Search

International audience; Two surveys were conducted in September 2014 and July 2015 in important vegetable-growing areas in Azerbaijan. Cucurbit and solanaceous plants exhibiting symptoms of mosaic, yellowing, leaf curl or necrosis were collected and tested serologically and molecularly for the presence of the major viruses infecting these crops. For cucurbits, the most common viruses in both sampling sets were aphid-transmitted ones, including potyviruses (watermelon mosaic virus, WMV, zucchini yellow mosaic virus, ZYMV), cucumoviruses (cucumber mosaic virus, CMV) and poleroviruses (cucurbit aphid-borne yellows virus, CABYV). Eggplant mottled dwarf (EMDV) was also detected for the first time in Azerbaijan on cucumber at a low prevalence. In solanaceous crops, CMV was the most common virus detected, followed by potato virus Y (PVY). Tomato spotted wilt virus (TSWV), alfalfa mosaic virus (AMV) and tobamoviruses (tomato mosaic virus (TMV), pepper mild mottle virus (PMMoV)) were also detected in 2015. The begomovirus tomato yellow leaf curl virus (TYLCV) was present on tomato only in the Absheron area, where it had a high prevalence and induced important losses. TYLCV-like symptoms on tomato in other areas of Azerbaijan were due to phytoplasma diseases.

The recent identification of a new nanovirus, pea necrotic yellow dwarf virus, from pea in Germany prompted us to survey wild and cultivated legumes for nanovirus infections in several European countries. This led to the identification of two new nanoviruses: black medic leaf roll virus (BMLRV) and pea yellow stunt virus (PYSV), each considered a putative new species. The complete genomes of a PYSV isolate from Austria and three BMLRV isolates from Austria, Azerbaijan and Sweden were sequenced. In addition, the genomes of five isolates of faba bean necrotic yellows virus (FBNYV) from Azerbaijan and Spain and those of four faba bean necrotic stunt virus (FBNSV) isolates from Azerbaijan were completely sequenced, leading to the first identification of FBNSV occurring in Europe. Sequence analyses uncovered evolutionary relationships, extensive reassortment and potential remnants of mixed nanovirus infections, as well as intra- and intercomponent recombination events within the nanovirus genomes. In some virus isolates, diverse types of the same genome component (paralogues) were observed, a type of genome complexity not described previously for any member of the family Nanoviridae. Moreover, infectious and aphid-transmissible nanoviruses from cloned genomic DNAs of FBNYV and BMLRV were reconstituted that, for the first time, allow experimental reassortments for studying the genome functions and evolution of these nanoviruses.

International audience

Virus like symptoms appeared on most watermelon plants grown at different locations in southern provinces of Iran. The symptoms included chlorotic patches on leaves, vein yellowing and stunting of watermelon plants. The causal agent of watermelon chlorotic stunt disease was transmitted to healthy watermelon, jimson-weed and bean by the whitefly Bemisia tabaci, but not by sap inoculation. Coat protein and nucleic acid of Watermelon chlorotic stunt virus (WmCSV) were detected in infected plants using a dot-immunobinding assay (DIBA) and squash-blot hybridization, respectively. The data obtained confirmed that watermelon chlorotic stunt disease in Iran is caused by WmCSV. Agroinoculation of some plant species by the cloned genomic components (DNA-A and DNA-B) of a non-sap-transmissible Iranian isolate of WmCSV (WmCSV-Ir) has been demonstrated. Host range studies using agroinoculation indicated that most plant species in the Cucurbitaceae and some species of the Solanaceae are susceptible to WmCSV-Ir. Infection of agroinoculated plants was confirmed by DIBA and polymerase chain reaction. The virus from agroinfected plants was transmissible by the whitefly Bemisia tabaci. Results obtained from a limited survey during 1997-2000 indicated the presence of WmCSV-Ir in some watermelon-growing provinces of southern but not in northern, central, and north-eastern provinces of Iran. WmCSV has apparently not yet spread to these regions.

