Simultaneous detection and genetic variability of stone fruit viroids in the Czech Republic

In this paper, we report a large-scale survey for the incidence of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) in stone fruit collections and commercial orchards in the Czech Republic. From the 645 samples analysed, PLMVd was detected in 80 (26.6%) of peaches and the HSVd in 3 (1.3%) of apricot and 1 (0.33%) of peach trees. Sixty-seven accession of peach (44.6%) from the Czech Clonal GeneBank were infected by PLMVd. In addition, we used naturally infected trees to standardise the simultaneous detection of PLMVd and HSVd plus host mRNA as the control by means of one-step multiplex RTC-PCR. Eleven PLMVd and two HSVd isolates were sequenced and analysed. All the PLMVd variants were highly homologous (97–100%) to previously reported PLMVd variants from Tunisian peach and almond trees, and clustered together in the previously reported phylogenetic group III. The HSVd variants obtained from apricot and peach trees were included in the previously proposed recombinant group PH/cit3.     

Molecular characterization of Chinese Hop stunt viroid isolates reveals a new phylogenetic group and possible cross transmission between grapevine and stone fruits

A survey for the incidence of Hop stunt viroid (HSVd) infection in China was conducted using dot-blot hybridization. Out of 553 tested samples, 127 samples of stone fruits (apricot, peach, plum and almond), grapevine and hop were positive for HSVd, giving a mean infection rate of 23?%. Phylogenetic analysis revealed that most of the HSVd variants isolated from stone fruits, grapevine and hop were clustered into known hop and plum groups. However, two grapevine variants, HSVd.g50 and HSVd.g57, could not be clustered into any known groups, indicating a previously unknown phylogenetic group of HSVd isolates. HSVd.g38 was the single grapevine variant that clustered with the plum group isolates, supporting cross transmission between grapevine and stone fruits and the heterogeneity of grapevine isolates.     

侵染肥城桃的病毒和类病毒的分子检测与鉴定

为明确山东肥城桃种植区桃树上主要存在的病毒和类病毒及其发生情况,采集具有花叶、斑驳和皱缩典型症状的肥城桃样品,提取叶片总RNA后,分别选用桃树上已报道的啤酒花矮化类病毒Hopstuntviroid(HSVd)、桃潜隐花叶类病毒Peach latent mosaic viroid(PLMVd)、苹果褪绿叶斑病毒Apple chlorotic leaf spot virus(ACLSV)、樱桃锉叶病毒Cherry rasp leaf virus(CRLV)、桃花叶病毒Peach mosaic virus(PMV)、李属坏死环斑病毒Prunus necrotic ringspot virus(PNRSV)、李痘病毒Plum pox virus(PPV)、李矮缩病毒Prunus dwarf virus(PDV)、樱桃绿环斑驳病毒Cherry green ring mottle virus(CGRMV)、杏假褪绿叶斑病毒Apricot pseudo-chlorotic leaf spot virus(APCLSV)、李树皮坏死茎纹孔伴随病毒Plum bark necrosis stem pitting-associated virus(PBNSPaV)和小樱桃病毒1号Little cherry virus 1(LchV1)的特异性引物进行RT-PCR检测。PCR结果显示仅HSVd、PLMVd、ACLSV、PNRSV和PBNSPaV的扩增产物中得到了预期大小的目的片段,将目的片段克隆测序后,经NCBI BLAST比对发现,山东肥城桃分离物HSVd、PLMVd、ACLSV、PNRSV和PBNSPaV与GenBank已报道分离物序列一致性均达90%以上。表明山东肥城桃已感染HSVd、PLMVd 2种类病毒和ACLSV、PNRSV、PBNSPaV 3种病毒。     

Identification and characterization of known and novel viroid variants in the Greek national citrus germplasm collection: threats to the industry

Seventy four percent of the budwood tree sources samples from the Greek national citrus germplasm foundation collection were positive for one or more viroids. Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), the two potentially damaging viroids for the Greek citriculture, especially after transitioning to Citrus tristeza virus resistant/tolerant rootstocks and scions, were detected along with Citrus bark cracking viroid, Citrus bent leaf viroid (CBLVd), and Citrus dwarfing viroid (CDVd). All samples tested negative for Citrus viroid V (CVd-V), CVd-VI and CVd-I-LSS (CBLVd variant). An HSVd isolate related to the non-cachexia variant contained two critical cachexia-related nucleotide changes, while two more isolates were unique among the previously reported HSVds. Unusual CDVd isolates with altered RNA secondary structure were identified in trees additionally co-infected with CEVd and HSVd. Budwood sources that had previously undergone therapy tested negative for all targeted viroids, suggesting that budwood sources in Greece can be protected against graft-transmissible pathogens, even under severe inoculum pressure. Therapied and tested citrus propagative material requires a comprehensive program not available currently in Greece, involving regulators, scientists, and the private sector, for the establishment and successful operation of a national citrus germplasm collection.     

