Identification and effects of mixed infection of Potyvirus isolates with Cucumber mosaic virus in cucurbits.

Mixed infections in cucurbits are frequently observed in natural conditions between viruses from the Potyvirus genus and Cucumber mosaic virus (CMV), which significantly decreases productivity. The objectives of the present study was to compare the host range of PRSV-W, WMV, and ZYMV isolates and evaluate the effects of mixed infections with CMV in zucchini plants (Cucurbita pepo L.). Host range studies comprising 23 plant species confirmed some similarities and biological differences among the isolates of PRSV-W, ZYMV, and WMV. RT-PCR confirmed the amplification of DNA fragments of the PRSV-W, WMV, and ZYMV coat protein gene (cp) and cytoplasm inclusion gene (ci). The virus interaction studies in zucchini Caserta plants indicated synergistic interactions, particularly among species from the Potyvirus genus, and some CMV interference with some virus combinations.

Detecção de Cucumber mosaic virus do subgrupo- IB em feijão-de-metro no estado do Pará.

O feijão-de-metro é uma hortaliça amplamente cultivada nos municípios da região metropolitana de Belém. Diversas doenças podem comprometer a sua produtividade, dentre elas as viroses. Recentemente, foi detectado o Cucumber mosaic virus (CMV) em vagens de feijão-de-metro provenientes do município de Castanhal-PA. Este trabalho teve como objetivo identificar o subgrupo do CMV detectado em vagens de feijão-de-metro, por meio de RT-PCR, sequenciamento do ácido nucléico e análise utilizando o programa Blast, ClustalW e MEGA 7.0. Para isso, foi feita a extração de ácidos nucleicos total a partir de folhas de fumo inoculado com o isolado. Posteriormente, foi realizado o RT-PCR utilizando os primers específicos (CMV-CPR e CMV-CPF). A partir da análise da filogenia foi observado que o isolado formou um clado com os acessos do subgrupo IB de CMV.

A Survey of Viruses Affecting Dry Bean and Cowpea in Lebanon

A survey was conducted to identify viruses affecting dry bean (Phaseolus vulgaris) and cowpea (Vigna unguiculata) in Lebanon. Three hundred and thirty four samples exhibiting virus-like symptoms were collected from 13 different locations during the fall growing season of 1984. Samples were stored at 20 deg C until they were tested by the enzyme-linked immunosorbent assay (ELISA) for the presence of blackeye cowpea mosaic virus (B1CMV), bean yellow mosaic virus (BYMV), bean common mosaic virus (BCMV) and cucumber mosaic virus (CMV). In preliminary tests, the extraction buffer 0.1M phosphate + O.1MEDTA, pH 7.4 was found to be far better than the standard extraction buffer and, accordingly, was used for virus extraction for all field samples. Results obtained indicated that around 50% of the bean samples tested were infected with B1CMV. Incidence of BCMV, BYMV and CMV in the samples tested were 4,4 and 1.7%, respectively. B1CMV was detected in 10 locations, whereas, BYMV, BCMV and CMV were found in 1,4 and 4 locations, respectively. Mixed infections such as BCMV, BICMV, BCMV+CMV, BYMV+CMV and BICMV+BCMV+CMV were detected. In 35% of the samples assayed, the causal virus was not identified

Broad Bean Mottle Virus: Identification, Serology, Host Range, and Occurrence on Faba Bean (Vicia Faba) in West Asia and Norm Africa

