Transmission of double-stranded RNA and a disease factor in Ophiostoma ulmi

A diseased isolate of Ophiostoma ulmi was found to contain 10 segments of double-stranded RNA (dsRNA) with molecular weights ranging from 2.40 x 10⁶ to 0.23 x 10⁶. In contrast seven other healthy isolates in the same vegetative compatibility group contained either no dsRNA or up to four dsRNA segments. Transmission of the disease to healthy isolates by hyphal anastomosis was accompanied by-transmission of the 10 dsRNA segments. In a genetic cross in which the diseased isolate acted as the female parent single-ascospore progeny were healthy and either contained no dsRNA or only one segment of dsRNA. When elm trees were inoculated with diseased isolates, subsequent reisolations were healthy and retained only two to seven of the dsRNA segments or were diseased and retained all 10 dsRNA segments. Following conidiogenesis a diseased isolate gave rise to single-conidial progeny which were either slow growing and diseased, like their parent, with all 10 dsRNA segments, or faster growing like healthy isolates. Some of the faster growing conidial isolates retained only two to seven of the dsRNA segments and were disease-free. However a majority of the faster growing conidial isolates retained all 10 dsRN A segments and were shown to carry the disease in a latent form. The possibility that the disease of O. ulmi is conferred by specific segments of dsRNA and the potential of d-factors for the control of Dutch elm disease are discussed.     

Detection of double-stranded RNA and virus-like particles in Australian isolates of Pythium irregulare

Mycelial extracts of Pythium irregulare were processed using CF-11 chromatography and analysed by gel electrophoresis for double-stranded RNA (dsRNA) content. DsRNA molecules of between 1 and 6 kb were found in 33 of 39 isolates tested. The dsRNA profiles of individual isolates remained unchanged after repeated subculturing of hyphal tips, after storage under water for 2 years, or when single zoospores were used to generate subcultures. However, dsRNA profiles varied between isolates, and even within individual populations; in the one case eight different dsRNA types were recovered upon testing 17 isolates from a single wheatfield. Isometric virus-like particles (VLPs) of approximately 45 nm were found in isolates containing dsRNA, while no such particles could be found in isolates in which dsRNA was not detected. Electrophoretic profiles of dsRNA extracted from partial purifications of VLPs were identical to those of dsRNA extracted from whole mycelium, suggesting that the dsRNA is at least partially encapsidated. Attempts to transmit dsRNA between isolates by hyphal anastomosis or by coinfection in wheat plants were unsuccessful, with only parental types being recovered. Analysis of 29 isolates representing 14 other Pythium species failed to detect dsRNA, even when co-isolated with P. irregulare known to contain dsRNA     

Association of three RNA molecules with potato mop-top virus

Summary Potato mop-top virus particles, purified from systemically infectedNicotiana benthamiana plants and then disrupted by heating with sodium dodecyl sulphate and 2-mercaptoethanol, contained only a single polypeptide of M_r 19 100 detectable by polyacrylamide gel electrophoresis. Single-stranded RNA preparations from virus particles, when subjected to electrophoresis in an agarose gel containing methylmercuric hydroxide as a denaturant, were shown to contain approximately equal proportions of three RNAs of 6.5, 3.2 and 2.5 kb. Double-stranded RNA preparations extracted from systemically infectedN. benthamiana leaves or from locally infectedN. debneyi leaves, was shown by polyacrylamide gel electrophoresis to contain a major species of 3.2 kbp and two minor species of 6.5 and 2.4 kbp.     

Histopathological changes associated with silverleaf syndrome in squash

Silverleaf syndrome on squash ( Cucurbita pepo ) is characterized by vein clearing, and leaf whitening, and has been associated with the presence of nymphs of Bemisia tabaci (sweetpotato whitefly), and the presence of two double-stranded RNAs (4·2 and 4·6 kb) in symptomatic leaves. However, the aetiology of the disorder remains unknown. Anatomical studies using transverse and paradermal sections from symptomatic leaves of C. pepo cv. Dixie showed turgescent, aligned adaxial epidermal cell layer, flat cuticle and the presence of sub-epidermal spaces. Histochemical studies failed to reveal any breakdown in the cellulose cell walls; however, the cuticular layer, the pectin layer and cellulose wall of epidermal cells formed a fiat compact structure. Some changes were observed in the lipid content, with the presence of lipid bodies near cell wall membranes. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of preparations of symptomatic squash leaves, partially purified by differential centrifugation, revealed a protein with a molecular mass of about 48 k Da that was not found in comparable preparations of asymptomatic leaves. This protein was localized by immunological staining in the spongy mesophyll cells and in the layer of bundle sheath associated with sieve elements and companion cells of the leaf phloem.     

