| 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. |
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