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.     

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.     

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.     

Interactions between citrus viroids affect symptom expression and field performance of clementine trees grafted on trifoliate orange

ABSTRACT Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), a noncachexia variant of Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), and Citrus viroid IV (CVd-IV) were co-inoculated as two-, three-, four-, and five-viroid mixtures to Clementine trees grafted on trifoliate orange to evaluate their effect on symptom expression, tree growth, and fruit yield. Most trees infected with CEVd-containing viroid mixtures developed exocortis scaling symptoms, as did CEVd alone, whereas most trees infected with HSVd- or CVd-IV-containing mixtures developed bark-cracking symptoms. Trees infected with mixtures containing both CEVd and CVd-IV revealed the existence of antagonism between these two viroids in terms of the expected bark-scaling and cracking symptoms. Synergistic interactions also were identified in trees infected with certain viroid combinations that, in spite of lacking CEVd, expressed exocortis-like scaling symptoms. Viroid interactions also affected the expected response of trees in terms of vegetative growth and fruit yield. Trees infected with viroid combinations containing CEVd or CVd-III were smaller and produced less fruit than trees infected with mixtures not containing these viroids. Viroid interactions on scion circumference and cumulative fruit yield, in terms of additivity of their effects, were statistically confirmed using a factorial analysis of variance model with two mean estimation approaches. In single-viroid infections, CEVd, CVd-III, and, to a lesser extent, CBLVd consistently and significantly reduced tree size and fruit yield. Conversely, HSVd and CVd-IV slightly increased fruit yield and reduced scion circumference. Rare and not consistent significant interactions were detected with the five-, four-, and three-viroid combinations. Antagonistic interactions between CEVd and CVd-III or CBLVd and CVd-III were revealed over the years with consistent significance. The antagonistic interaction between CEVd and CVd-IV was highly significant over the years when additional viroids were present; however, this antagonism appeared much later in the case of an exclusive interaction. HSVd and CVd-IV showed a consistent and significant synergistic interaction on yield only when both viroids were exclusively present. These results demonstrate antagonistic or synergistic relationships between citrus viroids depending on the viroid mixtures present in the host.     

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.     

Molecular characterization and variability analysis of <Emphasis Type="Italic">Apple scar skin viroid</Emphasis> in India

Apple scar skin viroid (ASSVd) infection is a major limitation to apple fruit quality and causes huge economic losses. In surveys of apple orchards in the northern Indian state of Himachal Pradesh, fruits with dappling symptoms were noticed. ASSVd was detected from these fruits and molecularly characterized. Ten clones from three isolates were sequenced, of which seven were new sequence variants of ASSVd. The clones had significant sequence variability (94–100%) with each other. Variability was more common in the pathogenic domain of the viroid genome. Four of the clones were 330 nucleotides (nt) long, and the other six had an additional nucleotide. Phylogenetic analysis showed close affinity of the present isolates with some Chinese and Korean isolates. The study reports seven new variants of ASSVd and also provides the first molecular evidence of viroid infection (ASSVd) in apple in India.     

Goat horns: Platforms for viroid transmission to fruit trees?

Mechanical inoculations with contaminating tools and propagation of infected budwood were considered the main causes for the omnipresence of multiple viroid species among citrus and other Middle Eastern and Mediterranean fruit trees and grapevines. However, neither means could explain viroid infections of wild trees — scattered on terrains inaccessible to humans — nor the finding of similar viroids among graft-incompatible plants. Northern hybridization of RNA extracts made of scrapings from the surfaces of goat (Capra hircus) horns that were rubbed against etrog (Citrus medica) stems infected with a citrus viroids complex, revealed accumulation of considerable amounts ofCitrus exocortis viroids (CEVd) andHop stunt viroids (HSVd). Experimental transmission of both CEVd and HSVd was obtained by rubbing healthy citrus plants with goat horns that had been rubbed 24 h earlier on infected etrog stems. These results implicate goats as possible vectors of viroids. Transmissionvia goats could have facilitated the long-range spread of viroids among cultivated and wild plants andvice versa and also among graft-incompatible plants.     

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.     

