<Emphasis Type="Italic">In situ</Emphasis> localization of <Emphasis Type="Italic">Grapevine fanleaf virus</Emphasis> and phloem-restricted viruses in embryogenic callus of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

In grapevine, somatic embryogenesis is particularly effective in eliminating several important virus diseases. However, the mechanism whereby regenerated somatic embryos are freed of the viruses is not clear. The distribution of Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated virus-3 (GLRaV-3) and Grapevine virus A (GVA) in embryogenic callus of grapevine was investigated by in situ hybridization using digoxygenin-labelled oligonucleotide probes. Four months after culture initiation, in callus originated by GFLV-infected explants we observed a mosaic of infected and uninfected cells, with high concentrations of viruses in some cell groups in peripheral zones of the callus. In addition some abnormal somatic embryos showed a high hybridization signal. In callus originated by GVA- and GLRaV-3-infected explants the viruses were concentrated in few cells surrounded by areas of virus-free cells. The two viruses were generally localized in different clusters of cells inside the callus and the levels of infection were lower than those observed in GFLV-infected callus. No virus was detected in callus nor in somatic embryos after 6 months of culture. The results highlight the difficulties of some viruses at stably invading callus tissues and the differential ability of GFLV to spread in the callus cells compared to the phloem-limited viruses.     

<Emphasis Type="Italic">Odontoglossum ringspot virus</Emphasis> causing flower crinkle in <Emphasis Type="Italic">Phalaenopsis</Emphasis> hybrids

A new disorder exhibiting flower crinkle on Phalaenopsis orchids bearing white flowers has been observed in Taiwan, China and Japan for several years. This disorder decreased the flower longevity and was considered as a physiological syndrome. The objective of this study was to identify and characterize the real causal agent of this new Phalaenopsis disorder. Five plants of Phalaenopsis hybrids “V3” (Phal. Yukimai × Phal. Taisuco Kochdian) with flower crinkle symptoms were collected and tested by enzyme-linked immunosorbent assay with antisera against 18 viruses. The extract of leaves and flowers from one diseased plant (96-Ph-16) reacted positively only to antiserum against Odontoglossum ringspot virus (ORSV), while those from the other four plants (96-Ph-7, 96-Ph-17, 96-Ph-18 and 96-Ph-19) reacted positively to the antisera against ORSV and Cymbidium mosaic virus (CymMV). Five ORSV isolates, one each from flowers of those five diseased Phalaenopsis orchids, were established in Chenopodium quinoa. A CymMV culture was isolated from the flowers of one of the ORSV/CymMV mix-infected Phalaenopsis orchids (96-Ph-19). To determine the causal agent of the flower crinkle disease, healthy Phalaenopsis seedlings were singly or doubly inoculated with the isolated ORSV and/or CymMV. Results of back inoculation indicated that ORSV is the sole causal agent of the crinkle symptom on petals of Phalaenopsis orchid. The CP gene of the ORSV isolates from this study shared 97.3–100% nucleotide identity and 96.2–100% amino acid identity with those of 41 ORSV isolates available in GenBank. This is the first report demonstrating ORSV as the sole virus causing flower crinkle disease on Phalaenopsis orchids.     

Immunodetection of the replicative complex of <Emphasis Type="Italic">Barley yellow dwarf</Emphasis><Emphasis Type="Italic">virus</Emphasis>-PAV <Emphasis Type="Italic">in vivo</Emphasis>

The genomic fragments of two open reading frames (ORFs) 1 and 2 of German and Canadian PAV isolates of Barley yellow dwarf virus (BYDV-PAV) were sequenced. Sequences only slightly differed from previously published sequences of this virus. Two polyclonal antisera against proteins encoded by ORFs 1 and 2 of a German ASL-1 isolate were developed using recombinant antigens expressed in E. coli as a fusion either to His₆− or thioredoxin-tags. In Western blot analysis with total protein extracts from BYDV infected plants, antisera efficiently recognized the 99 kDa fusion protein expressed from ORF1 and ORF2 (P1–P2 protein). Later in infection the P1–P2 protein disappeared and two smaller proteins, revealing sizes of 39 and 60 kDa, could be detected.     

