MPPOL

Introduction
Materials & Methods
Results
Discussion
Acknowledgements
References

Molecular Plant Pathology On-line http://www.bspp.org.uk/mppol/1997/0612nadeem

Cotton leaf crumple virus and cotton leaf curl virus are two distantly related geminiviruses

Nadeem A1, Weng Z, Nelson MR, Xiong Z

Department of Plant Pathology, Forbes 204, University of Arizona, Tucson, AZ 85721, USA.

1Current address: Central Cotton Research Institute, Old Shujahabad Road, Multan, Pakistan.



Corresponding
author


Z. Xiong, Department of Plant Pathology, Forbes 204, University of Arizona, Tucson, AZ 85721, USA.
Telephone: 520-621-9869 Fax 520-621-9290 email: zxiong@ag.arizona.edu



ABSTRACT



Cotton leaf crumple virus (CLCrV) and cotton leaf curl virus (CLCuV) are two whitefly-transmitted geminiviruses that infect cotton. Using a polymerase chain reaction (PCR)-based technique, the complete DNA A component of each virus was amplified as two DNA fragments from total nucleic acids extracted from infected cotton leaves and subsequently cloned. Two pairs of PCR primers were designed according to conserved regions of several published, whitefly-transmitted geminivirus sequences. Electrophoretic analysis of the PCR products indicated that the DNA A of CLCrV and CLCuV was approximately 2.6 kb and 2.7 kb, respectively. Southern hybridization analyses of the cloned PCR fragments showed that CLCrV DNA and CLCuV DNA did not hybridize with each other under high stringency conditions whereas they hybridized weakly with each other under low stringency conditions. Riboprobes prepared from cloned DNA fragments also hybridized with their respective single-stranded virion DNA and the double-stranded replicative form extracted from infected cotton leaves. A smaller-than-genomic viral DNA, presumably a subgenomic DNA, was detected in CLCuV-infected cotton, but not in CLCrV-infected cotton by the hybridization. The hybridization data are consistent with partial sequence analysis of the AC1 gene and the capsid protein gene. Although both viruses share a high degree of nucleic acid identity, CLCrV is more closely related to the New World geminiviruses such as Abutilon mosaic virus, sida golden mosiac virus, bean dwarf mosaic virus, and tomato mottle virus; whereas CLCuV is more closely related to the Old World viruses such as ageratum yellow vein virus, tomato leaf curl virus, African cassava mosaic virus, and tomato yellow leaf curl virus. Our data indicate that CLCrV and CLCuV are two distinct and distantly related geminiviruses.




INTRODUCTION


Figure 1A


Figure 1B



Recent changes in the sweet potato whitefly (Bemisia tabaci Genn.) populations have led to increased incidences of two important viral diseases of cotton (Gossypium hirsutum L.) caused by cotton leaf crumple virus (CLCrV) (A. Nadeem, unpublished data) and cotton leaf curl virus (CLCuV) (Hameed et al., 1994; Mansor et al., 1993). CLCrV was first reported from California (Dickerson et al., 1954) and in Arizona a few years later (Allen et al., 1960). CLCrV symptoms are characterized by floral distortion, hypertrophy of interveinal tissue resulting in downward curling of leaves, and foliar mosaic accompanied by vein clearing and frequent vein distortion (Figures 1A and 1B) (Brown et al., 1987). Losses resulting from CLCrV infection range from 21 to 86%, depending on the age of plants at the time of infection (Allen et al., 1960; Brown et al., 1987; van Schaik et al., 1962). CLCrV can be transmitted experimentally by B. tabaci to numerous species within the Malvaceae and Fabaceae families (Brown and Nelson, 1987). Typical geminivirus particles were observed in partially purified CLCrV preparations (Brown and Nelson, 1984).



