Zaofengwang’ the least. The results of indoor tests were consistent with those in the field.更多还原