Detection of Huanglongbing (Citrus Greening) Disease by Nucleic Acid Spot Hybridization Kuraba Gopala,*, Sundeep Sudarsanb, Venati Gopia, Latchireddy Naram Naiduc, Maniyaram Ramaiaha, Yasodam Sreenivasulua, and Edward Wesleyb a Molecular Diagnostics Laboratory, All India Co-ordinated Research Project on Citrus, Citrus Research Station, Andhra Pradesh Horticultural University, Tirupati – 517 502 (A. P.), India. E-mail: [email protected] b Department of Biotechnology, Muthayammal College of Arts & Science, Rasipuram – 637 408 (Tamilnadu), India c Horticultural Research Station, Sithampet, Srikakulam District (A. P.), India * Author for correspondence and reprint requests Z. Naturforsch. 64 c, 711 – 716 (2009); received February 12/April 30, 2009 Polymerase chain reaction (PCR) amplifi cation with primers specifi c to the rDNA region successfully amplifi ed the 1160-bp DNA fragment from a Huanglongbing (HLB)-infected sweet orange sample with mottling symptoms leaves, but not from healthy sweet orange plants. The PCR product of 1160-bp was used as probe labeled with biotin for detection of the HLB pathogen in the nucleic acid spot hybridization (NASH) test. It was found that the HLB pathogen could be detected up to 1:100 dilution in HLB-infected tissue. Total DNA extracted from HLB-infected tissue was diluted 2-fold as 900 ng in TE buffer and spotted on a nitrocellulose membrane. Strong signals were observed up to 225 ng of DNA dilution, whereas a moderate signal was recorded at 112 ng. No hybridization signal was observed in the healthy samples, while strong signals were observed in the positive control. Key words: Huanglongbing, PCR Detection, Nucleic Acid Spot Hybridization, Non-Radio- active Probes Introduction teria. HLB is caused by Candidatus Liberibacter africanums in Africa, L. asiatiums in Asia (Garnier Sweet orange (Citrus sinensis Osbeck) cv. Sath- et al., 2000) and Ca. L. americanus in Brazil (Teix- gudi is an important commercial cultivar in South eira et al., 2005). Two psyllid vectors transmit the India, and Andhra Pradesh (A. P.) occupies the HLB disease. The Asian form of the HLB disease fi rst place in cultivation of Sathgudi. In India is spread rapidly by the Asian psyllid Diaphorina A. P. tops in terms of area followed by Mahar- citri, while the African form of the HLB disease is ashtra, Punjab and Karnataka. Citrus fruits are transmitted by the African psyllid Trioza erytreae. rich in vitamins and minerals and, namely used It is widely accepted that both species of bacte- as fresh juice, for fl avouring dishes of vegetables, ria multiply in both of the psyllid vectors, but this fi sh, meat and salads. The citrus species is prone has not been demonstrated with molecular evi- to 150 diseases and disorders caused by fungi, vi- dence. However, Moll and Martin (1973) noticed ruses, bacterial and phytoplasmal infections. Bud- marked increases in the number of HLB bacteria transmissible diseases like Huanglongbing (HLB), in T. erytreae vectors over 9 days and concluded tristeza, citrus yellow mosaic, exo-cortis and citrus that bacteria were multiplied in these vectors. ring spot cause huge loss in fruit production in all Since the bacterial nature of the HLB organ- the citrus-growing areas. ism was established, Jagoueix et al. (1996) used The HLB disease is easily transmissible by universal primers for general amplifi cation of graft and psyllid vectors from citrus to citrus. It prokaryotic 16S rDNA. Based on the sequence can also be transmitted by dodder, Cuscuta refl - information, primers have been developed to exa. The bacterium-like organism is restricted to amplify a 1160-bp region of ribosomal DNA for phloem sieve tube elements. This bacterium has a detection of the HLB disease by polymerase membranous cell wall of the Gram-negative type chain reaction (PCR). Ribosomal DNA primers and belongs to the subdivision of the Proteobac- have been widely used for detection of all forms 0939 – 5075/2009/0900 – 0711 $ 06.00 © 2009 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D NNC_9_10_2009.indbC_9_10_2009.indb 711711 006.10.20096.10.2009 113:04:273:04:27 712 K. Gopal et al. · Citrus Greening Disease of HLB. These primers have been shown not to tained on different citrus species seedlings in amplify 16S ribosomal sequences of other citrus an insect proof glasshouse. Available glasshouse pathogens (Jagoueix et al., 1996). isolates of the HLB disease were also used for The HLB bacterium infects citrus trees of al- detection of the HLB disease by PCR and nu- most all cultivars and causes substantial econom- cleic acid spot hybridization (NASH) test using ic losses to the citrus industry by shortening the the 726-bp HLB DNA fragment cloned in the lifespan. It is an important epidemic disease and pRTZ5 vector. is diffi cult to control in India and several other Asian countries. Furthermore, the HLB disease DNA extraction has a long incubation period and many latently The total DNA was extracted from midribs of infected citrus plants occur in the fi eld (Mc Clean, HLB-infected sweet orange cv. Sathgudi and from 1970; Ahlawat et al., 1995). The development of healthy plants. The samples were ground to a pow- diagnostics for virus and virus-like diseases of der in liquid nitrogen. 