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.     

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.     

Viruses and viroids of stone fruits in Syria

Field surveys were carried out in the main stone fruit-growing areas of Syria to evaluate the sanitary status of mother blocks, varietal collections and commercial orchards. The presence of virus and virus-like diseases was checked by enzyme-linked immunosorbent assay (ELISA), sap transmission to herbaceous hosts, testing on the woody indicators Prunus persica cv. GF 305 and Prunus serrulata cv. Kwanzan and dot-blot hybridization tests. A total of 1337 samples was tested by ELISA (444 apricot, 283 peach, 246 cherry, 222 almond and 142 plum). The overall mean infection rate was 13%, and the percentage infection level of single species was: peach 24%, cherry 16%, almond 13.5%, apricot 6%, plum 5%. The following viruses and viroids were detected: PNRSV, PDV, ACLSV, PPV, ApMV, PLMVd and HSVd ¹ .     

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。     

Epidemiology of sharka disease in Spain

PPV was first detected in Spain in 1984 in Japanese plum ( Prunus salicina Lindl) cv. Red Beaut and spread very quickly to other Japanese and European plums and apricot cultivars but left peach cultivars unaffected. In the years following the detection of PPV, the predominant aphid species visiting Prunus orchards in Mediterranean areas were Aphis gossypii followed by Aphis spiraecola , the latter being the main aphid species found at present. Both species are considered to be the main vectors of PPV in Spanish early Prunus growing areas. Spatial analysis of the spread of PPV-D in Japanese plum and apricot trees confirmed the lack of significant association between immediately adjacent trees. The observed spatial pattern of sharka suggests a lack of movement of PPV-viruliferous aphid vectors to immediately adjacent trees and indicates their preferential movement to trees several tree spaces away. PPV-D is the only type currently present in Spain, with the exception of a PPV-M outbreak that was detected in and successfully eradicated from Aragón in 2002. The short-distance spread of PPV-M infection occurred as far as 12 m along the rows of peach trees. However, PPV-D has not been observed to spread through peach cultivars, despite being grown in the vicinity of heavily infected plots of apricot or Japanese plum trees.     

Detection of plum pox virus in Spain

Until recently, plum pox (sharka) virus (PPV) was never detected in Spain on any of the material analysed by the enzyme-linked immunosorbent assay (ELISA). It was only in June 1984 that the virus was first detected by two different antiscra in Japanese plum trees ( Prunus salicina ), cv. Red Beaut, showing typical symptoms of the disease. The detection was later confirmed by graft-transmission to GF-305 peach seedlings, and also by immunoelectron microscopy. The PPV was experimentally transmitted from GF-305 to GF-305 by aphids and from GF-305 to herbaceous plants by mechanical inoculation. Thus far (January 1985), PPV has been detected basically in Japanese plum trees in Sevilla, Murcia, Valencia and Castellón, in apricot in Castellón, and in peach trees in Sevilla and Lérida.
De nombreuses analyses par la méthode ELISA n'ont, pendant longtemps, pas permis de détecter le plum pox virus (agent de la sharka) en Espagne. Ce n'est qu'en juin 1984 que la présence du virus a été confirmée, par l'utilisation de deux antiséra différents, chez des pruniers japonais ( Prunus salicina ) cv. Red Beaut qui manifestaient des symptôines typiques de la maladie. La détection a été confirmée par greffage sur des plants du pêcher GF-305, ainsi que par microscopie immuno-électronique. La transmission du PPV de GF-305 à GF-305 a été réalisée à l'aide de pucerons et de GF-305 à des plantes herbacées mécaniquement. A cette date (janvier 1985). le PPV n'a été détecté quc dans des prunicrs japonais dans, les provinces de Sevilla, Murcia, Valencia et Castellón, ainsi que dans des abricotiers en Castellón et dans des pêchers en Sevilla et en Lérida.     

Evaluation of apricot (Prunus armeniaca L.) resistance to Apple chlorotic leaf spot virus in controlled greenhouse conditions

Apple chlorotic leaf spot virus (ACLSV) seems to be the causal agent of apricot viruela disease. This disease has become an important problem for apricot production in Spain, mainly affecting the ‘Búlida’ cultivar, although no information is available about the behaviour of other cultivars with regards to ACLSV. In this study, the behaviour of 29 apricot cultivars against ACLSV (Apr 62 isolate) was evaluated under controlled conditions in an insect-proof greenhouse. Three different rootstocks, ‘GF305’ peach, ‘Real Fino’ apricot and ‘Adesoto’ plum, were first inoculated by grafting ACLSV-infected bark and were later grafted with the apricot cultivar to be evaluated. Apricot cultivars were evaluated during three cycles of study. ACLSV was asymptomatic on the leaves of all cultivars and rootstocks, so level of susceptibility or resistance was determined by virus detection through RT-PCR. ‘GF305’ rootstock showed a greater susceptibility level than ‘Real Fino’ and ‘Adesoto’. Most of the cultivars were susceptible to ACLSV with different levels of susceptibility, and only ‘Bergeron’ and ‘Mauricio’ were resistant.     

