Plant
Viruses and Viroids in Tunisia: development of a certification programme for
plant propagation materials
By H. Fakhfakh, F. Gorsane, H. Acheche, A. Elleuch, F. Djilani,
I. Fekih-Hassen, M. Makni & M. Marrakchi
Laboratory of
Molecular Genetics, Immunology and Biotechnology
Faculty of Sciences of Tunis, 2092 Elmanar Tunis, Tunisia
Introduction
In Tunisia, during the last decade, vegetable (pepper, potato, tomato and lettuce), grape and citrus production have been developed through increased cultivated areas of field and greenhouse crops. Heavy losses caused by viral infections are observed mainly in field crops, but also in early greenhouse winter crops, because seedlings are not raised under insect-proof conditions and are exposed to infection. The severity of losses also appears to be directly related to the lack of certified seed and to cultivation methods.
Several Tunisian growing regions were surveyed, and serological and molecular techniques were used to detect viral and viroidal infections in the crops and to characterise the biological and molecular variability of a collection of isolates, selected so as to cover the geographical diversity of these pathogens in Tunisia. These experiments demonstrated the presence of both viruses and viroids isolates in Tunisia and revealed a worrying situation for the future of vegetable, grape and citrus cultivation in this country, if appropriate control measures are not taken.
1. Vegetable, grape and citrus cultivation
Vegetables, grapes and citrus fruits are among the most important horticultural commodities in Tunisia. These crops are a vital source of human nutrition and contribute significantly to the cash income of farmers in Tunisia. The process of exchanging plant materials is laborious, expensive and time consuming. In most cases, quarantine procedures significantly delay the release of imported materials. Traditional procedures for examining plant materials are quite slow and often insufficient to detect diseases, especially virus and viroid diseases.
Currently, there is an increasing demand in Tunisia
for locally produced fruits and vegetables and for their propagation materials,
both for export and local markets. There are two different laboratories producing
tissue culture propagation materials which are supposed to be disease-free.
These laboratories contribute towards producing a small proportion of the needed
vegetable material, grape seedlings and potato tubers. In addition, there is
a continuous demand in Tunisia for new commercial cultivars and an expanded
germplasm base for breeding programmes. Pathogen-testing programmes to ensure
that only disease-free material will be distributed through local nurseries
are of critical importance, but at the same time they are a hindrance to effective
production. Viral and viroidal infections present an almost intractable problem
to the Tunisian government, as several months or even years would be required
to complete testing for these pathogens in locally produced or imported germplasm
and produce virus or viroid-free plants. Thus, it is vital that pathogen testing
and control procedures be improved. The development of processes to detect viruses
and viroids rapidly and accurately using molecular biology and biotechnology
techniques will provide regulatory agencies with the necessary tools to shorten
and improve their procedures for detection and diagnosis.
Tunisia produces 190,000, 700,000 and 30,000 tons of pepper, tomato and potato,
respectively. For citrus and grapes, these values are estimated at 240,000 tons
and 12 Q/ha, respectively. Tunisia imports potato tubers, vegetable seeds, bulbs
and seedlings of ornamental plants and fruit tree seedlings.
Most, if not all, economically important crop plants are subject to attack by
several viruses and viroids which reduce both the yield and quality of the crop.
Vegetatively propagated plants, such as the potato, are severely damaged when
grown from infected plant materials. Viruses transmitted through true seeds,
or through distributed seedlings such as those of lettuce and tomatoes, can
cause even greater losses in a crop, especially when they are also transmitted
by insects. The use of virus- and viroid-free plants and the elimination of
virus and viroid sources by early diagnosis have already made a considerable
positive impact worldwide on yield and quality of some crops. The most important
virus problems affecting agricultural crops in Tunisia are: for tomatoes and
peppers: tomato yellow leaf curl virus (TYLCV), pepper veinal mottle virus (PVMV),
potato virus Y (PVY), tomato spotted wilt virus (TSWV), tomato mosaic virus
(ToMV), cucumber mosaic virus (CMV); for potatoes: potato virus Y (PVY), potato
leafroll virus (PLRV); for lettuce: lettuce mosaic virus (LMV); for grapes:
australian grapevine viroid (AGVd), grapevine yellow speckle viroid (GYSVd),
for citrus : citrus exocortis viroid (CEVd) and hop stunt viroid (HSVd).