Phylogeny of Egyptian isolates of <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> (CMV) and <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> (ToMV) infecting <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>

Four Cucumber mosaic virus (CMV) (CMV-HM 1–4) and nine Tomato mosaic virus (ToMV) (ToMV AH 1–9) isolates detected in tomato samples collected from different governorates in Egypt during 2014, were here characterized. According to the coat protein gene sequence and to the complete nucleotide sequence of total genomic RNA1, RNA2 and RNA3 of CMV-HM3 the new Egyptian isolates are related to members of the CMV subgroup IB. The nine ToMV Egyptian isolates were characterized by sequence analysis of the coat protein and the movement protein genes. All isolates were grouped within the same branch and showed high relatedness to all considered isolates (98–99%). Complete nucleotide sequence of total genomic RNA of ToMV AH4 isolate was obtained and its comparison showed a closer degree of relatedness to isolate 99–1 from the USA (99%). To our knowledge, this is the first report of CMV isolates from subgroup IB in Egypt and the first full length sequencing of an ToMV Egyptian isolate.     

Competence of <Emphasis Type="Italic">Frankliniella occidentalis</Emphasis> and <Emphasis Type="Italic">Frankliniella intonsa</Emphasis> strains as vectors for <Emphasis Type="Italic">Chrysanthemum stem necrosis virus</Emphasis>

The vector competence of Frankliniella occidentalis for Chrysanthemum stem necrosis virus (CSNV) was evaluated. Three vector strains with distinct competences for Tomato spotted wilt virus (TSWV) transmission were investigated, including an artificially selected strain (TsH) that has a particularly high competence (>90 %). Newly hatched larvae of F. occidentalis were given an acquisition access period of 5 days on CSNV-infected D. stramonium leaves, and reared to maturity. Their transmission efficiencies were examined using a leaf disk assay using Petunia x hybrida leaves. Following the leaf disk assay, the virus accumulation in the vectors was examined via a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) of their bodies. The results showed that the CSNV acquisition and transmission efficiency of the TsH strain did not differ from those of the others, indicating that the competence of F. occidentalis as a vector for CSNV is not related to that for TSWV. The CSNV transmission and acquisition efficiencies of two F. intonsa strains (Hiroshima and Fukuoka) were also evaluated. In Hiroshima strain, 35 % of adults were viruliferous, but only two transmitters (3 %) were observed. In Fukuoka strain, 6 % were viruliferous, and no transmitters were observed. These results indicate that F. intonsa cannot be a major vector for CSNV. The accumulation of CSNV in the adults of F. occidentalis and F. intonsa evaluated using DAS-ELISA showed a significant difference in ELISA values among transmitter, viruliferous non-transmitter, and non-viruliferous individuals. These results clearly demonstrated that only transmitters that accumulated a threshold quantity of virus can transmit CSNV to plants.     

Natural infection of <Emphasis Type="Italic">Nicandra physaloides</Emphasis> by <Emphasis Type="Italic">Tomato severe rugose virus</Emphasis> in Brazil

Nicandra physaloides, a common weed in South America, was found to be infected by an isolate of Tomato severe rugose virus (ToSRV), a bipartite begomovirus. The plants developed severe yellow rugose mosaic and were collected in São Paulo State, Brazil. This isolate of ToSRV was transmitted by Bemisia tabaci B biotype from infected plants of N. physaloides to healthy plants of N. physaloides and tomato in a glasshouse. This is the first report of natural infection of N. physaloides by ToSRV in Brazil.     

Performances and application of antisera produced by recombinant capsid proteins of <Emphasis Type="Italic">Cymbidium mosaic virus</Emphasis> and <Emphasis Type="Italic">Odontoglossum ringspot virus</Emphasis>

Antisera against important orchid viruses, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV), were separately produced using bacterially expressed recombinant capsid proteins (CP), instead of purified virus particles, as immunogens. These antisera were then designated as home-made CymMV CP antiserum (HM-Cy) and home-made ORSV CP antiserum (HM-OR). The high specificity of HM-Cy and HM-OR were confirmed by immunoblot. Their detection limits were determined using indirect-enzyme-linked immunosorbent assay (I-ELISA). Both HM-Cy and HM-OR showed low background reactivity to healthy plants and thus displayed a high S/H ratio (sample OD405/healthy control OD405) in tested orchids. The data indicated that our antisera were efficient and accurate in determination of negative and positive results in ELISA test as commercial antibodies. Therefore, these home-made antisera of CymMV and ORSV are suitable for the certification programme of orchids due to their low cost and high specificity. HM-Cy and HM-OR were further used for a field survey to study the incidence of CymMV and ORSV. The results showed that CymMV is more prevalent than ORSV in Taiwan.     

