Bhaju K. Tamot, Department of Plant Agriculture, University of Guelph and Peter M. Gresshoff, Plant Molecular Genetics, Institute of Agriculture, The University of Tennessee
A DNA sequence for a putative causative organism of citrus greening disease was detected by DNA Amplification Fingerprinting (DAF). Forty arbitrary oligonucleotide primers ranging front live to twelve nucleotides in length were screened for polymorphisms of DNA from greening disease infected and apparently healthy Citrus reticulata Blanco and C sinensis Osbeck trees. One printer (5’ GGGTAACGCC 3’) generated a pronounced diagnostic band of 200 bp from infected plants which was not present in healthy trees. The diagnostic polymorphic marker band was excised from the silver stained polyacrylamide gel, cloned, and DNA sequenced. The sequence of the DNA amplification product from infected C. sinensis shows homology with bacteria. 200 base pairs are a small fragment of DNA to conclude a possible cause causative organism of Citrus Greening Disease (CGD) yet it shows maximum homology (>80%) with bacteria.
Citrus greening disease (CGD) is a devastating disease problem causing extensive damage to citrus trees in many Asian and African countries. Infection by the greening pathogen becomes apparent only at the fruiting stage when it is beyond control. Fruits remain small, lopsided in shape and green on the shaded side, even after normal period of maturation. A phloem-colonizing microbe has been identified as a bacterium by electron microscopy (Gamier et a!., 1984). Recently, the bacterium was identified as Liberobacler asiaticum in Asian countries and L. africum in South Africa by cloning 16S rRNA gene (Jagoueix et al., 1997). However, it has not yet been culturable in synthetic cell-free media. Accordingly, development of disease symptoms has not been demonstrated in citrus plants by any culturable causative organisms. The greening pathogen is transmitted in Asia by the insect Diaphorina citrii Kuwayama (Rcgnii and Lama, 1988) as well as by means of planting and grafting materials (Jagoueix et al., 1994).
Early diagnosis is not only a critical step towards applying efficient eradication and vector control measures when epidemics occur, but equally important as a measure to preserve germplasm collections of citrus. Over the past 20 years different techniques, such as serological and histo-chemical analysis, antibiotic sensitivity test, indexing techniques, and electron microscopy have been used to detect possible causative agents of the greening disease (Da-Graca, 1991). These techniques are cumbersome and technically complex. Therefore, a rapid, low cost method for early-detecting citrus greening disease without the use of labeled probes and obtaining pure culture is of utmost importance, especially if it is to be applicable to lesser developed countries.
Villechanoux et al. (1992) detected greening disease from different parts of Asia by DNA hybridization with three radio-labeled probes developed by differential hybridization between healthy was carried out in a twin-block Thermocycler (Ericomp Inc., San Diego, CA). Thermocycling was carried with a temperature profile of two step cycles of I s at 96°C and I s at 30°C 35 cycles and 72°C for 5 minutes. Ericomp cyclers are of moderate ramping speed (13 – 18°C per minute).
DNA separation and silver staining
DNA Amplification products were analyzed by polyacrylamide gel electrophoresis (PAGE) (Mini Protein Gel II, Bio Rad). Polyacrylamide gel electrophoresis was carried out in 0.45mm thick gel slab of 4.5% polyacrylamide with a l9:l ratio of acrylamide to the cross-linker piperazine di-acrylamide (Bio Rad), 7 M Urea, TBE (100 mM Tris HCl, 83 mM boric acid and 1 mM Na2EDTA pH 8.3), 10% ammonium per-sulfate and N-N-N’-N’-Tetra-methylethylene-diainine (TEMED). Gels were usually cast onto a gel bond PAG polyester backing film (FMC, Rockland, MB) which was used to support the gel and for further handling. Sample (3.5 ul of approximately 30 ng/ul) was loaded with equal amount of loading buffer (5 M urea and 0.02% xylene cyanole FF) in gels which has been prerun for about 10 min in 1 M TBE electrophoresis buffer (Caetano-Anollés et al., 1991). Electrophoresis was carried out at 118 V for approximately 75 min.
After electrophoresis, the gels were treated with 7.5% acetic acid for 15 min, rinsed in double distilled water three times for two minutes, impregnated in AgNO3 (1 g/liter), 1.5 ml 37% HCOH/liter for 25 min rinsed quickly with water and developed by Na2CO3 (30 g/liter), 2.5 ml 37% HCOH/liter, Na2S2O3.5H2O (2 mg/l) at 8-10°C. The staining reaction was stopped by using cold 7.5% acetic acid (Bassam et al., 1991, Caetano-Anolles and Gresshoff, 1994) and gels were air-dried for preservation.
Isolation, cloning and sequencing of the disease specific DNA amplification product
A-200 bp band size was excised from PAGE gels and subsequently re-amplified with the same primer (5 GGGTAACGCC 3) in a 25 ul reaction volume (Weaver et al., 1994), six times, to produce a single copy band. The amplified DNA product (100 ng) in 7.5 ul water was treated with 1 ul of 10x ligation buffer, 1 ul of l0x dNTP (1 mM each), and 0.5 ul Klenow fragment, incubated at 37°C for 30 minutes for filling blunt ends of DNA for cloning. Klenow fragment was deactivated at 65°C for 10 minutes. Recently, we developed a more efficient band re-isolation method (Men and Gresshoff, 1998).
