Genetic diversity and biological variation among California isolates of Cucumber mosaic virus

Han-Xin Lin, Luis Rubio{dagger}, Ashleigh Smythe{ddagger}, Manuel Jiminez§ and Bryce W. Falk

Department of Plant Pathology, University of California, One Shields Avenue, Davis, CA 95616, USA

Correspondence
Bryce Falk
bwfalk{at}ucdavis.edu


   ABSTRACT
Top
ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
Genetic diversity and biological variation were compared for California isolates of Cucumber mosaic virus (CMV). These fell into five pathotypes based on their reactions on three cucurbits including a susceptible squash, a melon with conventional resistance and a commercial CMV-resistant transgenic squash. Thirty-three isolates infected and caused symptoms on CMV-resistant transgenic squash. Forty-two isolates infected the CMV-resistant melon, but only 25 isolates infected both. Single-strand conformation polymorphism (SSCP) analysis was used to differentiate 81 California isolates into 14 groups, and the coat protein (CP) genes of 27 isolates with distinct and indistinguishable SSCP patterns were sequenced. Fourteen isolates corresponding to the different SSCP patterns were also used for phylogenetic analysis. Seventy-nine isolates belonged to CMV subgroup IA, but two belonged to CMV subgroup IB. This is the first report of subgroup IB isolates in the Americas. All CMV isolates had a nucleotide identity greater than or equal to 93·24 %. There was no correlation between CP gene variation and geographical origin, collection year, original host plant, or between the degree of CP amino acid sequence identity and the capacity to overcome transgenic and/or conventional resistance. SSCP and sequence analyses were used to compare 33 CMV isolates on CMV-resistant transgenic squash and susceptible pumpkin plants. One isolate showed sequence differences between these two hosts, but this was not due to recombination or selection pressure of transgenic resistance. CMV isolates capable of infecting cucurbits with conventional and transgenic CMV resistance were present in California, even before CMV transgenic material was available.

{dagger}Present address: Instituto Valenciano de Investigaciones Agrarias (IVIA), 46113 Moncada, Valencia, Spain.

{ddagger}Present address: Department of Nematology, University of California, Davis, CA 95616, USA.

§Present address: University of California Cooperative Extension, 4437 S. Laspina St, Ste B, Tulare, CA 93274, USA.


   Introduction
Top
ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
Genetically engineered transgenic resistance has been used recently in attempts to control plant virus diseases (Beachy, 1997; Gonsalves, 1998). Despite the numerous potential benefits offered by the use of transgenic plants for virus disease control, concerns have been raised regarding their widespread use. One concern has been the possible development and/or emergence of new viruses and/or virus genotypes that could overcome the engineered resistance and subsequently affect virus and host plant ecology (Aaziz & Tepfer, 1999; Robinson, 1996; Tepfer, 1993). This could potentially result from many different causes. First, several studies have shown that recombination can occur between an infecting mutant virus and transgenic plants expressing wild-type genes of the same virus (Allison et al., 1996; Gal et al., 1992; Greene & Allison, 1994, 1996; Schoelz & Wintermantel, 1993; Wintermantel & Schoelz, 1996), suggesting that new virus genotypes might be generated via such events. Secondly, it is possible that virus genotypes that are able to infect transgenically derived resistant plants may already exist in natural virus populations and they could gradually take advantage of the niches available after the deployment of resistant plants. Thirdly, RNA viruses have potential for high genetic variation due to the absence of proofreading ability of the RNA replicase. A single virus isolate does not consist of a single RNA sequence, but of a population of related sequence variants, often referred to as quasispecies (Domingo et al., 1995; Eigen, 1996; Holland et al., 1992). The quasispecies nature of RNA viruses implies a high adaptive potential, allowing for the rapid selection of biologically distinct sequence variants with the highest fitness in new environments. Thus, if present, sequence variant(s) capable of overcoming transgenic resistance could rapidly come to dominate the virus population allowing for a resistance-breaking phenotype. Therefore, knowledge of virus population diversity relative to transgenic (and even conventional) resistance is needed.