One of the faba bean viruses found in West Asia and North Africa was identified as broad bean mottle virus (BBMV) by host reactions, particle morphology and size, serology, and granular, often vesiculated cytoplasmic inclusions. Detailed research on four isolates, one each from Morocco, Tunisia, Sudan and Syria, provided new information on the virus. The isolates, though indistinguishable in ELISA or gel-diffusion tests, differed slightly in host range and symptoms. Twenty-one species (12 legumes and 9 non-legumes) out of 27 tested were systemically infected, and 14 of these by all four isolates. Infection in several species was symptomless, but major legumes such as chickpea, lentil and especially pea, suffered severely from infection. All 23 genotypes of faba bean, 2 of chickpea, 4 of lentil, 11 out of 21 of Phaseolus bean, and 16 out of 17 of pea were systemically sensitive to the virus. Twelve plant species were found to be new potential hosts and cucumber a new local-lesion test plant of the virus. BBMV particles occurred in faba bean plants in very high concentrations and seed transmission in this species (1.37%) was confirmed. An isolate from Syria was purified and two antisera were produced, one of which was used in ELISA to detect BBMV in faba bean field samples. Two hundred and three out of the 789 samples with symptoms suggestive of virus infection collected in 1985, 1986 and 1987, were found infected with BBMV: 4 out of 70 (4/70) tested samples from Egypt, 0/44 from Lebanon, 1/15 from Morocco, 46/254 from Sudan, 72/269 from Syria and 80/137 from Tunisia. This is the first report on its occurrence in Egypt, Syria and Tunisia. The virus is a potential threat to crop improvement in the region.

Survey of Viruses Affecting Faba Bean in Six Arab Countries

A field survey of faba bean (Vicia [aba L.) for viruses in six Arab countries showed the presence of nine viruses. Bean leaf roll virus (BLRV), bean yellow mosaic virus (BYMV), broad bean mottle virus (BBMV) and to a lesser extent broad bean stain virus (BBSV) were the most common. When testing with ELISA 789 samples with symptoms suggestive of virus infection collected from Egypt, Lebanon, Morocco, Sudan, Syria and Tunisia, BBMV was detected in 203 samples, BBSV in 151, broad bean true mosaic virus (BBTMV) in 7, broad bean wilt virus (BBWV) in 47, BYMV in 314, cucumber mosaic virus (CMV) in 96, pea enation mosaic virus (PEMV) in 31, and pea seed-borne mosaic virus (PSbMV) in 49 samples. Identity of selected field isolates was confirmed by electron microscopy and host reaction studies. In a yield experiment, infection with BYMV, BBMV and BBSV 11 weeks after sowing (pre-flowering) led to 81, 54 and 84% yield loss, respectively. Inoculation with the same viruses 15 weeks after sowing (flowering) and 20 weeks after sowing (pod setting) led to 56, 84 and 18%, and 39, 37 and 18% yield loss, respectively.

Detection of banana bunchy top virus in virus-infected plants using polymerase chain reaction

Polymerase chain reaction (PCR) was developed for the detection of Banana bunchy top virus (BBTV) at maximum after 210 min and at minimum after 90 min using Pc-1 and Pc-2, respectively. PCR detection of BBTV in crude sap indicated that the freezing of banana tissue in liquid nitrogen (LN2) before extraction was more effective than using sand as the extraction technique. BBTV was also detected using PCR assay in 69 healthy and diseased plants using Na-PO4 buffer containing 1 % SDS. PCR detection of BBTV in nucleic acid extracts using seven different extraction buffers to adapt the use of PCR in routine detection in the field was studied. Results proved that BBTV was detected with high sensitivity in nucleic acid extracts more than in infectious sap. The results also suggested the common aetiology for the BBTV by the PCR reactions of BBTV in nucleic acid extracts from Australia, Burundi, Egypt, France, Gabon, Philippines and Taiwan. Results also proved a positive relation between the Egyptian-BBTV isolate and abaca bunchy top isolate from the Philippines, but there no relation was found with the Cucumber mosaic cucumovirus (CMV) isolates from Egypt and Philippines and Banana bract mosaic virus (BBMV) were found.

On the role of RNA silencing in the pathogenicity and evolution of viroids and viral satellites

Viroids and most viral satellites have small, noncoding, and highly structured RNA genomes. How they cause disease symptoms without encoding proteins and why they have characteristic secondary structures are two longstanding questions. Recent studies have shown that both viroids and satellites are capable of inducing RNA silencing, suggesting a possible role of this mechanism in the pathology and evolution of these subviral RNAs. Here we show that preventing RNA silencing in tobacco, using a silencing suppressor, greatly reduces the symptoms caused by the Y satellite of cucumber mosaic virus. Furthermore, tomato plants expressing hairpin RNA, derived from potato spindle tuber viroid, developed symptoms similar to those of potato spindle tuber viroid infection. These results provide evidence suggesting that viroids and satellites cause disease symptoms by directing RNA silencing against physiologically important host genes. We also show that viroid and satellite RNAs are significantly resistant to RNA silencing-mediated degradation, suggesting that RNA silencing is an important selection pressure shaping the evolution of the secondary structures of these pathogens.