Figure 1C


Figure 1D


CLCuV was first reported in Africa (Nour and Nour, 1963). Characteristic symptoms of CLCuV-infected cotton include upward or downward curling of leaves, vein distortion and thickening, and enations on the underside of the leaves (Figure 1C and 1D) (Mansor et al., 1993). Infected plants bear few flowers and yield reduction varies with the age of plants at the initial infection. Severe epidemics of CLCuV have occurred in Pakistan in the past few years, with yield losses as high as 100% in fields where infection occurred early in the growing season (personal observations, Hameed et al., 1994). The virus can be experimentally transmitted by B. tabaci to cotton, Nicotiana benthamiana, cowpea, soybean, and okra. Using the polymerase chain reaction (PCR) with geminivirus-specific oligonucleotide primers, Mansor et al. (1993) amplified a DNA fragment from CLCuV infected N. benthamiana. This PCR fragment hybridized with a DNA A probe of African cassava mosaic virus (ACMV). Extracts of CLCuV infected experimental hosts and naturally infected cotton reacted positively with polyclonal antibodies to ACMV and with monoclonal antibodies to Indian cassava mosaic virus (ICMV) (Hameed et al., 1994). Together, these data suggest that CLCuV is a member of Geminiviridae.



Figure 2


Geminiviridae consists of a diverse group of circular, single-stranded DNA viruses. Three genera of geminiviruses have been established on the basis of host ranges, vector relationships, and the number of genomic DNA components (Murphy et al., 1995; Padidam et al., 1995; Rybicki, 1994). Genus "Subgroup III Geminivirus" consists of all the whitefly-transmitted geminiviruses that infect dicotelydonous plants. With the exception of some tomato yellow leaf curl virus (TYLCV) isolates (Navot et al., 1991), the genome of this geminivirus subgroup consists of two DNA components, DNA A and DNA B. All the subgroup III geminiviruses with bipartite genomes sequenced to date have a similar genome organization (for review, see Lazarowitz, 1992; Padidam et al., 1995). The virion strand of DNA A encodes the capsid protein gene while the complementary strand encodes AC1, AC2, and AC3 that are important for viral DNA replication and transcriptional regulation (Figure 2). DNA B contains two genes that affect disease symptoms and viral movement in infected plants (Lazarowitz, 1992). There is a considerable degree of homology at the DNA level and the protein level within this geminivirus genus. Based on geographic distribution and variations in the genome organization, subgroup III geminiviruses can be further divided into the Old World geminiviruses and the New World geminiviruses (Howarth and Vandermark, 1989). The former group occurs frequently in Africa and Asia and is represented by ACMV (Morris et al., 1990). The later group occurs mainly in North and South America and is represented by bean golden mosaic virus (BGMV) (Howarth et al., 1985).



Both CLCrV and CLCuV infect dicotelydonous plants and are whitefly-transmitted (Brown et al., 1983; Mansor et al., 1993). Previous studies (Brown and Nelson, 1984; 1987; Hameed et al., 1994; Mansor et al., 1993) suggested that they belong to the subgroup III geminiviruses. However, little information is available on the relationship of these two viruses with each other and with other subgroup III geminiviruses. In this paper, we report nucleic acid hybridization and partial sequencing analysis of the PCR amplified fragments of CLCrV and CLCuV DNA A components. Our study indicates that CLCrV and CLCuV are two distinct and distantly related subgroup III geminiviruses.



MATERIALS
& METHODS



Virus isolates
The CLCrV isolate used in this study was originally collected and maintained on greenhouse cotton plants in Arizona. The CLCuV isolate was originally collected in Multan, Pakistan and maintained on greenhouse cotton plants at the National Agricultural Research Center, Islamabad, Pakistan.

DNA extraction
A mini-DNA isolation technique procedure described by Gawel and Jarret (1991) was used to extract total nucleic acids from cotton leaves with the following modifications. Young leaves were harvested 24 h before extraction. Cotton leaves were then pulverized in liquid nitrogen and subsequently resuspended in 20 volumes of the extraction buffer (2% CTAB, 20 mM EDTA, 1.4 M NaCl, 100 mM Tris-HCl, pH 8.0) preheated to 65°C. Incubation of the homogenate at 65°C, extraction with chloroform-isoamyl alcohol, and precipitation of nucleic acids were carried out as previously described (Gawel and Jarret, 1991). The final pellet was resuspended in TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA) containing 50 µg/ml RNase A.



