抗生素对桉树内生细菌群落及青枯病抗性的影响

Effects of antibiotics on endophytic bacterial communities in Eucalyptus urophylla and eucalypt resistance to bacterial wilt

为了探明桉树内生细菌群落变化与桉树对青枯病抗性间的关系,试验用加入抗生素的培养基继代培养尾叶桉组培苗,采用PCR-DGGE技术检测了经抗生素处理后桉苗内生细菌的变化,并测定其对青枯病的抗性。结果显示,浓度为60mg·L^(-1)的氯霉素和150 mg·L^(-1)的氨苄青霉素显著降低了桉树组培苗根部内生细菌的数量,但对茎、叶中内生细菌的数量没有影响。DGGE图谱显示,氨苄青霉素处理的桉苗与对照苗的条带基本一致,氯霉素处理后与对照条带有明显的区别,表明氨苄青霉素只影响了桉苗内生细菌的数量,氯霉素使桉苗内生细菌的优势菌发生了变化。对DGGE条带克隆测序和Blast比对发现,A、B、C条带的序列(KU363009、KU363010和KU363011)与GenBank中不可培养细菌的序列(KJ655389.1、KF006350.1和FJ832152.1)相似性分别为100%、99%和99%;D条带序列(KU363012)与不可培养的色球藻属(Chroococcidiopsis sp.)序列(LN878320.1)相似性达到98%。氨苄青霉素和氯霉素处理后,桉苗青枯病的初发病时间与对照相比延后1~2 d,氨苄青霉素处理的桉苗发病率显著低于对照。氯霉素处理的前6 d发病率显著低于对照,之后发病率急剧上升与对照无显著差异。上述结果表明,氨苄青霉素和氯霉素可以减少桉苗根中内生细菌的数量,并推迟桉树青枯病的发病时间。

In order to investigate the relationship between the variation of endophytic bacterial communities in Eucalyptus urophylla and the resistance of eucalypt to bacterial wilt caused by Ralstonia solanacearum, E. urophylla seedlings were cultured on media supplemented with antibiotics. PCR-DGGE were applied for the detection of community changes of endophytic bacteria in eucalypt seedlings treated with antibiotics, and the resistance of the antibiotic-treated eucalypt seedlings to R. solanacearum was also determined. The results showed that the population of endophytic bacteria in the roots of E. urophylla seedlings, treated with either chloramphenicol at 60 mg · L-1 or ampicillin at 150 mg· L-1, were significantly decreased. However, both of the two antibiotic treatments had no effects on the population of endophytic bacteria in the stems and leaves of E. urophylla seedlings. The DGGE pattern of endophytic bacteria in seedlings treated with chloramphenicol was significantly different from that of the non-treated control, whereas treatment with ampicillin did not cause obvious changes in DGGE profiles. The results indicated that ampicillin could only affect the number of endophytic bacteria in eucalypt seedlings, and chloramphenicol caused the change of dominant species in eucalypt seedlings. The Blast-search results of cloning sequence from the DGGE dominant bands demonstrated that bands A, B and C （ Accession No. KU363009, KU363010 and KU363011, respectively） had a high similarity to those unidentified and uncultured bacteria in GenBank （KJ655389.1, KF006350.1 and FJ832152.1） at the homology levels of 100%, 99% and 99%, respectively. The sequence of band D showed a similarity of 98% to the partial 16S rRNA gene （LN878320.1） of uncultured Chroococcidiopsis sp. in GenBank. Compared with the control, eucalypt seedlings treated with either of the antibiotics showed delayed wilting symptoms by 1-2 days. The incidence of bacterial wilt in seedlings treated with ampicillin was significantly lower than that of control, while the incidence treated with chloramphenicol was significantly lower than that of the control for the first 6 days, and since the 7th day the percentage of wilted seedlings showed a dramatic increase and had no significant difference from the control. These results demonstrated that both ampicillin and chloramphenicol could decrease the population of endophytic bacteria in the roots of E. urophylla seedlings and delay the onset of eucalypt bacterial wilt.