Incidence and genetic diversity of raspberry bushy dwarf virus (RBDV) in Rubus spp. in Turkey

Raspberry bushy dwarf virus (RBDV), recently renamed to Idaeovirus rubi, is one of the most common viruses infecting Rubus species worldwide but there is still a limited number of genome sequences available in the GenBank database and the majority of the sequences include partial sequences of RNA-1 and RNA-2. The distribution and incidence of RBDV in main raspberry and blackberry growing provinces in Turkey were monitored during 2015–2019 and 537 Rubus spp. samples were tested by both DAS-ELISA and RT-PCR. Among the tested samples, 36 samples tested positive for RBDV by DAS-ELISA and 67 samples by RT-PCR. There was relatively low nucleotide diversity among the Turkish isolates. Turkish isolates shared 93%–97.7%, 84.3%–98.9%, and 85%–99.2% nucleotide sequence identities with available sequences in the GenBank, in partial RNA-1, movement protein (MP) and coat protein (CP) genes, respectively. In the phylogenetic tree constructed for RNA-1, MP, and CP sequences, all Turkish raspberry isolates were clustered in a distinct clade. However, the blackberry isolates showed considerable variation in nucleotide sequences and were placed in three distinct groups. The divergent blackberry isolates showed high variability in MP (84.5%–89.3%) and CP (85.5%–89.7%) regions and were placed in a distinct group. The rest of blackberry isolates clustered together with sweet cherry RBDV isolates adjacent to the grapevine clade or together with raspberry isolates. The comparative analysis conducted on three RNA segments of RBDV highlighted the high sequence diversity of Turkish RBDV isolates. This study also emphasizes the importance of regular monitoring of RBDV infections in Turkey, with special regard to those Rubus spp. and grapevine accessions employed in conservation and selection programmes. In particular, the presence of new RBDV genetic variants and infection of Rubus species must be taken into account to choose a correct detection protocol and management strategy.     

Rubus host range of rubus yellow net virus and its involvement with other aphid-borne latent viruses in inducing raspberry veinbanding mosaic disease

After graft inoculation with rubus yellow net virus (RYNV), 12 of 34 Rubus species and cultivars developed noticeable symptoms. R. macraei developed the most conspicuous symptoms and is recommended as an improved indicator plant. In attempts to determine the cause of raspberry veinbanding mosaic, a disease in which RYNV is involved, several European and North American red raspberry cvs were graft-inoculated with RYNV and three other aphid-borne viruses, black raspberry necrosis (BRNV), raspberry leaf mottle (RLMV) and raspberry leaf spot, singly and in all combinations. In periods of up to 4 yr, classical veinbanding mosaic symptoms developed in sensitive cvs only when they contained both RYNV and RLMV. These symptoms were intensified in plants co-infected with additional viruses. Veinbanding mosaic disease did not develop in any of 11 cvs infected with RYNV + BRNV, the combination of viruses previously assumed to be responsible for this disease in Britain and North America.     

Identification of resistance to Peanut bud necrosis virus (PBNV) in wild Arachis germplasm

Eighty three wild Arachis germplasm accessions, belonging to 24 species of five sections and one natural hybrid derivative of a cross between the cultivated and a wild Arachis species, were evaluated along with a susceptible groundnut cultivar for resistance to Peanut bud necrosis virus (PBNV) in a replicated field trial at ICRISAT, Patancheru, India. Thirty days after sowing, the percentage of infected plants were recorded for all the accessions and subsequently young leaflets from all these accessions were tested for the presence of the virus by enzyme linked immunosorbent assay (ELISA). One accession each of A. benensis and A. cardenasii, and two accessions of A. villosa, in the section Arachis, two accessions of A. appressipila in the section Procumbentes, and one accession of A. triseminata under section Triseminatae were not infected by PBNV. These seven field‐resistant accessions were tested under glasshouse conditions for virus resistance by mechanical sap inoculations. One accession of A. cardenasii and two accessions of A. villosa did not show systemic infection. Similarly, in another glasshouse test, where 13 A. cardenasii accessions of section Arachis were evaluated, two accessions did not show systemic infection. In all these resistant accessions, the inoculated leaves showed infection, but the systemic leaves did not show the presence of virus in spite of repeated mechanical sap inoculations. So, the resistance in these accessions appears to be due to a block in systemic movement of the virus. To our knowledge this is the first report on the identification of resistance to PBNV in wild Arachis species. Since both A. cardenasii and A. villosa are the progenitors of cultivated groundnut and can be hybridised with the latter, the resistant accessions are being utilised in conventional breeding programmes to transfer PBNV resistance to widely adapted groundnut cultivars.     

