Analysis of the temporal and spatial disease progress of Bemisia tabaci-transmitted Cucurbit yellow stunting disorder virus and Cucumber vein yellowing virus in cucumber

Cucurbit yellow stunting disorder virus (CYSDV) has been present in greenhouse-grown cucumber in Spain since 1992. However, in the autumn of 2000 Cucumber vein yellowing virus (CVYV) was introduced, leading to mixed infections of both Bemisia tabaci -transmitted viruses. The temporal and spatial spread of disease symptoms were monitored in experimental plastic-covered greenhouses during six consecutive cucumber plantings from 2000 to 2002. Using linear regression analysis of 46 disease-progress curves, the Gompertz model best described the CYSDV epidemics in 2000, whereas the logistic model best described the development of CYSDV and CVYV epidemics in 2001 and 2002. The fitted models were used to calculate the amount of degree Celsius-days at half-maximum infection in the greenhouses (° D _0·5). After multiple regression analysis, 56% of the variation in ° D _0·5 of CYSDV was related to the numbers of whiteflies infesting the cucumber crops, and was independent of the mean temperatures in the greenhouses. In contrast, 76% of the variation in ° D _0·5 of CVYV was related to both the numbers of vectors present and maximum temperature. Symptom expression in cucumbers mechanically inoculated with CVYV was most prevalent when plants were grown at regimes of at least 28°C day temperature. According to analysis of spread using Taylor's power law, beta-binomial distribution fitting, and the ordinary runs test, the prevalence of CVYV showed significant overdispersion, whereas that of CYSDV did not. The χ² test of independence and Spearman's rank correlation coefficient were used to measure co-occurrence and covariation, respectively, during the first half of the cultivation period. These results showed that the two diseases were not associated.     

Yellowing disease in zucchini squash produced by mixed infections of Cucurbit yellow stunting disorder virus and Cucumber vein yellowing virus

Zucchini squash is host to Cucurbit yellow stunting disorder virus (CYSDV), a member of the genus Crinivirus, and Cucumber vein yellowing virus (CVYV), a member of the genus Ipomovirus, both transmitted by the whitefly Bemisia tabaci. Field observations suggest the appearance of new symptoms observed on leaves of zucchini squash crops when both viruses were present. When infected during controlled experiments with CYSDV only, zucchini plants showed no obvious symptoms and the virus titer decreased between 15 and 45 days postinoculation (dpi), after which it was no longer detected. CVYV caused inconspicuous symptoms restricted to vein clearing on some of the apical leaves and the virus accumulated progressively between 15 and 60 dpi. Similar accumulations of virus followed single inoculations with the potyvirus Zucchini yellow mosaic virus (ZYMV) and plants showed severe stunting, leaf deformation, and mosaic yellowing. However, in mixed infections with CYSDV and CVYV, intermediate leaves showed chlorotic mottling which evolved later to rolling, brittleness, and complete yellowing of the leaf lamina, with exception of the veins. No consistent alteration of CVYV accumulation was detected but the amounts of CYSDV increased ≈100-fold and remained detectable at 60 dpi. Such synergistic effects on the titer of the crinivirus and symptom expression were not observed when co-infected with ZYMV.     

Incidence of five viruses infecting sweetpotatoes in Uganda; the first evidence of Sweet potato caulimo-like virus in Africa

In a survey of most sweetpotato-growing areas of Uganda, virus-like diseases were observed in all districts surveyed. Out of 338 fields sampled in 35 of the then 42 districts, 219 (65%) had some plants with symptoms. The most common symptoms included vein clearing, mottling, leaf distortion, yellowing, stunting and leaf strapping. Particularly high virus-like disease incidences (means of 34–86%) were encountered in districts around Lake Victoria and in the Rift Valley in southern and western parts of Uganda; particularly low incidences were encountered in the east and north of Uganda. Using four formats of enzyme-linked immunosorbent assay in combination with immunoelectron microscopy and polymerase chain reaction assays, five viruses were identified. Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV) were most commonly detected, being found in about 90% of samples. Sweet potato mild mottle virus at 10%, Sweet potato chlorotic fleck virus (SPCFV) at 8% and Sweet potato caulimo-like virus (SPCaLV) at 0·07% were more rarely detected. Most infections were multiple, SPCSV + SPFMV constituting > 90% of all double infections. Triple infections, involving mainly SPFMV, SPCSV and either SPMMV or SPCFV, and quadruple infections of SPFMV + SPCSV + SPMMV + SPCFV were observed in < 10% of the diseased samples. The identification of SPCaLV is the first evidence of its occurrence in Africa.     

