Determining of Molecular Characterization of Citrus Greening Disease in Southern Kerman

HLB (Huanglongbing ex greening) caused by three species of bacterium Candidatus Liberibacter asiaticus, Ca. L. africanus and Ca. L. americanus, is the most important citrus disease over the globe. The causal agent is transmitted by two psyllids Diaphorina citri and Trioza erytreae and infected bud woods. The causal agent of HLB disease was identified as a phloem-restricted, Gram-negative bacterium belonging to a new genus in the α-Proteobacteria subdivision. In Asia, the pathogen of HLB was categorized as the Candidatus Liberibacter asiaticus transmitted by the Asian citrus psyllid (Diaphorina citri). This was also reported in the south of Iran over 2000. Diagnosis of HLB disease can be difficult under field conditions when relying on visual surveys. This is due to its low concentration in its citrus hosts and the nonspecific nature of HLB symptoms, similarity of its symptoms to micronutrient deficiency such as zinc, magnesium, and iron and virus-like disease symptoms such as stubborn caused by Spiroplasma citri. Therefore, distinguishing causal agents of similar symptoms such as nutritional or stress related symptoms from HLB disease needs a robust procedure. Many detection methods have been used to detect Candidatus Liberibacter spp. including biological indexing (grafting and vector), electronic microscopy (EM), DNA probe specific to the bacterium, enzyme- linked Immunosorbent assay (ELISA). However, TEM methods are less practical, because ultra-thin sectioning is tedious and requires expensive equipment. Transmission tests are of limited value due to the latency and the long incubation period in insect and plant. Development of conventional polymerase chain reaction (PCR) test has great advantages of analyzing the bacterium at genetic level. The objective of this study was the detection of HLB in symptomatic citrus plants based on conventional polymerase chain reaction assay (PCR) in the south of Iran and the comparison of Iranian strains with other strains on the globe. In order to detect, identify and characterize the diseases, leaf and fruit samples were collected from some suspected sites situated in the southern Kerman. Samples were stored in plastic bags and transferred into the laboratory and conserved at low temperature (60C) before DNA extraction. Total DNA was extracted from symptomatic samples on the basis of the method/protocol of Murray and Thompson (1980) with minor modifications. Briefly, 5 to 10 symptomatic leaves were washed with sterile water and dried on paper. The leaf midrib tissue derived from field plants was cut out by sterile scalpel and CTAB buffer added with addition of B-mercaptoethanol. The extract was transferred to a new tube and incubated at 65 0c for 15min. The other steps were conducted according to the original protocol. Existence of pathogen in samples was confirmed using PCR with primers A2 / J5 and OI1 / OI2c. The PCR products were analyzed by gel electrophoresis using a 1% agarose in TBE buffer (Tris base, boric acid and 0.5M EDTA [pH 8.0]) and stained with ethidium bromide. Gel was visualized and analyzed by the GEL documentation.12 of 48 samples amplified with primers above and the disease was confirmed. By sequencing of PCR products with length of 1077 bp and comparison with the strains positive control, also production two fragments of 640 bp and 520 bp resulting from the digestion with XbaI PCR products, the agent of disease was found to be Candidatus Liberibacter asiaticus. The agent showed 100% homology with standard HLB Asiatic type. BLAST analysis showed that the nucleotide sequences obtained for the ribosomal protein (GenBank Accessions No. GN 049632) had 100% identity with sequences of ‘Ca. L. asiaticus’ from China (DQ431997), Taiwan (AB555707), Indonesia (AB480102), Florida (CP001677), and Brazil (AY91933). The Asian vector of HLB, Diaphorina citri was reported in 2000, therefore, the diseases might be distributed in other areas in the southern Iran. Thus, detection of HLB disease in young citrus plants is important to prevent a widespread outbreak of this disease. The results also showed that Iranian strain belongs to Asian type of Liberibacter and nominated Candidatus Liberibacter asiaticus. In the southern Iran, Diaphorina citri with high ability to spread the Candidatus Liberibacter asiaticus is found in most of citrus cultivating areas which implies a high risk of rapid dissemination. Therefore, the survey of the disease by an accurate and sensitive method is recommended for the disease detection in new areas and eradication of infected trees.