The occurrence of maize mosaic virus on sorghum in India1

A leaf disease of sorghum (Sorghum bicolor) characterised by fine discontinuous chlorotic streaks between the veins, was observed on sorghum grown during the 1987/88 post-rainy season in peninsular India. Early-infected plants were stunted, had shortened internodes, and produced poorly developed panicles. The virus was transmitted by the delphacid planthopper, Peregrinus maidis. Negatively stained leaf dip preparations contained bullet-shaped virus particles (208 ± 4.4 × 66 ± 1.0 nm) resembling those of rhabdoviruses. In ultrathin sections, the particles budded through the inner nuclear membrane and were present in the cytoplasm within membrane-bound vesicles that were apparently contiguous with the distended outer nuclear membrane. A method for purifying the virus was developed utilising polyethylene glycol (PEG) precipitation, Celite filtration and sucrose densitygradient centrifugation. An antiserum was produced in rabbits with a titre of 1/2650 in the precipitin ring interphase test. The virus could be detected in infected sorghum leaf tissues using a direct antigen coating form of enzyme-linked immunosorbent assay (DAC-ELISA). In immuno-double diffusion tests, the virus reacted positively with antisera to maize mosaic virus (MMV) from Reunion (MMV-RN) and Hawaii (MMV-HI), but not with antisera to barley yellow striate mosaic (BYSMV), cereal chlorotic mottle (CCMV), and cynodon chlorotic streak (CCSV) viruses. Thus, the virus isolated from sorghum is designated the MMV-S isolate. In DAC-ELISA tests, MMV-S reacted positively with antisera to MMV-R, MMV-HI, MMV-Florida isolate, CCSV, and CCMV, and weakly with antiserum to BYSMV. SDS-polyacrylamide gel electrophoresis revealed four major proteins of relative mass M_r 70 000, 59 000, 32 000 and 28 000. In electro-blot immunoassay, MMV and CCSV antisera detected the G and N proteins. These data suggest that MMV-S should be placed in the sonchus yellow net virus subgroup of plant rhabdoviruses.     

Purification and Serology of the Iranian Maize Mosaic Rhabdovirus1)

Leaves of maize infected with the Iranian maize mosaic rhabdovirus (IMMRV) were homogenized in 0.1 M citrate-0.04 M Na₂SO₃ buffer, pH 5.4, containing 10 % sucrose and the extract was subjected to low speed and high speed centrifugation followed by resuspension in 0.05 M potassium phosphate buffer, pH 7.2, containing 10 % sucrose. Partially purified preparation was obtained by density-gradient centrifugation, removal of the virus zones and their concentration by high speed centrifugation. Two virus specific bands were observed in density-gradient columns. An antiserum with a titer of 128 was prepared by injecting partially purified virus into rabbits. In agar-gel-diffusion tests, the antiserum produced one or two precipitin lines against diseased maize extract but none against healthy maize extract. IMMRV was not related to barley yellow striate mosaic (BYSMV), cereal chlorotic mottle (CCMV), Cynodon chlorotic streak (CCSV), Festuca leaf streak, and maize mosaic (MMV) viruses as well as to two unidentified rhabdoviruses occurring in wheat and Bermuda grass in the vicinity of Shiraz, when these viruses were tested against IMMRV antiserum in agar-gel-diffusion and enzyme-linked immunosorbent assay. Likewise, IMMRV did not react with antisera to BYSMV, CCMV, CCSV and MMV in agar-gel-diffusion tests. IMMRV appears to be different from most reported rhabdoviruses of cereals.     