Transgene-mediated resistance to <Emphasis Type="Italic">Papaya ringspot virus</Emphasis>: challenges and solutions

Global papaya production is severely affected by papaya ringspot disease caused by Papaya ringspot virus (PRSV). Management of this potyvirus is challenging, due to 1) its non-persistent transmission by numerous aphid species and 2) the diversity of PRSV strains that exists within a country or between different geographical regions. Papaya cultivars with transgenic resistance have reduced the impact of the disease. There are no effective alternatives to transgenic resistance available in areas where disease pressure is high. In Hawaii, transgenic papayas such as “SunUp” and “Rainbow” have remained resistant to PRSV more than two decades saving the commercial papaya industry. Following the success in Hawaii, researchers from other countries have focused on developing PRSV-resistant transgenic papaya. These transgenic cultivars often demonstrated an initial transitory resistance that was ultimately overcome by the virus. For other cases, resistance was inconsistent. That is, some transgenic lines were resistant while others were not. Transgenic cultivars are now losing PRSV-resistance for various reasons in China and Taiwan. In this review, we present an update on work with transgenic papaya with resistance to PRSV. The focus is on factors affecting transgenic resistance in papaya and our attempt to explain why the Hawaiian scenario of complete and durable resistance has not been replicated in other regions. The utilization of more recent technologies to the development of virus resistance in papaya is also discussed.     

Suppression of <Emphasis Type="Italic">Papaya ringspot virus</Emphasis> infection in <Emphasis Type="Italic">Carica papaya</Emphasis> with CAP-34, a systemic antiviral resistance inducing protein from <Emphasis Type="Italic">Clerodendrum aculeatum</Emphasis>

CAP-34, a protein from Clerodendrum aculeatum inducing systemic antiviral resistance was evaluated for control of Papaya ringspot virus (PRSV) infection in Carica papaya. In control plants (treated with CAP-34 extraction buffer) systemic mosaic became visible around 20 days that intensified up to 30 days in 56% plants. During this period, CAP-34-treated papaya did not show any symptoms. Between 30 and 60 days, 95% control plants exhibited symptoms ranging from mosaic to filiformy. In the treated set during the same period, symptoms appeared in only 10% plants, but were restricted to mild mosaic. Presence of PRSV was determined in induced-resistant papaya at the respective observation times by bioassay, plate ELISA, immunoblot and RT-PCR. Back-inoculation with sap from inoculated resistant plants onto Chenopodium quinoa did not show presence of virus. The difference between control and treated sets was also evident in plate-ELISA and immunoblot using antiserum raised against PRSV. PRSV RNA was not detectable in treated plants that did not show symptoms by RT-PCR. Control plants at the same time showed a high intensity band similar to the positive control. We therefore suggest that the absence/delayed appearance of symptoms in treated plants could be due to suppressed virus replication.     

Genetic diversity of <Emphasis Type="Italic">Rhizobium vitis</Emphasis> strains in Japan based on multilocus sequence analysis of <Emphasis Type="Italic">pyrG</Emphasis>, <Emphasis Type="Italic">recA</Emphasis> and <Emphasis Type="Italic">rpoD</Emphasis>

Forty-one strains of Rhizobium vitis, either tumorigenic (Ti) or nonpathogenic, were characterized using multilocus sequence analysis (MLSA) of the partial nucleotide sequences of pyrG, recA, and rpoD. The strains separated into seven clades. Rhizobium vitis (Ti) strains isolated from Japan were divided into five genetic groups (A to E), and nonpathogenic R. vitis strains were divided into two genetic groups (F and G). This result suggests that there are new genetic groups of R. vitis in Japan. Among these groups, members of A and B groups are widely distributed throughout Japan.     

Resistance of <Emphasis Type="Italic">Solanum</Emphasis> species to <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> subgroup IA and its vector <Emphasis Type="Italic">Myzus persicae</Emphasis>

