Transcript Biological And Molecular Detection Of Some Viroids Infecting Citrus

Biological and Molecular Detection of Some Viroids
in Egypt
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.
RESULTIS & DISSCUSION
INTRODUCTION
Citrus 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.
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.
Symptoms distincted CCaVd in Parsons showing
small gum spots on park tissue
Sever symptoms of CEVd in Etrog/ mexicanlime showing sever
epinasty.
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 ).
MATERIAL AND METHODS
Source 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.
Return polyacrylamid gel electropherosis(R-PAGE)
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).
Dot blot hybridization for detection of
CEVd ( A) and CCaVd (B) from infected Plant using
Non radioactive technique
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.
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