Similarities in the Genomic Sequence and Coat Protein-Structure of Plant Viruses

The genomic sequences of several RNA plant viruses including cucumber mosaic virus, brome mosaic virus, alfalfa mosaic virus and tobacco mosaic virus have become available recently. The former two viruses are icosahedral while the latter two are bullet and rod shaped, respectively in particle morphology. The non-structural 3a proteins of cucumber mosaic virus and brome mosaic virus have an amino acid sequence homology of 35% and hence are evolutionarily related. In contrast, the coat proteins exhibit little homology, although the circular dichroism spectrum of these viruses are similar. The non-coding regions of the genome also exhibit variable but extensive homology. Comparison of the brome mosaic virus and alfalfa mosaic virus sequences reveals that they are probably related although with a much larger evolutionary distance. The polypeptide folds of the coat protein of three biologically distinct isometric plant viruses, tomato bushy stunt virus, southern bean mosaic virus and satellite tobacco necrosis virus have been shown to display a striking resemblance. All of them consist of a topologically similar 8-standard β-barrel. The implications of these studies to the understanding of the evolution of plant viruses will be discussed.

Evaluating new insecticides for the control of Helicoverpa spp. (Lepidoptera: Noctuidae) on capsicum and zucchini

The efficacy of insecticides in controlling Helicoverpa spp., predominantly H. armigera (Hubner), on capsicum and zucchini was tested in small plot trials. Indoxacarb, methoxyfenozide, spinosad, emamectin benzoate and novaluron provided control, as measured by the percentage of damaged fruit, equal to or better than standard treatments of methomyl or methomyl alternated with methamidophos on capsicum. The Helicoverpa nucleopolyhedrovirus gave control equivalent to the standard treatment, as did Bacillus thuringiensis aizawai, but B. thuringiensis kurstaki was ineffective. Helicoverpa armigera larvae were present in zucchini flowers but did little damage to the fruit. None of the insecticides significantly reduced the percentage of damaged zucchini fruit compared with the untreated control. Bifenthrin, spinosad, emamectin benzoate and methoxyfenozide were effective in controlling larvae in flowers, while methomyl, B. thuringiensis aizawai, B. thuringiensis kurstaki and novaluron were not effective. Data indicated that all the insecticides effectively controlled larvae of Diaphania indica (Saunders), cucumber moth, in the zucchini flowers. There has been a limited range of insecticides available to manage Helicoverpa spp. in these vegetable crops, but these trials demonstrate the effectiveness of a number of newer insecticides that could be used and that would be compatible with integrated pest management programs in the crops.

Isolation and Estimation of Cytokinins and Cytokinin-Containing Transfer RNAs from Cucumis

N6-({Delta}2-Isopentenyl) adenosine antibodies were used for the isolation of free cytokinins and cytokinin-containing tRNAs from parts of Cucumis sativus L. var. Guntur seedlings and for the estimation of cytokinins in them. Immobilized N6-({Delta}2-isopentenyl) adenosine antibodies retained tRNAs containing N6-({Delta}2-isopentenyl) adenosine and N6-(4-hydroxy-3-methylbut-2-enyl) adenosine with equal efficiencies. There were at least five cytokinins in the free form in cucumber seedlings. N6-(4-Hydroxy-3-methylbut-2-enyl) adenosine, N6-({Delta}2-isopentenyl) adenosine, and N6-({Delta}2-isopentenyl) adenine were present at least to the extent of 80, 23, and 9 nanograms, respectively, in the cotyledons and 40, 6, and 3 nanograms, respectively, in the decotyledonated seedlings per gram of tissue. Only two cytokinins were found in the tRNAs of cucumber cotyledons, namely N6-({Delta}2-isopentenyl) adenosine and N6-(4-hydroxy-3-methylbut-2-enyl) adenosine in amounts of 12 and 318 nanograms, respectively, per gram of tissue. Immunoaffinity chromatographic analysis of radiolabeled aminoacyl tRNAs from cucumber cotyledons showed that tRNAPhe and tRNATyr contained cytokinins whereas tRNAAla did not.

