Deng X, Zhou G, Li H, Chen J, Civerolo E L
Laboratory of Citrus Huanglongbing Research, Department of Plant Pathology, South China Agricultural University, Guangzhou, Guangdong 510642, P. R. China.
Crop Diseases, Pests, and Genetics Research Unit, San Joaquin Valley Agricultural Sciences Center, United States Department of Agriculture-Agricultural Research Service, Parlier, CA 93648.
Plant Dis. 2007 Aug;91(8):1051. doi: 10.1094/PDIS-91-8-1051C.
Murraya paniculata (orange jasmine) is a popular ornamental rutaceaous plant and is known to be a preferred host for the Asian citrus psyllid, Diaphorina citri (Kuwayana), the primary vector of 'Candidatus Liberibacter spp.' that causes citrus Huanglongbing (HLB). HLB is a highly destructive citrus disease worldwide. However, the presence of 'Ca. Liberibacter spp.' in M. paniculata remains uncertain (2). Clarification of M. paniculata as a host of 'Ca. Liberibacter spp.' has direct impact on HLB control programs. During June of 2006, we identified three M. paniculata trees near a mandarin orchard affected by HLB in Luoding City and two trees from Guangzhou City, Guangdong Province, People's Republic of China. All trees had leaves showing yellowing and mottling symptoms characteristic of HLB. Both symptomatic and asymptomatic leaves were collected. DNA was extracted using the CTAB (cetyltrimethylammoniumbromide) method and assayed by nested-PCR. The general bacterial 16S rDNA primer set fDl/rD1 (3) was used for the first round of amplification. Amplification was conducted as previously described (1), and 2 μl of PCR reaction product were used for a second round of amplification using the same procedure but with 35 PCR cycles with primer set OI1/OI2c (3,4). After agarose gel electrophoresis and staining with ethidium bromide, a 1.1-kb DNA band was unambiguously associated with symptomatic but not asymptomatic leaf samples. Nonnested-PCR using primer set OI1/OI2c alone did not yield a target DNA band or yielded a very weak DNA band. XbaI digestion of the nested-PCR DNA product yielded two fragments, 520 and 640 bp long, characteristic of 'Ca. L. asiaticus'. PCR amplicons were sequenced and were 1,095 bp long. This sequence shared >98% similarity to sequences of 'Ca. L. asiaticus' in the GenBank database. We observed that nested-PCR is necessary for consistent amplification of DNA from 'Ca. L. asiaticus' from M. paniculata. We excluded the possible nonspecific amplification associated with nested-PCR by XbaI restriction enzyme digestion and by nucleotide sequence analysis. Our data indicate that M. paniculata is a host of 'Ca. L. asiaticus' but the bacterial titer might be low. References: (1) X. Deng et al. Online publication. doi:10.1094/PHP-2007-0419-01-BR. Plant Health Progress, 2007. (2) M. Garnier and J. Bove. Huanglongbing (Greening). Page 46 in: Compendium of Citrus Diseases. 2nd ed. L. W. Timmer et al., eds. The American Phytopathological Society, St. Paul, MN, 2000. (3) S. Jagoueix et al. Int. J. Syst. Bacteriol. 44:379, 1994. (4) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996.
九里香是一种受欢迎的芸香科观赏植物，已知它是亚洲柑橘木虱（Diaphorina citri (Kuwayana)）的首选寄主，亚洲柑橘木虱是引起柑橘黄龙病（HLB）的“类立克次氏体菌属（Candidatus Liberibacter spp.）”的主要传播媒介。HLB是一种在全球范围内极具破坏性的柑橘病害。然而，九里香中是否存在“类立克次氏体菌属”仍不确定（2）。明确九里香作为“类立克次氏体菌属”的寄主对HLB防控计划有直接影响。2006年6月，我们在广东省罗定市一个受HLB影响的柑橘园附近鉴定出3株九里香树，并从中华人民共和国广东省广州市鉴定出2株。所有植株的叶片均呈现出HLB特有的黄化和斑驳症状。采集了有症状和无症状的叶片。使用CTAB（十六烷基三甲基溴化铵）法提取DNA，并通过巢式PCR进行检测。第一轮扩增使用通用细菌16S rDNA引物对fD1/rD1（3）。扩增按照先前描述的方法进行（1），取2 μl PCR反应产物用于第二轮扩增，使用相同程序，但使用引物对OI1/OI2c进行35个PCR循环（3,4）。经琼脂糖凝胶电泳并用溴化乙锭染色后，一条1.1 kb的DNA条带明确与有症状而非无症状的叶片样本相关。单独使用引物对OI1/OI2c进行非巢式PCR未产生目标DNA条带或产生非常微弱的DNA条带。巢式PCR DNA产物经XbaI酶切产生两个片段，长度分别为520和640 bp，这是“亚洲类立克次氏体菌（Ca. L. asiaticus）”的特征。对PCR扩增产物进行测序，长度为1,095 bp。该序列与GenBank数据库中“亚洲类立克次氏体菌”的序列相似度>98%。我们观察到巢式PCR对于从九里香中一致扩增“亚洲类立克次氏体菌”的DNA是必要的。我们通过XbaI限制性内切酶消化和核苷酸序列分析排除了与巢式PCR相关的可能非特异性扩增。我们的数据表明九里香是“亚洲类立克次氏体菌”的寄主，但细菌滴度可能较低。参考文献：（1）X. Deng等人。在线发表。doi:10.1094/PHP - 2007 - 0419 - 01 - BR。《植物健康进展》，2007年。（2）M. Garnier和J. Bove。黄龙病（黄化病）。见：《柑橘病害汇编》第2版。L. W. Timmer等人编。美国植物病理学会，明尼苏达州圣保罗，2000年，第46页。（3）S. Jagoueix等人。《国际系统细菌学杂志》44:379，1994年。（4）S. Jagoueix等人。《分子细胞探针》10:43，1996年。