Sixty-nine tomato genotypes representing nine Solanum species were evaluated for resistance to Cucumber mosaic virus (CMV) subgroup IA and its aphid vector Myzus persicae. Resistance was assessed by visual scoring of symptoms in the field under natural conditions, and in the greenhouse by artificial inoculations through aphid M. persicae and mechanical transmissions in the year 2007 and 2009. Considerable variation in responses was observed among the evaluation methods used. Field evaluations were found liable to errors as different levels were observed for the same genotypes in the different years, however mechanical inoculation was found to be the most useful in identifying CMV subgroup IA resistance, in contrast aphid transmission was most useful in identifying insect transmission resistance. All genotypes observed as highly resistant to CMV subgroup IA in the field or through vector transmission became systemically infected through mechanical inoculations. Using mechanical inoculation, six genotypes (TMS-1 of S. lycopersicum, LA1963 and L06049 of S. chilense, LA1353, L06145 and L06223 of S. habrochaites) were found resistant and another six (L06188 and L06238 of S. neorickii, L06219 of S. habrochaites, L05763, L05776 and L06240 of S. pennellii) were found tolerant showing mild symptoms with severity index (SI) ranging 1-2 and with delayed disease development after a latent period (LP) of 18–30 days. However, these genotypes were found to be resistant to highly resistant in the field and through inoculation by M. persicae; and they also supported low population levels of M. persicae except TMS-1. Another nine genotypes (LA2184 of S. pimpinellifolium L., LA2727 of S. neorickii, LA0111, L06221, L06127 and L06231 of S. peruvianum L., LA1306, L06057 and L06208 of S. chmielewskii) showing a susceptible response after mechanical inoculation were highly resistant, resistant and tolerant after M. persicae transmission. The resistant genotypes, identified in the present study can be exploited in the breeding programmes aimed at developing tomato varieties resistant to CMV subgroup IA and broadening the genetic base of CMV-resistant germplasm. The differences observed between mechanical and aphid transmission suggests that one should consider both evaluation methods for tomato germplasm screening against CMV subgroup IA.     

Relationships of <Emphasis Type="Italic">Barley yellow dwarf virus</Emphasis>-PAV and <Emphasis Type="Italic">Cereal yellow dwarf virus</Emphasis>-RPV from Iran with viruses of the family <Emphasis Type="Italic">Luteoviridae</Emphasis>

Barley yellow dwarf disease is one of the most important problems confronting cereal production in Iran. Barley yellow dwarf virus-PAV (BYDV-PAV) and Cereal yellow dwarf virus-RPV (CYDV-RPV) are the predominant viruses associated with the disease. One isolate of BYDV-PAV from wheat (PAV-IR) and one isolate of CYDV-RPV from barley (RPV-IR) were selected for molecular characterisations. A genome segment of each isolate was amplified by PCR. The PAV-IR fragment (1264 nt) covered a region containing partial genes for coat protein (CP), read through protein (RTP) and movement protein (MP). PAV-IR showed a high sequence identity to PAV isolates from USA, France and Japan (96–97%). In a phylogenetic analysis it was placed into PAV group I together with PAV isolates from barley and oats. The fragment of RPV-IR (719 nt) contained partial genes for CP, RTP and MP. The sequence information confirmed its identity as CYDV. However, RPV-IR showed 90–91% identity with both RPV and Cereal yellow dwarf virus-RPS (CYDV-RPS). Phylogenetic analyses suggested that it was more closely related to RPS. These data comprise the first attempt to characterise BYD-causing viruses in Iran and southwest Asia. The nucleotide sequence data reported appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers AY450425 and AY450454     

<Emphasis Type="Italic">Corythucha ciliata</Emphasis>, a new <Emphasis Type="Italic">Platanus</Emphasis> pest in Turkey

An invasive Tingidae, the platanus lace bug Corythucha ciliata (Say, 1832) (Hemiptera: Tingidae), which specializes on Platanus spp., was found for the first time in Turkey in 2007; it was recorded from a 120 km² area in the northwestern part of the country. Infestations occurred in an area between Taşkesti and Abant in Bolu Province, which is located near major cities and two main motorways. The pest species is newly spreading in Turkey, causing noticeable damage to Platanus orientalis trees.     

First report of a <Emphasis Type="Italic">Grapevine Algerian latent virus</Emphasis> disease on statice plants (<Emphasis Type="Italic">Limonium sinuatum</Emphasis>) in Japan

A viral disease was found in Nagano Prefecture, Japan, on statice (Limonium sinuatum) with chlorotic leaf spot, necrotic stunt, and dwarfing. Spherical virus particles 30 nm in diameter were isolated from infected plants and statice seedlings and caused identical symptoms 4 weeks after mechanical inoculation. Nucleotide and deduced amino acid sequences of the coat protein showed 98% and 98.7% identities with those of Grapevine Algerian latent virus (GALV) nipplefruit strain. This is the first report in Japan of a viral disease on statice caused by GALV. The nucleotide sequence data reported here are available in the DDBJ/EMBL/GenBank databases under accession AB461854.     

