Development of multiplex RT-PCR assay for simultaneous detection of four viruses infecting apple (Malus domestica)

The major viruses infecting apple cultivars throughout the world including India are apple mosaic virus (ApMV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV), and recently, a new virus, apple necrotic mosaic virus (ApNMV), was reported from mosaic-infected apple cultivars in India. The aim of this study was to detect the ApNMV virus along with the other three viruses (ApMV, ASPV and ASGV) simultaneously by multiplex RT-PCR. Four primer-pair-produced amplicons of 670, 550, 350 and 210 bp corresponding to ApNMV, ApMV, ASPV and ASGV, respectively, were found to be specific for these viruses when tested individually. The annealing temperature (55°C), primer concentration (0·8 µl) and other components of the master mix were standardized for the development of one-step m-RT-PCR assay. The m-RT-PCR protocol developed was further validated with 30 samples from seven symptomatic or asymptomatic apple cultivars, which revealed the presence of more than one virus in these cultivars. Most of the viruses were found to be present either alone or in mixed infection; however, ASPV was more common in tested cultivars. An easy, cost-effective and rapid multiplex RT-RCR protocol was developed to detect the four viruses, which infect apple plants either in individually or together in the field. This assay will help in the surveying and indexing of apple germplasm and the distribution of all four viruses in the apple growing regions of India.     

Association of Tobacco Ringspot Virus with a Union Incompatibility of Apple

A virus-like disease of apple was observed for the first time in Canada in 1974 in a three year old planting. The disease was characterised by stem pitting, necrosis and breaking or separation of scion/rootstock at the graft union. Foliage was sparse and leaves were chlorotic and diffusely mottled. Using both frozen and freshly harvested leaves of infected apple trees from rootstock suckers as inoculum, a virus was transmitted to herbaceous indicator hosts. Based on host range symptoms, serology and electron microscopy, this virus was identified as tobacco ringspot virus. Initial counts of nematodes in the rhizophere of apple trees affected with union necrosis showed high densities of Xiphinema americanum Cobb., a known vector of nepoviruses. Reports of similar symptoms have indicated that the disease, apple union necrosis and decline was associated with the recovery of tobacco ringspot virus from infected rootstocks. This is the first report associating a union incompatibility condition of apple and tobacco ringspot virus.     

Pepino mosaic virus, a new potexvirus from pepino (Solanum muricatum)

Pepino mosaic virus (PepMV), a previously undescribed virus, was found in fields of pepino (Solanum muricatum) in the Canete valley in coastal Peru. PepMV was transmitted by inoculation of sap to 32 species from three families out of 47 species from nine families tested. It caused a yellow mosaic in young leaves of pepino and either a mild mosaic or symptomless infection in 12 wild potato species, five potato cultivars and potato clone USDA 41956 but S. stoloniferum and potato cultivars Merpata and Revolucion reacted with severe systemic necrotic symptoms. The virus was transmitted by plant contact but not by Myzus persicae. It was best propagated and assayed in Nicotiana glutinosa. Sap from infected N. glutinosa was infective after dilution to 10^-1 but not 10^-6, after 10 min at 65°C but not 70°C and after 3 months at 20°C. PepMV had filamentous particles with a normal length of 508 nm; the ends of some seemed damaged. Ultra-thin sections of infected leaves of N. glutinosa revealed many inclusions containing arrays of virus-like particles some of which were banded or whorled; small aggregates of virus-like particles were also common. The virus was purified by extracting sap from infected leaves in a solution containing 0·065 M disodium tetraborate, 0·435 M boric acid, 0·2% ascorbic acid and 0·2% sodium sulphite at pH 7·8, adding silver nitrate solution to the extract, and precipitating the virus with polyethylene glycol followed by two cycles of differential centrifugation. Particles of PepMV normally yielded two proteins with molecular weights of 26 600 and 23 200, but virus obtained from infective sap aged overnight yielded only the smaller protein suggesting that it was a product of degradation of the larger one. The virus is serologically related to two potexviruses, narcissus mosaic and cactus X and its properties are typical of the potexvirus group.     

Partial purification and some properties of wineberry latent, a virus obtained from Rubus phoenicolasius

Wineberry latent virus (WLV) was obtained from a single symptomless plant of American wineberry (Rubus phoenicolasius) originally imported from the United States of America. On graft inoculation, WLV infected but induced no distinctive symptoms in several Rubus species including those used as indicators for known Rubus viruses. It was not seed-borne in wineberry. WLV was mechanically transmitted to several herbaceous species but induced local lesions in only a few; it was weakly systemic in some Chenopodium species. Infective C. quinoa sap lost infectivity after diluting to 10^-4, heating for 10 min at 70°C, and storage either for 8 days at 18°C or for 32 days at 4°C. Sap from infected plants contained flexuous filamentous particles c. 510°12 nm. WLV was partially purified by extracting infected C. quinoa leaves in 0·05 M tris-HCl buffer (pH ₇) containing 0·2% thio-glycerol and 10% (v/v) chloroform and concentrating virus by precipitation with 7% (w/v) polyethylene glycol (PEG, mol. wt 6000) and 0·1 NaCl. The virus was then pelleted through a 30% (w/v) sucrose pad containing 7% PEG+0·1 M NaCl and finally sedimented through a sucrose density-gradient. These preparations had A_260/280 ratios of 1·26, contained end to end aggregates of WLV particles and formed a partly polydispersed peak in the analytical ultracentrifuge. WLV did not react with antisera to four potex-viruses, or to apple chlorotic leaf spot or apple stem grooving viruses.     

