Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/17/2008
Publication Date: 2/10/2009
Citation: Chen, J., Deng,, X., Liu,, S., Pu,, X., Li,, H., Civerolo, E.L. 2009. A phytoplasma related to Candidatus Phytoplasma asteri is associated with citrus showing Huanglongbing (yellow shoot disease) symptoms in Guangdong, P. R. China. Phytopathology. 99:236-242. Interpretive Summary: Citrus Huanglongbing (HLB), meaning yellow shoot disease, was first described in Guangdong, P. R. China. It is also called greening disease in South Africa. HLB is a highly destructive in citrus production worldwide. HLB was found in Florida in 2005, but not in any other area of U.S. Studies on HLB are challenging because the true nature of the HLB pathogen remains unclear. The best data currently available point to a group of unculturable bacteria, Candidatus Liberibacter spp., that are associated with HLB. While detection of Ca. Liberibacter spp. has been helpful in diagnosis of HLB, many researchers have experienced failure of Ca. Liberibacter spp. detection in citrus trees showing typical HLB symptoms. In this study, we carried out two surveys in Guangdong of China. We found that 14.2 percent samples were positive for Ca. Liberibacter asiaticus only, 29.1 percent were positive for phytoplasma only, 48.9 percent were positive for both Ca. Liberibacter asiaticus and phytoplasma and 7.8 percent of the samples were negative for both Ca. Liberibacter asiaticus and phytoplasma. A total of 78.0 percent of samples were phytoplasma positive and a total of 63.1 percent of samples were Ca. Liberibacter positive. Data from this study showed that in addition to Ca. L. asiaticus, a phytoplasma related to Ca. P. asteri was also associated with citrus HLB in Guangdong, P. R. China.
Technical Abstract: Huanglongbing (HLB) or yellow shoot disease (ex. greening disease) is highly destructive to citrus production worldwide. Understanding the etiology of HLB is critical for managing the disease. HLB is currently known to be associated with infection by Candidatus Liberibacter asiaticus in China. However, Koch’s postulates have not been fulfilled demonstrating that Ca. L. asiaticus is the etiological agent of HLB. In addition, it remains unclear if other plant pathogens are also involved in HLB. In a survey performed in Guangdong Province, P. R. China in 2006 and 2007, 141 citrus samples showing typical symptoms of HLB from 11 different cities were collected. PCR using phytoplasma specific primer sets fU5/rU3 nested with primer set P1/P7 identified 110 (78.0%) positive samples. A 1,785 bp amplicon was obtained with primer set P1/P7. Analysis showed a 100% identity of this sequence to three strains of Candidatus Phytoplasma asteri [onion yellows (Japan), aster yellows ‘watercress’ (Hawaii), and valeriana yellows (Lithuania)] in the region of 16S rDNA and 16S-23S intergenic spacer. Meanwhile, a total of 89 (63.1%) samples were positive for Ca. Liberibacter asiaticus. When both phytoplasma and Ca. Liberibacter asiaticus were considered, 14.2% samples were positive for Ca. Liberibacter asiaticus only, 29.1% were positive for phytoplasma only, 48.9% were positive for both and 7.8% of the samples were negative for both. Examination by transmission electron microscopy (TEM) showed the presence of both walled and wall-less bodies in phloem of symptomatic citrus. The association of phytoplasma with HLB was further demonstrated using the model host periwinkle [Catharanthus roseus (L.) G. Don.]. HLB-associated bacteria were transmitted from symptomatic citrus to periwinkle through dodder (Cuscuta campestris Yunck). PCR detected the same phytoplasma in the affected periwinkle, along with Ca. L. asiaticus. In addition to yellowing/mottling, the infected periwinkle showed typical symptoms of virescence and phyllody that are commonly associated with phytoplasmal diseases. TEM analysis of affected periwinkle revealed bacteria-like organisms with pleomorphic morphology, characteristic of phytoplasmas. Data from this study showed that in addition to Ca. L. asiaticus, a phytoplasma related to Ca. P. asteri was also associated with citrus HLB in Guangdong.