Occurrence of yellowing viruses (Beet pseudo-yellows virus, Cucurbit yellow stunting disorder virus and Cucurbit aphid-borne yellows virus) affecting cucurbits in Greece

A survey of the incidence of yellowing viruses in Greek glasshouse (and occasional field) cucumber and melon crops was carried out during 2000–03. In most cases disease incidence ranged from 50 to 80%. Simplex RT-PCR was used for the detection of Beet pseudo-yellows virus (BPYV) and Cucurbit yellow stunting disorder virus (CYSDV), and DAS-ELISA for the detection of Cucurbit aphid-borne yellows virus (CABYV). The results showed that BPYV was the predominant virus in cucumber and melon crops, whereas CYSDV, reported for first time in Greece, was isolated only in three regions of southern Greece: Rhodes, Crete and Arkadia. CABYV was detected only in three cucumber glasshouses in Pella (Macedonia). A simplified triplex RT-PCR method using a simple sample-preparation protocol was developed to allow rapid, sensitive and simultaneous detection of the three viruses. Sequence comparisons of the PCR products of BPYV and CYSDV revealed 98·7 and 100% amino acid identity, respectively, with previously reported sequences. The arable weed species Amaranthus retroflexus , Selosia cristata , Sonchus oleraceus and Sonchus sp. were identified as potential BPYV reservoirs.     

Development and use of detection methods specific for <Emphasis Type="Italic">Cucumber vein yellowing virus</Emphasis> (CVYV)

Two methods for the detection of Cucumber vein yellowing virus (CVYV) on infected plants were developed, based on the information provided by cDNA clones covering the 3-end of the genome of a Spanish isolate (CVYV-AILM). The sequenced portion of the CVYV-AILM genome showed a 96.6% aminoacid identity with that of a reported sequence of another CVYV isolate from Israel (Lecoq et al., 2000). The first detection method used a RNA specific probe for hybridization with nucleic acids extracted from infected plants. The probe was complementary to a portion of the CVYV genome including the C-terminal part of the NIb and most of the coat protein (CP) coding regions. The second detection method employed polyclonal antisera raised against recombinant viral CP expressed in bacteria. The specific antibodies were used to detect the presence of virus particles in plant extracts. Both procedures resulted in a highly specific detection of CVYV in plants infected with different isolates of the virus. No interference was observed with other cucurbit-infecting viruses. Sensitivities achieved were sufficient for routine diagnosis of the presence of the virus in plants.     

Inheritance of resistance to angular leaf spot (Pseudomonas syringae pv. lachrymans) in cucumber and identification of molecular markers linked to resistance

Angular leaf spot is a common disease of cucumber ( Cucumis sativus ) caused by Pseudomonas syringae pv. lachrymans . Genetics of resistance to this disease was investigated using two sets of parameters: (i) disease severity, i.e. the number and size of necrotic and chlorotic lesions on the infected leaves, and (ii) presence or absence of a chlorotic halo around the necrotic spots on the infected leaves. Disease severity appears to be controlled by multiple genes and the heritability of the resistance was estimated to be 53%. The presence or absence of the chlorotic halo was determined to be governed by a single gene, with the presence of the halo (the susceptible phenotype) being a dominant character. A RAPD marker linked to the gene conferring the chlorotic halo was identified. Genetic distance between this marker, OP-AO07, a polymorphic 420 bp amplicon in the DNA of the susceptible plants, and the locus encoding the chlorotic halo was estimated to be 13 cM.     