植物抗病毒基因工程研究进展

病毒作为一种依赖于宿主细胞代谢的病原体对全球农业造成了重大经济损失。虽然目前已利用多种防治策略来控制病毒病,例如培育抗病毒品种、使用化学杀菌剂、切断病毒的感染途径、组织脱毒、传统农业防治等,但都无法从根本上控制病毒病的危害。近年来的研究表明,基因工程手段能够有效对抗植物病毒病害。本文综述了基因工程手段改造植物抗病毒能力的技术和方法。     

Biology of the European large raspberry aphid (Amphorophora idaei): its role in virus transmission and resistance breakdown in red raspberry

1 The European large raspberry aphid Amphorophora idaei Börner is the most important vector of viral diseases afflicting commercially grown red raspberry ( Rubus idaeus L.) in Northern Europe, with European raspberry production amounting to 416 000 tonnes per annum. This review synthesizes existing knowledge on its biology and interactions with other organisms, including its host plant and the viral pathogens it vectors.
2 Information about trophic interactions with other insect herbivores and natural enemies is reviewed. Vine weevils Otiorhynchus sulcatus compromise aphid resistance in some raspberry cultivars, increasing A.   idaei abundance by 80%. Parasitoids show mixed success in parasitizing A.   idaei , although Aphidius ervi attack rates more than doubled when A.   idaei fed on a partially susceptible raspberry cultivar, compared with a resistant variety. These findings are discussed in the context of potential biological control as part of an integrated pest and disease management framework.
3  Amphorophora idaei transmits four known viruses: Black raspberry necrosis virus, Raspberry leaf mottle virus, Raspberry leaf spot virus and Rubus yellow net virus , with A.   idaei taking as little as 2 min to transmit some viruses.
4 Existing control strategies, including resistant cultivars, insecticides and eradication of disease from parent plants, are described. In particular, strong selection pressures have resulted in A .  idaei overcoming genetic resistance in many raspberry cultivars and most insecticides are now ineffective.
5 Future directions for the sustained control of A.   idaei are suggested, taking into consideration the possible effects of climate change and also changes in agronomic practices in U.K. agriculture.     

RNA N6-methyladenosine modification suppresses replication of rice black streaked dwarf virus and is associated with virus persistence in its insect vector

N⁶ methylation of adenosine (m⁶A) was recently discovered to play a role in regulating the life cycle of various viruses by modifying viral and host RNAs. However, different studies on m⁶A effects on the same or different viruses have revealed contradictory roles for m⁶A in the viral life cycle. In this study, we sought to define the role of m⁶A on infection by rice black streaked dwarf virus (RBSDV), a double-stranded RNA virus, of its vector small brown planthopper (SBPH). Infection by RBSDV decreased the level of m⁶A in midgut cells of SBPHs. We then cloned two genes (LsMETTL3 and LsMETTL14) that encode m⁶A RNA methyltransferase in SBPHs. After interference with expression of the two genes, the titre of RBSDV in the midgut cells of SBPHs increased significantly, suggesting that m⁶A levels were negatively correlated with virus replication. More importantly, our results revealed that m⁶A modification might be the epigenetic mechanism that regulates RBSDV replication in its insect vector and maintains a certain virus threshold required for persistent transmission.     

Purification and properties of blackberry chlorotic ringspot, a new virus species in subgroup 1 of the genus Ilarvirus found naturally infecting blackberry in the UK

A mechanically transmissible virus was isolated from Bedford Giant blackberry plants showing chlorotic mottling and ringspot symptoms growing in Scotland. It infected several herbaceous test plants, many of them symptomlessly. This virus was also transmitted to several Rubus species and cultivars by graft inoculation with scions from the field‐infected Bedford Giant plant. Most grafted plants were infected symptomlessly, but Himalaya Giant blackberry and the hybrid berry Tayberry developed symptoms similar to those in the infected Bedford Giant plant. In the sap of infected Chenopodium quinoa, the virus lost infectivity when diluted 10^?4 but not 10^?3, after 6 h and 48 h when kept at 20°C and 4°C, respectively, but was infective for more than 8 days when kept at ?15°C. Preparations of purified virus from infected C. quinoa or spinach sedimented as three major nucleoprotein components and consisted of quasi‐isometric particles that varied in size from 24 to 32 nm in diameter and that were not penetrated by negative stain. Such virus particle preparations contained a major polypeptide of ca 28 kDa and three single‐stranded RNA species of estimated size 3.2, 2.8 and 2.1 kb. The complete sequence of the largest RNA (RNA 1, 3478 nt) and the partial sequence of the other RNAs (1863 and 2102 nt long, respectively) were determined and compared with sequences in databases. These findings, together with the biological and biochemical properties of this virus, indicate that it should be regarded as a distinct species in subgroup 1 of the genus Ilarvirus even though it was serologically unrelated to existing members of this subgroup. The virus showed a very distant serological relationship with prune dwarf virus (PDV) but differed significantly from it in the amino acid sequence of its coat protein, experimental host range and symptomatology and was unrelated to PDV at the molecular level. The virus, tentatively named blackberry chlorotic ringspot virus, is therefore a newly described virus and the first ilarvirus found naturally infecting Rubus in the UK.     

