【作者】 刘利平；

【导师】 邓子牛；

【作者基本信息】 湖南农业大学 ， 园艺学， 2013， 博士

【摘要】 柑橘溃疡病(Xanthomonas axonopdis pv. citri)是世界性的检疫性病害,为柑橘最重要的病害之一,已对多数柑橘主产国造成严重危害。湖南是中国主要的柑橘产区之一,全省推广的冰糖橙和脐橙等主栽甜橙品种受溃疡病的危害尤为严重。国内外对柑橘溃疡病的研究已开展了大量工作,但是溃疡病的致病机理尚不清楚,因此田间的防控效果不理想。因此,本研究从病原、寄主和环境三方面进行多因素的全面分析,探讨影响溃疡病菌在不同品种上侵染力差异的各项因素,同时开展致病相关因子的研究,以期对溃疡病的田间防治提供指导,对溃疡病菌与寄主互作及致病机理的研究提供理论基础。本文从病原和寄主的生物学特性与侵染力的关系分析到转录因子对病原菌致病性调控进行了较为系统的研究,基本达到了预期的研究目标,其主要研究结果如下：1.溃疡病菌在离体培养条件下培养12-18h时致病力最强,培养60h以后致病力明显下降。浓度是决定发病速度和程度的关键,在寄主器官没有伤口时,需要高浓度(108CFU/ml及以上)的足量病原菌(人工接种5μL)才能引起典型的溃疡病症状,并且浓度越高,发病越快。寄主受机械损伤有利于病原菌的侵染,注射接种或者通过针刺、摩擦等方法给寄主造成创伤后接种,只需102CFU/ml浓度的溃疡病菌就足以引发病害。因此,病原菌本身的生长发育状态,以及接触到寄主表面时的病原浓度是决定溃疡病菌在寄主上侵染程度的关键因子,而机械损伤则有利于增加溃疡病菌在寄主上侵染的严重性。2.采用多种接种方法接种不同柑橘种类品种叶片,冰糖橙、脐橙等甜橙品种最易感病,田间较少感病的温州蜜柑等宽皮柑橘人工接种时还是会表现典型的溃疡病症状,田间一般不感病的金柑,接种后仍不易感病,抗病的枸橼C-05在接种后,接种处全部出现褐化死亡的过敏反应,表现出主动抗病。所以,品种感病性差异与自身的遗传因素相关。3.在温室条件下,采用108CFU/ml的溃疡病菌进行喷雾接种,冰糖橙新抽生而未展开的叶片感病率较低；展开达到最大叶面积1/4的叶片和1/2的叶片感病最为严重；完全展开达到最大叶面积但未转绿的叶片也有发病,且发病率较低；完全展开达到最大叶面积且已转绿的叶片几乎没有病斑产生。而抗病的枸橼C-05仅在处于幼嫩阶段的新叶上出现少数褐化死亡的斑点,所有的叶片均无典型溃疡病斑产生。该结果说明抗病品种的抗病性不受组织器官的发育阶段影响,而溃疡病在感病品种上的侵染力差异受叶龄影响。4.冰糖橙、纽荷尔脐橙、糖橙、宫本温州蜜柑、浏阳金柑和枸橼C-05等6个柑橘属种类品种中,以浏阳金柑气孔密度最小,为353.85个/mm2,其它5个感病性差异较大的种类品种气孔密度在515-551个/mm2之间,无显著差异,且在相同环境条件下,品种之间的气孔开张度也没有显著差异。因此,品种间的气孔的密度和开张度与品种感病性的差异没有必然的联系。5.在叶面不受伤时,喷雾接种108CFU/ml的溃疡病菌后,扫描电镜和EGFP标记菌示踪均观察到溃疡病菌在感病的冰糖橙和抗病的枸橼C-05叶面均能生长,菌体在叶面随机分布,没有气孔趋向性,没有识别气孔的能力。因此,根据以上结果认为柑橘气孔的形态特征和开闭特性不宜作为评价寄主对溃疡病的感病性或抗病性的指标。6.喷雾接种EGFP标记病原菌后,在激光共聚焦显微镜下观察到溃疡病菌在冰糖橙和枸橼叶面均能正常生长,其分布与扫描电镜所观察到的结果一致：但是,通过注谢和针刺接种EGFP标记菌时,在激光共聚焦显微镜下观察到溃疡病菌在冰糖橙叶片的叶肉组织增殖迅速,很快引发病害,而在枸橼C-05的叶肉组织中,溃疡病菌的生长受到抑制,最终未能诱发病症。结合其他接种结果分析,说明溃疡病菌的侵染确实没有气孔偏好性,且枸橼C-05在叶表面没有阻止细菌进入的物理障碍,而是在细菌进入叶肉组织后抑制其进一步的繁衍定殖。7.根据气象因素与发病规律的分析,病害发生前均需要雨水,平均气温接近20℃时,春梢与夏秋梢一样发病,在发病期间,一直都伴随着不同大小的刮风天气。因此,水和温度是柑橘溃疡病发生的必要条件,风则是加重病害发生的辅助条件。所以,当温度即回升至20℃时,在降雨之前必须进行药剂喷施以预防柑橘溃疡病。8.根据已公布的柑橘溃疡病菌基因组序列的信息,通过生物学相关软件进行分析,发现柑橘溃疡病菌基因组中的7个GntR转录因子,分属4类亚家族,其中有2个(XAC-0568和XAC0877)属于FadR亚家族,3个(XAC0711.XAC1640和XAC3532)属于HutC亚家族；1个(XAC0737)属于MocR亚家族,1个(XAC1548)属于YtrA亚家族。通过对同源基因的分析,发现转录因子XAC1548与十字花科中的转录调控因子XC2736相似率高达98%,该因子证实参与了致病性调控；同时,在XAC1548基因上游启动子序列有一个不严谨的与致病相关的植物诱导启动子(PIP box),这一切表明,XAC1548可能参与了溃疡病的致病性调控。9.将柑橘溃疡病菌GntR基因家族的XA C1548命名为gntR1Xac,且成功构建了gntR1Xac基因敲除载体,获得了gntR1Xac缺失的溃疡病菌,活体接种试验表明缺失突变体丧失了在感病泳糖橙上的致病性,这意味着XAC1548转录因子在溃疡病的致病过程中参与了调控作用。更多还原

【Abstract】 Citrus canker（Xanthomonas axonopdis pv. citri, Xac） is a quarantine disease worldwide, one of the most serious diseases to citrus industry causing great damages to numerous citrus producing countries. Especially, the infected fruits loss their commercial value and are restricted to export to the fresh fruit market where citrus grows. Hunan province is one of the main citrus producers in China, and the rapidly expanding sweet orange cultivars, like’Bingtang’and navel orange, have been seriously infected by canker disease in the field. There has been difficulty to effectively control the disease, because the reason of heavy disease outbreak on sweet orange is not clear. A great number of researches have been carried out on citrus canker disease, the results, however, are often contradictory in the point of evaluating the susceptibility and resistance of the host plants. The mechanism, therefore, of pathogenesis and resistance related to the disease, and the interaction between the pathogen and the host have remained