Cucumber mosaic virus (CMV) is one of the most economically important plant viruses and has a very wide host range including plants from approximately 365 genera and at least 85 families (Palukaitis et al., 1992). A number of CMV isolates have been described previously and classified into two subgroups, I and II, according to serological relationships, peptide mapping of the coat protein (CP), nucleic acid hybridization and nucleotide sequence identity (Palukaitis et al., 1992). More recently, phylogenetic analysis of a number of CMV isolates led to a further subdivision of subgroup I into subgroups IA and IB (Roossinck, 2002; Roossinck et al., 1999). CMV is endemic in cucurbit planting areas in California and other parts of the world, and effective genetic resistance is lacking in most cucurbits (Provvidenti, 1993). Transgenic yellow crookneck squash (Cucurbita pepo cv. Destiny III) plants engineered with the CP genes of CMV, Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV) have been developed and sold commercially in the USA, and have shown good resistance against a number of CMV isolates (Tricoli et al., 1995). However, whether resistance-breaking CMV isolates/variants already exist in virus populations, and whether or not specific CMV genotypes may be associated with transgenic or conventional host plant resistance, are unknown. In this paper, we used host range analysis, RT-PCR, single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses to estimate the biological and genetic diversity of California CMV isolates collected before and after the development of CMV-resistant transgenic Destiny III plants.


   Methods
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ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
Virus isolates.
Eighty-one California CMV isolates were analysed (see Tables 1 and 2). We consider an isolate as a virus culture derived from a single infected plant, and the isolates used here represented two groups. The first, group {alpha}, consisted of 63 CMV isolates collected from cucurbit trials containing transgenic and non-transgenic squash lines and cultivars at the Kearney Agricultural Center (Parlier, CA, USA), in 1999. Group {alpha} CMV isolates were collected directly from field plants. Symptomatic leaves were either harvested and directly frozen at -20 °C until analysis, or inoculated to indicator plants in the greenhouse. Leaves from these indicator plants were then vacuum-dried and stored at -20 °C until used for analysis. The second group, group {beta}, consisted of 18 CMV isolates collected from various host plants and different areas during 1985–1994 (see Table 2). These represented a collection of diverse isolates and exact records of their collection and/or maintenance were not always available. Some group {beta} isolates were mechanically transferred in the greenhouse to various host plants at different times. In these cases, leaf samples were vacuum-dried and the date stored was noted. For some of these isolates, two or three samples stored at different times were available and these were used here to evaluate the stability of the isolate over time (see Table 2). Well-characterized CMV strains including CMV-Fny (subgroup I) and CMV-Ls and -Q (subgroup II) (kindly provided by K. Perry, Purdue University) were maintained on plants of small sugar pumpkin (C. pepo L. cv. Small sugar) and used for comparison.


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Table 1. Virus incidence in field-grown transgenic and non-transgenic squash

Plants were grown in replicated field trials at the Kearney Agriculture Center (Parlier, CA, USA). The trials included non-transgenic squash (cultivars Fortune, Enterprise, Sunglo, Gentry, Cougar, Sunny delight, Sun Ray, Fancy Crook and experimental line PS31195), transgenic squash Liberator III, Prelude II and Destiny III, and non-transgenic zucchini experimental breeding lines and the cultivars Ambassador, Counselor, Dividend, Raven and Falcon. Liberator III and Destiny III have transgenic resistance to ZYMV, WMV and CMV; Prelude II has transgenic resistance to ZYMV and WMV.

 

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Table 2. California CMV isolates used in this work

 
Biological assay.
All biological tests were carried out by mechanical inoculation with infected leaf tissue ground in potassium phosphate buffer (0·03 M potassium phosphate, pH 7·4, containing 0·05 % Na2SO3) (1:10, w/v). The sap was gently rubbed on the cotyledons of test plants pre-dusted with celite. The inoculated plants were maintained in an insect-proof greenhouse at 18–25 °C for up to 5 weeks post-inoculation. Three cucurbits were used for inoculation: Dixie (C. pepo cv. Dixie, susceptible) and Destiny III (transgenic resistance to WMV, ZYMV and CMV) yellow crookneck squash (kindly provided by Seminis Vegetable Seed Company), and Freeman cucumber [Cucumis melo var. Conomon, conventional resistance to CMV (Enzie, 1943), kindly provided by Harris Moran Seed Company]. Dixie and Destiny III plants are isogenic hybrids, the common male inbred parent was transformed with the WMV, ZYMV and CMV CP transgene cassette in order to create Destiny III (Seminis Vegetable Seed Company). For each of these host plants, one plant was used for initial inoculation with each virus isolate/strain. Plants of Destiny III were inoculated in two separate experiments (thus at least two plants per isolate/strain), and any CMV isolates that failed to infect plants of Freeman in initial inoculation were inoculated to two plants of Freeman in a second test. Thus, CMV isolates noted as not infecting plants of Freeman or Destiny III were evaluated on these plants in at least two experiments. If an isolate infected plants of Destiny III or Freeman, even one plant in only one experiment, we considered this isolate as capable of overcoming the resistance.

ELISA.
Indirect ELISA was carried out according to the method of Sasaya & Yamamoto (1995). Antibodies to CMV, WMV, ZYMV and Papaya ringspot virus (PRSV) were from our laboratory collection of polyclonal antisera.