1 ml of extraction buffer citrus and the production of virus-free planting (0.1 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1 M KCl, material is an important strategy and is the fi rst 0.65% sodium sulfi te) heated to 95 ºC was added step for the management of bud-transmissible dis- to the ground tissue in an Eppendorf tube and in- eases (Gopal et al., 2001). However, the detection cubated at 95 ºC for 10 min with occasional agita- of this fastidious bacterium is diffi cult because tion. The homogenate was placed on ice for 2 min of its non-culturability, its low concentration and and centrifuged for 10 min at 12000 × g. The su- uneven distribution in its natural hosts (Su and pernatant was treated with Rnase (100 µg/ml) and Chang, 1974). The disease is diagnosed by bio- the DNA was precipitated with 0.6 vol. of ice-cold logical indexing on indicator hosts, which is time- isopropanol. After centrifugation, sterile distilled consuming, and symptom expressions depend on water was added to the precipitate, and the mix- temperature. The disease, therefore, cannot be di- ture was heated briefl y to 65 ºC to completely dis- agnosed easily by conventional procedures such solve the DNA. DNA was re-precipitated with 2 as electron-microscopic examination of ultra-thin vol. of ethanol and 0.1 vol. of sodium acetate (pH sections and bioassay on indicator plants. As an 5.2) at – 20 ºC for 2 h. DNA was isolated by the alternative, a rapid and reliable detection proto- sodium sulfi te EDTA method used for detection col by PCR was developed. However, high levels of the HLB disease by PCR amplifi cation as re- of polyphenols and tannins in citrus leaves gener- ported by Gopal et al. (2007). ally interfere with obtaining good-quality DNA and thus affect the reliable detection of HLB or- PCR amplifi cation ganisms by PCR. Therefore a method was devel- PCR was performed in 50 µl reaction mixture, oped in which sodium sulfi te is added to the ex- using 1 µM of each primer, 200 µM each of dNTPs, traction buffer EDTA during extraction of DNA 0.05 U/µl of Taq DNA polymerase, 1× PCR re- from HLB-infected leaf midrib tissue (Gopal et action buffer, 1.5 mM of MgCl2 and 6 µl of DNA al., 2004). HLB isolates, namely sweet orange (EF template. The amplifi cation was performed in a 552698) and acid lime (EF 552699), are cloned, thermal cycler (Corbett Research, Mortlake, Aus- sequenced and deposited in the Genbank. Based tralia). PCR conditions used were as follows: 1 cy- on the sequence of the isolates a set of primers cle of 95 ºC for 5 min, 40 cycles of 95 ºC for 1 min, was designed and the amplifi ed product was used 52 ºC for 1 min, 72 ºC for 1 min, and 1 cycle of for preparation of biotin-labeled non-radioactive 72 ºC for 10 min. The PCR product was analyzed probes for detection of the HLB disease. The re- by 1% agarose gel electrophoresis in 1X TAE sults are reported in the present paper. (tri-acetate-EDTA) buffer containing ethidium bromide, and the gel was observed under a UV Material and Methods transilluminator and photographed. Detection of HLB PCR The bud sticks collected from sweet orange Detection of HLB by NASH cv. Sathgudi at All India Co-ordinated Research Non-radioactive labeling of DNA Project on Citrus, Tirupati, India were grafted on Non-radioactive labeling of DNA was done us- 1-year-old seedlings. The HLB culture was main- ing the biotin labeling kit and detection system NNC_9_10_2009.indbC_9_10_2009.indb 712712 006.10.20096.10.2009 113:04:273:04:27 K. Gopal et al. · Citrus Greening Disease 713 kit (Fermentas). All necessary reagents and buff- were removed and the membrane incubated at ers were provided in the kit, and the manufac- 42 ºC for 4 h in a hybridization oven with gentle turer’s protocol was followed as given below. rotation. Probe preparation Hybridization The IMP-agarose block containing the respec- The biotin-labeled DNA probe was denatured tive DNA fragments was cut from the gel and in boiling water for 5 min, added to pre-hybridi- purifi ed using the gel extraction kit (Eppendorf). zation solution (25 – 100 ng/ml) and incubated at The probe was prepared in 50 µl reaction mixture, 42 ºC over night in a hybridization oven with gen- using 10 µl of DNA template (100 ng to 1 µg), tle rotation.