backward. It is necessary to conduct series of researches to comprehensively investigate factors associated with pathogenicity in order to provide useful information for the disease control and technical background for further studies on the interaction between the pathogen and the host, and on the mechanism of pathogenensis. In this study, systematical investigations were performed starting from the biology of the pathogen and the host towards the regulation of pathogenesis by transcription factors. Expected research purpose has been achieved and the main results are following:1The bacteria cultured in vitro for12-18h have the highest virulence, while those cultured for60h substantially lose their pathogenicity. The inoculation concentration of the pathogen is determinant for the speed and severity of the disease development. When the host is not wounded, enough quantity （5ul） of higher concentration （108CFU/ml or more） should be inoculated to cause canker symptoms. The higher is the inoculumn concentration, the shorter time needs for disease development. Wounding the host tissues provided great assistance for the bacteria invasion. Only102CFU/ml of pathogen bacteria by infiltration inoculation can cause disease symptom. So. the development stage of pathogen and the bacteria concentration adhered on the host surface are the key factors to determine the severity of infection, and the mechanical damages is the important condition for the severity of infection.2With different inoculation methods on leaves at various developmental stages in greenhouse.’Bingtang’ sweet orange and’ Newhall’ navel orange showed severe disease symptoms;’Miyamoto’ satsuma. less infected in field, appeared typical symptoms; and Liuyang kumquat is hardly infected:no symptoms appeared on the leaves of citron C-05at all but with hypersensitive response to the inoculation. Therefore, the genetic background of the host determines the occurrence and the severity of the disease.3In greenhouse, leaves of’Bingtang’sweet orange at different ages on the new shoots were inoculated by spraying with10CFU/ml Xac suspensions. The results indicated that the young leaves un-expanded had low infection rate, those of1/2to1/4full size leaves were highly susceptible, while full size but not matured leaves were less susceptible; the mature leaves were not infected. This is a little different from the observation in the orchard, where the leaf reached full size but not mature is heavily infected. According to the results, for the susceptible varieties, the control of the disease should be performed from the bud burst through the whole shoot developmental period. The similar inoculation was done on citron C-05, only little brown and dead spots appeared in the young laves, but the leaves at all the developmental stages did not show any typical symptom, meaning that the resistance of this genotype is not related to shoot growing stage.4Among’ Bingtang’ sweet orange,’Newhall’navel orange,’Succari’ sweet orange.’Miyamoto’ satsuma mandarin, Liuyang kumquat and citron C-05, only Liuyang kumquat possesses significantly lower stoma density with No.353.85/mm2, the remaining5genotypes had No.515-551/mm" without any significant differences. Under uniform environmental conditions, all the citrus genotypes