Developing and communicating strategies for controlling virus diseases in vegetable cucurbit crops

Virus diseases cause serious yield and quality losses in field grown cucurbit crops worldwide. In Australia, the main viruses of cucurbits are Papaya ringspot virus (PRSV), Squash mosaic virus (SqMV), Watermelon mosaic virus (WMV) and Zucchini yellow mosaic virus (ZYMV). Plants infected early have severely distorted fruit. High infection incidences, of ZYMV and PRSV in crops cause losses of marketable fruit of up to 100% and infected crops are often abandoned. Two new alternative hosts of ZYMV were identified, the native cucurbit Cucumis maderaspatanus and wild legume Rhyncosia minima. No new alternative hosts of PRSV, SqMV or WMV were found in Western Australia or Queensland. Seed transmission of ZYMV (0.7%) was found in seedlings grown from ZYMV-infected fruit of zucchini but not of pumpkin. None was detected with PRSV or SqMV in zucchini or pumpkin seedlings, respectively. ZYMV spread to pumpkins by aphids was greater downwind than upwind of a virus source. Delaying sowing by 2 weeks decreased ZYMV spread. Millet non-host barriers between pumpkin plantings slowed ZYMV infection. Host resistance gene (zym) in cucumber cultivars was effective against ZYMV. Pumpkin cultivars with resistance gene (Zym) became infected under high virus pressure but leaf symptoms were milder and infected plants higher yielding with more market-acceptable fruit than those without Zym. Most zucchini cultivars with Zym developed severe leaf and fruit symptoms. ZYMV, PRSV, WMV and SqMV spread readily from infected to healthy cucurbit plants by direct leaf contact. ZYMV survives and remains infective on diverse surfaces for up to 6 hours but can be inactivated by some disinfectants. Phylogenetic analysis indicates at least three separate introductions of ZYMV into Australia, with new introductions rarely occurring. ZYMV isolates clustered into three groups according to collection location i) Kununurra, ii) Northern Territory and iii) Carnarvon, Qld and Vic. A multiplex Real-Time PCR was developed which distinguished between the three groups of Australian isolates. Integrated disease management (IDM) strategies for virus diseases of vegetable cucurbit crops grown in the field were improved incorporating the new information gathered. These strategies are aimed at causing using minimal extra expense, labour demands and disruption to normal practices.

Tuotantotekniikan ja säilytysajan vaikutus kasvihuonekurkkujen ja -tomaattien aistittavaan laatuun.

Finland has moved from growing vegetables by natural light to year-round greenhouse production using artificial lighting. Determination of sensory effects on greenhouse-grown vegetables is important as sensory evaluation provides information which chemical methods can not. It can tell us about the quality of samples which affects the consumers' behaviour. There are different opinions on how the quality of vegetables should be determined. The consumers are dissatisfied with the quality of vegetables and fruits, although the variety of products is larger than ever. The aim of this study was to find out how artificial lighting contributes to the sensory quality of greenhouse tomatoes and cucumbers compared to traditional natural lighting, and how storage affects the sensory attributes of the samples. In this study there were two sets of tomatoes and two sets of cucumbers, representing two different harvest seasons. Sensory evaluation involved two steps. The first step was to sort the samples and the second step was to generate a profile using descriptive analysis. Sorting was found to give some approximate information on differences between tomato and cucumber samples. MDS-maps dimensions were presented by age and lighting technique. The reliability of sorting results was quite good. The quality of the natural products was inconsistent. Production technology had more of an effect on cucumber samples than tomato samples. Natural light cucumbers were, for example sweeter and softer than artificial light cucumbers. Age had an especially large effect on cucumber appearance characteristics. There were less differences between tomato samples than cucumber samples. Production technology had less of an effect on tomato samples than age, e.g. hardness decreased during storage. In this study, it was found that artificial lighting has little effect on the sensory quality of Finnish greenhouse tomatoes compared with tomatoes grown under natural light.

