<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.     

Distinct association of an alphasatellite and a betasatellite with <Emphasis Type="Italic">Tomato leaf curl New Delhi virus</Emphasis> in field-infected cucurbit

Leaf samples of Cucurbita pepo with yellow mosaic disease symptoms were collected in 2012. Rolling circle amplification and PCR amplification with begomovirus-specific primers confirmed the presence of an Old World bipartite begomovirus, an alphasatellite and a betasatellite. Molecular analysis of full-length sequences showed that Tomato leaf curl New Delhi virus (DNA-A) is associated with its cognate DNA-B, Papaya leaf curl betasatellite and a novel alphasatellite. To the best of our knowledge, this is the first report of an alphasatellite and a betasatellite associated with a bipartite begomovirus.     

<Emphasis Type="Italic">Ageratum</Emphasis><Emphasis Type="Italic">yellow vein virus</Emphasis> isolated from tomato plants with leaf curl on Ishigaki Island,Okinawa, Japan

In 2005, severe leaf curling and yellowing were observed on tomato plants on Ishigaki Island. Because the symptoms were consistent with infection by a begomovirus, we used a polymerase chain reaction (PCR) with specific primers for begomovirus DNA-A and DNA satellite component (DNA-β) and detected products of the expected sizes from symptomatic tomato plant samples. DNA sequence analyses of the PCR products revealed that the symptomatic tomato plants were associated with Ageratum yellow vein virus (AYVV) infection. We confirmed AYVV transmission from the naturally infected weed host, Ageratum conyzoides, to healthy tomato plants by the insect vector Bemisia tabaci B biotype. This report is the first of AYVV occurrence in Japan.     

Co-transmission of <Emphasis Type="Italic">Tomato yellow leaf curl virus</Emphasis> (TYLCV)-Mld and TYLCV-IL by the whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

The ability of the whitefly Bemisia tabaci to transmit two strains of Tomato yellow leaf curl virus, the Israel and Mild strains, was studied after serial transfers of individual whiteflies that were viruliferous for both strains to tomato plants. After single whiteflies had successive acquisition feedings first on a single plant infected with one strain and then on a plant infected with the other strain, the single whiteflies later transmitted intermittently one, the other, or both strains to the test plants during serial transfers at 1-day intervals. Because both strains were found in the head, abdomen, and legs dissected from whiteflies during the retention period after the two successive acquisition feedings, both strains apparently circulate from midgut cells to salivary glands through the hemolymph.     

Pymetrozine interferes with transmission of<Emphasis Type="Italic">Tomato yellow leaf curl virus</Emphasis> by the whitefly<Emphasis Type="Italic">Bemisia tabaci</Emphasis>

Pymetrozine, a novel compound belonging to the class pyridine-azomethines, is a feeding inhibitor labeled for use against plant pests in the order Hemiptera. Pymetrozine was evaluated for its ability to interfere with whitefly transmission of the begomovirusTomato yellow leaf curl virus (TYLCV). Pymetrozine was applied as Fulfill ^TM 50 WG at two rates (0.291 and 0.582 g formulationl ⁻¹) to tomato seedlings with four to six true leaves. Viruliferous whiteflies (three to five per plant) were added 1, 4, 7 and 11 d after a single application of pymetrozine, and transmission rates were determined 4 wk after the addition of whiteflies. Pymetrozine provided protection against transmission of TYLCV by viruliferous whiteflies for up to 1 wk after a single apliation. No phytotoxicity was observed on tomato transplants. These results indicate that pymetrozine could be an effective tool for tomato transplant producers to protect susceptible transplants from infection by begomoviruses, such as TYLCV. Pymetrozine might also work well as part of an integrated approach to begomovirus management in greenhouse tomato fruit production. http://www.phytoparasitica.org positing Oct. 20, 2003.     