Hedgerow hawthorn (Crataegus spp.) and blackthorn (Prunus spinosa) as hosts of fruit tree viruses in Britain

Apple chlorotic leafspot virus (CLSV) was detected in 27 of 109 hawthorn and three of 67 blackthorn plants sampled in various parts of Britain. The CLSV isolates possessed similar properties to those isolated from other rosaceous species but differed in the severity of symptoms they induced in woody indicators. No seed or aphid transmission of CLSV was detected. Prunus necrotic ringspot (PNRV) and prune dwarf (PDV) viruses were detected in four and three respectively of 67 blackthorn plants. The PNRV and PDV isolates were serologically closely related to isolates from cherry. Arabis mosaic virus was detected in one blackthorn plant, but plum pox virus was not found in any of the tested plants.     

Identification of a new tospovirus causing necrotic ringspot on tomato in China

Background

Emerging tospoviruses cause significant yield losses and quality reduction in vegetables, ornamentals, and legumes throughout the world. So far, eight tospoviruses were reported in China. Tomato fruits displaying necrotic and concentric ringspot symptoms were found in Guizhou province of southwest China.

Finding

ELISA experiments showed that crude saps of the diseased tomato fruit samples reacted with antiserum against Tomato zonate spot virus (TZSV). Electron microscopy detected presence of quasi-spherical, enveloped particles of 80–100 nm in such saps. The putative virus isolate was designated 2009-GZT. Mechanical back-inoculation showed that 2009-GZT could infect systemically some solanaceous crop and non-crop plants including Capiscum annuum, Datura stramonium, Nicotiana benthamiana, N. rustica, N. tabacum and Solanum lycopersicum. The 3012 nt full-length sequence of 2009-GZT S RNA shared 68.2% nt identity with that of Calla lily chlorotic spot virus (CCSV), the highest among all compared viruses. This RNA was predicted to encode a non-structural protein (NSs) (459 aa, 51.7 kDa) and a nucleocapsid protein (N) (278 aa, 30.3 kDa). The N protein shared 85.8% amino acid identity with that of CCSV. The NSs protein shared 82.7% amino acid identity with that of Tomato zonate spot virus(TZSV).

Conclusion

Our results indicate that the isolate 2009-GZT is a new species of Tospovirus, which is named Tomato necrotic spot virus (TNSV). This finding suggests that a detailed survey in China is warranted to further understand the occurrence and distribution of tospoviruses.

     

Synergistic interactions of begomoviruses with Sweet potato chlorotic stunt virus (genus Crinivirus) in sweet potato (Ipomoea batatas L.)

Three hundred and ninety‐four sweet potato accessions from Latin America and East Africa were screened by polymerase chain reaction (PCR) for the presence of begomoviruses, and 46 were found to be positive. All were symptomless in sweet potato and generated leaf curling and/or chlorosis in Ipomoea setosa. The five most divergent isolates, based on complete genome sequences, were used to study interactions with Sweet potato chlorotic stunt virus (SPCSV), known to cause synergistic diseases with other viruses. Co‐infections led to increased titres of begomoviruses and decreased titres of SPCSV in all cases, although the extent of the changes varied notably between begomovirus isolates. Symptoms of leaf curling only developed temporarily in combination with isolate StV1 and coincided with the presence of the highest begomovirus concentrations in the plant. Small interfering RNA (siRNA) sequence analysis revealed that co‐infection of SPCSV with isolate StV1 led to relatively increased siRNA targeting of the central part of the SPCSV genome and a reduction in targeting of the genomic ends, but no changes to the targeting of StV1 relative to single infection of either virus. These changes were not observed in the interaction between SPCSV and the RNA virus Sweet potato feathery mottle virus (genus Potyvirus), implying specific effects of begomoviruses on RNA silencing of SPCSV in dually infected plants. Infection in RNase3‐expressing transgenic plants showed that this protein was sufficient to mediate this synergistic interaction with DNA viruses, similar to RNA viruses, but exposed distinct effects on RNA silencing when RNase3 was expressed from its native virus, or constitutively from a transgene, despite a similar pathogenic outcome.     