RT-PCR amplification.
Oligonucleotide primers were designed for the CMV CP gene and 3' non-translated region (3'NTR) based on the nucleotide sequences of CMV-Fny RNA 3 (subgroup I, GenBank accession no. D10538) and CMV-Q RNA 3 (subgroup II, GenBank accession no. M21464). These primers are highly conserved within each subgroup and are subgroup-specific. Primers F1 and F3 are complementary to the RNA 3 3'-terminal nucleotides 2197–2216 and nucleotides 1902–1922 of CMV-Fny, respectively. Primer F4 corresponds to nucleotides 1245–1266 of CMV-Fny RNA 3. Primers Q1 and Q3 are complementary to the RNA 3 3'-terminal nucleotides 2174–2193 and nucleotides 1850–1872 of CMV-Q, respectively. Primer Q4 corresponds to nucleotides 1235–1256 of CMV-Q RNA 3.

Total RNAs were extracted from healthy and CMV-infected plants using TRI reagent (Molecular Research Center) according to the manufacturer's instructions. First-strand cDNA was synthesized (primer F1 for subgroup I, primer Q1 for subgroup II) using total RNA as template. cDNA synthesis and PCR amplifications were carried out following the method of Rubio et al. (2001), except that the annealing temperature for both the CP gene (primers F3 and F4, or Q3 and Q4) and the CP+3'NTR (primers F1 and F4) were 45 °C.

SSCP analysis.
SSCP analysis was performed following the method of Rubio et al. (2001) with slight modifications. Five µl of the RT-PCR product was first digested by restriction endonuclease AflIII for the CP gene, or XhoI for the CP+3'NTR, in a 10 µl reaction volume at 37 °C for 60 min in order to obtain smaller size DNA fragments more suitable for SSCP analysis. Two µl of the digested DNA was mixed with 8 µl denaturing solution (95 % formamide, 20 mM EDTA, pH 8, 0·05 % bromophenol blue and 0·05 % xylene/cyanol) and denatured by heating at 95 °C for 10 min. Denatured DNA was subjected to electrophoresis at 4 °C in a non-denaturing 12 % polyacrylamide minigel (for the CP gene, the gel contained 5 % glycerol), using a constant voltage of 200 V for 3 h. The gels were stained with silver nitrate (Rubio et al., 2001).

cDNA cloning and nucleotide sequence analysis.
RT-PCR products of the CP gene were extracted using the QIAquick PCR extraction kit (Qiagen). These were then directly used for sequencing with the respective PCR primers, or were ligated into the pGEM-T vector (Promega) according to the manufacturer's instructions, followed by transformation into Escherichia coli DH5{alpha}. Positive colonies were screened by the PCR method using the same conditions as the PCR reaction above described. Sequences were determined in both directions by means of an ABI PRISM DNA sequencer 377 (Perkin-Elmer) in the DNA Sequence Facility of UC-Davis. Processing and multiple alignments of the nucleotide sequences (minus primer sequences) were done using the program CLUSTAL W (Thompson et al., 1994). Alignments were manually adjusted by MacClade 4.0. The nucleotide distance was estimated using the Jukes–Cantor method implemented in DNADIST of the PHYLIP package (Felsenstein, 1989). The nucleotide sequences of CMV-Fny, -Q and -C were also used for the estimation of nucleotide distance. The CP gene from CMV-C (GenBank accession no. D00462) was that used to engineer CMV resistance into Destiny III (Tricoli et al., 1995). Phylogenetic relationships were inferred using PAUP* version 4.0b10.0 based on the maximum-parsimony method with a 100 replicate bootstrap search. The first positions of each codon and the transversion substitutions were both weighted as 2. Two other members of the genus Cucumovirus, Peanut stunt virus (PSV) and Tomato aspermy virus (TAV) were defined as outgroups. All branches with <70 % bootstrap value were collapsed.


   Results
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ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
Viruses recovered from transgenic and non-transgenic plants
Viruses were collected from field cucurbit (zucchini, straightneck, scallop and yellow crookneck squash) trials in two separate plantings in 1999. The trials contained replicated plantings of virus-susceptible cucurbits, non-transgenic genotypes with reported levels of tolerance to one or more potyviruses and transgenic cultivars with resistance to two or three viruses (Table 1), and virus-infected plants were used as sources of naturally occurring CMV isolates. Visual observations showed that, compared with non-transgenic plants, very few symptomatic plants were found for the transgenic Prelude II, Destiny III or Liberator III plants. Plants showing virus symptoms were collected and tested by ELISA using antisera to CMV, ZYMV, WMV and PRSV (Table 1). Because so few transgenic plants showed symptoms, only eight samples were collected and analysed. Among four samples collected from plants of Prelude II (containing transgenes for WMV and ZYMV), three were infected by CMV, while one had a mixed infection of CMV and PRSV. Among four samples collected from plants of Destiny III and Liberator III (containing transgenes to WMV, ZYMV and CMV), only two were infected with PRSV. In contrast, among the 77 samples collected from non-transgenic plants, 59 had CMV, four had ZYMV, 29 had PRSV and 54 had WMV (Table 1).