Detection and quantification of <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> in irrigation waters

A quantitative RT real-time PCR method was developed for the detection and quantification of Tomato mosaic virus (ToMV) in irrigation waters. These have rarely been monitored for the presence of plant pathogenic viruses, mostly due to the lack of efficient and sensitive detection methods. The newly developed method presented here offers a novel approach in monitoring the health status of environmental waters. ToMV was reliably detected at as low as 12 viral particles per real-time PCR reaction, which corresponds to the initial concentration of approximately 4.2 × 10^?10 mg (6,300 viral particles) of ToMV per ml of sample. The sensitivity of the method was further improved by including the Convective Interaction Media^® (CIM) monolithic chromatographic columns for quick and efficient concentration of original water samples. Seven out of nine water sources from different locations in Slovenia tested positive for ToMV, after concentrating the sample. Four samples tested ToMV-positive without the concentrating procedure. The presence and integrity of infective ToMV particles in the original sample, as well as in the chromatographic fraction, was confirmed using different methods from test plants, DAS ELISA to electron microscopy and real-time PCR. In this study, we propose a unique and simple diagnostic scheme for rapid, efficient, and sensitive monitoring of irrigation waters that could also be adopted for other plant, human or animal viruses.     

First Report of <Emphasis Type="Italic">Pepper mottle virus</Emphasis> on <Emphasis Type="Italic">Capsicum annuum</Emphasis> in Japan

Pepper mottle virus, genus Potyvirus, was first identified in Japan based on particle morphology, host range, aphid transmission, and molecular classification using the nucleotide sequence of the coat protein gene and 3-untranslated region.     

<Emphasis Type="Italic">Cucurbit aphid-borne yellows virus</Emphasis> in Egypt

During 2010, yellowing symptoms were frequently observed in cultivated squash fields in Egypt. A total of 717 symptomatic squash leaf samples were collected from four regions where squash cultivation is of economic importance for the country: Kafrelsheikh, El-Behira, El-Sharkia and El-Ismailia. Serological analysis showed that 95.6% of the symptomatic squash samples were infected by Cucurbit aphid-borne yellows virus (CABYV), and visual estimation of the incidence of yellowing symptoms suggested a very high incidence of CABYV in the fields. Twelve CABYV isolates were characterized by sequencing two regions of the viral genome, open reading frame (ORF) 3 and ORFs 4/5. Overall, Egyptian isolates were very similar among them, and had higher similarity values with a French than with a Chinese isolate. The average nucleotide diversity for ORF 3 was significantly higher than for the other two regions, indicating that variability is not evenly distributed along the viral genome. The ratios between nucleotide diversity values in non-synonymous (d _N ) and synonymous (d _S) positions (d _N /d _S) for each ORF showed that the three ORFs are evolving under different pressures, although predominantly under purifying selection. Phylogenetic analyses revealed that these Egyptian isolates, with only one exception, shared the same clade with a French isolate. Moreover, these analyses suggested that Egyptian isolates belong to the Mediterranean group described previously.     

Identification and characterization of pathotypes in <Emphasis Type="Italic">Puccinia horiana</Emphasis>, a rust pathogen of <Emphasis Type="Italic">Chrysanthemum</Emphasis> x <Emphasis Type="Italic">morifolium</Emphasis>

Puccinia horiana is the causal agent of chrysanthemum white rust or Japanese rust. This microcyclic autoecious rust has a quarantine status and can cause major damage in the commercial production of Chrysanthemum x morifolium. Given the international and often trans-continental production of planting material and cut flowers of chrysanthemum and the decreasing availability of registered fungicides in specific regions, breeding for resistance against P. horiana will gain importance and will need to involve the appropriate resistance genes for the pathotypes that may be present. As pathotypes have not been well characterized in this system, the main objective was to build an international collection of isolates and screen these on a large collection of cultivars to identify different pathotypes. Using a robust and high throughput bioassay, we tested 36 selected cultivars with 22 individual single-pustule isolates of P. horiana. The isolates originated from three different continents over 4 different collection years and included some isolates from cultivars previously reported as resistant. In most cases the bioassays resulted in a clear scoring of interaction phenotypes as susceptible or resistant, while in several cases consistent intermediate phenotypes were found, often on specific cultivars. Twenty-four of the cultivars gave a differential interaction phenotype profile. All isolates produced a unique profile, infecting a minimum of 4 and a maximum of 19 differential cultivars. Based on the Person analysis of these profiles, this pathosystem contains at least seven resistance genes (and seven avirulence genes), demonstrating the highly complex race structure in this pathosystem.