Figure 2


PCR primer design
Two pairs of degenerate oligonucleotide primers were designed for PCR amplification of CLCrV and CLCuV DNA A from the total cotton DNA. Sequences of six whitefly-transmitted, bipartite geminiviruses (Figure 3) were aligned using the 'Pileup' programme in the Wisconsin Genetic Computer Group (GCG) Software Package. Two highly conserved regions were identified, one each in the capsid protein gene and in the AC1 gene of the geminivirus DNA A (Figure 2). A forward primer identical to and a reverse primer complementary to the sequence in each conserved region were designed. Primers F500 (forward) and R500 (reverse) correspond to 22 nucleotides from 492 to 513, and primers F1800 (forward) and R1800 (reverse) correspond to 20 nucleotides from 1797 to 1816 in the aligned multiple sequences. To accommodate non-conserved nucleotides at certain positions, 4-fold degeneracy was introduced to primers F500 and R500, and 6-fold degeneracy was introduced to primers F1800 and R1800 (Figure 3). Restriction sites XbaI and SstI were incorporated into the 5' termini of the reverse and forward primers, respectively, for the subsequent cloning experiments. Four additional non-viral nucleotides were added to the 5' of the restriction sites to facilitate digestion by XbaI and SstI. All the PCR and sequencing primers were synthesized by the DNA synthesis facility at the Arizona Research Laboratory, USA.



PCR amplification of viral DNA
PCR reactions were carried out in 50 µl of 10 mM Tris.HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, 0.1% Triton X-100, 0.25 mM dNTP, 2.5 U Taq DNA polymerase (Promega, Madison, WI, USA), 0.4 mM each of a forward primer and a reverse primer, and 100 ng of total cotton DNA. The mixture was overlaid with 50 µl mineral oil and amplified by 40 cycles of reactions in a Temp-tronic thermocycler (Thermolyne, Dubuque, IW, USA). Each cycle consisted of denaturation at 94°C for 1 min, annealing at 57°C for 30 s, and polymerization at 72°C for 1.5 min. The polymerization step was automatically increased by 3 s after each cycle.

Cloning of PCR fragments
PCR DNA fragments were excised from the agarose gel and recovered by binding to and elution from a silica matrix (GeneClean, Bio 101, La Jolla, CA, USA). CLCrV DNA fragments amplified with primers F500 and R1800 were first digested with SstI and XbaI and subsequently ligated into pBS(+) plasmid (Stratagene, La Jolla, CA, USA) digested with the same enzymes to yield plasmid pCLCrVL. Because of an internal XbaI site, the CLCrV DNA fragment amplified by R500 and F1800 primers was cloned by blunt-end ligation into the SmaI digested pBS(+) plasmid to yield plasmid pCLCrVU. For the cloning of CLCuV PCR DNA fragments, a TA cloning system (Marchuk et al., 1991) was used. pBluescript SK(+) plasmid (Stratagene, La Jolla, CA, USA) was first digested with EcoRV to yield blunt ends. An additional dT residue was added to the 3' end of the blunted DNA by incubation with Taq DNA polymerase in the presence of 2 mM dTTP for 2 h at 72°C. One microliter of the PCR product was directly ligated into the modified vector. The plasmids containing the CLCuV DNA fragments amplified with primers F500 and R1800 and with primer R500 and F1800 were designated as pCLCuVL and pCLCuVU, respectively. Unless stated otherwise, standard DNA manipulation techniques described by Sambrook et al (1989) were followed.