Unusual RNA plant virus integration in the soybean genome leads to the production of small RNAs

Horizontal gene transfer (HGT) is known to be a major force in genome evolution. The acquisition of genes from viruses by eukaryotic genomes is a well-studied example of HGT, including rare cases of non-retroviral RNA virus integration. The present study describes the integration of cucumber mosaic virus RNA-1 into soybean genome. After an initial metatranscriptomic analysis of small RNAs derived from soybean, the de novo assembly resulted a 3029-nt contig homologous to RNA-1. The integration of this sequence in the soybean genome was confirmed by DNA deep sequencing. The locus where the integration occurred harbors the full RNA-1 sequence followed by the partial sequence of an endogenous mRNA and another sequence of RNA-1 as an inverted repeat and allowing the formation of a hairpin structure. This region recombined into a retrotransposon located inside an exon of a soybean gene. The nucleotide similarity of the integrated sequence compared to other Cucumber mosaic virus sequences indicates that the integration event occurred recently. We described a rare event of non-retroviral RNA virus integration in soybean that leads to the production of a double-stranded RNA in a similar fashion to virus resistance RNAi plants.

Isolation Of A Cytokinin-Binding Protein From Cucumis-Sativus Cotyledons

A cytokinin-binding protein which exists as monomer and dimer was isolated from cucumber (Cucumis sativus L. var Guntur) cotyledons by affinity chromatography on AH-Sepharose-pi6 Ap /s> column. The protein bound to [3H]-N6-(Δ2-isopentenyl) adenosine trialcohol. On Sephadex G-50 chromatography it gave 2 peaks corresponding to molecular weight 4000 and 8000 daltons. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis, it gave only one band with an apparent molecular weight of 4000 daltons.

Molecular Characterization of Viruses Causing the Cassava Brown Streak Disease Epidemic in Eastern Africa