RNAi‐based enhanced resistance to Cowpea severe mosaic virus and Cowpea aphid‐borne mosaic virus in transgenic cowpea

Cowpea (Vigna unguiculata) is one of the most important legumes cultivated in many parts of the world. The diseases caused by Cowpea severe mosaic virus (CPSMV) and Cowpea aphid‐borne mosaic virus (CABMV) are considered among the most important constraints on yield and quality, especially in Latin America and Africa. Here, the concept of using an RNA interference construct to silence the CPSMV proteinase cofactor gene and the CABMV coat protein gene is explored, in order to generate resistant transgenic cowpea plants. Ten cowpea transgenic lines were produced, presenting a normal phenotype and transferring the transgene to the next generation. Plants were tested for resistance to both CABMV and CPSMV by mechanical co‐inoculation. Seven lines presented milder symptoms when compared to the control and three lines presented enhanced resistance to both viruses. Northern analyses were carried out to detect the transgene‐derived small interfering RNA (siRNA) in leaves and revealed no correlation between siRNA levels and virus resistance. Additionally, in the symptomless resistant lines the resistance was homozygosis‐dependent. Only homozygous plants remained uninfected while hemizygous plants presented milder symptoms.     

Evidence for long-term prevalence of cucumber vein yellowing virus in Sudan and genetic variation of the virus in Sudan and the Mediterranean Basin

Cucumber vein yellowing virus (CVYV) is emerging throughout the Mediterranean Basin, where it causes significant damage to cucumber and melon crops. It has been suggested that CVYV originated from the Middle East and has spread only recently to other areas. In this work, an isolate from Sudan was characterized, and surveys performed in that country between 1992 and 2012 revealed a long-term presence of CVYV with a high molecular variability, showing that the virus has long been endemic in sub-Saharan Africa. Comparison of the full-length sequences of 11 CVYV isolates from different geographic origins revealed recombination events in CVYV populations from the Mediterranean Basin and the Middle East, and evidence for different selection pressures along the genome. These results shed a new light on the evolution of CVYV.     

Genetic variation and evolutionary forces shaping Cucumber vein yellowing virus populations: risk of emergence of virulent isolates in Europe

The genetic variation and evolutionary mechanisms shaping Cucumber vein yellowing virus (CVYV) populations were investigated by analysis of nucleotide sequences coding for P1b, P1b/P3 and coat proteins (CP) from isolates collected in different countries. The complete genome sequence of isolate ISM from Israel was also determined and compared to those of isolates Jor from Jordan and ALM32 from Spain. This isolate had overall nucleotide identities of 94·23 and 94·96% with ALM32 and Jor, respectively. Nucleotide variation among isolates was not homogeneously distributed, with the 5′ half of the genome being more variable than the 3′ half. A Bayesian phylogenetic tree of the CP showed that CVYV isolates clustered into two main clades: isolates from the Middle East region (Lebanon, Israel and Jordan) clustered in both clades whereas the isolate from Tunisia clustered in clade I and the European isolates clustered as a homogeneous phylogroup in Clade II. A similar topology was observed for P1b but with incongruences with respect to the CP, suggesting genetic exchange among virus isolates, which were confirmed with recombination algorithms. The low genetic diversity within the European phylogroup with respect to the other isolates, neutralist tests and genetic differentiation analyses suggest that the Middle East region is the cradle of CVYV and that a unique virus introduction event occurred in Europe, where the virus spread rapidly. Taken together, these findings indicate a risk of emergence of virulent CVYV isolates in Europe either through migration from the Middle East or by genetic changes of the European isolates.     

