First Report of Cucumber vein yellowing virus on Cucumber, Melon, and Watermelon in Iran.

Several viral diseases are responsible for significant economic losses in commercial cucurbit production worldwide. During a survey conducted in July 2002 in cucurbit growing areas in southern Iran, vein-clearing symptoms and leaf chlorosis on older leaves were observed on a cucumber plant near Jiroft (Kerman Province). These symptoms were similar to those caused by Cucumber vein yellowing virus (CVYV, genus Ipomovirus, family Potyviridae), a virus first described in Israel (1) and now widespread in cucurbit crops in the Middle East and Mediterranean Regions (2). The identification of CVYV was established through differential host range reaction and immunosorbent electron microscopy (IEM) experiments. Typical vein-clearing symptoms were observed following mechanical inoculation of cucumber and melon plantlets, but no symptoms were observed in Chenopodium quinoa, C. amaranticolor, Nicotiana tabacum, or Vigna sinensis. Numerous, slightly flexuous, elongated virus particles were observed in infected plant extracts. The particles were decorated by a polyclonal antiserum raised against a Sudanese isolate of CVYV. To confirm CVYV identification, total RNA extracts (TRI-Reagent, Sigma Chemical, St. Louis, MO) were obtained from the original cucumber sample. Reverse transcription-polymerase chain reactions (RT-PCR) were carried out using CVYV-specific primers CVYV-CP-5': 5'-GCTTCTGGTTCTCAAGTGGA-3' and CVYV-CP-3': 5'-GATGCATCAGTTGTCAGATG-3' designed according to the partial sequence of the coat protein gene of CVYV-Isr (GenBank Accession No. AF233429) (2). A 540-bp fragment corresponding to the central region of CVYV coat protein was obtained from extracts of infected plants but not from healthy plant extracts. Additional watermelon (n = 6) and melon (n = 4) leaf samples collected from plants growing in the same farm were tested for the presence of CVYV using RT-PCR. All samples reacted positively for CVYV. However, a sample of Citrullus colocynthis, a wild relative of watermelon growing nearby, was negative. CVYV was not detected using RT-PCR in 123 additional cucurbit samples collected from the eastern and central regions of Iran during a survey conducted in 2002. To our knowledge, this is the first report of the occurrence of CVYV in Iran. Additional surveys in southern regions where Bemisia tabaci, the vector of CVYV, is abundant are required to better estimate the prevalence of this virus in cucurbit crops in Iran. References: (1) S. Cohen and F. E. Nitzany. Phytopathol. Mediterr. 1:44, 1960 (2) H. Lecoq et al. J. Gen. Virol. 81:2289, 2000.

First Report of Cucurbit aphid-borne yellows virus in Iran Causing Yellows on Four Cucurbit Crops.

A survey was conducted from 2001 to 2004 in the major cucurbit-growing areas in Iran to reassess the relative incidence of cucurbit viruses. Severe yellowing symptoms were observed frequently on older leaves of cucurbit plants in various regions in outdoor crops, suggesting the presence of Cucurbit aphid-borne yellows virus (CABYV, genus Polerovirus, family Luteoviridae) (1,2). Leaf samples (n = 1019) were collected from plants of melon (Cucumis melo L.), cucumber (C. sativus L.), squash (Cucurbita sp.), and watermelon (Citrullus lanatus L.) showing various virus-like symptoms (mosaic, leaf deformation, yellowing). All samples, collected from 15 provinces, were screened for the presence of CABYV by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with IgGs and alkaline phosphatase-conjugated IgGs against a CABYV reference isolate (1). Of the 1,019 samples tested, 471 were positive for CABYV using DAS-ELISA. Some of the positive samples had typical severe yellowing symptoms while symptoms in other samples were masked by mosaic or leaf deformations caused by other viruses frequently found in mixed infections (data not shown). During the entire survey, CABYV was detected by DAS-ELISA in 201 of 503 melon samples, 72 of 129 cucumber samples, 158 of 249 squash samples, and 40 of 138 watermelon samples. These results indicate that CABYV is widely distributed on four cucurbit species in the major growing areas of Iran. In order to confirm CABYV identification, total RNA extracts (TRI-Reagent, Sigma Chemical, St Louis, MO) were obtained from 25 samples that were positive using DAS-ELISA originating from Khorasan (n = 4), Esfahan (n = 6), Teheran (n = 3), Hormozgan (n = 4), Azerbaiejan-E-Sharqi (n = 4), and Kerman (n = 4). Reverse transcription-polymerase chain reactions (RT-PCR) were carried out using forward (5'-CGCGTGGTTGTGG-TCAACCC-3') and reverse (5'-CCYGCAACCGAGGAAGATCC-3') primers designed in conserved regions of the coat protein gene according to the sequence of a CABYV reference isolate (3) and three other unpublished CABYV sequences. RT-PCR experiments yielded an expected 479-bp product similar to the fragment amplified with extracts from the reference isolate. No amplification of the product occurred from healthy plant extracts. To our knowledge, this is the first report of the occurrence of CABYV in Iran on various cucurbit species. The high frequency (46.2%) with which CABYV was detected in the samples assayed indicates that this virus is one of the most common virus infecting cucurbits in Iran. References: (1) H. Lecoq et al. Plant Pathol. 41:749, 1992 (2) M. A. Mayo and C. J. D'Arcy. Page 15 in: The Luteoviridae. H. G. Smith and H. Barker, eds. CAB International Mycological Institute, Wallingford, UK, 1999. (3) H. Guilley et al. Virology 202:1012, 1994.