Investigation of biological properties, double-stranded RNA patterns and antigen concentration in citrus species infected with citrus tristeza virus

Biological properties and dsRNA patterns of one Cyprus and three Turkish isolates of citrus tristeza virus (CTV) were investigated. In addition, CTV antigen concentration and effect of tissue sampling time from naturally infected Shamouti sweet orange trees grown in the field of Icel Province, Turkey, were also determined. The Cyprus isolate showed vein clearing symptoms on grapefruit, ‘Madam Vinous’ and Mexican lime and stem pitting symptoms on Mexican lime. The three Turkish isolates showed only vein clearing symptoms on Mexican lime. All four isolates showed a full-length major double-stranded RNA (dsRNA) band of 13.3 × 10⁶ Da mol. wt in extracts from infected Madam Vinous sweet orange trees, and major or minor dsRNA bands with 2.0. 0.8 and 0.5 × 10⁶ mol.wt. All seven different citrus varieties inoculated with the Igdir (D) strain contained full-length dsRNA. The additional two dsRNA of 0.8 and 0.5 × 10⁶ mol.wt were also detected as clearly as full-length dsRNA in these hosts, but were weaker inCitrus exelsa and ‘Interdonat’ lemon. Madam Vinous, rough lemon and Mexican lime were the best hosts for dsRNA analysis. ELISA values were highest in April (OD_405nm =0.476), decreased steadily until August, and then increased gradually through December. ELISA values were lowest in July and August (OD_405nm =0.157 and 0.141, respectively). dsRNA recovery from a field tree infected with isolate Igdir D was good in March, April and May and poor in January and February. No dsRNA band was detected in August or September. http://www.phytoparasitica.org posting July 9, 2002.     

Variability among Spanish citrus tristeza virus isolates revealed by double-stranded RNA analysis

A survey for citrus tristeza virus (CTV) strains, based on double-stranded RNA (dsRNA) analysis, was carried out in five locations on the eastern citrus-growing area of Spain. CTV was recovered from 137 trees of different ages, citrus species and varieties, sampled in 53 orchards. The best months for dsRNA recovery were April, May, September, October, and November, and the highest dsRNA yield was obtained from sweet orange cultivars. Sixteen dsRNA profiles differing by the number and/or position of subgenomic bands were detected. One of these profiles was detected in more than half the trees analysed. Maximum diversity of dsRNA patterns was found in the location with the oldest citrus orchards and the highest CTV incidence (Alzira-Carcaixent). In many instances, several dsRNA profiles were detected in neighbouring trees of the same orchard, notably in Alzira-Carcaixent, where 70% of the plots sampled contained more than one profile. The possible causes for the diversity of CTV isolates found in this specific area are discussed.     

侵染中国陕西南瓜的中国南瓜曲叶病毒的鉴定和全基因组分析

为明确南瓜叶片上卷、黄化的症状是否由病毒侵染引起,本研究采用小RNA深度测序对采集自陕西地区的南瓜叶片样品进行了鉴定。结果显示,侵染南瓜样品的病毒可能是中国南瓜曲叶病毒(squash leaf curl China virus, SLCCNV)。经PCR扩增并且克隆测序获得了病毒的DNA-A和DNA-B组分的全基因组序列。序列比对发现,所克隆的DNA-A组分与SLCCNV海南分离物(SLCCNV-Hn61)DNA-A的一致性最高,为99.1%;DNA-B组分与SLCCNV-Hn61和三亚分离物SLCCNV-SY的DNA-B组分一致性最高,为96.8%。系统进化树分析发现所克隆的DNA-A和DNA-B组分分别与SLCCNV-Hn61和SLCCNV-SY的亲缘关系最近。以上研究结果表明侵染陕西南瓜叶片的病毒是SLCCNV的分离物。这是首次报道SLCCNV在陕西地区的危害,研究结果为当地经济作物南瓜的病害防控提供参考。     