2b Protein is essential to induce a novel gradual cell death in Zucchini yellow mosaic virus-inoculated cucumber cotyledon co-infected with Cucumber mosaic virus

Cucumber cotyledons inoculated with Cucumber mosaic virus (CMV, Pepo strain) or Zucchini yellow mosaic virus (ZYMV, Z5-1 isolate) developed either mild chlorotic spots or no symptoms. Cotyledons treated with CMV plus ZYMV also developed mild chlorotic spots. However, plants ZYMV-inoculated cotyledons had veinal yellowing and gradual cell death by 20 days postinoculation (dpi) when co-inoculated with CMV on the other cotyledon. When analyzing this synergism, an enzyme-linked immunosorbent assay showed that CMV gradually increased in CMV-inoculated cotyledons of plants, with the other cotyledon mock- or ZYMV-inoculated. However, CMV significantly increased at 9 to 14 dpi in the ZYMV-inoculated cotyledons of plants co-infected with CMV. ZYMV similarly increased in cotyledon pairs of both co-infected and singly infected plants. Inoculation with PepoΔ2b, a modified Pepo-CMV that lacks translation of the 2b protein, revealed that PepoΔ2b without the 2b protein systemically infected cucumber but induced no symptoms on cotyledons or true leaves. Plants with a ZYMV-inoculated cotyledon and co-infected with PepoΔ2b did not undergo cell death; nevertheless, PepoΔ2b was at high levels comparable to levels of CMV in the ZYMV-inoculated cotyledon. The 2b protein thus seems essential for induction of the novel gradual cell death in ZYMV-inoculated cotyledons of cucumbers co-infected with CMV.     

Zucchini yellow mosaic virus; two outbreaks in the Netherlands and seed transmissibility

In 1983 and 1987/88 two limited outbreaks of zucchini yellow mosaic virus in cucumber and zucchini squash occurred in the Westland glasshouse district in the Netherlands, mainly in glasshouses. The disease could be eradicated and has not recurred so far. In both cases a relatively mild but still highly pathogenic strain of the virus was involved. Diseased plants of zucchini yielded severely distorted or no fruits and it was difficult to obtain seeds from infected plants. Two out of 4196 seedlings grown in isolation from seed from inoculated zucchini plants showed symptoms and contained the virus, indicating that the virus can be transmitted via seed but at very low rate. This explains the erratic incidence and international distribution of the virus.     

Resistance to Plum pox virus in plants expressing cytosolic and nuclear single‐chain antibodies against the viral RNA NIb replicase

The expression of engineered single‐chain variable fragments specific to the NIb RNA replicase of Plum pox virus (PPV) (scFv2A) in transgenic plants was successfully used as a strategy to interfere with viral infection. Different scFv2A fusion proteins were constructed to target those subcellular compartments, such as the cytosol, endoplasmic reticulum (ER) membrane structures and the nucleus, where NIb protein presumably accumulates. Several transgenic lines of Nicotiana benthamiana plants expressing the scFv2A targeted to the cytosol (2A lines), ER (6K2 lines) and nucleus (NLS lines) were obtained. The protective effect of scFv expression was determined by mechanical virus inoculation in five 2A, three 6K2 and four NLS transgenic lines. The strongest resistance was afforded with the 2A‐3 (six non‐infected plants out of 10), 6K2‐1 (17 out of 33) and NLS‐11 (16 out of 19) transgenic lines. The success of this interference with PPV infection opens new possibilities for the control of this RNA virus and could be exploited not only to confer resistance in transgenic plants, but also to elucidate the role of the non‐structural NIb protein in different cell compartments during viral infection.     

Cucumber mosaic virus infection of chickpea stands: temporal and spatial patterns of spread and yield‐limiting potential