Southern hybridization
An enhanced chemiluminescent (ECL, Amersham, Arlington Heights, IL, USA) technique was used in Southern hybridization analysis. Plasmids containing cotton geminivirus DNA fragments were purified using a PEG technique (Sambrook et al., 1989). Following digestion with appropriate restriction enzymes, viral DNA inserts were fractionated by agarose gel electrophoresis and subsequently transferred to nylon membrane (Sigma, St. Louis, MO, USA) using a semi-dry transfer apparatus (Biorad, Richmond, CA, USA). Probes specific for CLCrV and CLCuV were prepared in a standard PCR reaction by incorporating Fluorescence(Fl)-dUTP (Amersham, Arlington Heights, IL, USA) into the amplified products. The PCR conditions were essentially identical as described above except that an equal molar ratio of Fl-dUTP and dTTP was used along with the other deoxyribonucleotides. Prehybridization, hybridization with Fl-dUTP labelled probes, and the subsequent chemiluminescence detection of the hybridized probes with anti-Fl-UTP-peroxidase conjugates were carried out as described by the manufacturer. For the hybridization of total cotton DNA, riboprobes were prepared using cloned PCR fragments as templates. Plasmids pCLCrVL and pCLCuVL were linearized with XbaI. 32P-labelled riboprobes complementary to the viral genomic DNA were synthesized using T7 RNA polymerase (Life Technologies, Gaithersburg, Maryland). The transcription-labelling reaction was carried out in a 20 µl volume containing 40 mM Tris-HCl, pH 8.0, 8 mM MgCl2, 2 mM spermidine-(HCl)3, 25 mM NaCl, 10 mM DTT, 0.5 mM each of ATP, CTP, and GTP, 15 mM UTP, 50 µCi a-32P- UTP (800 Ci/mmol), 25 U T7 RNA polymerase, and 0.5 µg linearized plasmid DNA. After incubation at 37°C for 1.5 h, DNA templates were removed by digestion with RNase-free DNase. Hybridization of the 32P-labelled riboprobes with total cotton DNA immobilized on nylon membrane was carried out essentially as described by Sambrook et al. (1989) except that the hybridization temperature was increased from 42°C to 58°C.

Nucleic acid sequencing
Nucleotide sequences were determined by the dideoxynucleotide chain termination technique (Sanger et al., 1977) with Sequenase II (United State Biochemical, Cleveland, OH) according to manufacturer's instructions. Purified double-stranded plasmid DNA containing cotton geminivirus inserts was denatured and used directly as templates for sequencing. DNA inserts in clones pCLCrVL, pCLCrVU, pCLCuVL, and pCLCuVU were flanked by T7 and T3 primer sites. The terminal sequences of the DNA inserts in each of the four clones were obtained using either T3 or T7 primer.

Sequence analysis
Sequence fragments of CLCrV and CLCuV were assembled with the GELASSEMBLE program in the GCG Software Package to produce the partial sequence for the capsid protein and AC1 ORFs. The assembled sequences were compared with each other, and with the available geminivirus sequences in the Genbank database (release 97.0 including non- redundant EMBL entries) with the FASTA program (Pearson and Lipman, 1988) available in the same software package. Parameters for the FASTA multiple sequence comparison were as following: word size of 6, gap weight of 12.0, and gap penalty of 4.0. To normalize comparison scores, an equal number of nucleotides was used to analyse the sequences of the capsid protein gene and the AC1 gene regions from both viruses.



RESULTS


Figure 4



Total cotton DNA extraction and PCR amplification of viral DNA
The mini DNA extraction procedure described in this paper consistently yielded high quality DNA preparations. Purified total genomic DNA migrated as a single, sharp DNA band of high molecular weight when analysed by agarose gel electrophoresis (Figure 4, lane 8). An important factor in the successful extraction of cotton DNA was the high buffer:tissue ratio, usually 20:1. Lower buffer:tissue ratios resulted in a significant reduction in both the quality and quantity of the DNA obtained (data not shown).



Figure 7


The effectiveness of the extracted DNA as PCR template was determined with both primer pairs. A single DNA fragment was amplified with each primer pair from the CLCrV-infected cotton DNA (Figure 4). No DNA products were amplified from DNA samples extracted from healthy cotton (lanes 3 and 6) or from negative controls with no exogenous DNA template (lanes 4 and 7). Subsequent Southern hybridization analysis showed that these DNA fragments were amplified from CLCrV DNA (Figure 7). This result indicated that geminivirus-specific DNA fragments can be amplified from total DNA prepared with the described procedure.