First report of blueberry red ringspot disease caused by <Emphasis Type="Italic">Blueberry</Emphasis><Emphasis Type="Italic">red</Emphasis><Emphasis Type="Italic">ringspot</Emphasis><Emphasis Type="Italic">virus</Emphasis> in Japan

Virus-like symptoms—red ringspots on stems and leaves, circular blotches or pale spots on fruit—were found on commercial highbush blueberry (Vaccinium corymbosum) cultivars Blueray, Weymouth, Duke and Sierra in Japan. In PCR testing, single DNA fragments were amplified from total nucleic acid samples of the diseased blueberry bushes using primers specific to Blueberry red ringspot virus (BRRV). Sequencing analysis of the amplified products revealed 95.7–97.7% nucleotide sequence identity with the BRRV genome. This paper is the first report of blueberry red ringspot disease caused by BRRV in Japan. The nucleotide sequence data reported in this paper are available in the GenBank/EMBL/DDBJ database as accessions AB469884 to AB469893 for BRRV isolates from Japan.     

Detection and characterization of <Emphasis Type="Italic">Ageratum enation virus</Emphasis> and a nanovirus-like satellite DNA1 from zinnia causing leaf curl symptoms in India

Leaf curl in Zinnia elegans was observed in a subtemperate region in northern India, and the causal organism was identified as Ageratum enation virus (AEV). The virus appeared to be monopartite and associated with nanovirus-like satellite DNA1. The whole genome was amplified by rolling circle amplification, cloned, and sequenced using a primer walking strategy. The genome (FN543099) was composed of 2753 nucleotides (nt) and that of DNA1 (FN543100) of 1374 nt. DNA-β was not detected from any of the infected samples. The study presents the first report of a begomovirus from Zinnia spp. in India and the complete nucleotide sequence characterization of AEV associated with nanovirus-like satellite DNA1.     

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.     

Identification and characterization of <Emphasis Type="Italic">Apple stem grooving virus</Emphasis> causing leaf distortion on pear (<Emphasis Type="Italic">Pyrus pyrifolia</Emphasis>) in Taiwan

A putative virus-induced disease of pear (Pyrus pyrifolia var. Hengshen) showing symptoms of reduced size of foliage and leaf distortion was observed in orchards in central Taiwan in 2004. The sap of symptomatic leaf samples reacted positively to an antiserum against Apple stem grooving virus (ASGV). Two virus cultures, designated as TS1 and TS2, were isolated from symptomatic pears. Flexuous filamentous virions of ∼ 12 × 600 nm were observed in symptomatic pear leaves and purified virus preparations. Results of back inoculation of pear seedlings with TS1 revealed that ASGV was the causal agent of the disease. Sequence analyses of the cloned coat protein (CP) genes of TS1 and TS2 shared 88–92.4% nucleotide and 90.7–97.1% amino acid identities with those of other ASGV isolates available in GenBank. The polyclonal antibody generated against ASGV TS1 has been routinely used for the detection of the ASGV-infection in the imported pear scions for quarantine purpose via enzyme-linked immunosorbent assays (ELISAs). One of 1,199 samples of pear scions imported from Japan during 2005–2007 was identified as ASGV-positive and the virus was designated as AGJP-22. The CP gene amplified from this AGJP-22 shared 97.9–98.3% amino acid identities to those of the domestic isolates and they were closely related phylogenetically. To date, these data present for the first time conclusive evidence revealing that ASGV is indeed the causal agent of the pear disease displaying symptoms of reduced size of foliage and leaf distortion in Taiwan.     

Characterization of <Emphasis Type="Italic">Pepper yellow leaf curl virus,</Emphasis> a tentative new <Emphasis Type="Italic">Polerovirus</Emphasis> species causing a yellowing disease of pepper

Pepper (Capsicum annuum) is an important crop worldwide. In Israel, approximately 2,500 ha are grown all year round for the local and export markets. Herein, we report the identification of a viral pathogen causing a new devastating disease in pepper crops. The disease syndrome includes shortening of stem internodes, interveinal yellowing, and upward rolling of the leaf blade, accompanied by fruit discoloration and size reduction. Virus purification from infected plants yielded isometric particles, 25 nm in diameter. The causal agent of the disease was tentatively named Pepper yellow leaf curl virus (PYLCV). The virus cross-reacted in DAS ELISA with antisera against Cucurbit aphid-borne yellows virus and Potato leafroll virus, members of the Polerovirus genus. The partial nucleotide sequence obtained from the cloned viral coat protein and movement protein genes indicated 92% identity at the amino acid level with Tobacco vein distortion virus (TVDV), another member of the Polerovirus genus. However, the host range of PYLCV is significantly different from the host range described for TVDV. Based on our findings, the taxonomic status of PYLCV is discussed.     