Variation for isozymes and double-stranded RNA among isolates of Puccinia striiformis and two other cereal rusts

Gel electrophoresis was used to examine the variation in isozymes and dsRNA within and between the rust species Puccinia striiformis, P. recondita and P. hordei. No differences in isozyme phenotype were found among 29 diverse isolates of the wheat-attacking form of P. striiformis (WYR). Smaller numbers of isolates of the barley-attacking form (BYR) of P. recondita and of P. hordei showed similar intra-group uniformity. There were major differences in isozyme phenotypes between the three species, while WYR and BYR differed for two of 12 enzymes. Double-stranded RNA was detected in each species and in all 26 isolates examined. For WYR and BYR, all isolates within each group had the same or a similar phenotype. In contrast, each isolate of P. recondita and P. hordei had a unique phenotype. There were differences in dsRNA phenotypes both between the three species and between WYR and BYR.
The uniformity of these rust populations and species for isozyme phenotype is contrasted with their variability in pathogenicity and with the variability in isozymes encountered in higher organisms. Uniformity may result from a feature of the biology of the rust species and populations examined, or from the relative homogeneity of the environment of biotrophic pathogens. Consistent differences in isozyme and dsRNA phenotype between the WYR and BYR isolates of P. striiformis indicate that these are two discrete populations, supporting the view that they should be recognized as f.sp. tritici and f.sp. hordei , respectively.     

Occurrence of squash mosaic virus in melons in Greece

Squash mosaic virus (SqMV) was isolated from melon plants raised from imported seed. Virus identification was based on host range, aphid and seed transmission tests, electron microscopy and serology. Seed testing by ELISA detected SqMV only in the cultivar Touralia imported from the USA.     

二化螟dsRNA降解酶基因的克隆与表达分析

为解析二化螟Chilo suppressalis体内参与降解双链RNA(double-stranded RNA,dsRNA)的关键核酸酶的功能,克隆二化螟不同的非专一性核酸酶（non-specific nuclease,NUC）基因,并对这些基因进行生物信息学分析和组织定量表达分析,同时对dsRNA降解酶（dsRNA degrading nuclease,dsRNase）活力的组织分布进行研究。结果显示,共克隆获得5个NUC基因,其中有4个编码dsRNase亚家族基因（CsdsRNase1～CsdsRNase4）和1个编码Endonuclease G亚家族基因（CsEndoG）。5个NUC基因的开放阅读框核苷酸序列长度范围为828～1 338 bp,编码275～445个氨基酸残基,其分子量大小为31.68～49.57 kD,预测等电点为5.48～9.42。CsdsRNase1和CsdsRNase2含有信号肽序列,两者相似度极高,且与家蚕Bombyx mori和斜纹夜蛾Spodoptera litura中具有dsRNA降解酶活力的dsRNase同源聚类;CsdsRNase1和CsdsRN...     

Soil amendments with Brassica cover crops for management of Phytophthora blight on squash

BACKGROUND: Phytophthora blight induced by Phytophthora capsici is responsible for serious yield loss in vegetable production in the United States and other countries. This study was conducted to evaluate the efficacy of Brassica cover crops used as soil amendments for managing Phytophthora blight of squash. RESULTS: In greenhouse studies, disease incidence on squash plants was significantly reduced by soil amendment with mustard shoots or roots used at 1 and 2.5% (plant tissue/soil, w/w). The shoots of canola used at 1 or 2.5% also suppressed disease, while the roots of canola or other crops did not reduce disease significantly. In field studies, soil amendments with mustard and canola provided the greatest disease reduction and increased squash yield significantly compared with the non‐treated control. Mustard and canola did not appear to be susceptible to P. capsici. CONCLUSION: The results indicated that some Brassica crops, particularly mustard and canola, had the potential to significantly reduce Phytophthora blight on squash when used as soil amendments. As P. capsici has a remarkable ability to develop resistance to chemical fungicides, use of effective Brassica cover crops could be a biorational alternative to fungicides and a valuable component in developing integrated disease management programs. Copyright © 2011 Society of Chemical Industry     