The DNA fragment was cloned into the Smal site of vector pGEM-3Z according to Promega Technical Bulletin, transferred into JM 109 strain bacteria (Promega) and white colonies containing recombinant plasmids were selected from Isopropylthio- D-galactoside (IPTG) and 5-Bromo-4- chloro-3-indolyl -D-galactosidase (Xgal) containing media. Plasmid DNA of the pGEM-3Z vector was digested with XbaI and KpnI, blotted onto Zeta probe blotting paper (Bio Rad) and hybridized overnight at 65°C with a 32P-adATP labeled amplification product probe, using the random primer labeling kit (Boehringer, Germany).
Plasmid DNA was isolated from 10 ml overnight culture according to the mini—preparation protocol, extracted once with Phenol/ Chloroform/ Isoamyl and once with Chloroform/Isoamyl Alcohol (Sambrook eta!., 1989). DNA was precipitated by 0.1 volume of 3 M NaOAc (pH 4.8) and 0.6 volume of cold isopropanol (-20°C) and incubated in liquid nitrogen for 10 minutes. DNA was pelleted at 4°C and dissolved in 1 ml water, precipitated with 0.5 ml of 30% PEG-8000 in 30 mM MgCl2, incubated 10 minutes at room temperature and spun down at maximum speed for 10 minutes (Nicoletti and Condorell, 1993). The PEG-precipitated DNA was denatured by 2 M NaOH, 2 mM EDTA at room temperature for 5 minutes and neutralized with 3 M NaOAc pH 4.8 for sequencing (Wang and Sodja, 1991). The sequencing reaction was done using the Ml3 reverse primer, 32P-adATP (3000 ci/mmol DuPont NEN, Boston, MA) and sequenase version 2 according to Protocol supplied by United States Biochemical (Cleveland, OH). The reaction product was separated in 5%
Figure 2(a). DNA profiles of greening disease infected (I) and non-infected (NI) citrus plants: Lane C. reticulata (NI); Lane 2, C. reticulate (I), Lane 3, C. sinensis (NI) and Lane 4, C. sinensis (I). Genomic DNA of these plants was amplified with Amplitaq Stoffel Fragment and 5GGGTAACGCC 3’ primer. The line in-between lanes is drawn for quick detection of band of interest. M (Marker-200 bp)
Figure 2(b) DNA profile of greening disease infected and non-infected: Lane I C. reticulata (NI), Lane 2, C reticulata (I), Lane 3, C. sinensis (NI), Lane 4, C sinensis (I), 5 Lane, C. reticulata (I) Pakistan, amplified with Amplitaq DNA polymerase and primer (5’GGGTAACGCC 3’). The line in between lanes is drawn for quick detection of band of interest. M (Marker bp).
It was necessary to confirm whether the polymorphic band (200 bp) both in infected mandarin and orange (Fig. 2a) was a product of the pathogen and not of host plant. Therefore, the amplified DNA was sequenced as described by Weaver et al., 1994. Sequence homology of the 200 bp fragment was searched by using gene bank BLASTn program. The maximum percentage (>80%) of similarity was found with bacteria.
There are no typical symptoms of citrus greening disease for field identification. Zinc and iron deficiency and other pathogens also duplicate greening symptoms. A potentially causative pathogen for citrus greening disease so far was only confirmed by electron microscopy (Villechanoux et al., 1992) and by cloning (Jagoueix et al., 1994 and 1997). Here we have demonstrated that a putative pathogen causing citrus greening disease could be identified by DNA amplification fingerprinting.
The use of silver stained polyacrylamide gel for analyzing DNA amplification products resulted in high resolution of especially lower sized fragments (50-300 bp) of both major and minor bands with a consistent re-production in banding pattern. However, we could not detect such small amplification products in 1.2 % agarose gels. The sensitivity arid reproducibility of the DAF technique for generating fingerprinting of bacteria, fungi and plant and bacteria in symbiotic healthy samples from different regions which indicates that the product was from DNA of causative organism. Since an amplification product (588 bp) was observed only in infected plant, we hybridized the product with five non-infected samples from Pokhara, Lumle and Knoxville and two infected plants of C. reticulata and C. sinensis. Southern blot hybridization of the 32P labeled 588 bp PCR product to total genomic DNA from infected and healthy citrus trees showed preferential hybridization only to infected samples and virtually no signals in healthy plants (data not shown). Our result clearly suggests one type of causative organism which causes severe-damage to citrus plants in South East Asia.
This work was supported byNational Science Foundation (USA) Grant # NSF-INT 9311802. We thank Dr. Iqrar A. Khan for samples from Pakistan and Dr. Prakash M. Pradhanang for helping sample collection in Pokhara and Lumle.
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