Department of Life Science, Fu Jen Catholic University, Hsin Chuang 24205, Taipei, Taiwan, Republic of China.
Appl Environ Microbiol. 2009 Nov;75(21):6831-8. doi: 10.1128/AEM.01653-09. Epub 2009 Sep 11.
A xanthomonad differential medium (designated Xan-D medium) was developed, on which streaks and colonies of xanthomonads, including 13 species of the genus Xanthomonas, turned wet-shining yellow-green and were surrounded with a smaller milky zone and a bigger clear zone in 3 to 4 days. The characteristics could easily be differentiated from those of yellow nonxanthomonads and other bacteria. The mechanism of color change and formation of a milky zone on the medium are mainly due to the Tween 80 hydrolytic capacity of xanthomonads. The gene, estA, responsible for Tween 80 hydrolysis was cloned and expressed in Escherichia coli, which acquired a capacity to hydrolyze Tween 80 and could turn green and form a milky zone on the Xan-D medium. The nucleotide sequence of estA is highly conserved in the xanthomonads, and the sequence was used to design a specific PCR primer set. The PCR amplification using the primer set amplified a 777-bp specific DNA fragment for all xanthomonad strains tested. The Xan-D medium was used to isolate and differentiate Xanthomonas campestris pv. campestris from naturally infected cabbages with black rot symptoms for a rapid diagnosis. All isolated X. campestris pv. campestris strains developed characteristic colonies and were positive in the PCR with the estA primer set. The Xan-D medium was further amended with antibiotics and successfully used for the detection of viable X. campestris pv. campestris cells from plant seeds. Although some yellow nonxanthomonads and other saprophytic bacteria from plant seeds could still grow on the medium, they did not interfere with the color development of X. campestris pv. campestris. However, Stenotrophomonas maltophilia, which is closely related to xanthomonads, existing in a seed lot could also develop yellow-green color but had different colony morphology and was negative in the PCR with the estA primer set. Accordingly, the combination of the Xan-D medium with the estA-specific PCR is a useful and reliable method for the isolation and detection of viable xanthomonad cells from plant materials.
开发了一种黄单胞菌鉴别培养基（命名为 Xan-D 培养基），其上的黄单胞菌条纹和菌落，包括 13 种黄单胞菌属的物种，在 3 至 4 天内变成湿亮的黄绿色，并伴有较小的乳白色区和较大的透明区。这些特征很容易与非黄单胞菌和其他细菌的特征区分开来。培养基上颜色变化和乳白色区形成的机制主要归因于黄单胞菌对吐温 80 的水解能力。负责吐温 80 水解的基因 estA 被克隆并在大肠杆菌中表达，该菌获得了水解吐温 80 的能力，并能在 Xan-D 培养基上变绿并形成乳白色区。estA 的核苷酸序列在黄单胞菌中高度保守，并且该序列被用于设计特定的 PCR 引物对。使用该引物对进行的 PCR 扩增可扩增出所有测试的黄单胞菌菌株的 777bp 特异性 DNA 片段。Xan-D 培养基用于从自然感染黑腐病的白菜中分离和鉴别野油菜黄单胞菌白菜亚种，以便快速诊断。所有分离的野油菜黄单胞菌白菜亚种都形成了特征性的菌落，并且在用 estA 引物对进行的 PCR 中呈阳性。Xan-D 培养基进一步添加了抗生素，并成功用于从植物种子中检测有活力的野油菜黄单胞菌白菜亚种细胞。尽管一些来自植物种子的黄色非黄单胞菌和其他腐生细菌仍能在培养基上生长，但它们不会干扰野油菜黄单胞菌白菜亚种的颜色发育。然而，与黄单胞菌密切相关的嗜麦芽寡养单胞菌也能产生黄绿色，但菌落形态不同，并且在用 estA 引物对进行的 PCR 中呈阴性。因此，Xan-D 培养基与 estA 特异性 PCR 的结合是一种从植物材料中分离和检测有活力的黄单胞菌细胞的有用且可靠的方法。