Characterization of a new Apple dimple fruit viroid variant that causes yellow dimple fruit formation in ‘Fuji’ apple trees

A 303-nucleotide viroid was isolated from an apple tree (Malus × domestica, ‘Fuji’) cultivated in Japan. The viroid had 84.9% overall nucleotide sequence homology to Apple dimple fruit viroid (ADFVd), a member of Pospiviroidae, reported from Italy. This viroid differed from the Italian variant by 47 mutations (38 substitutions, six deletions and three insertions), and most of these mutations occurred on either side of the central conserved region. The leaves and branches of the infected trees did not have any disease symptoms, but the fruits were dimpled and yellow. The infected scions were top-grafted onto a healthy ‘Fuji’ apple tree, which tested positive for this viroid in a northern hybridization analysis, and yellow dimple fruits were produced in the second growing season. We propose that this viroid is a new variant of ADFVd and causes yellow dimple fruit formation in ‘Fuji’ apple trees.     

Detection and characterization of phytoplasmas in diseased stone fruits and pear by PCR-RFLP analysis in Turkey

During the late summer-early autumn of 2002, surveys were carried out in Turkey to determine the presence of phytoplasma diseases in fruit trees. Phytoplasmas were detected and characterized by PCR-RFLP analysis and TEM technique in stone fruit and pear trees in the eastern Mediterranean region of the country. Six out of 24 samples, including almond, apricot, peach, pear and plum, gave positive results in PCR assays. RFLP analysis usingSspI andBsaAI enzymes of PCR products obtained with primer pair f01/r01 enabled identification of the phytoplasmas involved in the diseases. Stone fruit trees, including a local apricot variety (‘Sakıt’) and a pear sample, were found to be infected with European stone fruit yellows (ESFY, 16SrX-B) and pear decline (PD, 16SrX-C) phytoplasmas, respectively. This is the first report in Turkey of PD phytoplasma infecting pear and of ESFY phytoplasma infecting almond, apricot, myrobalan plum and peach; ESFY phytoplasma infecting Japanese plum was previously reported. http://www.phytoparasitica.org posting July 21, 2005.     

Species of<Emphasis Type="Italic">Oligonychus</Emphasis> infesting date palm cultivars in the Southern Arava Valley of Israel

In a study of date fruit damage caused byOligonychus spp., we investigated whether the cultivar affects phenology, and on what hosts the mites over-winter. Samples were taken from ‘Deglet Noor’, ‘Barhi’ and ‘Medjool’ trees from mid-April through mid-September during the years 1999–2002. In the ground-cover mites were monitored by collecting Bermuda grass (Cynodon dactylon) under each sampling tree. Over 99% of the mites collected on Deglet Noor and Barhi fruit were identified asO. afrasiaticus. Mean population levels ofO. afrasiaticus reached ten mites or more (initiation of infestation) on Medjool in the second half of May, whereas on Deglet Noor this did not occur before the first week of July. On Barhi the initiation of infestation varied between plots and years, ranging from the second half of May to the beginning of July, but always occurred earlier than Deglet Noor. Mite populations on the pinnae remained low from June through October, not exceeding seven mites per pinna, whereas on fruit strands they reached peak populations of approximately 4000 mites per strand. The sex ratio (proportion of females) ofO. afrasiaticus on fruit of all three cultivars was highly female-biased, usually above 0.85. During winter,O. afrasiaticus was found on Bermuda grass in the orchard ground-cover as well as on fronds of all three cultivars. http://www.phytoparasitica.org posting Jan. 30, 2003.     

Improved control of moldy-core decay (<Emphasis Type="Italic">Alternaria alternata</Emphasis>) in Red Delicious apple fruit by mixtures of DMI fungicides and captan

The sterol biosynthesis inhibitors bromuconazole and difenoconazole and tank mixes of each fungicide with captan were applied to apples and evaluated as controls for moldy-core and fruit decay caused by Alternaria alternata. Effectiveness of a mixture of bromuconazole and captan in controlling colonization by the fungus was also evaluated. Decay formation by A. alternata on mature detached fruits was partially inhibited by bromuconazole at 0.5 μg ml⁻¹ and was completely inhibited at 50 μg ml⁻¹; it was significantly affected by either bromoconazole at 5 μg ml⁻¹ or captan at 1,250 μg ml⁻¹, and was completely inhibited by their mixture. In general, three foliar applications of bromuconazole or difenoconazole in the field, during the bloom period, reduced the numbers of infected fruits by 40–60% compared with untreated control trees. However, tank mixes of either fungicide with captan improved control of moldy-core in fruits at harvest. Tank mixtures of bromuconazole and captan also significantly reduced the percentage of fruits colonized by A. alternata when sampled at various days after full bloom. Artificial inoculations in the orchard at full bloom did not change the inhibitory effects of the tank mixtures. Large-scale demonstration trials in commercial orchards supported these findings. The inhibitory effects of tank mixes on decay development in detached fruits, and on moldy-core in the field indicate that a control programme based on mixtures of either bromuconazole or difenoconazole with captan during the bloom period can effectively reduce moldy-core on Red Delicious apples.     