Studies on the diagnosis of hop stunt viroid in fruit trees: Identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents

A non-radioactive digoxigenin-labelled RNA probe specific for hop stunt viroid (HSVd) diagnosis has been developed. The high sensitivity and specificity of this RNA probe in dot blot hybridizations to nucleic acids from field samples, allowed the confirmation of the presence of HSVd in apricot (Prunus armeniaca L.) and its detection in two fruit tree species not previously described as hosts of this pathogen, almond (Prunus dulcis Miller) and pomegranate (Punica granatum L.). This result supports and extends the notion of the world wide distribution of HSVd, infecting cultivated fruit trees. HSVd was also found to accumulate to much higher levels in mature apricot fruits than in leaves. Additionally, a sample processing procedure which does not involve the use of organic solvents was demonstrated to render faithful results when used for viroid detection. The combined reliability and facility of use of both this extraction procedure and the non-radioactive probe will benefit agronomic investigations addressing the detection and eradication of HSVd. Other applications of the work described here, as the study of possible causal relations between specific disorders and HSVd infection, are also discussed.     

Sanitary status of stone-fruit trees in East Anatolia (Turkey) with particular reference to apricot

Field surveys were carried out in the main stone-fruit-growing areas of East Anatolia (Turkey) to assess the sanitary status of varietal collections, mother blocks and commercial orchards. The presence of virus and virus-like diseases was ascertained by enzyme-linked immunosorbent assay (ELISA), sap transmission to herbaceous hosts, graft transmission to peach cv. GF305 and molecular hybridization tests. A total of 1019 samples was tested by ELISA (859 apricot, 120 cherry, 21 almond and 19 peach). The sanitary status of apricot was extremely satisfactory, as the infection level was less than 0.3%. Cherry and almond, however, showed 21% and 33% infection respectively. The viruses identified were apple chlorotic leaf spot trichovirus (ACLSV), prune dwarf ilarvirus (PDV) and prunus necrotic ringspot ilarvirus (PNRSV). The commonest virus was PDV. Plum pox potyvirus (PPV), apple mosaic ilarvirus (ApMV) and the nepoviruses tomato black ring (TBRV), raspberry ringspot (RpRSV), strawberry latent ringspot (SLRV), cherry leaf roll (CLRV), arabis mosaic (ArMV) and tomato ringspot (ToRSV) were not encountered. Peach latent mosaic viroid (PLMVd) and hop stunt viroid (HSVd) were not detected either.     

Current status of sharka disease in Puglia (Italy) and implementation of an eradication programme1

Plum pox potyvirus (PPV) was introduced into Puglia (IT) a few years ago with infected propagative material coming from nurseries outside the region. Infections were detected in commercial plum and apricot orchards, but not in local nurseries. Extensive surveys were carried out in young apricot, plum and peach orchards to assess the distribution, incidence and spread of PPV infections, with a view to possibly enforcing an eradication programme. Surveys were based on visual inspections of about 300 commercial orchards and nurseries (covering an area of more than 550 ha) in the whole of Puglia, by ELISA and IBM tests, and by biological testing on GF305 of field samples. A total of 23,000 plants were individually checked by ELISA and IEM. The eradication involved 13 plum (35 ha) and 2 apricot (5 ha) orchards showing different levels of PPV infection. Six of the plum plantings showed infection exceeding 30% and were completely uprooted; in the remaining orchards, infection did not exceed 10% and uprooting was limited to infected trees. PPV was never detected in peach. The high incidence of PPV in some plum orchards was due to the heavy initial contamination of propagating material. Secondary virus spread was monitored in apricot orchards and found to occur at a low rate.     

Resistance to plum pox potyvirus in apricots1

The susceptibility of 115 apricot cultivars to plum pox potyvirus (PPV) has been examined, since 1981, in the experimental plots of the Pomology Institute at Naoussa and Skydra, Makedonia (GR). Inoculation was assured by aphids, transmitting strain PPV-M (Marcus) from naturally infected trees in adjacent peach orchards. For each cultivar, four to six trees were examined for at least 4 years. Observations on symptoms were made on leaves early in May and on fruits at maturity. Most cultivars expressed severe disease symptoms. Those without symptoms were inoculated by grafting onto heavily infected old apricot trees. The grafted shoots were tested for PPV in the following year by ELISA and on the woody indicator GF305. The cultivars which were rated as resistant after artificial inoculation and ELISA came from North America: Early Orange, Stella, NJA2, Sunglo, Veecot, Harlayne, Goldrich and Henderson. Most of these have been crossed with quality cultivars for the creation of resistant hybrids. The PPV resistance of large numbers of these apricot hybrids is now under investigation.     