Biological diversity of CMV isolates on transgenic and non-transgenic plants
Although no CMV isolates were recovered from the transgenic, CMV-resistant Destiny III field plants, we still evaluated the biological diversity of several CMV isolates collected from the surrounding non-transgenic plants or plants with transgenic resistance to WMV and ZYMV (Prelude II). Sixty-three CMV group {alpha} isolates identified above, plus 18 group {beta} CMV isolates collected from various host plants and different geographic areas within California during 1985–1994 (Table 2), as well as the standard strains, CMV-Fny (subgroup I), -Q and –Ls (subgroup II), were compared by inoculating plants of susceptible (Dixie), conventional-resistant (Freeman) and the transgenic-resistant Destiny III. All CMV isolates were first inoculated onto plants of small sugar pumpkin. However, as most of the field-collected group {alpha} isolates were mixed infections with other viruses (e.g. WMV, ZYMV and/or PRSV; Table 1), they could not be used directly for biological comparisons. Therefore, they were first inoculated to Nicotiana benthamiana plants (a non-host for PRSV) to eliminate PRSV. Our ELISA data showed that PRSV was eliminated, as were most of WMV and/or ZYMV. Only 68 CMV isolates free of the potyviruses were used for further biological analyses (but all 81 isolates were used for initial sequence comparisons, see below). Based on their abilities to infect and cause symptoms on plants of Dixie, Destiny III and Freeman, the 68 California CMV isolates could be classified into five pathotypes (Table 3).


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Table 3. Pathogenicity assay of 68 California CMV isolates using susceptible and resistant cucurbits

 
All CMV isolates infected plants of Dixie, most producing severe symptoms including systemic mosaic and stunting with or without leaf crinkling and curling. However, isolate Ca induced only very mild symptoms, i.e. plant stunting, one or two curled leaves with a few irregular yellowing patches along and around veins and a few necrotic lesions on the petioles. Isolate Ca did not infect plants of either Freeman or Destiny III. Identical results were obtained for three repeated experiments. Isolate Ca was grouped into pathotype I because of its unique symptom phenotype on plants of Dixie. Pathotype II consisted of 17 isolates infecting plants of Dixie, but not plants of Destiny III or Freeman (Table 3). However, unlike isolate Ca, these isolates induced severe symptoms on plants of Dixie. Only for pathotypes I and II was the quality of symptoms used to differentiate pathotypes, separation of the remaining isolates being based on their abilities to infect and cause symptoms on the specific genotypes.

Thirty-three CMV isolates out of 68, including 17 group {alpha} (collected from Kearney Agricultural Center in 1999) and 16 group {beta} isolates (collected from different California areas during 1985–1994), infected transgenic Destiny III plants. Symptoms ranged from mild (very similar to the symptoms induced by Ca on Dixie plants; Fig. 1A), to moderate (stunted plants, more curled leaves with yellow veins and yellow patches around the veins and necrotic lesions on the petioles; Fig. 1B), to severe (mosaic, leaf curling, crinkling, stunting, more rapid symptom development; Fig. 1C). Among the Destiny III-infecting isolates, 25 could also infect Freeman plants and were grouped into pathotype III, whereas eight did not infect Freeman plants and were grouped into pathotype IV. Pathotype V contained 17 CMV isolates that infected both Freeman and Dixie plants, but not Destiny III plants. CMV-Fny showed similar pathogenicity to pathotype V, while Ls and Q were similar to isolate Ca, pathotype I. Taken together, these results show that California CMV isolates vary in biological diversity when compared by their ability to infect transgenic- and conventional-resistant plants. Interestingly, some of the Destiny III-infecting group {beta} CMV isolates were collected and stored before transgenic resistance was available, and thus before selection pressure due to transgenic resistance was present. Also of interest is that, even though no CMV was recovered from transgenic CMV-resistant Destiny III or Liberator III field plants, CMV isolates with the ability to infect transgenic-resistant Destiny III plants were recovered from non-transgenic plants in the same overall planting.



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Fig. 1. Different symptoms on Destiny III plants 30 days post-inoculation. (A) Mild symptoms (induced by isolate CK31), arrow shows a crinkled leaf. (B) Moderate symptoms (induced by isolate CK41), stunted plant, crinkled and curled leaves; yellow patches around veins. (C) Severe symptoms (induced by isolate SJ91), stunted plant, mosaic, curled and crinkled leaves.