showed similar stomata opening-closure trends in the whole day. So, the stomata structure does not have substantial effects on the susceptibility of host variety.5When leaf was not wounded, spray inoculation with108CFU/ml pathogen, it was observed under SEM and by monitoring with EGFP labeled Xac that the bacteria grew normally on the leaf surfaces of both suceptible’Bingtang’ sweet orange and the resistant citron C-05. The bacteria distributed randomly on the leaves without special choice to go through the stomata. These results indicated that the stomata characters are not the indicators of resistance or susceptibility of certain citrus genotype. 6With the analysis on the relationship between disease occurrence and the meteorological factors, it was observed that rainfall is necessary before the occurrence of canker disease, and the spring flash can be also infected, as well as summer and autumn shoots when the temperature reached about20℃. Wind seems co-exist with rainfall during the disease development period. Therefore, rainfall and temperature are the requirements for canker disease outbreak, while the wind is the supplementary condition. So, before rainfall arrives with temperature about20℃, practice should be performed to control the disease.7When the labeled bacteria were inoculated by spraying on leaf surface of’Bingtang’ sweet orange and citron C-05, the bacteria can grew normally and the diffusion of Xac on the leaf surface was the same as observed with SME. When the labeled pathogen was inoculated by infiltration into ’Bingtang’ sweet orange, the propagation of Xac inside the mesophyll was clearly observed after inoculation with high speed. While in citron inoculated by the same method, the bacteria growth were inhibited and the disease did not develop. Here it is clearer that the resistance of citron C-05does not rely on any physical obstacles on leaf surface to prevent the entrance of pathogen, but on the action to restrict the bacteria to grow in mesophyll.8Based on the Xac genomic information, seven GntR family transcription factors were detected in Xac genome with bioinformatic analysis software. These seven factors are further classified into four sub-families, i.e. XAC-0568and XAC0877in FadR subfamily; XAC0711, XAC1640and XAC3532in HutC subfamily; XAC0737in MocR subfamily and XAC1548in YtrA subfamilies. Based on homological researches, it was found that XAC1548possesses98%of homology with XC₂736from Brassica and the later was proven to play an important role in the pathogenic process. Meanwhile, there is a PIP （Plant-inducible promoter） box, which is related to pathogenesis, in the promoter sequence upstream the XAC1548gene. All these results indicated that XAC1548may participate in pathogenesis of Xac. This is the first characterized GntR transcription factor in Xac.9The gene of XAC1548is named gntR1Xac-and the knock out of the gene was realized in Xac. The gntR1Xac-mutated Xac was obtained. By in vivo inoculation assay, it was observed that the Xac mutant lost its pathogenecity, implying the role of the XAC1548in pathogenesis of Xac. This is the first Xac GntR transcription factor that is verified for its regulation function of pathogenesis. This result will provide basis of further researches on pathogenesis in Xac.更多还原