Cassava brown streak disease (CBSD) was described for the first time in Tanganyika (now Tanzania) about seven decades ago. Tanganyika (now Tanzania) about seven decades ago. It was endemic in the lowland areas of East Africa and inland parts of Malawi and caused by Cassava brown streak virus (CBSV; genus Ipomovirus; Potyviridae). However, in 1990s CBSD was observed at high altitude areas in Uganda. The causes for spread to new locations were not known.The present work was thus initiated to generate information on genetic variability, clarify the taxonomy of the virus or viruses associated with CBSD in Eastern Africa as well as to understand the evolutionary forces acting on their genes. It also sought to develop a molecular based diagnostic tool for detection of CBSD-associated virus isolates. Comparison of the CP-encoding sequences of CBSD-associated virus isolates collected from Uganda and north-western Tanzania in 2007 and the partial sequences available in Genbank revealed occurrence of two genetically distinct groups of isolates. Two isolates were selected to represent the two groups. The complete genomes of isolates MLB3 (TZ:Mlb3:07) and Kor6 (TZ:Kor6:08) obtained from North-Western (Kagera) and North-Eastern (Tanga) Tanzania, respectively, were sequenced. The genomes were 9069 and 8995 nucleotides (nt), respectively. They translated into polyproteins that were predicted to yield ten mature proteins after cleavage. Nine proteins were typical in the family Potyviridae, namely P1, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb and CP, but the viruses did not contain HC-Pro. Interestingly, genomes of both isolates contained a Maf/HAM1-like sequence (HAM1h; 678 nucleotides, 25 kDa) recombined between the NIb and CP domains in the 3’-proximal part of the genomes. HAM1h was also identified in Euphorbia ringspot virus (EuRSV) whose sequence was in GenBank. The HAM1 gene is widely spread in both prokaryotes and eukaryotes. In yeast (Saccharomyces cerevisiae) it is known to be a nucleoside triphosphate (NTP) pyrophosphatase. Novel information was obtained on the structural variation at the N-termini of polyproteins of viruses in the genus Ipomovirus. Cucumber vein yellowing virus (CVYV) and Squash vein yellowing virus (SqVYV) contain a duplicated P1 (P1a and P1b) but lack the HC-Pro. On the other hand, Sweet potato mild mottle virus (SPMMV), has a single but large P1 and has HC-Pro. Both virus isolates (TZ:Mlb3:07 & TZ:Kor6:08) characterized in this study contained a single P1 and lacked the HC-Pro which indicates unique evolution in the family Potyviridae. Comparison of 12 complete genomes of CBSD-associated viruses which included two genomes characterized in this study, revealed genetic identity of 69.0–70.3% (nt) and amino acid (aa) identities of 73.6–74.4% at polyprotein level. Comparison was also made among 68 complete CP sequences, which indicated 69.0-70.3 and 73.6-74.4 % identity at nt and aa levels, respectively. The genetic variation was large enough for dermacation of CBSD-associated virus isolates into two distinct species. The name CBSV was retained for isolates that were related to CBSV isolates available in database whereas the new virus described for the first time in this study was named Ugandan cassava brown streak virus (UCBSV) by the International Committee on Virus Taxonomy (ICTV). The isolates TZ:Mlb3:07 and TZ:Kor6:08 belong to UCBSV and CBSV, respectively. The isolates of CBSV and UCBSV were 79.3-95.5% and 86.3-99.3 % identitical at nt level, respectively, suggesting more variation amongst CBSV isolates. The main sources of variation in plant viruses are mutations and recombination. Signals for recombination events were detected in 50% of isolates of each virus. Recombination events were detected in coding and non-coding (3’-UTR) sequences except in the 5’UTR and P3. There was no evidence for recombination between isolates of CBSV and UCBSV. The non-synonomous (dN) to synonomous (dS) nucleotide substitution ratio (ω) for the HAM1h and CP domains of both viruses were ≤ 0.184 suggesting that most sites of these proteins were evolving under strong purifying selection. However, there were individual amino acid sites that were submitted to adaptive evolution. For instance, adaptive evolution was detected in the HAM1h of UCBSV (n=15) where 12 aa sites were under positive selection (P< 0.05) but not in CBSV (n=12). The CP of CBSV (n=23) contained 12 aa sites (p<0.01) while only 5 aa sites in the CP gene of UCBSV were predicted to be submitted to positive selection pressure (p<0.01). The advantages offered by the aa sites under positive selection could not be established but occurrence of such sites in the terminal ends of UCBSV-HAMIh, for example, was interpreted as a requirement for proteolysis during polyprotein processing. Two different primer pairs that simultaneously detect UCBSV and CBSV isolates were developed in this study. They were used successfully to study distribution of CBSV, UCBSV and their mixed infections in Tanzania and Uganda. It was established that the two viruses co-infect cassava and that incidences of co-infection could be as high as 50% around Lake Victoria on the Tanzanian side. Furthermore, it was revealed for the first time that both UCBSV and CBSV were widely distributed in Eastern Africa. The primer pair was also used to confirm infection in a close relative of cassava, Manihot glaziovii (Müller Arg.) with CBSV. DNA barcoding of M. glaziovii was done by sequencing the matK gene. Two out of seven M. glaziovii from the coastal areas of Korogwe and Kibaha in north eastern Tanzania were shown to be infected by CBSV but not UCBSV isolates. Detection in M. glaziovii has an implication in control and management of CBSD as it is likely to serve as virus reservoir. This study has contributed to the understanding of evolution of CBSV and UCBSV, which cause CBSD epidemic in Eastern Africa. The detection tools developed in this work will be useful in plant breeding, verification of the phytosanitary status of materials in regional and international movement of germplasm, and in all diagnostic activities related to management of CBSD. Whereas there are still many issues to be resolved such as the function and biological significance of HAM1h and its origin, this work has laid a foundation upon which the studies on these aspects can be based.