 
Genetic differentiation by SSCP analysis
By using subgroup-specific primers F1 and Q1 to synthesize cDNA and then performing PCR using primers for the CP gene, we could differentiate CMV isolates belonging to subgroups I and II. CMV-Fny (subgroup I), and CMV-Q and -Ls (subgroup II) yielded a single DNA fragment of about 678 bp (subgroup I) and 638 bp (subgroup II), respectively, as expected. No RT-PCR products were obtained from extracts of healthy plants using either primer set, or from plants infected by subgroup I or II isolates using the heterologous primers (data not shown). Considering that subgroup I isolates are more common in California (Daniels & Campbell, 1992; Rodriguez-Alvarado et al., 1995), we first used primer F1 to synthesize cDNA and then performed PCR with primers F3 and F4. All 81 isolates (from the initial inoculated plants of small sugar pumpkin) yielded a single DNA fragment of about 678 bp (data not shown), suggesting that they belonged to subgroup I. The RT-PCR products were further used for SSCP analysis and nucleotide sequencing.

We next used SSCP analysis of the CP gene RT-PCR product as a first approach to differentiate CMV isolates having different nucleotide sequences. To achieve greater sensitivity and resolution, we used the restriction endonuclease AflIII to cleave the CP DNA product into two smaller DNA fragments of 214 bp and 464 bp. Fourteen distinct SSCP patterns were obtained after analysing the AflIII-digested PCR products of all 81 CMV isolates (Fig. 2). When SSCP patterns for all isolates were examined together, patterns F and I were the most predominant, found for 23 and 40 isolates, respectively. Among 63 group {alpha} isolates, 23 showed pattern F and 32 showed pattern I (Fig. 2). Pattern I also represented the majority of group {beta} isolates (8 of 18 isolates, Fig. 2). Samples of the same group {beta} isolates, but dried and stored at different times, showed the same SSCP pattern (e.g. see isolates 144I, I90, 116, SJ91, 113 and 160, Table 2). This suggested that these isolates had not changed significantly during laboratory and greenhouse manipulations, at least in CP sequence.



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Fig. 2. SSCP analysis of AflIII-digested RT-PCR products of the coat protein (CP) gene of California CMV isolates. (A) Map depicting CMV genome segment 3. Boxes indicate open reading frames (ORFs) for the 3a movement protein (MP) and the CP, and NTR indicates the 3'NTR. Positions of RT-PCR primers, the resulting RT-PCR products and the AflIII and XhoI restriction sites used for digesting DNA fragments are shown. (B) The 14 SSCP patterns resulting from SSCP analysis of the 81 CMV isolates. Letters at the top designate the specific, different SSCP patterns. Numbers at the bottom indicate the number of isolates having the corresponding SSCP pattern. CMV isolates corresponding to respective SSCP patterns are: pattern A: C94M3; pattern B: C18; pattern C: V27; pattern D: I90 (A, B), C92, RNC; pattern E: 113 (A, B), C94T5; pattern F: CK (4, 5, 7, 8, 9, 12, 14, 18, 33, 34, 36, 37, 38, 43, 45, 48, 49, 50, 52, 55, 56, 58, 62); pattern G: CK57; pattern H: CK41; pattern I: CK (1, 2, 3, 6, 10, 11, 13, 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 28, 29, 30, 31, 32, 35, 39, 40, 42, 46, 47, 59–61, 63), 144I (A, B), C94M7, C94T1, 116 (A, B), SJ91 (A, B, C), C96, CH, 160; pattern J: MD284; pattern K: CK26; pattern L: Ca; pattern M: CK (44, 51, 53, 54); pattern N: CK27.

 
Nucleotide sequence variation
RT-PCR products of 27 CMV isolates (see Table 2) with different or indistinguishable SSCP patterns were sequenced in order to assess the sensitivity of our SSCP analysis and to estimate the genetic distance between 81 CMV isolates, and between samples of the same isolates dried at different times (i.e. SJ91 A, B and C). Nucleotide sequences were also analysed for the possible correlation between the CP gene sequence and ability of the CMV isolates to infect plants of Destiny III and/or Freeman. Comparison of nucleotide sequences of those isolates having different SSCP patterns showed that even one base substitution in a 678 bp DNA sequence could be detected by SSCP analysis. The nucleotide distance between 14 isolates representing each different SSCP pattern ranged from 1·6x10-2 to 6·76x10-2 (Table 4). The average nucleotide distance between isolates with different SSCP patterns was 2·95±1·74x10-2. Eleven isolates having the same SSCP pattern F showed very high nucleotide identity with only 0–3 nucleotide substitutions per sequence. The average nucleotide distance between isolates with pattern F was 3·1±1·76x10-3, tenfold less than for those with different SSCP profiles. These results indicated that our SSCP analysis was very sensitive for detecting minor sequence variations. Thus, even though not all RT-PCR products for all isolates were sequenced, the nucleotide distance between isolates with different SSCP patterns most likely gave a good estimation of the nucleotide distance of all CMV isolates analysed here.