An important new apricot disease in Spain is associated with <Emphasis Type="Italic">Hop stunt viroid</Emphasis> infection

The province of Murcia (Murcia Region) is the one of the most important apricot growing regions in Europe. In recent years a fruit disorder named by growers as “degeneración” has been detected in apricot commercial orchards of this region, mainly in the variety Velázquez Fino. Affected fruits are characterized by changes in their external appearance involving rugosity and a loss of organoleptic characteristics, which leads to an unmarketable product. In order to identify the causal agent of this disorder, the presence of the most important viruses affecting stone fruit trees and Hop stunt viroid (HSVd) was tested. While negative results were obtained for all viruses analysed, the viroid was detected in all the symptomatic trees. Within a single tree, the viroid was restricted to branches bearing fruits with the characteristic symptoms but was usually absent from the rest of the tree. Sequence analysis of several isolates of HSVd obtained from these affected trees revealed the presence of two variants previously detected in other apricot cultivars. Taken together, these results suggest that degeneration is associated with HSVd.     

Somatic embryogenesis efficiently eliminates viroid infections from grapevines

Indirect somatic embryogenesis is effective at eliminating the most important viruses affecting grapevines. Accordingly, this technique was tested as a method for eradicating two widespread viroids, Grapevine yellow speckle viroid 1 (GYSVd-1) and Hop stunt viroid (HSVd), from four grapevine cultivars. Both viroids were detected by RT-PCR in grapevine floral explants used for initiating embryogenic cultures, as well as in undifferentiated cells of embryogenic and non-embryogenic calli from anthers and ovaries. In contrast, somatic embryos differentiated from these infected calli were viroid-free, and viroids were not detected in embryo-derived plantlets even 3 years after their transfer to greenhouse conditions. A wider spatial distribution of HSVd than GYSVd-1 within proliferating calli was revealed by in situ hybridization, whereas no hybridization signal was detected in the somatic embryos. In addition, GYSVd-1 and HSVd were localised in the nucleus of infected cells, conclusively showing the nuclear accumulation of representative members of Apscaviroid and Hostuviroid genera, which has been only an assumption so far. Somatic embryogenesis was compared to in vitro thermotherapy, a technique routinely used for virus eradication. After thermotherapy, HSVd and GYSVd-1 were detected in all in vitro plantlets of the cultivar Roussan, and in all lines analysed after 3 years of culture in greenhouse. The high efficiency with which somatic embryogenesis may eliminate viroids and viruses from several infected grapevine cultivars, should allow the availability of virus- and viroid-free material, which would be useful not only for sanitary selection but also for basic research on plant-virus and plant-viroid interactions in grapevine.     

Survey on viroids infecting grapevine in Italy: identification and characterization of Australian grapevine viroid and Grapevine yellow speckle viroid 2

Five viroid species have been reported from grapevine. Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd-1) are distributed worldwide, whereas Grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd) and Citrus exocortis viroid (CEVd) are found only sporadically. However, the presence of AGVd and GYSVd-2 in several countries, including China, Turkey and Tunisia, suggests a wider dissemination, possibly also in Europe, where AGVd has never been found and GYSVd-2 has been occasionally identified in Italy. Taking advantage of a multiplex RT-PCR assay recently developed for detecting simultaneously these five viroids, vines growing in Italy in commercial vineyards and germplasm collections were surveyed. Besides confirming the widespread presence of HSVd and GYSVd-1 in the field, GYSVd-2 and/or AGVd were identified in two grapevine table cultivars (Sultanina Bianca and Red Globe) from germplasm collections. Tests extended to vines cultivated in southern Italy confirmed the presence of both viroids, which were further characterized. No major sequence divergences between the AGVd and GYSVd-2 variants from Italy and those previously described from other countries were observed. Phylogenetic analysis supported the close relationships among AGVd variants from Italy, Tunisia and Australia. To our knowledge this is the first report of AGVd in Europe and the first molecular characterization of GYSVd-2 isolates from a European country.     

Viruses and viroids of stone fruits in Jordan

Field surveys were carried out in the main stonefruit-growing areas of Jordan to assess the sanitary status of varietal collections, mother plant blocks and commercial orchards. The presence of virus and virus-like diseases was determined by ELISA, sap transmission to herbaceous hosts, graft transmission to Prunus persica cv. GF 305 and P. serrulata cv. Kwanzan, and molecular hybridization tests. A total of 1312 samples was tested by ELISA (531 peach, 361 plum, 218 apricot, 135 almond and 67 cherry trees). The overall mean level of infection was about 14%, indicating an acceptable sanitary status as a whole, considering that no sanitary selection has ever been carried out in Jordan. The infection level of different species was: peach (18%), cherry (15%), almond (14%), apricot (11%) and plum (10%). The following viruses and viroids were identified: Plum pox potyvirus (PPV), Prunus necrotic ringspot ilarvirus (PNRSV), Prune dwarf ilarvirus (PDV), Apple mosaic ilarvirus (ApMV), Apple chlorotic leaf spot trichovirus (ACLSV), Hop stunt viroid (HSVd) and Peach latent mosaic viroid (PLMVd). Most of these agents (ApMV, ACLSV, PLMVd and HSVd) are reported for the first time from Jordan.     