The need for releasing and producing virus and viroid-free plant material either
from imported or from local stocks requires the development and improvment of
the certification program. Until now, the quarantine and control service of
Tunisia undertakes certification of such products using biological or serological
methods. Such methods are not expensive but time-consuming and subject to many
problems of sensitivity. Recently, improved knowledge of pathogen genomes and
optimisation of molecular biology and biotechnology techniques applied to the
diagnosis of plant pathogens have stimulated studies in order to develop diagnostic
methods that are sensitive, reliable and less time-consuming for detecting,
and ultimately controlling, economically important pathogens affecting selected
agricultural crops. The plant pathogen detection team of our laboratory is working
on standardizing the developed protocols and transferring them to the quarantine
service, together with the acquisition of skills in their use through training.
This will enable Tunisia to streamline its indexing programmes and quarantine
procedures.
2. Major viruses and viroids of the selected crops
Lettuce
Lettuce mosaic virus (LMV) is potentially the most destructive virus
of lettuce (Lactuca sativa L.) and has a worldwide distribution. It is
transmitted through seed and by aphids in a non-persistent manner (Dinant and
Lot, 1992; Zerbini et al., 1995; Tomlinson, 1970). Field symptoms include
vein clearing, mosaic or mottling and/or leaf deformation and necrosis. Infection
can severely damage the crop and considerably affect the yield. LMV can be controlled
in areas where preventive measures can be applied; however, it remains a damaging
virus to lettuce in regions where such programmes are lacking. LMV is a member
of the genus Potyvirus family Potyviridae (Tomlinson, 1964). Its
single-stranded genomic RNA is typically about 10kb and contains a single large
open reading frame encoding a polyprotein. It has a viral-encoded protein (VPg)
linked to its 5' end and is polyadenylated at its 3' end (Shukla et al.,
1994). Resistance to LMV in lettuce is linked to the recessive genes g
and mo, considered to be allelic or very closely linked (Pink et al.,
1992) and now denoted mo1¹ and mo1². LMV is biologically variable
and its isolates have been classified into pathotypes, according to their virulence
to particular lettuce varieties (Pink et al., 1992; Dinant and Lot, 1992;
Bos et al., 1994; Revers et al., 1997a). Of particular concern
are the seed-transmissible isolates, here referred to as MOST (mo-breaking,
seed-transmitted), that are able completely to overcome mo1¹ and mo1²
resistance genes.
Potato
Potato leaf roll virus (PLRV), a Luteovirus, can reduce yields, tuber
size and internal quality. The genome of PLRV consists of a single molecule
of positive sense ssRNA with a 5' linked VPg and no poly (A) sequence (Mayo
et al., 1982). This virus is detected in Tunisia. Many studies are carried
on this virus in order to characterise Tunisian isolates.
Potato virus Y (PVY), a Potyvirus, is a ubiquitous plant virus
capable of infecting a large range of plants in several families such as the
Solanaceae (potato, tobacco, tomato and pepper). PVY isolates can be classified
into pathotypes such as O, N, C, NTN (Barnett, 1992). The PVY genome is a positive-sense,
single-stranded RNA of approximately 9.7 kb with a genome-linked protein (VPg)
at the 5'terminus and a poly (A) tail at the 3' terminus of the RNA molecule
(Hari, 1995). This virus, also infecting peppers and tomatoes, has been studied.
Molecular variability was performed by sequence analysis (Fakhfakh et al.,
1995), and monoclonal antibodies have been produced that enable the differentiation
of many isolates.
Tomatoes and Peppers
In the last few years, tomato and pepper crops in Tunisia have frequently been
infected with viruses, causing heavy losses and a dramatic reduction of cropping
areas. Major viruses responsible for these losses are cucumber mosaic virus
(CMV), pepper veinal mottle virus (PVMV), and tomato yellow leaf curl virus
(TYLCV).
Cucumber mosaic virus (CMV) is a tripartite, single-stranded (+)-sense RNA virus (Kaper and Waterworth, 1981; Palukaitis et al., 1992). Its three genomic RNAs have been designated as RNA1, 2 and 3, and are 3.4, 3.0, and 2.2 kb in length, respectively (Peden and Symons, 1973). CMV has the largest host range of any virus, infecting 775 plant species in 85 families and 365 genera, including both monocotyledons and dicotyledons (Kaper and Waterworth, 1981). CMV is transmitted in a non-persistent manner by more than 60 aphid species. Tissue culture, ELISA and RT-PCR are techniques used in combative strategies. An RT-PCR assay was developed for the detection of CMV.