Figure 5


Figure 2


Size comparison of PCR amplified CLCrV and CLCuV DNA
To compare the size of the DNA A components of CLCrV and CLCuV, virus-specific DNA fragments were amplified by PCR from total DNA extracted from infected cotton leaves, and subsequently analysed by agarose gel electrophoresis (Figure 5). DNA fragments of approximately 1.2 kb and 1.4 kb were amplified from CLCrV-infected cotton DNA with the primer pair F500 and R1800 (lane 5) and with the primer pair R500 and F1800 (lane 3), respectively. DNA fragments of approximately 1.2 kb and a slightly larger fragment of 1.5 kb were amplified from CLCuV-infected cotton DNA with the same two pairs of primers, respectively (lanes 4 and 2). The PCR primers were designed in such a way that the total size of the two amplified DNA fragments was approximately equal to the size of the DNA A component (Figure 2). Therefore, on the basis of gel electrophoresis data, the size of CLCrV DNA A was estimated to be 2.6 kb while that of CLCuV DNA A was estimated to be 2.7 kb.



Figure 6


Differential hybridization of CLCrV and CLCuV DNA A components
To further differentiate these two viruses, four probes labelled with Fl-dUTP were synthesized by PCR. These four probes corresponded to the upper and lower fragments amplified from the two viruses. The labelling reactions were carried out with the same two pairs of primers and with DNA extracted from infected plants as templates. Plasmids pCLCrVL and pCLCrVU, containing the lower and upper fragments of CLCrV DNA A, and plasmids pCLCuVL and pCLCuVU, containing the lower and upper fragments of CLCuV DNA A, were used in the hybridization. Viral DNA inserts were released by restriction digestion, fractionated in a 0.8% agarose gel (Figure 6A), transferred to nylon membrane, and hybridized with Fl-dUTP labelled probes. The nylon membranes were subsequently washed under two conditions. Low stringency washing consisted of two 15-minute washes in 1X SSC containing 0.1% SDS at room temperature, followed by two 15-minute washes in 0.5 X SSC containing 0.1% SDS at 65°C. In the high stringency washing, the last two washes were carried out in 0.1X SSC containing 0.1% SDS. Under the low stringency conditions, each probe hybridized strongly with its own DNA fragments (Figure 6B). Probes representing the lower fragment of both CLCrV and CLCuV DNA A also hybridized weakly with the corresponding DNA fragment of the other virus, but probes representing the upper fragment of the DNA A did not. Under high stringency conditions, each probe only hybridized with its own DNA. Little hybridization signal was detected with heterologous DNA fragments (Figure 6C).



Figure 7


Hybridization of PCR-amplified DNA fragment with total cotton DNA
In order to confirm that the DNA fragments amplified by PCR represent the geminiviral DNA in the infected cotton tissue, Southern hybridization analysis of the total DNA from infected cotton leaves was conducted with 32P-labelled riboprobes. Clones pCLCrVU and pCLCuVU, representing the upper half of the respective geminivirus DNA A were used as templates for the transcription reaction. Each clone was sequenced partially to determine the orientation of the virion-sense strand. Riboprobes complementary to the virion-sense strand of CLCrV and CLCuV were synthesized by run-off transcription with T7 RNA polymerase from each clone linearized with XbaI. Approximately 5 µg of total cotton DNA, undigested or digested with EcoRV, were fractionated in a 0.8% agarose gel. The fractionated DNA was subsequently transferred to nylon membrane and hybridized with the riboprobes (Figure 7).