Factors in the suppression of weeds by squash interplanted in corn

In order to examine the weed-suppressive properties of squash interplanted in corn, we compared different planting densities and weeding regimens, measuring the weed biomass, light interception by crop canopy, and yield. The corn was machine-planted at a normal monocrop density, while the squash was hand-planted at or above a normal monocrop density in an irrigated and otherwise mechanized system. A simulated intercrop consisting of corn with an artificial squash canopy was included. Interplanted squash reduced the biomass of the total weeds and of the dominant weeds, Amaranthus retroflexus and Convolvulus arvensis . The corn yield was not necessarily reduced in the intercrop, except at a higher squash density or under limited moisture. Shading by the squash was the major mechanism of weed suppression, but the analysis suggested that other factors, such as allelopathy, might contribute. Additional hand weeding had no effect on the corn yield.     

Gentian Kobu-sho-associated virus: a tentative, novel double-stranded RNA virus that is relevant to gentian Kobu-sho syndrome

A virus-like dsRNA of about 23 kbp was detected in gentian plants showing Kobu-sho syndrome including stunting, shortened internodes, and tumors on stems, nodes and roots. Nucleotide sequence analysis has suggested that this dsRNA is related to Pestivirus species but not to any other plant viruses. It was protected from externally added RNase, suggesting that the dsRNA is encapsidated. The dsRNA was co-extracted in a crude homogenate of glutaraldehyde-fixed tissue with the virus-like particles that have been associated previously with Kobu-sho syndrome in gentian (Usugi et al. Jpn J Phytopathol 76:21–24, 2010). The RNA sequence was detected in more than 99 % of Kobu-sho gentian individuals but in less than 20 % of apparently healthy gentian individuals from fields affected with Kobu-sho. Thus, we propose naming the virus Gentian Kobu-sho-associated virus.     

Association of citrus psorosis B symptoms with a sequence variant of the Citrus psorosis virus RNA 2

Citrus psorosis virus (CPsV), the type species of genus Ophiovirus, is the presumed causal agent of a bark scaling disease in citrus plants. CPsV virions are kinked filaments composed of three negative‐strand RNA molecules and a ～48‐kDa coat protein. The virus induces two different syndromes: psorosis A (PsA), characterized by limited bark scaling lesions in the trunk and main limbs, and a more aggressive form of the disease called psorosis B (PsB) with rampant bark lesions affecting even thin branches and chlorotic blotches in old leaves. In the greenhouse, the PsA and PsB syndromes can be induced by graft inoculating healthy citrus seedlings with non‐lesion or with lesion bark inoculum from PsA‐affected field trees. PsA‐ and PsB‐inducing CPsV sub‐isolates obtained by this procedure from the same tree showed identical single‐strand conformation polymorphism (SSCP) profiles in homologous segments of the RNAs 1 and 3, whereas segments of the RNA 2 enabled discrimination between PsA‐ and PsB‐associated sequence variants. SSCP analysis of the RNA 2 population present in different tissues of psorosis‐infected plants showed that: (i) PsA‐inducing isolates contain PsB‐associated sequence variants at low frequency, (ii) the PsB‐associated sequence variant is predominant in blistered twigs and gummy pustules affecting old leaves, characteristic of PsB isolates, and (iii) the PsB‐associated sequence variant accumulates preferentially in bark lesions of the trunk and limbs. SSCP analysis of the RNA 2 population also enabled monitoring of interference between PsA‐ and PsB‐associated variants in plants co‐inoculated with both psorosis types.     

Pink root rot of squash caused by Setophoma terrestris in Japan

Pink root rot of squash (Cucurbita moschata) caused by Setophoma terrestris was found in Maebashi, Gunma Prefecture, Japan in July 2007. Cucumber grafted on the squash first developed wilt and finally blight. These symptoms followed a severe pink root rot of the squash rootstock. The fungal isolates from diseased roots were identified as S. terrestris based on morphological characteristics and nucleotide sequences. One isolate induced a similar pink root rot but not entire wilting of the cucumber vine. We propose the name “pink root rot” (koshoku-negusare-byo in Japanese) of squash for the new disease.