PCR-DGGE指纹图谱技术分析发病龙须菜附生菌菌群组成

本研究以07-2品系龙须菜(Gracilaria lemaneiformis)为实验材料,提取自然条件下代表不同病变程度的健康、病间、白化藻体的附生菌DNA,经PCR扩增16S r DNA基因的V3可变区并进行变性梯度凝胶电泳(DGGE),分离V3片段,再对目的条带进行克隆鉴定。实验结果经数据化后计算龙须菜附生菌数目并比较附生菌菌群多样性,通过非度量多维尺度分析(n MDS)及多元梯度分析(RDA)来比较附生菌菌群组成相似性及优势菌群。实验结果表明,随着龙须菜病变程度的加深,附生菌菌落数及菌群多样性逐渐增加(P0.05)。n MDS分析结果表明,健康藻体与白化藻体在附生菌菌群组成上存在差异,病间藻体在附生菌菌群组成上与健康藻体更为接近。RDA分析结果表明,龙须菜健康藻体附生菌可能的优势菌群是海洋单胞菌属(Marinomonas),病间藻体附生菌可能的优势菌群是单胞菌属(Alteromonas)、红杆菌科(Rhodobacteraceae)、假交替单胞菌属(Pseudoalteromonas),白化藻体附生菌可能的优势菌未能鉴定到,而白化残留藻体附生菌鉴定到的优势菌有Maribacter、红杆菌科、玫瑰变色菌属(Roseovarius)。从上述结果可以看出,龙须菜健康藻体与发病藻体在附生菌菌群组成及优势菌上存在较大差异,而介于两者之间的藻体在菌群组成上更偏向于健康藻体。

Analysis of the bacterial community composition of the epiphytes on diseased Gracilaria lemaneiformis using PCR-DGGE fingerprinting technology

Gracilaria lemaneiformis, one of the important sources of agar, is the third most widely cultivated seaweed in China, after Laminaria and Laver. In recent years, there has been a large-scale decrease in the cultivation of G. lemaneiformis, resulting in huge economic losses for the seaweed cultivation industry. Epiphytes grow as an integral part on the surfaces of large algae, and the variations in the seaweed-associated microbial community compositions are typically associated with disease occurrence. In this study, the G. lemaneiformis 07-2 strain was used as experimental material. DNA of epiphytes from the healthy, adjacent, and bleached fronds was extracted, and the V3 variables amplified from 16S rDNA genes were carried out by denaturing gradient gel electrophoresis (DGGE). Finally, the 23 objective bands were cloned and identified. The number and diversity index of the bacterial community of the epiphytes from the three fronds were calculated after digitization of the experimental results. The similarity among the dominant species in the bacterial communities were compared by non-metric multidimensional scaling (nMDS) and redundancy analysis (RDA). The results show that the colony forming units and bacterial community diversity increased gradually with the aggravation in the degree of lesion in G. lemaneiformis (P<0.05). The nMDS results indicate that the bacterial community composition of the epiphytes varies between the healthy and bleached fronds, while the composition of adjacent fronds is closer compared with that between healthy fronds. The RDA results reveal that the potential dominant bacteria of the epiphytes of the healthy and adjacent fronds are Marinomonas and Alteromonas, Rhodobacteraceae, and Pseudoalteromonas, respectively. However, the potential dominant bacteria of the epiphytes of the bleached fronds were not identified. The dominant bacteria of the epiphytes of the bleached attached fronds were Maribacter, Rhodobacteraceae, and Roseovarius. It can be seen from the above results that there is a significant difference between the healthy fronds and the diseased fronds of G. lemaneiformis in terms of the bacterial community composition and dominant bacteria of the epiphytes, while the epiphytic bacterial community composition of the adjacent fronds was more similar than that of the healthy fronds.