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Table 4. Nucleotide distances of the CP gene among 14 CMV isolates showing different SSCP patterns

 
When nucleotide sequences of the groups {alpha} and {beta} CMV isolates were compared, we found that all group {alpha} isolates showed very high sequence identity (>98·84 %), despite the fact that some isolates had distinct SSCP patterns. In contrast, group {beta} isolates were more diverse. The nucleotide identity of group {beta} isolates was as low as 93·24 %. However, half the group {beta} isolates (SSCP patterns I and J) had very high identity (>98·77 %) with group {alpha} isolates, suggesting that the genetic variation of CMV in California was not correlated with the geographic region, harvest date or host plant from which the virus was originally isolated. Also, we found no association between the nucleotide sequence and the biological characteristics, especially the ability to infect plants of Freeman and/or Destiny III. When the deduced amino acid sequences of those isolates infecting resistant or susceptible plants were compared, there were no differences.

Phylogenetic analysis of the CP gene
To analyse and compare these CMV isolates further, phylogenetic analysis of the CP gene was performed for the 14 isolates that represented different SSCP patterns, together with 13 nucleotide sequences from GenBank, as well as two other members of the genus Cucumovirus, PSV and TAV, defined as outgroups. The result confirmed our RT-PCR data showing that these 14 CMV isolates belonged to subgroup I (Fig. 3). In addition, and somewhat surprisingly, we found that these isolates were split into two subgroups. Isolate 113B belonged to subgroup IB and was most closely related to isolates NT9 and Tfn from Taiwan and Italy, respectively. These three isolates formed a single clade with 100 % bootstrap support. Isolate C94T5, which showed the same SSCP pattern as isolate 113B (see Fig. 2), was then also sequenced and used for phylogenetic analysis. Isolate C94T5 was found to have only one nucleotide change from 113B, and by phylogenetic analysis it also fell into the same clade as isolates 113B, NT9 and Tfn (data not shown). The remaining 13 CMV isolates fell into subgroup IA. Among these, isolates C94M3 and V27 formed a clade having 88 % bootstrap support. All group {alpha} isolates (CK26, 27, 31, 33, 41, 51 and 57) and a group {beta} isolate (MD284) were closely related to each other and formed a clade having 98 % bootstrap support. Overall, group {beta} isolates were more diverse than group {alpha} isolates (Fig. 3).



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Fig. 3. Phylogram trees of the CP genes for California and reference CMV isolates reconstructed by maximum-parsimony analysis with 100 bootstrap replicates. Isolates belonging to different subgroups (IA, IB and II) are labelled. Bootstrap values are shown below the branches, and the number of character changes (branch length) is shown above the branch lines. The GenBank accession numbers of the reference CMV isolates used here are: Fny (D10538), IA (AB042294), Ixora (U20219), Leg (D16405), Ls (AF127976), Mf (AJ276481), NT9 (D28780), Q (M21464), S (AF063610), SD (AB008777), Tfn (Y16926), Trk 7 (L15336), Y(D12499), ER_PSV (U15730), V_TAV (L79972). California CMV isolates are indicated (see Table 2 for description and Table 4 for GenBank accession numbers).

 
Comparison of major sequences from CMV-infected transgenic and non-transgenic plants
RT-PCR products, as used here, represent the mixture of virus sequence variants with the major sequence reflected as the prominent bands in the SSCP patterns (Rubio et al., 1999, 2001). To assess whether the major sequence variants of 33 CMV isolates from CMV-infected plants of Destiny III were different from those in the original inocula (CMV-infected plants of small sugar pumpkin), SSCP analysis of the XhoI-digested RT-PCR products of the CP+3'NTR region (972 bp, see Fig. 2; this region was used for transformation to create Destiny III) was performed. Our results showed that the major SSCP bands for 32 of the 33 CMV isolates were indistinguishable for a given isolate when the SSCP patterns of CMV-infected plants of Destiny III and pumpkin were compared (Fig. 4, and data not shown). Thus, significant changes in the major sequences were not detected for most isolates. However, one isolate, CK41, did show obviously different SSCP patterns for RT-PCR products from plants of Destiny III compared with those from plants of pumpkin (Fig. 4). The SSCP bands seen for CK41-infected Destiny III plants appeared to be a subpopulation of those in the CK41-infected pumpkin SSCP profile.