Dissecting the multifunctional role of the N‐terminal domain of the Melon necrotic spot virus coat protein in RNA packaging,viral movement and interference with antiviral plant defence

The coat protein (CP) of Melon necrotic spot virus (MNSV) is structurally composed of three major domains. The middle S‐domain builds a robust protein shell around the viral genome, whereas the C‐terminal protruding domain, or P‐domain, is involved in the attachment of virions to the transmission vector. Here, we have shown that the N‐terminal domain, or R‐domain, and the arm region, which connects the R‐domain and S‐domain, are involved in different key steps of the viral cycle, such as cell‐to‐cell movement and the suppression of RNA silencing and pathogenesis through their RNA‐binding capabilities. Deletion mutants revealed that the CP RNA‐binding ability was abolished only after complete, but not partial, deletion of the R‐domain and the arm region. However, a comparison of the apparent dissociation constants for the CP RNA‐binding reaction of several partial deletion mutants showed that the arm region played a more relevant role than the R‐domain in in vitro RNA binding. Similar results were obtained in in vivo assays, although, in this case, full‐length CPs were required to encapsidate full‐length genomes. We also found that the R‐domain carboxyl portion and the arm region were essential for efficient cell‐to‐cell movement, for enhancement of Potato virus X pathogenicity, for suppression of systemic RNA silencing and for binding of small RNAs. Therefore, unlike other carmovirus CPs, the R‐domain and the arm region of MNSV CP have acquired, in addition to other essential functions such as genome binding and encapsidation functions, the ability to suppress RNA silencing by preventing systemic small RNA transport.     

Molecular genetic analysis of virus isolates from wild and cultivated plants demonstrates that East Africa is a hotspot for the evolution and diversification of Sweet potato feathery mottle virus

Sweet potato feathery mottle virus (SPFMV, genus Potyvirus) is globally the most common pathogen of cultivated sweet potatoes (Ipomoea batatas; Convolvulaceae). Although more than 150 SPFMV isolates have been sequence‐characterized from cultivated sweet potatos across the world, little is known about SPFMV isolates from wild hosts and the evolutionary forces shaping SPFMV population structures. In this study, 46 SPFMV isolates from 14 wild species of genera Ipomoea, Hewittia and Lepistemon (barcoded for the matK gene in this study) and 13 isolates from cultivated sweet potatoes were partially sequenced. Wild plants were infected with the EA, C or O strain, or co‐infected with the EA and C strains of SPFMV. In East Africa, SPFMV populations in wild species and sweet potato were genetically undifferentiated, suggesting inter‐host transmission of SPFMV. Globally, spatial diversification of the 178 isolates analysed was observed, strain EA being largely geographically restricted to East Africa. Recombination was frequently detected in the 6K2‐VPg‐NIaPro region of the EA strain, demonstrating a recombination ‘hotspot’. Recombination between strains EA and C was rare, despite their frequent co‐infections in wild plants, suggesting purifying selection against strain EA/C recombinants. Positive selection was predicted on 17 amino acids distributed over the entire coat protein in the globally distributed strain C, as compared to only four amino acids in the coat protein N‐terminus of the EA strain. This selection implies a more recent introduction of the C strain and a higher adaptation of the EA strain to the local ecosystem. Thus, East Africa appears as a hotspot for evolution and diversification of SPFMV.     