Bulb and Root Rot in Lily (Lilium longiflorum) and Onion (Allium cepa) in Israel

In the past 10 years, there has been a substantial increase in reports, from growers and extension personnel, on bulb and root rots in lily (Lilium longiflorum) in Israel. Rot in these plants, when grown as cut flowers, caused serious economic damage expressed in reduction in yield and quality. In lily, the fungal pathogens involved in the rot were characterized as binucleate Rhizoctonia AG‐A, Rhizoctonia solani, Pythium oligandrum, Fusarium proliferatum (white and purple isolates) and F. oxysporum, using morphological and molecular criteria. These fungi were the prevalent pathogens in diseased plants collected from commercial greenhouses. Pathogenicity trials were conducted on lily bulbs and onion seedlings under controlled conditions in a greenhouse to complete Koch's postulates. Disease symptoms on lily were most severe in treatments inoculated with binucleate Rhizoctonia AG‐A, P. oligandrum and F. proliferatum. Plant height was lower in the above treatments compared with the control plants. The least aggressive fungus was R. solani. In artificial inoculations of onion, seedling survival was significantly affected by all fungi. The most pathogenic fungus was F. proliferatum w and the least were isolates of F. oxysporum (II and III). All fungi were successfully re‐isolated from the inoculated plants.     

Virulence and Vegetative Compatibility of Dutch and Italian Isolates of Fusarium oxysporum f. sp. lilii

The virulence and vegetative compatibility of eight Dutch and four Italian isolates of Fusarium oxysporum obtained from lily were compared. The virulence was tested by determination of the specific interaction between the Fusarium isolates and eight lily cultivars. A specific interaction was not found, so the existence of races was not demonstrated. Six of the twelve isolates turned out to be non-pathogenic for lily. The pathogenic isolates fell in four vegetative compatibility groups. No vegetative compatibility was found between isolates of F. oxysporum f. sp. lilii and those of f. sp. gladioli.     

Vorkommen von Tobacco Rattle-Virus in Roggen (Secale cereale) in der Bundesrepublik Deutschland

Natural occurrence of tobacco rattle virus in rye (Secale cereale) in the Federal Republic of Germany Near Bergen/Dumme in northern Germany, in an area known for the occurrence of tobacco rattle virus (TRV) on potatoes, 5–10 % of rye plants of one experimental plot were severely stunted and showed distinct chlorotic mottling of leaves. In these plants TRV was identified on the basis of particle morphology and serology (Immunoelectron microscopy and ELISA). TRV was also detected in Capsella bursa-pastoris and Stellaria media from an area adjacent to the rye plot but not in nearby commercial rye fields.     

Responses of potato cultivars (Solanum tuberosum) to infection with Cucumber mosaic virus isolated from different host species

Eight isolates of Cucumber mosaic virus (CMV) isolated from seven different host species were tested for their virulence on potato cv. Desirée. Three isolates caused a systemic infection, of which one isolate from Asiatic lily (CMV-P26) and one from cucumber (CMV-J) appeared to be highly virulent, in contrast to the third isolate (CMV-M) that originated from cucumber and caused mild symptoms only. These three isolates were transmitted to 26 additional potato cultivars by mechanical inoculation in a greenhouse. All cultivars were infected with at least one CMV isolate and developed local chlorotic symptoms, but only 17 cultivars (including Desiree) developed primary systemic symptoms including necrosis, mosaic and/or malformation of leaves. Furthermore, in only five cultivars (including Desiree) CMV was transmitted to tubers and was subsequently detected in plants of the first and second vegetative progeny, the secondary symptoms of these plants being severe. The observed phenotypic responses of potatoes to CMV were not associated with the maturity type (early or late) or resistance to other viruses. Results of this study indicate a high level of biological variability among CMV isolates and that infection in potato depends on CMV isolate and potato cultivar.     