Patterns of spread and yield losses were examined when migrant aphid vectors transmitted Cucumber mosaic virus (CMV) within chickpea (Cicer arietinum) plots. When numbers of chickpea plant infection foci were varied to provide initial infection incidences of 0·3–2%, rate of virus spread and its final incidence increased in proportion to initial virus incidence and pathogen progress curves reflected a polycyclic pattern of spread. Chickpea seed yields decreased by 44–45% when CMV incidence reached 61–74% at final assessment in plots with simulated 1–2% initial incidence. In chickpea plots with or without introduced lupin plant infection foci, cumulative spatial data for diseased plants were assessed using spatial analysis by distance indices (sadie ). When CMV spread within a plot, infection was concentrated in large patch clusters and was consistent with comprehensive localized spread around primary infection foci. When introduced infection foci were absent, there was more diffuse spread with many isolated clusters. In individual plants that developed CMV symptoms at different growth stages, shoot and pod dry weight were reduced by 60–65% and 77–79% (early infection) and 44 and 66% (late infection), respectively. Seed yield losses were 78–80% (early) and 65–67% (late), and reduction in 50‐seed weight was 20–25% regardless of time of infection, so seed number was reduced more by early than late infection. Infection also reduced seed quality as, in addition to smaller seed size, seed coats were discoloured and malformed: the proportions of malformed seeds were 9–11% (early), 3–6% (late) and 0·5% (healthy).     

Cucumber mosaic virus populations in Tunisian pepper crops are mainly composed of virus reassortants with resistance‐breaking properties

Cucumber mosaic virus is one of the most prevalent viruses in Tunisian pepper crops, where it has been detected in 68% of plants developing mosaic symptoms, making it essential to characterize the molecular and biological properties of local CMV populations. Two hundred and seventy‐eight isolates collected in the late 1990s, 2006 and 2008–2010 were characterized genetically. Isolates belonging to the three phylogenetic subgroups of CMV (IA, IB and II) were detected, but surprisingly, 90% of the isolates were reassortants between subgroups IA and IB, with two predominant haplotypes, IB‐IA‐IA and IB‐IA‐IB (nomenclature according to the subgrouping of the three genomic RNAs). The IB‐IA‐IA haplotype was present in all regions surveyed, while IB‐IA‐IB was observed only in northern Tunisia. This situation was unexpected, because CMV reassortants were previously thought to be counterselected in nature, and this raises the questions of the origin of IB strains in Tunisia and of the widespread distribution of these two reassortant types. Phylogenetic studies revealed low diversity within haplotypes, whatever the locality or the year of sampling. However, analysis of haplotype frequencies revealed a high genetic differentiation between CMV populations, which was better explained by the localities of sampling than by years. Geographic distances affected the differentiation of CMV populations, mainly between north and central Tunisia. When tested against a polygenic resistance to CMV movement in pepper, 55 of 57 isolates tested were able to break the resistance, indicating that this resistance would not be useful for controlling CMV in Tunisian pepper fields.     

Analysis of Iris yellow spot virus replication in vector and non‐vector thrips species

Iris yellow spot virus (IYSV, genus Tospovirus) is a viral disease of bulb and seed onion crops and is transmitted by Thrips tabaci. Foliage damage of up to 75% has been reported in Kenya and Uganda. In this study, the rate of IYSV replication in the larva, pupa and adult stages of T. tabaci and other non‐vector thrips species and colour forms such as Frankliniella occidentalis, F. schultzei (dark) and F. schultzei (pale) was evaluated by monitoring relative levels of nucleocapsid (N) and non‐structural (NSs) proteins using N‐ and NSs‐specific antibodies. The effect of IYSV replication on mortality of thrips was also determined. N protein levels increased in all three stages of IYSV‐fed T. tabaci, indicating replication of IYSV. In IYSV‐fed non‐vector thrips, the increase of N protein levels in the larval stage was lower than IYSV‐fed T. tabaci but higher than their healthy counterparts. The N protein levels did not increase at pupal and adult stages. NSs protein was not detected in first instar of either vector or non‐vector thrips species. After a 4 h post‐acquisition period, a significant increase in NSs proteins was only observed in IYSV‐fed T. tabaci, clearly differentiating vectors and non‐vectors of IYSV. IYSV replication did not influence the survival of the vector thrips species, T. tabaci populations or the non‐vector thrips species. This study indicates the effectiveness of monitoring non‐structural proteins such as NSs, compared to nucleocapsid proteins, for differentiating vectors and non‐vectors of IYSV.