Alternaria Brown Spot of Minneola in Greece; Evaluation of Citrus Species Susceptibility

During the last three years, a new disease was observed in northwestern Greece on Minneola trees, hybrid of mandarin and grapefruit. On May small brown necrotic leaf spots surrounded by yellow halo areas of various sizes appeared and covered a major portion of the leaves with extension of necrosis into the veins. On young fruits small, slightly depressed black spots were the first symptoms, which later became 2–7 mm in diameter. Brown spots were observed on the leaves and fruits in several orchards in the same area, causing leaves and fruits to drop. In some orchards over 50% of the fruits were affected. From the fruit and leaf spots the typical small-spore species Alternaria alternata was isolated. Pathogenicity tests were performed by artificially inoculating fruits of Minneola, common mandarin and Clementine. The symptoms of the disease were reproduced only on fruits of Minneola hybrids by the specific strain of the fungus Alternaria alternata pv. citri. Different citrus susceptibility tests indicated that mandarins Minneola, Nova and Page were very susceptible to tested isolates while Clementine SRA and Poros Clementine were not. All lemons and lime Seedless were not susceptible. Grapefruit New Hall was not susceptible, while the Star Ruby was. Orange Lane Late, Navel Late, Oval Poros, Olinda, Navel Athos were not susceptible and only Moro showed reaction being slightly susceptible only to one isolate.     

Peach red marbling and peach sooty ringspot, two new virus-like degenerative diseases of Prunus

More than 600 Prunus samples were examined by using a nonradioactive digoxigenin-labelled RNA probe specific for hop stunt viroid (HSVd). Prunus salicina and Prunus armeniaca appeared to be better hosts than Prunus persica . The weak viroid concentration in flowers and young leaves of peach trees growing in the field did not permit its detection in such samples. The diagnosis was more reliable (about 85%) with bark and leaves aged 4 months and more, from regrowths of GF 305 peach seedlings inoculated and kept in the greenhouse. Detection of HSVd in leaves and bark of apricot and Japanese plum plants aged 3 months or more also proved reliable (about 80% and 90%, respectively). HSVd could be transmitted in apricot, peach and plum nucleic acid preparations to GF 305 peach seedlings by repeated stem slashing, and to cherries ( Prunus avium and Prunus serrulata ) by approach grafting with an infected P. salicina source. The viroid was eliminated from 18% of the clones obtained after thermotherapy.
In the course of this study, 25 selected Prunus accessions suspected to be infected by unusual diseases were analysed by hybridization with a HSVd-specific probe and by indexing on GF 305 peach seedlings in the greenhouse. Fifteen of these accessions were found to be infected by HSVd, 19 induced reddish marbling, and four induced small blackish spots on the leaves aged about 4 months. Repeated assays showed that these foliar symptoms were not caused by the viroid. Peach red marbling (PRMa) has not been associated with any known virus and seems to be caused by an infectious agent not yet described. That could also be the case with the agent of peach sooty ringspot (PSRS). PRMa and PSRS symptoms were reproduced by grafting and indexing, and their causal agents eliminated by thermotherapy in a significant fraction of the treated plants. They behave like viral agents and can infect the different Prunus species studied.     

Effect of cultural practices on the development of arabica coffee berry disease,caused by <Emphasis Type="Italic">Colletotrichum kahawae</Emphasis>

In the high altitude regions of Africa, coffee berry disease (CBD), caused by Colletotrichum kahawae, is the main constraint for arabica coffee (Coffea arabica) production. However, certain agricultural practices can reduce losses caused by the disease and thereby promote optimum production. On small family farms in Cameroon, mixed cropping with fruit trees, intercropping with food crops and maintenance pruning of coffee trees are very widespread agricultural practices that can affect CBD epidemics. Consequently, an epidemiological study was conducted to assess how cultural practices affected the disease in an arabica coffee smallholding in Cameroon. The disease was monitored on a weekly basis over four successive years (2002–2005) on coffee trees in diverse cultural situations. Cultural practices likely to reduce losses due to CBD were identified. The infection rate was significantly lower on coffee trees grown intensively than on coffee trees grown in the traditional manner. Coffee trees located under the shade of fruit trees were significantly less attacked than those located in full sunlight. In addition, berries on the leafless parts of branches, near the main trunk of the coffee tree, were less infected than those on leafy sections. These results show that maintenance pruning, removal of mummified berries, and mixed cropping with shade plants are cultural practices which create environmental conditions that limit CBD development.