Molecular Analysis of the Coat Protein of Potato virus Y Isolates in Greece Suggests Multiple Introduction from Different Genetic Pools

The phylogenetic relationships among Potato virus Y (PVY) isolates from northern and southern Greece were investigated. A large part of coat protein gene of 49 tobacco isolates and three from pepper was examined. The analysis showed that all 52 isolates consisted of 34 distinct haplotypes, with only one haplotype found in both northern and southern regions. The southern population was more diverse than that from the north. The phylogenetic analyses of the Greek haplotypes alone or in combination with isolates from other countries using the maximum likelihood method classified unambiguously almost all the haplotypes examined. Nine tobacco haplotypes from the south were classified as C‐like (particularly C1), whereas 22 haplotypes from tobacco and two from pepper from both north and south were classified as N‐like. One tobacco haplotype from the south was found recombinant between N‐like and C1 lineages. The pattern of molecular evolution was examined using the fixed‐effects likelihood and the single‐likelihood ancestor counting methods. The analysis indicated that the evolution of PVY isolates appeared to be conservative (purifying selection and neutral evolution). These findings are discussed in relation to the introduction of PVY in the tobacco crop in Greece and the between region dispersal. A scenario of multiple introductions of PVY isolates in north and south Greece from different genetic pools and low or nil between region spread of the virus isolates was proposed.     

Natural infection of sugar beet with tomato bushy stunt virus

A virus transmissible toChenopodium quinoa was isolated from leaves of sugar beet showing large chlorotic ring spots and line pattern. The virus was serologically unrelated to tobacco necrosis virus and tomato black ring virus or to its beet ringspot strain either. A positive result was obtained with antiserum against tomato bushy stunt virus. Reactions of herbaceous indicators and properties of the virus in crude sap were in accordance with the serological diagnosis. A survey of natural hosts of tomato bushy stunt virus demonstrated recently by the authors is given.     

Problems Encountered with the Selection of Cucumber Mosaic Virus (CMV) Isolates for Resistance Breeding Programs

Among the Chili breeding lines from the Asian Vegetable Research Center, two were chosen for the screening of a larger selection of Cucumber mosaic virus (CMV) isolates, mainly from Asian countries. The chili line (VC246) showed a resistance against several CMV‐isolates and was compared with chili line VC27a that was susceptible to CMV infection. Among the 28 CMV isolates, five were identified as resistance breaking (AN‐like) and non‐resistance breaking (P3613‐like) for the line VC246, whereas all isolates could establish a systemic infection on VC27a. However, further testing revealed that resistance in VC246 was also dependent on the way of inoculation and the inoculums itself. Graft inoculation could overcome the resistance, and the inoculation with isolated viral RNA resulted in no infection at all on the resistant chili line, independent of the virus isolate. Using a pseudo‐recombinant approach, we identified RNA2 of resistance breaking isolates as responsible for systemic infection and confined the area within RNA2 to the 3′ terminal part including the ORF 2b. Sequence alignments of that area revealed eight distinct mutations on amino acid level, which was present either in resistance or non‐resistance breaking isolates. A reversion from the P3613‐like to the AN‐like sequence of two of these mutations induced no effect on Capsicum sp., but induced symptoms on several tobacco species distinct from those induced by the wild‐type virus. However, pseudorecombinants, each generated from sets of two different AN‐like isolates, which were expected to infect VC246 systemically, did not indicating that probably RNA2 must be in a specific context to have the effect. In this case, a generalized attribution of functions to single amino acid exchanges might be impossible or at least extremely difficult.     

广东省烟草花叶病病原病毒的鉴定

烟草花叶病是广东省产烟区的主要病害之一。我们在南雄等八个县进行调查,1983年至1984年的一般发病率为2～20％。根据血清学反应、病毒粒体形态、鉴别寄主反应及寄主范围、媒介昆虫种类、物理性质、交互保护反应等各项检验结果,鉴定广东省烟草花叶病病原是:三个黄瓜花叶病毒可能株系(普通株系CMV-C,烟草坏死株系CMV-TN,烟草黄色坏死株系(CMV-TYN),烟草花叶病毒(TMV),马铃薯病毒Y(PVY),烟草脉带花叶病毒(TVBMV)和烟草褪绿斑驳病毒(TCMV)(暂定)。     