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Fig. 4. Comparison of SSCP patterns of XhoI-digested RT-PCR products of the CP+3'NTR region of CMV-infected plants of pumpkin and Destiny III (see Fig. 2). Isolate names (see Table 2) are shown at the tops of lanes. Plants used: P, C. pepo cv. Small sugar (pumpkin); De, C. pepo cv. Destiny III.

 
To determine whether the SSCP profiles seen above for CK41-infected Destiny III plants were due to selective effects of the transgenic resistance, we used CK41-infected small sugar pumpkin plants to inoculate Destiny III and non-transgenic plants, including Dixie (which is of the same genetic background as Destiny III, but is not transgenic) and analysed the major CMV CP sequences from each plant. We first compared RT-PCR products of the CP+3'NTR by SSCP analysis, and then cloned the RT-PCR products of the CP gene and compared sequences for ten clones from CK41-infected pumpkin, N. benthamiana, Dixie, Destiny III and Freeman plants. These analyses showed that the original CK41-infected pumpkin plant contained two dominant CMV sequences (designated as sequences A and B) that were only 98·97 % identical over the CP region (seven nucleotide differences). Sequence A was the dominant sequence in CK41-infected N. benthamiana, Dixie and Destiny III plants, and sequence B was the dominant sequence in CK41-infected Freeman plants. Thus, the SSCP profile for CK41-infected Destiny III plants (Fig. 4) was not due to recombination between the transgene and infecting virus nor due the selective effects of the transgenic resistance, because the same change was also seen for non-transgenic plants.


   Discussion
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ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
We present data here on the biological and genetic diversity of California CMV isolates. Using biological analysis we identified 33 CMV isolates capable of infecting CMV-resistant transgenic squash. It is interesting to note that 16 of these isolates were collected and/or stored prior to the development of the transgenic CMV-resistant Destiny III. Thus, CMV isolates capable of infecting transgenic resistant plants were already present and did not arise in the population as a result of selection pressure imposed by the transgenic resistance. Also of interest is that 17 CMV isolates capable of infecting transgenic-resistant squash came from the field trials where Destiny III and other virus-resistant transgenic plants were grown. However, none of these isolates came from field-grown plants of Destiny III (or Liberator III), but were from plants of non-transgenic cultivars grown in the same trial, or plants with transgenic resistance to WMV and ZYMV (Prelude II) but not CMV (Table 1). All of the plants of Destiny III in the field trial and tested by us were CMV-negative, indicating that, despite the local presence of CMV isolates capable of infecting the transgenic Destiny III, the transgenic CMV-resistant plants exhibited effective field resistance. It should be noted that the inoculation method we used in the greenhouse was mechanical inoculation, so that the challenge pressure (i.e. the concentration and dose of virus inoculum) is most likely much higher than occurred in the field, and we consistently inoculated young plants. At least some of these factors have been shown to affect resistance and susceptibility of transgenic, virus-resistant plants (Ferreira et al., 2002). The plants in the field are generally exposed to various levels of virus pressure by aphid inoculation at different ages, and previous work has shown that the protection conferred by the CP-mediated transgenic plants was effective against aphid inoculations of CMV (Quemada et al., 1991).

Our results also showed that 42 CMV isolates were able to overcome the conventional resistance of Freeman plants (Table 3). Among these isolates, 17 did not infect transgenic Destiny III (pathotype V), and among 33 isolates capable of overcoming the transgenic resistance, eight did not infect plants of Freeman (pathotype IV). These results suggest that the mechanisms involved in overcoming Freeman resistance and transgenic Destiny III resistance are different, and that California CMV populations are biologically diverse and contain variants with the ability to overcome both types of resistance.

A number of studies have shown that CP-mediated transgenic resistance generally provides more effective protection against challenging viruses with higher amino acid sequence identity as opposed to distantly related viruses or virus strains (Anderson et al., 1989; Nelson et al., 1987, 1988; Van Dun & Bol, 1988; Van Dun et al., 1988). The resistance of plants of Destiny III is believed to be mediated at the CP protein level, and not by post-transcriptional gene silencing (Quemada & Tricoli, 1995). Therefore, if this scenario applies to plants of Destiny III expressing the CMV-C CP gene, it would be expected that plants of Destiny III would show better protection against infection by closely related isolates (i.e. subgroup I isolates) than by distantly related isolates (i.e. subgroup II isolates). This trend was observed when Destiny III was challenged by CMV-Fny, which has 98·69 % amino acid identity with CMV-C. However, Destiny III also showed high protection against infection by CMV-Q and CMV-Ls (subgroup II), which only have 80·51 % amino acid sequence identity with CMV-C. When the group {alpha} isolates having 100 % amino acid sequence identity with one another, but only 97·37 % amino acid sequence identity with CMV-C, were compared, we found that some group {alpha} isolates could infect plants of Destiny III (or Freeman) and some could not. Thus, there was no direct correlation in our studies between the CP amino acid sequence identity of the CMV isolates used here and their ability to infect plants of Destiny III or Freeman. The lack of correlation between the degree of amino acid sequence identity and the level of protection was also observed in the transgenic tobacco plants expressing the CP gene of CMV-C (Quemada et al., 1991). Thus, other factors (e.g. non CP-viral genomic sequences, CMV satellite RNAs) could contribute to the biological variation.