Complete nucleotide sequence of Iranian tomato yellow leaf curl virus isolate: further evidence for natural recombination amongst begomoviruses. Brief Report.

Complete nucleotide sequence of the Iranian strain of tomato yellow leaf curl virus (TYLCV-IR) was determined and compared with some begomoviruses. The complete sequence of TYLCV-IR clustered together with TYLCV and TYLCV-MId from Israel. A similar relationship holds when the deduced amino acid sequences of V1, V2, C2 and C3 and nucleotide sequences of IR, and RIR were compared. In contrast, phylogenetic analyses of amino acid sequences of C4, C1, and nucleotide sequences of LIR revealed that TYLCV-IR clustered with TLCIRV and two Indian species: ToLCBV- [Ban4], and ToLCKV. The phylogenetic analyses, Recombination Detection Program analyses, and sequence alignment survey provided evidence of the occurrence of recombination between an Israeli TYLCV-MId, as major parent, and TLCIRV, as minor parent. In this recombination event, a region (from nt 2149 to 2766) of TYLCV-MId genome were replaced with corresponding genome sequences of TLCIRV (RDP P-value = 5.976 x 10(-72)), which include LIR, C4, and N-terminal of C1. Infectivity of the cloned TYLCV-IR genome was demonstrated by successful agroinoculation of tomato ( Lycopersicon esculentum) and other plant species. The disease was transmitted by the natural vector Bemisia tabaci from agroinoculated plants to test plants, reproducing in this way the full biological cycle and proving that the genome of TYLCV-IR consists of only one circular single-stranded DNA molecule.

Watermelon chlorotic stunt virus from the Sudan and Iran: Sequence Comparisons and Identification of a Whitefly-Transmission Determinant.

ABSTRACT The genomes of two Watermelon chlorotic stunt virus (WmCSV) isolates, one from the Sudan and one from Iran, were cloned and sequenced. Sequence relationship with other geminiviruses characterizes WmCSV as a typical Eastern Hemisphere geminivirus with a bipartite genome. The two geographically distant WmCSV isolates from Africa and the Middle East share a very high overall sequence similarity: 98% between their DNA-A and 96% between their DNA-B components, and their respective capsid proteins are identical. A single amino acid change in the capsid protein (N131D) renders WmCSV whitefly nontransmissible. This region of the capsid is also implicated in transmission by Bemisia tabaci of Tomato yellow leaf curl virus.

Genetic analysis of the monopartite tomato yellow leaf curl geminivirus: roles of V1, V2, and C2 ORFs in viral pathogenesis.

Tomato yellow leaf curl virus (TYLCV) is a whitefly-transmitted geminivirus with a monopartite genome. We have investigated the functions of the V1, V2, and C2 ORFs by mutational analysis. We analyzed the ability of TYLCV mutants containing disrupted ORFs V1, V2, or C2 to replicate, spread, and cause symptoms in Nicotiana benthamiana and tomato plants. All the mutants retained the capability of autonomous replication in protoplast-derived cells of tomato and leaf discs of N. benthamiana, although both V1 and V2 gene products appeared to play a role in the accumulation of viral single-stranded DNA. In contrast, none of the mutants was able to systemically infect tomato plants, demonstrating that the V1, V2, and C2 gene products are all required for a successful infection process in this host. The effect of the mutation in ORF C2 appeared to be host-specific, since N. benthamiana plants were systemically infected, although symptom development was attenuated.