Western Flower Thrips (Frankliniella occidentalis) Transmits Maize Chlorotic Mottle Virus

Maize chlorotic mottle virus (MCMV) is one of the co‐infection pathogens that cause corn (maize) lethal necrosis, but the transmission mechanism of MCMV is not yet clear. In order to determine the ability of western flower thrips (Frankliniella occidentalis; WFT) to transmit MCMV, imported maize seeds from Thailand were germinated in an insect‐free greenhouse and the seedlings were tested for the transmission by WFT of chlorotic mottle virus disease. The thrips (WFT), starved for 48 h then allowed to feed for 30 min on maize plants infected with MCMV or asymptomatic maize plants, were transferred to healthy seedlings. After 35 days, the seedlings with WFT from diseased maize plants showed chlorotic mottle symptoms, whereas seedlings with WFT from asymptomatic maize plants remained healthy. A single band of 711 bp was amplified by RT‐PCR using primers MCMV‐F/MCMV‐R from the MCMV‐infected plants and WFT collected from the diseased plants. Sequencing of the amplified product and further sequence comparison indicated that the two viruses from both sources showed 99% similarity of nucleotides and they should be regarded as identical. In addition, isometric particles c. 30 nm in diameter, characteristic of MCMV, were found in the WFT samples from diseased maize plants. Thus, it is concluded that WFT transmits MCMV. Our findings suggest that corn lethal necrosis disease can be controlled or minimized by the eradication of WFT from the field or greenhouses.     

Konjac mosaic virus Naturally Infecting Three Aroid Plant Species in Andhra Pradesh,India

The genomes of three potyvirus isolates from, respectively, naturally infected Colocasia esculenta, Caladium spp. and Dieffenbachia spp. in Andhra Pradesh, India, were amplified by RT‐PCR using degenerate potyvirus primers. Sequence analysis of RT‐PCR amplicons (1599 nucleotides) showed maximum identity of 97% with the KoMV‐Zan isolate of Konjac mosaic virus (KoMV) from Taiwan (A/C AF332872). The three isolates had a maximum identity of 99.4%. The length of coat protein (CP) gene of three isolates was 846 nucleotides encoding 282 amino acids with a deduced size of 32.25 kDa. The CP gene of the isolates had, respectively, 78.1–95.7% and 88.2–96.4% identity at nucleotide and amino acid levels with KoMV isolates. The CP gene of the three isolates had 93.1–100% (nucleotide) and 98.2–100% (amino acid) identity. The 3′‐UTR of the three isolates showed maximum identity of 91.1–100% identity between and with other KoMV isolates. In the CP amino acid–based phylogenetic analyses, the isolates branched as a distinct cluster along with known KoMV isolates. The three potyvirus isolates associated with mosaic, chlorotic feathery mottling, chlorotic spots, leaf deformation and chlorotic ring spots on three aroids were identified as isolates of KoMV for the first time from Andhra Pradesh, India.     

Phylogenetic Analysis of Iris yellow spot virus Isolates from Onion (Allium cepa) in Georgia (USA) and Peru

Iris yellow spot virus (IYSV) was first observed in sweet onions in Georgia (USA) in 2003 in the Vidalia region. The virus had been reported in the onion‐growing regions in western USA several years before being detected in Georgia in the east. Although symptoms were observed on onions in Peru several years earlier, the presence of IYSV was not confirmed in Peru until after the virus was detected in Georgia. We characterized nine isolates of IYSV recovered from sweet onions in both Georgia (four isolates) and Peru (five isolates) by sequencing the nucleocapsid (N) gene and compared those sequences with sequences available in GenBank. Sequence divergence between IYSV isolates from Georgia and Peru was low with 1.1%, and comparisons with IYSV isolates from other regions showed divergence of up to 11.4%. Bootstrap analysis indicated with a high degree of confidence that the Georgia and Peruvian isolates fell into the same clade and were different from known isolates from western USA that fell into sister clades. The high degree of similarity between Georgia and Peruvian isolates suggests that gene flow occurred from Peru into Georgia.     