<Emphasis Type="Italic">Neofusicoccum parvum</Emphasis> associated with fruit rot and shoot blight of peaches in Greece

Shoot blights and fruit rots comprise the most serious diseases of peaches in Greece. In this study, the importance of the fungus Neofusicoccum parvum as a casual agent of a fruit rot and shoot blight of peach trees in Greece was investigated. This pathogen was isolated from both immature and mature peach fruit of the cultivar “Catherine” and later on from mature fruit of the peach cultivars “Andross”, “RedHaven”, “Sun Crest” and “Sun Cloud”. In the first year of investigation, N. parvum was found causing preharvest fruit rot and shoot blights of peach trees only at the location “Ammos-Mesi-Meliki Verias” in the prefecture of Imathia (the main peach production area of Greece) at incidences of 30 and 8%, respectively. However, in 2006 N. parvum was isolated from more locations such as Diavatos, Veria, Kopanos and Agia Marina in the prefecture of Imathia, but only at less than 3% of the total surveyed rotted peach fruit and blighted shoots. The pathogen overwintered as sub-epidermal pycnidia in blighted shoots or mummified fruit that remained on peach trees. This study also showed that the optimum temperature for mycelial growth and conidial germination of N. parvum was 25 oC. Pathogenicity tests using peach fruit showed that isolates of N. parvum and Diplodia seriata (isolated from pistachio grown in the same region) showed no significant differences in their virulence. In laboratory inoculation tests using detached shoots from 25 peach and nectarine cultivars, N. parvum isolates obtained from rotted peaches caused different size cankers on these cultivars. The cultivar Big Top was the most susceptible while the cultivar Maria Bianca the least susceptible.     

A preliminary account of the sanitary status of stone-fruit trees in Morocco

Field surveys were carried out in the main stone-fruit growing areas of Morocco to evaluate the sanitary status of commercial orchards, varietal collections and nurseries. The presence of virus and virus-like diseases was checked by ELISA, sap transmission to herbaceous hosts, testing on woody indicators and molecular hybridization (dot-blot and tissue-printing). 1211 samples (382 almond, 339 peach, 291 plum, 150 apricot and 49 cherry) were tested by ELISA for the presence of Prunus necrotic ring spot virus (PNRSV), Prune dwarf virus (PDV), Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV) and Plum pox virus (PPV). The overall average of virus infection rate was 16.4%, whereas that of single species was: 22.6% for almond, 17.8% for plum, 15% for peach, 10.2% for cherry, and 2.7% for apricot. The following viruses were detected: PNRSV, PDV, ACLSV and ApMV. 565 samples were tested by dot-blot and tissue-printing hybridization for the presence of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd). 48 samples were infected, 41 by PLMVd and 7 by HSVd. In addition, nested-PCR tests identified Plum bark necrosis and stem-pitting associated virus (PBNSPaV) in a few almond trees affected by stem pitting.     

桃树上啤酒花矮化类病毒(Hop stunt viroid)的检测及序列分析

 2005年8月和2006年2月从中国北京、陕西、河北、山东、广西等地共采集76个无明显症状的桃树样品,经斑点杂交、RT-PCR以及生物学鉴定检测,来自北京和陕西的11个样品中检测到啤酒花矮化类病毒(Hop stunt viroid,HSVd),总感染率达14.5%。上述3种方法检测桃树上的HSVd具有一致性。将5个样品中的HSVd进行克隆测序,得到12条不同HSVd核酸序列,与GenBank中D13764序列(日本桃果实HSVd分离物)同源性为93.29%~100%。可以看出,国内桃树HSVd分离物核酸序列变异比较小,地域和品种间核酸序列无明显差异。这是首次比较系统地检测中国桃树上HSVd发生情况的报道。     

Development of a multiplex TaqMan real-time RT-PCR assay for simultaneous detection of Asian prunus viruses, plum bark necrosis stem pitting associated virus, and peach latent mosaic viroid

Asian prunus viruses (APV 1, APV 2 and APV 3), Plum bark necrosis stem pitting associated virus (PBNSPaV) and Peach latent mosaic viroid (PLMVd) are pathogens that infect Prunus species. A single-tube multiplex, TaqMan real-time RT-PCR assay was developed for the simultaneous detection and identification of these pathogens. The protocol includes amplification and detection of a fluorogenic cytochrome oxidase gene (COX) as an internal control. The results of the multiplex TaqMan RT-PCR assay correlated with those from conventional RT-PCR, with a 10-fold increase in sensitivity in the multiplex real-time format. The efficiency and accuracy of the assay was evaluated by testing stone fruit trees from positive control collections and several orchard locations. Several mixed infections of target pathogens were detected in peach orchard samples. This assay is simple, rapid and cost-effective and can be used by quarantine and certification programs where numerous stone fruit trees need to be tested for these pathogens.