Molecular characterisation of Onion yellow dwarf virus (OYDV) infecting garlic (Allium sativum L.) in Israel: Thermotherapy inhibits virus elimination by meristem tip culture

Garlic (cv. Shani) was tested using single step RT‐PCR and digoxygenin (DIG) labelled dot‐blot for a number of viruses. Following sequence analysis it was shown that at least three different polymorphs of the potyvirus Onion yellow dwarf virus (OYDV) infect the same plant simultaneously, together with the potyvirus Leek yellow stripe virus (LYSV), the carlavirus Garlic common latent virus (GCLV) and a multitude of allexiviruses (Shallot virus X (ShVX) related viruses]. Several garlic plants free of all the viruses tested were obtained through meristem‐tip culture. Plants infected with single viruses or with different combinations of viruses were similarly obtained. Meristem‐tip culture was confirmed as a satisfactory method of virus eradication, while thermotherapy treatment given to mother plantlets before meristem excision was found to specifically antagonise OYDV eradication. This work uses molecular methods for the first time to examine the effectiveness of meristem‐tip culture for the eradication of multiple viruses from garlic.     

烟草坏死病毒A诱导的基因沉默及其条件优化

以烟草坏死病毒A中国分离物(Tobacco necrosis virus A Chinese isolate,TNV-AC)侵染性cDNA克隆为基础,通过基因替换、基因插入策略构建获得多种重组TNV-AC,比较了外源基因片段插入位置、插入形式及接种植物培养温度对TNV-AC诱导的基因沉默的影响.外源基因片段替换CP基因19～828 nt的重组TNV-AC丧失了在本生烟中的系统移动能力,也不能有效诱导相应基因发生明显的沉默,说明替换策略不适合于TNV-AC.向CP基因终止密码子UAG附近插入外源基因片段后,TNV-AC仍可进行复制,但最适的插入位点位于UAG之后,且容纳外源片段的长度约为120 nt.当外源片段以反向重复的形式插入UAG之后,诱导基因沉默的效率较高.接种植物的培养温度也会显著影响基因沉默的效率以及插入片段的稳定性,低温(18℃)条件下诱导NbPDS基因沉默的效率明显高于高温(24℃)条件,且沉默表型可持续110天以上.除了本生烟PDS基因,TNV-AC沉默载体还可诱导本生烟sulfur基因Su和镁离子螯合酶H亚基基因ChlH发生沉默,以上结果说明,TNV-AC具有开发为本生烟基因功能鉴定的新VIGS载体的潜力.     

Detection and Molecular Variability of Turnip mosaic virus (TuMV) in Shaanxi,China

Turnip mosaic virus (TuMV) is one of the most devastating threats to oilseed rape by causing serious crop losses. A total of 86 leaf samples of oilseed rape from eight different locations in Shaanxi, China, were tested by RT‐PCR for TuMV; the results revealed an infection level of 43% by TuMV. The complete coat protein (CP) gene of 32 TuMV isolates was cloned and sequenced. Analysis of the CP gene with sequences from the database allowed the genetic classification of 170 TuMV isolates or sequences. Four genetic clusters were obtained: MB (mostly Brassica isolates), MR (mostly Radish isolates), IBR (mostly Intermediate between Brassica and Radish clusters) and OBR (mostly outside Brassica and Radish clusters). All subgroups were slightly related to the hosts, but unrelated to geographical origins. Most of Shaanxi TuMV isolates were on separate branches, compared with the 138 known isolates originating from other parts of the world. Our results help provide a better understanding of the genetic diversity of TuMV isolates infecting oilseed rape in Shaanxi, China.     

Molecular epidemiological investigation of infectious hypodermal and hematopoietic necrosis virus and Taura syndrome virus in <Emphasis Type="Italic">Penaeus Vannamei</Emphasis> cultured in China

The Infectious hypodermal and hematopoietic necrosis virus (IHHNV) and Taura syndrome virus (TSV) are two important shrimp viruses in cultured shrimp in America. These two viruses were transmitted to China at the beginning of the 21^st century. In this study, 214 shrimp samples of Penaeus vannamei were collected from seven different areas of China and tested by PCR for IHHNV and TSV infection. The results showed that there were a high prevalence of IHHNV (65.42%) and low prevalence of TSV (3.27%) in the tested samples. Several samples were found to be co-infected with these two viruses. A 3 kb fragment of 7 positive IHHNV samples and a structure protein region (ORF2) of three TSV positive samples were amplified and sequenced. The sequence comparison indicated that both IHHNV and TSV sequenced in China have a low genetic variations compared with the prototype IHHNV and TSV from Hawaii. Phylogenetic analysis showed that TSV isolates were clustered into two groups, Asia and America group, which was genetically correlated to geographic distribution. Foundation item: This work was supported by 973 project (2006CB101801)