Actinidia chlorotic ringspot‐associated virus: a novel emaravirus infecting kiwifruit plants

By integrating next‐generation sequencing (NGS), bioinformatics, electron microscopy and conventional molecular biology tools, a new virus infecting kiwifruit vines has been identified and characterized. Being associated with double‐membrane‐bound bodies in infected tissues and having a genome composed of RNA segments, each one containing a single open reading frame in negative polarity, this virus shows the typical features of members of the genus Emaravirus. Five genomic RNA segments were identified. Additional molecular signatures in the viral RNAs and in the proteins they encode, together with data from phylogenetic analyses, support the proposal of creating a new species in the genus Emaravirus to classify the novel virus, which is tentatively named Actinidia chlorotic ringspot‐associated virus (AcCRaV). Bioassays showed that AcCRaV is mechanically transmissible to Nicotiana benthamiana plants which, in turn, may develop chlorotic spots and ringspots. Field surveys disclosed the presence of AcCRaV in four different species of kiwifruit vines in five different provinces of central and western China, and support the association of the novel virus with symptoms of leaf chlorotic ringspots in Actinidia. Data on the molecular features of small RNAs of 21–24 nucleotides, derived from AcCRaV RNAs targeted by host RNA silencing mechanisms, are also reported, and possible molecular pathways involved in their biogenesis are discussed.     

Asaia lannaensis sp. nov., a new acetic acid bacterium in the Alphaproteobacteria

Asaia lannaensis sp. nov. was described for two strains isolated from flowers of the spider lily collected in Chiang Mai, Thailand. The isolates produced acetic acid from ethanol on ethanol/calcium carbonate agar, differing from the type strains of Asaia bogorensis, Asaia siamensis, and Asaia krungthepensis, but did not grow in the presence of 0.35% acetic acid (v/v). The new species is the fourth of the genus Asaia, the family Acetobacteraceae.     

Differences among isolates of simian hemorrhagic fever (SHF) virus

Simian hemorrhagic fever (SHF) virus is a member of the Togaviridae family which currently is unclassified to genus. We have studied the relatedness of four different SHF virus isolates obtained from infected macaque or patas monkeys. Differences were found among isolates in type and severity of disease produced in patas monkeys, cell sensitivity to infection, viral antigens, and levels of specific antibody induced in patas monkeys. Based on these criteria, the four isolates have been grouped in two categories: those producing acute infections in patas monkeys (LVR, P-180) and those producing persistent infections (P-248, P-741). The P-180 isolate induced the most severe disease in experimentally infected patas monkeys, but only occasionally were their infections fatal. Persistently infected patas monkeys were viremic over a period of years, but showed no signs or symptoms of infection. All four isolates were found to be antigenically related by use of enzyme-linked immunosorbent assay (ELISA); the P-248 isolate showing the weakest antigenic relationship. However, none of the four isolates induced cross-neutralizing antibodies in infected patas monkeys. High titers of specific IgG antibody (up to 31,250 as determined by ELISA) were induced in acutely infected patas monkeys (LVR, P-180), but antibody was barely detectable (less than or equal to 50) in persistently infected patas monkeys (P-248, P-741). LVR lytically infected USU-104 cells, patas monkey peritoneal macrophages (PMAC), and rhesus monkey PMAC. The P-180 isolate lytically infected both patas monkey PMAC and rhesus monkey PMAC, but not USU-104 cells. The isolates producing persistent infections (P-248, P-741) lytically infected only rhesus monkey PMAC. These results show that marked differences exist among isolates of SHF virus from naturally infected animals. These differences should be useful in categorizing new isolates.