M.A. Amer1, H.Fahmy2 , A. El-Hawary2 and Kh.A. El-Dougdoug3

1 Molecular Biology Lab., Virus and Phytoplasma Dept., Plant Pathology Research Inst., ARC. 2 Citrus Certification Center, Bahteem and 3 Depart. of Microbiology, Fac. of Agric., Ain Shams Univ., Cairo,

Egypt.

INTRODUCTIONCitrus Exocortis Viroid (CEVd) , the causal agent of citrus Exocortis

diseases is an infectious single stranded circular RNA (Semanick, 1980 ). The disease affects Poncirus trifoliate and most of its hybrids, especially the citranges, Rangpur lime, Palestine sweet lime, some citrons and lemons and a number of other citrus varieties. It is symptom-less in many varieties, such as sweet and sour oranges, grapefruits, mandarins and rough lemon. Symptoms of Exocortis includes stunting , bark splitting and scaling of the rootstock portion of the tree. (Roistacher , 1995). Citrus cachexia Viroid ( CCaVd) , the causal agent of cachexia (Xyloporosis ) disease of citrus, is also an infectious single stranded circular RNA (Roistacher , 1988 ; Semancik et al., 1988). CCaVd ( also known a citrus viroid II b ) ( CVIIb) is a few nucleotides shorter than the closely related citrus viroid IIa( CVIIa) which causes a mild form of citrus exocortis disease (Semancik et al., 1988). Both CCaVd and CVIIa are variants of the hop stunt viroid (HSVd) ( Diener et al., 1988; Duran-Vila et al., 1989). In Egypt it has been found that both viroids among other graft transmissible diseases are affecting citrus industry. (Nour El-Din, 1959 ). Diagnosis of those diseases were depending on visual observation and in some cases biological indexing. Recently new techniques of molecular biology has been applied to detect the causal agent for those diseases such as sPAGE , rPAGE, RT-PCR and hybridization.(El-Dougdoug et al., 1997; Hala, 1999 ; Fahmy et al., 2001) .The highly rate of infection by viroids in citrus that has been reported. (Fahmy et al., 2001), can be referred to infection transmitted by contaminated tools which is linked with agricultural practices in Egypt which could be avoided by using hypochlorite to sterilized tools before use it in field.(Roistacher , 1993). The aim of this work to apply biological indexing and molecular techniques to detect viroids affecting two varieties that has been selected as a primary candidate for citrus certification programme from the varietals collection of horticultural Research Institute in Giza.

MATERIAL AND METHODSSource of Viroid isolates:

Two candidate , Cleopatra Mandarin and Balady orange from Horticultural Research Institute ( varietals collection ) were sampled as reported by Roistacher , 1995. Biological indexing to woody indicators :Four budsticks were collected from each quadrant of the suspected tree. Tools were dipped with a 1% sodium hypochlorite solution when going from tree to tree. The Etrog citron bud is then grafted to a vigorous seedling rootstock of rough and Volkameriana lemon. This is the same procedure used for forcing the Parson’s Special mandarin bud in the Cachexia index. Two positive controls were used ( California ). They were kept under temperatures 32-38°C maximum during the day and 27-30°C minimum at night. (Roistacher, 1993).Infected mandarin and Navel orange leaves were extracted and mechanically inoculated on the herbaceous plants Chrysanthemum monifolium , Gynura arantica , Lycopersicum esculantum cv. Castle rock and Potinia hybrida plants by multiple puncture through an inoculum droplet on the emerging true leaves. They were kept under temperatures 32-38°C maximum during the day and 27-30°C minimum at night. (Denier, 1979). RNA extraction for return polyacrylamide gel electrophoresis(R-PAGE)

Low molecular weight RNA was extracted from infected citrus tree leaves by phenol /chloroform extraction and LiCl fractionation (Owens and Diener, 1984; Schumacher et al.,1986; Singh and Boucher, 1987).cDNA synthesis , amplification of cEVd and CCaVd :

one ul of total RNA was added to the RT-PCR reaction mixture containing 5 ul of 10 X PCR buffer, 3 ul of 25 mM MgCl2, 3 ul of 10 mMdNTPs, 1 ul of reverse and forwered primer for CEVd and CCaVd ( Yang et al., 1992) , I ul RNaasin , 1 ul M-MLVarse ( 200 U/ ul) , 1 ul Taq DNA polymerase ( 5U/ul) and nuclease free water to a final volume of 50 ul in a PCR tube. PCR tubes were transferred to the thermocyler set with the following parameters : denaturation at 94 C for 30 sec, annealing at 55 C for 30 sec and extenstions at 72 C for 45 sec, for 35 cyles with a final extenstion at 72 C for 10 min. Analysis of RT-PCR amplified products:

Aliquots of 5 ul of RT-PCR amplified products were analysed on 1 % agarose gel in TBE buffer at 100 V for 1 h. gel was stained with ethedium bromide. 50 bp PCR marker (promega) was used to determine the size of RT-PCR amplified products. Preparation of cDNA probes and dot blot hybridization:

The obtained insert from RT-PCR products were labeled using the following PCR technique using the GeniusTM System (Boehringer Mannheim Corp.) according to the manufacturer’s instructions.The preparation of tissue extracts for dot blots was carried out according to Loebenstein et al.,1997. Prehybridization and hybridization :

The applied technique was performed according to Sambrook et al., 1982 using the GeniusTM System (Boehringer Mannheim Corp.) according to the manufacturer’s instructions.

CONCLUSION

Citrus exocortis Pospiviroid(CEVd) and Citrus cachexia Hostuviroid (CCaVd) were detected in Naval Orange (Citrus sinensis L.) and Mandarin (C. relicalata Blanco) exhibited citrus viroid like symptoms respectively , using biological indexing and molecular techniques. The results of indexing showing twisting and epinasty on Etrog (C. medica ) and Parson`s Special Warm Manaren . As well mechanically inoculated on Chrysanthemum monifolium , Gynura arantica , Lycopersicum esculantum cv. Castle rock and Potinia hybrida plants with clarified extracts of infected citrus leaves, symptoms were nearly the same (leaf rugosity, twisting and dark color on top leaves). A low molecular weigh ribonucleic acid ( RNA) with structural properties of viroids were detected in puified RNA using return polyacrylamide gel electrophoresis (R-PAGE). Using reverse transcription polymerase chain reaction (RT-PCR) and specific primers for CEVd and hop stunt Hostuviroid for CCaVd, a major of cDNA product of approximately 370 and 300 nucleotides ( full length) respectively were detected in purified RNA. Molecular dot blot hybridization assay using digoxigenin labeled cDNA probe for CEVd and CCaVd has been applied for the detection of CEVd and CCaVd in a small amounts of infected tissues. No hybridization reaction was observed between probes and uninfected plant materials.

Return polyacrylamid gel electropherosis(R-PAGE) analysis of CEVd and CCaVd . RNA extracted from infected citron tree. In certain cases, a viroid like

nucleic acid could be visual in low molecular weight RNA prearations from diseased citrus. R-PAGE

analysis of one such RNA preparation revealed the presence of a viroid whose mobility was very similar to

that of the CEVd ( lane 1&3 ) and CCaVd ( lane 4).Uninfected plant materia ( lane 3 ).

Agarose gel electrophersis (%) analysis for CEVd cDNA and HSVd related CCaVd amplified products obtained from 1 ug were analysed on 1 % agarose gel electropherosis. Results shows that the full leangth size of CEVd.cDNA (approximately 375 bp) as a sharp band (Lane 4 &5). The same procedures were carried out on HSVd related CCaVd cDNA. Results shows that the full leangth of CCaVD cDNA (approximately 300 bp ( Lane 1&2)) were detected in infected tissues. Uninfected plant material ( Lane 3).

Symptoms distincted CCaVd on citrus trees naturally infection . (A) small gum spots on bark tissue of naval tree

and (B) pink (pits and coloration on scion xylem) on mandarin tree.

Biological and Molecular Detection of Some Viroids in Egypt

REFERENCES

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Dot blot hybridization for detection of CEVd ( A) and CCaVd (B) from infected Plant using Non radioactive technique

RESULTIS & DISSCUSION

Sever symptoms of CEVd in Etrog/ mexicanlime showing sever

epinasty.

Symptoms distincted CCaVd in Parsons showing small gum spots on park tissue