Only one DNA species from CLCrV-infected cotton sample hybridized strongly with the riboprobe prepared from pCLCrVU (Figure 7A, lane 1). When the same DNA sample was digested with EcoRV, an additional species of slowly migrating DNA was detected (Figure 7A, lane 2). The faster migrating DNA species is presumably the single-stranded CLCrV genomic DNA since it is the major DNA species and its mobility is not affected by the restriction digestion. The large DNA species was presumably the double-stranded (ds), replicative form of CLCrV DNA as its hybridization signals were intensified by the restriction digestion. This DNA species was calculated to have a mobility corresponding to a 2.6 kb ds DNA. The result also suggests that the uncut replicative form co-migrates with the single-stranded virion DNA. Digestion with EcoRV altered the mobility of the replicative form and allowed it to migrate slower than the single-stranded DNA. The riboprobe derived from pCLCrVU did not hybridize with cotton DNA extracted from healthy (Figure 7A, lanes 5 and 6) and CLCuV infected cotton leaves (Figure 7A, lanes 3 and 4).

In contrast, riboprobes prepared from pCLCuVU plasmid hybridized with two species of undigested DNA from CLCuV-infected cotton leaves (Figure 7B, lane 3). The larger species co-migrated with the CLCrV genomic DNA and presumably represents the single-stranded genomic DNA of CLCuV. The smaller DNA species that hybridized with riboprobes specific for CLCuV DNA A is presumably a subgenomic DNA. Hybridization with EcoRV-digested cotton DNA yielded a higher molecular weight DNA species that may represent the replicative form of CLCuV DNA A (Figure 7B, lane 4). The same riboprobe hybridized neither with DNA from healthy cotton leaves (Figure 7B, lanes 5 and 6) nor with DNA from CLCrV-infected leaves (Figure 7B, lanes 1 and 2).

Analysis of partial DNA A sequences of CLCrV and CLCuV
Partial sequences for the coat protein gene and AC1 gene of CLCrV and CLCuV were determined from their respective DNA clones and assembled (Figure 8). Nucleotide sequences of CLCrV and CLCuV were aligned with each other using the FASTA program (Pearson and Lipman, 1988). At the nucleic acid level, capsid protein regions showed the highest homology with 74% identity between CLCrV and CLCuV (Figure 8A), whereas the identity in AC1 regions was about 67% (Figure 8B).

In order to determine the relationship of these two viruses to other geminiviruses, partial sequences of AC1 gene and coat protein gene of CLCrV and CLCuV were compared to all geminiviruses available in the GenBank and EMBL databases. The compiled results of FASTA search are shown in Table 1. Similarity scores between two nucleic acid sequences were calculated as described by Pearson and Lipman (1988). Higher scores indicate more closely related viruses. All the geminiviruses listed in the table were sorted according to the similarity scores in the AC1 gene region. When CLCrV sequences were used as query sequences, Abutilon mosaic virus (AbMV), sida golden mosiac virus (SGMV), tomato mottle virus (ToMoV), and bean dwarf mosaic virus (BDMV) produced highest scores (Table 1A). All these viruses were classified as the New World geminiviruses (Howarth and Vandermark, 1989; Padidam et al., 1995; Rybicky, 1994). On the contrary, when CLCuV sequences were used as query sequences, ageratum yellow vein virus (AYVV), tomato leaf curl virus (TLCV), Nigerian and Kenyan strains of ACMV, and tomato yellow leaf curl virus (TYLCV) produced the highest scores. All of these virus are classified as the Old World viruses (Howarth and Vandermark, 1989; Padidam et al., 1995; Rybicki, 1994). These data suggest that CLCrV is more closely related to the geminiviruses in the New World and CLCuV is more closely related to geminiviruses of the Old World. The FASTA scores in reciprocal tests between CLCrV and CLCuV are the lowest among all the group III geminiviruses, indicating that they are the least related among the geminiviruses compared.



DISCUSSION


Figure 7



We have reported here for the first time the PCR amplification of geminiviral DNA associated with both CLCrV and CLCuV-infected cotton plants. Although we have not conclusively proved that the viruses are the causal agent for these two diseases, nucleic acid hybridization of the amplified CLCrV and CLCuV DNA fragments with total DNA extracted from diseased cotton plants suggests that they are. Virion sense and the replicative forms of geminiviral DNA in CLCrV and CLCuV-infected plants were easily detected with riboprobes synthesized from the CLCrV and CLCuV DNA fragments (Figure 7). This easy detection of the geminiviral DNA indicated that the viral DNA represented by the PCR amplified DNA fragments was abundant in the diseased tissues.