Pepper veinal mottle virus (PVMV), a member of the Potyvirus genus, is an endemic virus in pepper and other solanaceous crops mainly in West African countries. It is readily sap transmissible to a narrow range of hosts. Transmission by aphids occurs in a non-persistant manner. Its has a positive single-stranded polyadenylated RNA genome of about 10 Kb and a VPg protein covalently linked to the 5' end. PVMV shares several properties with other potyviruses such as the formation of viral inclusion bodies called "pinwheels" in the cytoplasm of infected cells. However, PVMV is serologically unrelated to several pepper infecting potyviruses such as PVY, type member of this genus, CVMV, TEV, and PeMV.
Tomato yellow leaf curl virus (TYLCV) has been extensively studied in the Middle East, where losses of nearly 100% have been commonly reported. The most serious viral disease of tomatoes in the Mediterranean region, the virus is transmitted by the whitefly (Bemisia tabaci). This virus is a ssDNA geminivirus spreading in Middle Eastern countries (Navot et al.,1991; Nakhla et al., 1994). Represented by isolates different from those spreading in the Middle East, this virus is also present in Tunisia. Molecular methods for its detection by both DNA hybridization and PCR have been developed, providing evidence that this virus has been introduced into the Western Hemisphere, causing heavy losses. Using specific primers, this virus was identified in Tunisia. According to sequence analysis, Tunisian isolates are homologous to Sardinian ones. This work is conducted in collaboration with the department of plant pathology of the Wisconsin-Madison University.
Grapes
The most important diseases of grapevine in Tunisia are: fanleaf, caused by
GFLV; leafroll, caused by GLRaV; stem pitting complex, particularly Kobber Stem
Grooving, caused by GVA; and Corky Bark, caused by GVB (Acheche et al.,
1998).
Citrus and Grapes
Viroids are the smallest known pathogens and have only been found in plants.
They consist of a small, circular, single-stranded infective RNA, ranging in
size from 246 nucleotides in Avocado Sun Blotch Viroid (ASBVd) (Symons, 1981)
to 399 in Chrysanthemum Chlorotic Mottle Viroid (ChCMVd) (Navarro and Flores,
1997). Unlike viruses, they do not have a protein coat protein and are not known
to encode any proteins; hence they must rely on host enzymes for their biological
function (Wan Chow Wash and Symons, 1997). Viroids cause serious diseases in
economically important crops such as potato, tomato, fruit trees (mainly palms,
citrus, apple, peach, grapevine) and ornamental plants. Five viroids can infect
grapevine (Rezain et al., 1992). These are Hop Stunt Viroid (HSVd), Citrus
Exocortis Viroid (CEVd), the two grapevine yellow speckle viroids (GYSVd-1 and
GYSVd-2), and Australian Grapevine Viroid (AGVd). Grapevine viroids are subdivided
into three groups based on their homology within the central domain of the viroid
molecule (Szychowski et al., 1998). They have never been reported in
North Africa. Expression of yellow speckle is ephemeral and mostly evident at
the end of summer, indicating that symptoms are strongly influenced by climatic
conditions. Experiments have shown that Vein-Banding disease results from a
synergistic reaction between grapevine viroids and Grapevine Fan Leaf Virus
(GFLV) (Szychowski et al., 1998).
The various viroids detected in citrus plants are divided into five groups based on physical properties, migration rates in gel electrophoresis, nucleotide sequence similarity and biological properties. These groups are: citrus exocortis viroid group (CEVd), citrus viroid group I (CVd-I), citrus viroid group II (CVd-II), citrus viroid group III (CVd-III) and citrus viroid group IV. Citrus exocortis is a disease known to be caused by the viroid complex in which CEVd is the main component. Exocortis is distributed worldwide. Infected susceptible plants show symptoms of bark scaling on the rootstock and general stunting.
Since most measures for the control of virus and viroidal diseases are based on prevention rather than cure, it is essential to have reliable and sensitive methods for pathogen detection.
