| Degradation of coral floras by bleaching had been observed in many different reefs in the South China Sea, and the coverage of live coral had declined by more than30%over the past few decades. In this report, we firstly did some pathological research on the yellow inflammatory-like disease of Porites pukoensis and White Syndrome of P. andrewsi,which distributed in Xisha Archioelago, South China Sea. In addition, we analysed the16S-23S rDNA intergenic spaces (IGS) of the pathogens of the White Syndrome P. andrewsi,The results were as follows:The the yellow inflammatory-like disease was divided into A, B, C, D four levels according to the characteristics of polyp growth, coral skeletal injuries, the appearance of yellow mucus in the early stage and the time it took to restored normal in the late stage. The incidence of survey area was0.13±0.04in May;0.16±0.04in June;0.13±0.04in early July;0.13±0.06in late July;0.14±0.09in early August;0.08±0.02in late August. From May to August, the relationship between incidence and time was irregular, because the physical damage occurred with strong randomness. The results of PCR amplification of the bacteria metagenomic from the yellow mucus using primers16S rDNA and bacterial isolation on the2216E agar plates showed that there were few bacterias in the mucus. The scanning electron microscopy (SEM) figures showed that the lesion of sample was covered with yellow mucus, debris and sediment, but the surface of healthy parts were very clean and coral skeleton and mucus were clearly visible. The yellow mucus were firstly observed in the lesion parts, which were wrapped by new tissues after30days. The results showed that the yellow mucus, probably secreted by polyp, might played a key role in the process of the self-healing of the yellow inflammatory-like disease of P. pukoensis.The results of the transmission experiment in the field showed:no replicates responded in the first five days; seven out of eight (87.5%) responding replicates exhibited symptoms within the first two weeks. The rates of bleaching ranged from0.90to10.76cm2d-1with a mean of5.40±3.34cm2d-1(mean±SD). To identify the coral pathogen according to Koch’s postulates, a highly selective enrichment medium for marine Vibrio spp., thiosulphate citrate bile sucrose (TCBS) agar, was used to isolate test strains from bleached P. Andrewsi. Furthermore, to rapidly isolate highly virulent strains, two-step immersion was selected for the inoculating mixed bacterial suspension, and one-step immersion was utilized for inoculating single bacterial suspensions. After screening, two virulent strains XSBZ03and XSBZ14were identified. The morphology of the dominant colony (more than80%) isolated from the inoculated bleached tissue was the same as that of the original strain used for inoculation. All of the re-isolated colonies of strains XSBZ03and XSBZ14independently caused the same disease sign (bleaching) on the coral in the one-step immersion, respectively. Through field and laboratory examination, two strains of Vibrio alginolyticus were identified as the causative agents of P. andrewsi White Syndrome in the Xisha Archipelago, South China Sea. After pin-pointing the "guilty" group, a second virulence test on each member of that group was conducted. In this way, the efficiency of accurate identification of the bacterial pathogens of coral disease was greatly improved. Our method might be useful for bacterial coral disease studies in the future.Based on the conservative sequences flaning the3’end of the16S and the5’end of the23S rDNAs of Escherichia coli, the authors designed primers and PCR-amplified the16S-23S rDNA intergenic spacers(IGS) of4strains of Vibrio alginolyticus, and cloned the purified PCR production with pMD-T19vector. Different clones were selected for sequencing. Characteristics of the sequences determined by the16S-23S rDNA IGS were examined against GenBank BLAST and http://lowelab.ucsc.edu/tRNAscan-SE/function. Then the ClustalX2, DNAman, Megalign and Editseq were used to analyse them. The results showed: there were33IGSs sequences with different length.Each IGS contained0-4tRNAs and6types of polymorphic IGSs which were recogined,namely, IGS0, IGSG,IGSIA, IGSAG, IGSGLV, IGSGLAV.IGS0,IGSG, IGSIA, IGSAG and IGSGLV appeared to be the most prevalent form of IGS and were found in four strains of V.alginolytics, but IGSGLAV was only belonged to the HN08155. Not only heteromorphic IGSs were various because of the different tRNAs in them, but homotypic-IGSs were also multifarious. For example, IGS0was a major IGS type with the size ranging from419to628bp. The difference of homotypic-IGS between pathogenic bacteria in coral and strains in control included deletion, insertion and substitution of one or more bases and base fragments:the deletion of T in200site of XSBZ014IGS0(429bp), the deletion of TTTTT in517-522site and TCTA in533-538site of XSBZ014IGSAG(637bp), the insertion of TTTTT in239site of XSBZ014IGS0(429bp), the insertion of AGTTTGGTT in142-151site of XSBZ03IGSGLV(842bp), A replaced by T in202site of XSBZ014IGS0(429b p).IGSGLAV, IGSGLV and IGSAG of V. alginolytics were reported for the first time in the world. The structural variation of the spacers would provided the basis of developing diagostic methods at the level of srains. |
PDF Full Text Request