MR. HAJIMORAD'*, A. KHEYR-POUR", V. ALAVJ", A. AHOONMANESH^,M.BAHAR'^t, M. A.REZAIAN'* and B. GRONENBORN''^ Plant Pests and Diseases Research Institute, PO Box 1454, Tehran-19395: and ^Department of PlantPathology, Esfahan University ofTeehnology. Esfahan; Iran"IfLstitut dcs Seiences Vegetales. CNRS. 91198 Gif-sur-Yvette Cedex, Franee^ of Horticulture, CSIRO. GPO Box 350, South Australia 5001. AustraliaA virtis causing a disease of tomato, prevaletit in the southern provinces of Iran, with symptoms of leaf-curling, stunting, reduction of leaf size, leaf corrugation, shortening of internodes and severe reductionin fruit yield, was shown to be transmissible to healthy tomato plants by grafting and by whiteflies{Beniisia (abaci), but not by sap inoculation. Geminivirus DNA was detected in extracts of diseasedtomato plants by dot-blot hybridization assays using as probes full-length cloned DNA of Australian,Italian {Sardinian) or Jordanian strains of tomato yellow leaf curl virus (TYLCV). Geminivirus coatprotein was detected in whitefly inoculated plants by dot immunobinding assay using polyclonalantibody raised against Jordanian TYLCV. A limited survey using the dot-blot hybridization assay forvirus detection indicated the presence of the virus in tomato-growing provinces of southern but notnorthern Iran. Whitefly transmission experiments to tomato under controlled greenhouse conditionsshowed that some isolates of TYLCV-like geminiviruses from different parts of Iran difFer insymptomatology.INTRODUCTIONA serious virus disease, which exhibits variousprominent symptoms such as upward curling ofleaf margins, stunting, reduction of leaf size atthe top of the plant, corrugated leaf, shorteningof internodes and severe reduction in fruit yield,has become prevalent in tomato fields all over theMiddle East (Makkouk & Laterrot, 1983).Similar symptoms have been described fromdiseased tomato plants in north and centralAfrica, south east Asia, Taiwan, Mexico, Italy(Sardinia), Spain, Australia, Dominican Repub-lic and Jamaica (Makkouk & Laterrot, 1983;Brown & Nelson, 1988; Czosnek et al., 1990;Kheyr-Pour et aL, 1991; Dry et al.. 1993;McGlashan et al., 1994; Noris et afA994\ Polston*Corresponding author.tPresent address: Department of Crop Protec-tion, Waitc Agricultural Research Institute, GlenOsmond, South Australia 5064, Australia.Accepted 15 November 1995.et al., 1994). The virus is transmitted by thewhitefly Bemisia tabaci (Cohen & Harpaz, 1964)but not mechanically (Cohen & Nitzany, 1966)and in all locations except Mexico has beencalled tomato yellow leaf curl geminivirus(TYLCV) (Brown & Nelson, 1988; Czosneket al., 1990; Kheyr-Pour et al., 1991; Dry et ai,1993; McGlashan et al.. 1994; Noris et al., 1994;Polston et ai., 1994). However, despite severalcommon characteristics between the virus foundin Mexico and TYLCV, the Mexican virus hasbeen called chino del tomate virus (Brown N Habibi, 1975; Javan-moghadam, 1993). In this report we describethe association of TYLCV with the disease inIran. We also indicate the distribution of the virusin Iran and describe symptom variation amongstvirus isolates from different parts of the country.

Accessions of the wild tomato species Lycopersicon chilense LA 1969 and L. htrsutum LA 1777 which are resistant to tomato yellow leaf curl virus (TYLCV) in field- and in whitefly-medi ated transmission tests were agroinoculated with a tandem repeat of the TYLCV genome. Large amounts of viral DNA started to accumulate in the agroinoculated L. chilense and L. hirsutum plants about 10 days after the agroinoculation. Yellowing and narrowing of the upper leaves were observed in the L. chilense plants but no curling as in susceptible L. esculentum cultivars. The agroinoculated L. hirsutum plants showed typical yellowing and curling of young leaves. These findings indicate that TYLCV introduced by means of agroinoculation leads to the breakdown of natural resistance mechanisms which prevent the replication, spread and expression of symptoms in resistant tomato genotypes.