Evidence of <Emphasis Type="Italic">olive mild mosaic virus</Emphasis> transmission by <Emphasis Type="Italic">Olpidium brassicae</Emphasis>

Transmission of three strains of OMMV by an Olpidium sp. was evaluated and compared. The three strains were 1) an OMMV wild type (WT) recovered from olive trees, 2) an OMMV variant (L11) obtained after 15 serial passages of single local lesions induced in Chenopodium murale plants, and 3) a construct OMMV/OMMVL11 in which the coat protein (CP) gene replaced that of the wild type. A single-sporangial culture derived from Chinese cabbage (Brassica pekinensis) used as a bait plant grown in soil of an olive orchard, was identified as Olpidium brassicae based on the size and sequence of the generated amplicon in PCR specific tests. Each of the three virus strains was soil transmitted to cabbage roots in the absence of the fungus at similar rates of 30 to 40%. Separate plant inoculation by O. brassicae zoospores incubated with each viral strain resulted in enhanced transmission of OMMV, reaching 86% of infection whereas that of the other two strains remained practically unaffected at ca. 34%. Binding assays showed that the amount of virus bound to zoospores, estimated spectrophotometrically, was 7% in the case of OMMV, and practically nil in the case of the other two viral strains. Substitution of the coat protein (CP) gene of OMMV by that of the OMMV L11 strain, drastically reduced viral transmissibility in the presence of zoospores to the level of that observed in their absence. Our data shows that OMMV soil transmission is greatly enhanced by O. brassicae zoospores and that the viral CP plays a significant role in this process, most likely by facilitating virus binding and later entrance into the host plant roots.     

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.     

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.     

First report of <Emphasis Type="Italic">Ageratum enation virus</Emphasis> infecting <Emphasis Type="Italic">Crassocephalum crepidioides</Emphasis> (Benth.) S. Moore and <Emphasis Type="Italic">Ageratum conyzoides</Emphasis> L. in India

In October 2009, vein yellowing disease was observed on the weeds Crassocephalum crepidioides and Ageratum conyzoides in a subtemperate region in northern India. Ageratum enation virus (AEV), along with a nanovirus like satellite DNA 1, was found to be associated with both weeds. The isolates had 99% identity with each other and with an isolate of AEV reported from Zinnia elegans from this region. To the best of our knowledge, this report is the first of any begomovirus infection in C. crepidioides in India and the first on AEV infecting C. crepidioides worldwide and A. conyzoides in India.     

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.     

First natural co-occurrence of tomato leaf curl New Delhi virus DNA-A and chili leaf curl betasatellite on tomato plants (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> L.) in India

Tomato leaf curl disease (ToLCD) affected 25% of the tomato crop in Chitrakoot, India and symptomatic leaves were collected for molecular assay. The complete sequences of bipartite begomovirus DNA-A and a betasatellite DNA were amplified. In a sequence analysis, begomovirus DNA-A and betasatellite shared highest sequence identity (91–99%) with Tomato leaf curl New Delhi virus (ToLCNDV) DNA-A and chili leaf curl betasatellite (ChLCB), respectively. The virus was transmitted by whitefly to tomato plants and caused ToLCD symptoms with 70% transmission rate. To our knowledge, this is the first report of the natural occurrence of ToLCNDV and ChLCB in India.     

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="BoldItalic">Didymella glomerata</Emphasis><Emphasis Type="Bold">causing leaf blight on pistachio</Emphasis>

During 2015 and 2016, we detected blighted leaves of pistachio (Pistacia vera) trees in different orchards in Arizona (USA). A Phoma-like species was isolated from pycnidia that appeared embedded in the leaf tissue. The pathogen was identified by means of morphological characteristics and DNA analysis (by sequencing of the ITS, BT, and EF regions) as Didymella glomerata. The optimum temperature for mycelial growth of the pathogen was around 25 °C. Inoculation tests were conducted on healthy and wounded pistachio leaves, fruits, and shoots of the cv. Kerman (female) and the cv. Peters (male). Overall, the pathogen was highly virulent on leaves of both pistachio cultivars and did not need injuries for infection. Conversely, the pathogen did not cause any macroscopic symptoms on the inoculated fruits and shoots but showed a certain endophytic behavior in the shoots. Also, data on the ability of different fungicides to inhibit the in vitro mycelial growth of the pathogen are presented.