The sizes of DNA A component for CLCrV and CLCuV were estimated to be 2.6 and 2.7 kb, respectively. This size difference indicates that CLCrV and CLCuV are probably two different viruses. Reciprocal hybridization of PCR-amplified DNA fragments further suggests that CLCrV and CLCuV are two distinct but related viruses. CLCrV and CLCuV DNA fragments did not hybridize with each other under high stringency conditions. Under low stringency conditions, only the DNA fragments representing the lower half of CLCrV and CLCuV DNA A hybridized weakly with each other.

Analysis of the partial viral sequences confirmed nucleic acid hybridization data that CLCrV and CLCuV are distantly related geminiviruses. Among the subgroup III geminiviruses considered in this analysis, CLCrV and CLCuV are among the least related (Table 1). CLCrV is more closely related to the New World geminiviruses; whereas CLCuV is more closely related to the Old World geminiviruses. According to Howarth and Vandermark (1989), CLCuV is likely to have originated in the Old World and CLCrV is probably a virus native to North America. These analyses are consistent with earlier studies on nucleic acid hybridization between ACMV DNA A and CLCuV DNA (Mansor et al., 1993), and on serological reactivity of ICMV and TYLCV antibodies with CLCuV-infected cotton and tobacco samples (Hameed et al., 1994). We have cloned only the DNA A components of these two viruses. Further research is needed to address whether DNA B components are present, particularly in CLCuV, as a number of Old World group III geminiviruses are monopartite (Padidam et al., 1995).



Figure 7


When riboprobes generated from the cloned PCR fragments were used to hybridize total DNA extracted from cotton tissues, both the virion sense DNA and the double-stranded replicative form of CLCrV and CLCuV were detected in the diseased leaf samples (Figure 7). In addition, a subgenomic DNA was detected in CLCuV-infected tissue but not in CLCrV-infected tissue. Subgenomic DNA of geminiviruses has been observed in plants naturally infected by ACMV (Stanley et al., 1986), tomato golden mosaic virus (MacDowell et al., 1986), wheat dwarf virus (Macdonald et al., 1988), TYLCV (Czosnek et al., 1989), and beet curly top virus (BCTV) (Frischmuth and Stanley, 1992; Stenger et al., 1992). In the case of ACMV, subgenomic DNA has been demonstrated to interfere with replication of the genomic DNA and is considered as a defective-interfering DNA (Stanley et al., 1990). It is noteworthy that the CLCuV subgenomic DNA hybridized with probes specific for the DNA A component (Figure 7) while nearly all the subgenomic DNA of the bipartite subgroup III geminiviruses were reportedly derived from the DNA B component. Although less likely, the CLCuV subgenomic DNA could also conceivably be derived from the DNA B component. The riboprobes used in this study contained CLCuV intergenic region, which could have hybridized with the intergenic region of the subgenomic DNA. Additional experiments are need to ascertain the origin of the CLCuV subgenomic DNA. The key to our successful PCR amplification of the geminiviral DNA from total cotton DNA was the high quality of the DNA preparation. Cotton contains a high level of phenolic terpenoid compounds and tannins (Katterman and Shattuck, 1983) that complicate DNA extraction. A critical factor in the successful DNA extraction is the high buffer:tissue ratio (20:1, v/w). Another advantage of this DNA extraction procedure is the short processing time and small amount of starting material. The extraction requires about 50 mg of leaf tissues, and the entire procedure can be accomplished in less than one hour. This saving in time and in materials will expedite large scale field survey of cotton geminiviruses by PCR.



Acknowledgements



This work was supported in part by a grant from USAID. We thank Dr Steven A. Lommel, North Carolina State University, for his critical review of the manuscript.



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