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.

Watermelon Chlorotic Stunt Virus (WmCSV) is a bipartite geminivirus residing in the genus Begomovirus (family Geminiviridae) and is whitefly transmitted (Bemisia tabaci). WmCSV infects members of the Cucurbitaceae, and represents a considerable threat to melon and watermelon crops in Sudan, Yemen, and Iran. One hundred twenty-eight melon accessions, mostly collected from Sudan, were screened in the open-field in Sudan under severe whitefly inoculation pressure in order to identify sources of resistance to WmCSV. One WmCSV resistant line HSD 2445-005 selected from this first screening along with 22 new melon accessions were agro-inoculated with an infectious Sudanese clone of WmCSV. Ten accessions exhibited resistance to WmCSV infection upon agro-inoculation. Six of them were confirmed to have complete resistance to WmCSV through graft-inoculation as well as multiple field trials in Sudan. These lines include the Sudanese accession HSD 2445-005, the South African accession PI 282448, and three Indian accessions: 90625, PI 124112, and PI 414723. Resistant plants exhibited no disease symptoms, and virus DNA could not be detected by hybridization with a WmCSV-specific probe.

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 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.

International audience

International audience; 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.

International audience

International audience

International audience

International audience

National audience

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.

Supplement

We have identified three alleles of the S-locus controlling self-incompatibility and their associated pistil proteins in Petunia inflata, a species that displays monofactorial gametophytic self-incompatibility. These S-allele-associated proteins (S-proteins) are pistil specific, and their levels are developmentally regulated. The amino-terminal sequences determined for the three S-proteins are highly conserved and show considerable homology to those of S-proteins from Petunia hybrida, Nicotiana alata and Lycopersicon peruvianum, three other species of the Solanaceae that also exhibit gametophytic self-incompatibility. cDNA clones encoding the three S-proteins were isolated and sequenced. Comparison of their deduced amino acid sequences reveals an average homology of 75.6%, with conserved and variable residue interspersed throughout the protein. Of the 137 conserved residues, 53 are also conserved in the N. alata S-proteins studies so far; of the 64 variable residues, 29 were identified as hypervariable based on calculation of the Similarity Index. There is only one hypervariable region of significant length, and it consists of eight consecutive hypervariable residues. This region correspond approximately to the hypervariable region HV2 identified in N. alata S-proteins. Of the two classes of N. alata S-proteins previously identified, one class exhibits greater homology to the three P. inflata S-proteins reported here than to the other class of N. alata S-proteins.

In order to study the extent and nature of differences among various S-allele-associated proteins in N. alata, we carried out comparative studies of seven such proteins. We first isolated and sequenced cDNA clones for the Sz-, SF11-, S1-, and Sa-alleles, and then we compared the deduced amino acid sequences both of these four S-proteins and of three previously published S2-, S3-, and S6-proteins. This comparison revealed (1) an average homology of 53.8% among the seven proteins and (2) two homology classes, with Sz and SF11 in one class and S1, S2, S3, and S6 in the other class. There are 60 conserved residues, including 9 cysteines. Of the 144 variable residues, 50 were identified as hypervariable based on a calculation of their Similarity Indices. Although conserved, variable, and hypervariable residues are dispersed throughout the protein, some are clustered to form five conserved, five hypervariable, and a number of variable regions. Those variable sites which contain residues conserved within one class of S-proteins but different between classes might provide a clue to the evolutionary relationship of these two classes of S-proteins. The hypervariable residues, which account for sequence variability, may contribute to allelic specificity.

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.