3. Survey of the present situation
LMV: The recent world-wide emergence of highly pathogenic and resistance-breaking isolates of LMV is alarming. These isolates, able to overcome the resistance genes mo1¹ or mo1², have a clear potential to be spread to new areas through the distribution of contaminated seed lots. This new situation has increased interest in developing tools for the study of the epidemiological properties of LMV isolates and for a better understanding of correlations among biological properties such as resistance-breaking, seed-transmissibility and molecular characteristics. We have tested a large number of samples collected in different lettuce-growing regions of Tunisia for the presence of LMV. The tissue-blotting assay proved simple, fast, reliable and very economical. As previously reported (Lin et al., 1990), this simple technique offers numerous advantages when a large number of samples need to be processed rapidly. In this respect, this technique should prove very useful for epidemiological studies of LMV in the future. The sanitary status of lettuce crops in Tunisia seems satisfactory, as none of 174 random samples of symptomless lettuce was infected with LMV. Analysis of the symptomatic samples indicated that at least during our survey season, LMV was the major virus infecting lettuce in Tunisia, since it was observed in half of the symptomatic samples. Comparison of the prevalence of symptoms in fields planted with susceptible or LMV-resistant varieties indicate that the mo1¹ and mo1² resistance genes still afford a significant degree of protection against LMV infection in Tunisia. Among the representative LMV isolates finally analysed, many of them induced very severe symptoms on the susceptible cv. Trocadéro and were able to overcome the mo1¹ resistance gene of cv. Mantilia. Using a combination of RT-PCR coupled with either RFLP analysis or direct sequencing proved equally effective for defining molecular affinities between LMV isolates. In particular, both identified all isolates as belonging to the Western Europe group, and within this group, many isolates were close to MOST ones. Whereas all other isolates were close to the common isolate and can be efficiently controlled by the deployment of varieties carrying either form of the mo1¹ resistance gene. Analysis of the phylogenetic affinities of the mo1¹-overcoming isolates shows that they are very closely related to known MOST isolates. The very close molecular relationships observed (sequence identity in the short region analysed) indicates that they are seed-transmitted. The presence of such isolates clearly represents a threat to the Tunisian lettuce industry. Improvement of the sanitary situation of lettuce crops in Tunisia calls for more widespread use of certified lettuce seed, if further entry of similar resistance-breaking isolates is to be averted (Fakhfakh et al., 2001; Krause-Sakate et al., 2002).
PVMV: Three Tunisian PVMV isolates identified in pepper and tomato fields and one isolate from Ivory Coast were subjected to biological and molecular analyses. Since these isolates induced different symptoms on tobacco, they were biologically clustered into two major groups according to their severity. As no PVMV sequence data are available, detailed sequence comparisons of coat protein gene (CP) and 3'-non translated region (3'NTR) were made. Results suggest that the 3'NTR region of the RNA genome may be directly involved in the modulation of disease symptoms (Gorsane et al., 1999 ; 2001).
CMV: Biological and molecular analyses, based on symptom expression and restriction enzyme patterns of coat protein PCR product, were carried out to differentiate Tunisian CMV isolates. Amplification of the coat protein gene of Tunisian CMV isolates was successfully performed using RT-PCR on viral ssRNA. This region is 870 nucleotides in size. Analysis of restriction fragments using HpaII digestions supports the assignment of the Tunisian CMV isolates to two distinct groups. The CMV-Tu1 isolate (severe subgroup, S-I) and six other Tunisian CMV isolates produced only two restriction fragments after HpaII digestion, but the CMV-Tu2 and CMV-Tu3 isolates (attenuate subgroup, S-II) and three other Tunisian CMV isolates produced many restriction fragments after HpaII digestion. This analysis revealed a correlation between HpaII restriction pattern and symptom expression. Hybridization with a specific probe showed that none of the three Tunisian CMV isolates, representing S-I and S-II subgroups, and none of twenty other isolates, was associated with a satellite RNA. Consequently, differences in symptom expression between the two Tunisian groups of isolates were not correlated with the presence of RNA satellite. This may be due to the ability of CMV Tunisian isolates neither to carry nor to support the accumulation of satellite RNA. The ultimate goal of this study is to develop a simple but reliable method for the diagnosis and control of CMV infection and classification of CMV isolates into groups. RT-PCR-RFLP could be seen as an appropriate method for detecting and typing CMV isolates. This method, when extended to many genome regions, will be helpful in detecting the emergence of new viral strains with new biological properties (Fakhfakh et al., 1999).