Replication of tomato yellow leaf curl virus (TYLCV) DNA in agroinoculated leaf discs from selected tomato genotypes.

The leaf disc agroinoculation system was applied to study tomato yellow leaf curl virus (TYLCV) replication in explants from susceptible and resistant tomato genotypes. This system was also evaluated as a potential selection tool in breeding programmes for TYLCV resistance. Leaf discs were incubated with a head-to-tail dimer of the TYLCV genome cloned into the Ti plasmid of Agrobacterium tumefaciens. In leaf discs from susceptible cultivars (Lycopersicon esculentum) TYLCV single-stranded genomic DNA and its double-stranded DNA forms appeared within 2-5 days after inoculation. Whiteflies (Bemisia tabaci) efficiently transmitted the TYLCV disease to tomato test plants following acquisition feeding on agroinoculated tomato leaf discs. This indicates that infective viral particles have been produced and have reached the phloem cells of the explant where they can be acquired by the insects. Plants regenerated from agroinfected leaf discs of sensitive tomato cultivars exhibited disease symptoms and contained TYLCV DNA concentrations similar to those present in field-infected tomato plants, indicating that TYLCV can move out from the leaf disc into the regenerating plant. Leaf discs from accessions of the wild tomato species immune to whitefly-mediated inoculation, L. chilense LA1969 and L. hirsutum LA1777, did not support TYLCV DNA replication. Leaf discs from plants tolerant to TYLCV issued from breeding programmes behaved like leaf discs from susceptible cultivars.

Tomato yellow leaf curl virus from Sardinia is a whitefly-transmitted monopartite geminivirus.

The genome of an isolate of tomato yellow leaf curl virus from Sardinia, Italy (TYLCV-S), a geminivirus transmitted by the whitefly Bemisia tabaci, has been cloned and sequenced. The single circular DNA molecule comprises 2770 nucleotides. Genome organisation closely resembles that of the DNA A component of the whitefly-transmitted geminiviruses with a bipartite genome. A 1.8 mer of the TYLCV-S genome in a binary vector of Agrobacterium tumefaciens is infectious upon agroinoculation of tomato plants. Typical tomato yellow leaf curl disease symptoms developed about three weeks after inoculation. The disease was transmitted by the natural vector B.tabaci from agroinfected plants to test plants, reproducing in this way the full biological cycle and proving that the genome of TYLCV-S consists of only one circular single-stranded DNA molecule. Contrary to the other whitefly-transmitted geminiviruses described so far, there is no evidence for the existence nor the necessity of a second component (B DNA) in the TYLCV-S genome.

Style proteins of a wild tomato (Lycopersicon peruvianum) associated with expression of self-incompatibility.

The identification, isolation and aminoterminal sequencing of two S-genotype-associated proteins from style extracts of Lycopersicon peruvianum Mill. is reported. There is a high level of homology between these two sequences and with the amino-terminal sequences of other S-allele-associated glycoproteins isolated from Nicotiana alata Link et Otto. These sequences were obtained by a new high-sensitivity method of selected twodimensional gel analysis followed by electroelution and purification of proteins by inverse-gradient high-performance liquid chromatography before sequencing.

Genetics of fertility restoration in the C-group of cytoplasmic male sterility in maize.

The genetics of fertility restoration of cms-C group cytoplasm of maize was studied using crosses involving stable maintainer lines and lines that restored full pollen fertility. Pollen fertility in the sources of cms-C sterile cytoplasms studied was restored by a single dominant restorer (Rf4) gene. The fertility restoration was sporophytic. Allelism tests among five restorer lines showed that they all apparently carried the same alleles (Rf4 Rf4). Similar tests also demonstrated that seven nonrestoring maintainer lines had apparently the same genotype (rf4 rf4), although a partial "late break" of fertility was observed at low levels in some maintainer crosses. Comparative studies among different cms-C sources (C, Bb, ES, PR and RB) indicated that similar inheritance of fertility restoration was involved. The data indicated that a single, dominant Rf gene is involved in the restoration of several C-group cytoplasms, at least in the lines studied here. This is the first single-gene, sporophytic restorer system described in maize to date.