Incidence and genetic diversity of Peach latent mosaic viroid and Hop stunt viroid in stone fruits in Serbia

Tissue-imprint hybridization (TIH) assay was validated for large-scale detection of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd). All 72 collected leaves (100%) from 2 PLMVd- and 2 HSVd-infected trees were positive in TIH, regardless of the geographic orientation of the scaffold, level of the canopy and position of the leaf in the shoot. In a large-scale survey in Serbia, we tested by TIH 871 trees of stone fruits, representing 602 cultivars from fruit collections in Belgrade, Čačak and Novi Sad. PLMVd was detected in 185 (50%) peach trees or 95 (54%) cultivars and HSVd in 2 apricot trees and cultivars (2%). The occurrence of HSVd is a new report for Serbia. No viroid infection was found in European plums, sweet cherries, sour cherries and wild Prunus spp. PLMVd-infected peach cultivars originated from the world’s main breeding centres of this crop. Western European and Asian cultivars were the most infected (58%) followed by those originating from North America (50%). Nine PLMVd and two HSVd isolates were sequenced and analyzed. All showed PMLVd sequences clustered together in the previously reported phylogenetic group III. Both HSVd isolates were found to be derived from recombinant events, but that of the cv. Saturn represented a putative new phylogenetic group of HSVd.     

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.     

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.     

Correlation of hop stunt viroid variants to cachexia and xyloporosis diseases of citrus

ABSTRACT Citrus viroid (CVd) group II is comprised of hop stunt viroid (HSVd)-related variants of 295 to 302 nucleotides. Included in this group are the cachexia-inducing agents citrus cachexia viroid (or CVd-IIb), CVd-IIc, Ca-903, and Ca-909 as well as the non-cachexia-inducing variant CVd-IIa. The cachexia indexing hosts 'Parson's Special' mandarin and 'Orlando' tangelo as well as Citrus macrophylla responded with symptoms of gumming, discoloration, and stem pitting when infected by CVd-IIb, CVd-IIc, or Ca-903. However, 'Palestine' sweet lime, the indicator host used to describe the xyloporosis disease, displayed a distinctly different fine-pitting reaction and no discoloration or gumming when infected by the same viroids. Cachexia-inducing variants contain a number of nucleotide changes more similar to hop-type HSVd sequences than to the citrus-type HSVd sequences, as typified by CVd-IIa. The nucleotide sequence of CVd-IIc was identical to CVd group II isolates common to trees expressing xyloporosis. Experimental evidence indicates that either CVd-IIb or CVd-IIc can cause citrus diseases known as cachexia and xyloporosis and that the two disease designations reflect the distinct responses of different indexing hosts to the same viroids.     

我国梅树上病毒及类病毒的检测和鉴定

目前, 我国梅树上的病毒种类及发生情况仍不完全清楚。本研究从北京、武汉、南京和无锡的梅园中采集了64份疑似感染病毒的叶片样品, 通过RT-PCR和斑点杂交, 对7种病毒和2种类病毒进行了检测。共检测到6种病毒和1种类病毒。其中, 李属坏死环斑病毒(prunus necrotic ringspot virus, PNRSV)和桃潜隐花叶类病毒(peach latent mosaic viroid, PLMVd)为我国梅树上的首次检出。PNRSV、亚洲李属病毒2号(Asian prunus virus 2, APV2)、桃叶痘伴随病毒(peach leaf pitting-associated virus, PLPaV)的检出率高于30%。综合考虑病毒的分布及检出率, PLPaV、APV2、PNRSV和李树皮坏死茎痘伴随病毒(plum bark necrosis stem pitting-associated virus, PBNSPaV)是武汉、南京和无锡梅树上的主要病毒。此外, 通过克隆和测序, 获得了PLMVd和梅树病毒A(mume virus A, MuVA)的基因组, PLPaV的RNA1组分和PNRSV外壳蛋白(CP)基因序列。序列比较分析显示, 我国PLMVd梅分离物和PNRSV梅分离物与我国桃分离物亲缘关系最近, 表明PLMVd和PNRSV可能在梅和桃树间交互侵染;我国MuVA梅分离物序列与日本梅分离物序列的相似性高达98.56%;PLPaV梅分离物与我国桃分离物之间序列变异较大。上述结果不仅进一步明确了我国梅树上的病毒及类病毒种类和分布情况, 而且有助于深入了解它们的流行与传播。     

Citrus viroid II variants associated with ‘Gummy Bark’ disease

A viroid etiology for citrus gummy bark (CGB) disease of sweet orange is supported by the similarity of symptom expression to cachexia disease of mandarins and tangelos caused by the hop stunt viroid (HSVd) related citrus viroid II (CVd-II), as well as the detection of CVd-II variants in CGB infected Washington navel and Dörtyol sweet orange, a Turkish cultivar. A survey was made of 67 clones of CVd-II related variants recovered from severe CGB symptomatic and non-symptomatic trees of the same cultivars growing in close proximity. Only CVd-IIa, a non-cachexia inducing variant, was found in non-symptomatic Washington navel trees and no CVd-II variants were recovered from the Dörtyol control. CGB infected sources contained a number of CVd-II related variants with the predominant species detected closely related to CVd-IIc, a known cachexia inducing viroid. Biological activity of representactive variants from CGB sources was determined by transmission to citron (Citrus medica) as well as by bioassay on the indexing host for cachexia, Parson's Special mandarin (Citrus reticulata).