Grapevine viruses: Detection of GLRaV3 in infected grapevine plants was performed. Viral RNA was successfully detected not only in total crude nucleic acid extracts of infected grapevine tissues but also in viruliferous mealybug vector extracts by RT-PCR. This detection was rapid, sensitive and specific without the occurrence of any background. Comparative ELISA, RT-PCR and IC-RT-PCR assays were carried out and revealed the greater sensitivity and specificity of PCR techniques (Acheche et al., 1999). Furthermore, we have optimized the experimental conditions for molecular procedures: IC-RT-PCR and RNA-capture RT-PCR. These techniques were effective to detect GFLV viruses with greater sensitivity and specificity than the direct RT-PCR and ELISA methods. A comparative study between IC-RT-PCR and RNA capture RT-PCR showed the efficiency of the nucleic acid capture procedure. Furthermore, we have identified the virus from this nematode vector using the optimized molecular techniques (Fattouch et al., 2001). All these viruses were detected in mixed infection on grapevine showing rugose wood (M'hirsi et al., 2001). Sequences of GLRaV3 and GFLV genome were cloned and analyzed by RFLP. These studies demonstrate the presence of molecular variants within these viruses. Expression of Coat viral protein in E. coli was also performed. This constitutes an excellent tool for the improvement of diagnosis methods.
Viroids: In order to develop rapid and specific detection techniques for viroids infecting citrus and grapevine cultures, we have compared biological and molecular protocols. Ideally, these procedures should allow the rapid screening of a large number of samples, and some of them should allow the detection of viroids maintained at low levels in the host plant.
Testing three protocols of nucleic acids extraction from tree leaves, our results showed that phenol protocol is the most laborious and time-consuming. The Trizol technique, reported for the first time for viroids purification, is rapid and simple but can be applied only for some plants. The cellulose procedure, which does not use organic solvents, gave a response with all the plants tested when coupled to the RT-PCR. Viroids amount as low as 50 pg RNA may be detected by R-PAGE in a routine test. The sensitivity of R-PAGE is similar to that of nucleic acid hybridization, and results are available within a day. Therefore, R-PAGE provides a suitable methodology for general screening of plant material for the presence of viroids. During double electrophoresis, viroids undergo a conformational transition by heat denaturation in which their highly base-paired, rod-like structure is transformed into an unfolded, circular, single-stranded RNA. This technique, although specific for small, circular RNAs, presents the disadvantage of its relatively low sensitivity and specificity. The polymerase chain reaction is a very powerful method. It has greatly facilitated detection of plant pathogens which would be difficult or time-consuming to detect using conventional techniques. PCR method offers a greater degree of specificity and involves fewer handling steps. The ubiquitous occurrence of viroids in Vitis varieties and rootstock selection both in California and Europe was noted by Szychowski et al., (1991). Given the wide exchange of grapevine material throughout the world, it is not surprising to find AGVd in Tunisia, even if it has not been reported before outside California and Australia. If AGVd turns out to be latent in these species, efforts should be addressed to the eradication of this pathogen, thus avoiding the establishment of field reservoirs that could be transmitted to susceptible crops. It must be remembered that AGVd results from recombinations among sequences of PSTVd, CEVd, ASSVd and GYSVd-1, cultivated close to each other in several areas around the world. It is the first example in which such rearrangements appear to have taken place between viroids within two separate viroid subgroups (Mc Innes and Symons, 1991). These data are confirmed by hybridization signals obtained when AGVd genome was used as probe. A simple and rapid method for nucleic acids extraction in a microcentrifuge tube scale is described, enabling the processing of a large number of samples. These techniques are very suitable for control of a viroidal disease which is induced by multiplication and the use of contaminated propagating material (Elleuch et al., 2002).
Acknowledgements
This work was partially supported by a CMCU collaborative project (CMCU 99/F0913) involving the Tunisian laboratory and INRA of Bordeaux (France), the "Ministère de l'Enseignement Supérieur de la Recherche Scientifique et de la Technologie" of Tunisia and the "Centre National de la Recherche Scientifique" of France.
References