SSCP proved here to be very sensitive for evaluating the molecular variation of CMV isolates. It was also used to compare the major sequences of the CMV isolates infecting transgenic Destiny III and non-transgenic plants. A rapid change of major sequence within a population of an individual isolate might result from selection of sequence variants with a higher adaptive potential after a host shift, or could be due to the appearance of a more competitive new genotype generated via recombination between the transgene and the infecting virus (see introduction). Our data showed that most CMV isolates analysed did not show significant major sequence changes after infecting transgenic Destiny III plants. For isolate CK41, which did show a different major sequence variant (SSCP pattern) in transgenic and non-transgenic plants, further analyses showed that this difference was due to neither recombination nor selection by transgenic resistance, but most likely reflected a random colonization on new host plants from a mixed population inoculum, because the subdivision of sequences A and B to different host plants was not consistently observed in three repeated inoculations and SSCP analyses (data not shown). More detailed evaluation of the effects of host species and transgenic resistance on the population structure within virus isolates is needed.

Sequence analysis of the CP genes of 27 CMV isolates with distinct and indistinguishable SSCP patterns (Table 2 and Fig. 2) showed that these CMV isolates all belonged to CMV subgroup I. Based on the sensitivity and accuracy of our SSCP analysis, it is reasonable to conclude that all 81 California CMV isolates analysed here belonged to subgroup I. It is unlikely that subgroup II isolates were also present as: (i) mixed infections with subgroups I and II isolates have not been reported from North America; and (ii) we also analysed the CMV MP gene of these same isolates by RT-PCR using CMV subgroup-universal primers and no subgroup II sequences were found by SSCP and nucleotide sequence analyses (unpublished data). Nucleotide sequence data also showed group {alpha} isolates collected from Kearney Agricultural Center in 1999 had very high sequence identity (>98·84 %; Table 4), and they formed a single clade in the phylogenetic analysis (Fig. 3), suggesting that the extant CMV population at this location was most likely derived from a common ancestor and only recently spread over the area. In contrast, group {beta} isolates were more diverse,with nucleotide identities as low as 93·24 %. This diversity was further confirmed by phylogenetic analysis (Fig. 3). It is also interesting to point out that two subgroup IB isolates (113B and C94T5) were found among the California CMV isolates analysed here, and they were most closely related to isolate NT9 from Taiwan and isolate Tfn from Italy. Indeed, these four isolates only had one to three nucleotide differences (Fig. 3 and data not shown). Subgroup IB isolates were believed to be only distributed in Asia (Roossinck et al., 1999) and Italy (Roossinck, 2002). Thus, this represents the first report that subgroup IB isolates occur in the Americas. The diversity seen here among group {beta} isolates is not so surprising as they were collected from different plant species, in different areas and in different years. However, the genetic variation of CMV observed here was not correlated with geographic region, collection date or plant species. This is similar to the population structure of CMV described in Spain, where the genetic structure of 17 CMV subpopulations was not correlated with location or year, but showed a metapopulation structure with local extinction and random recolonization from local or distant virus reservoirs (Fraile et al., 1997; Garcia-Arenal et al., 2001).


   ACKNOWLEDGEMENTS
 
We thank K. Ralston and M. Fon for technical assistance, and D. Tricoli and B. Copes for cucurbit seeds. We thank Kevin Cook for helpful discussions and are grateful to D. Tricoli and James Ng for critical reading of this manuscript, Keith Perry for providing CMV isolates Fny, Q and Ls, and M. J. Roossinck for helpful discussions, suggestions and comments on this manuscript. This work was in part supported by the Biotechnology Risk Assessment Research Grants Program (Award No. 99-33120-8293) from the US Department of Agriculture to B. W. F. H.-X. L. was partly supported by the China Scholarship Council from the Ministry of Education, PR China.


   REFERENCES
Top
ABSTRACT
Introduction
Methods
Results
Discussion
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Received 19 June 2002; accepted 11 September 2002.