Host range, purification and serological properties of heracleum latent virus

Heracleum latent virus (HLV occurs commonly in wild plants of Heracleum sphondylium (hogweed) in Scotland without causing symptoms. It was transmitted manually or by aphids (Cavariella aegopodii, C. pastinacae or C. theobaldi) to 37 of 105 species in 11 of 18 families (especially to members of the Amaranthaceae, Chenopodiaceae, Solanaceae and Umbelliferae), but was not transmitted through seed of four species tested. It has very flexuous filamentous particles c. 730 × 12 nm in phosphotungstate, with obvious cross-banding of pitch 3–8 nm. Leaf extracts lost infectivity after 1–2 days at 22°C, 10 min at 40–50°C and after dilution 10^-4-10^-5. Infectivity in leaf extracts was not stabilised by addition of Mg²⁺, Ca²⁺ or Ni²⁺, but was abolished by EDTA. HLV was purified by bentonite clarification, precipitation with polyethylene glycol (mol. wt 6000), and differential centrifugation. Its properties resemble those of the tentative closterovirus, apple chlorotic leaf spot (ACLSV), but no serological relationship was detected to this or to any of 18 other filamentous viruses, including six definitive closteroviruses. No cross-protection was observed between HLV, ACLSV and apple stem grooving virus.     

Occurrence and Diversity of Pome Fruit Viruses in Apple and Pear Orchards in Latvia

Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple mosaic virus are economically important viruses infecting fruit tree species worldwide. To evaluate the occurrence of these pome fruit viruses in Latvia, a large‐scale survey was carried out in 2007. Collected samples were tested for infection by DAS ELISA and multiplex RT‐PCR. The accuracy of the detection of the viruses in multiplex RT‐PCR was confirmed by sequencing amplified PCR fragments. The results showed a wide occurrence of viruses in apple and pear commercial orchards established from non‐tested planting material. More than 89% of the tested apple trees and more than 60% of pear trees were infected with one or more pome fruit viruses. Analyses showed that the high occurrence of viruses in several apple cultivars is due to the propagation of infected clonal rootstocks and scions from infected mother trees. Sequence analyses targeting the 3′‐terminal region of the tested viruses showed various degrees of genetic diversity within respective virus isolates. This is the first report of the occurrence of ACLSV, ASGV and ASPV in apple and pear trees in Latvia and demonstrates their genetic diversity in different host genotypes.     

Properties of a tomato isolate of Pelargonium zonate spot virus

A manually transmissible virus isolated from tomato plants with stunting, unfruitfulness, malformation and yellow rings and line patterns of the leaves was indistinguishable from Pelargonium zonate spot virus (PZSV) in biological, physico-chemical and serological properties. The tomato isolate (PZSV-T) of PZSV was seed transmitted in Nicotiana glutinosa and was detected in the pollen of this host. In sap of N. glutinosa PZSV-T lost infectivity after diluting 10^-1 to 10^-2, heating for 10 min at 35 to 40 °C or storage at 25 °C for 7 h. Virus particles were quasi-spherical with a diameter ranging between 25 and 35 nm with a modal value of 29 nm. Particles sedimented as three components (TV, MV and BV) with sedimentation coefficients of 80S (TV), 90S (MV) and 118S (BV); component BV is probably an aggregate of TV. Particles were unstable in CsCl and CS₂SO₄ but formaldehyde-stabilised particles banded at a common density of 1–268 g/cm³ in Cs₂SO₄. Particles contained a single protein species with mol. wt of c. 23000 and c. 18% single stranded RNA present as two species with mol. wts of c. 1.25 × 10⁶ (RNA-1) and 0.95 × 10⁶ (RNA-2). Mixtures of RNA-1 + RNA-2 were infectious and this infectivity was not enhanced by the addition of coat protein. Virus particles had a Tf (mid point of extinction when heated) of 63 °C and were readily dissociated by 0.1% SDS. PZSV-T was serologically unrelated to alfalfa mosaic and to 32 isometric viruses including five ilarviruses. Some properties of PZSV resemble those of ilarviruses but others are sufficiently different to suggest that it may not be a member of this virus group.     

Further Studies on Cynara Rhabdovirus

Cynara rhabdovirus (CyRV) was isolated from symptomless artichoke plants in southern Italy using Nicotiana langsdorffii as susceptible host and immune serum to artichoke latent virus to eliminate this virus from inoculum. CyRV can infect several solanaceous species, has thermal inactivation point of 40-45°C, dilution end point between 10^?2 and 10^?5 and longevity in vitro at 4 and 20°C of 4-5 days and 6-24 h, respectively. It was purified and used for preparing antisera with homologous titre varying from 1: 16 to 1: 64. In decoration tests, the virus did not react against antisera to eggplant mottled dwarf virus (EMDV) and its antiserum did not decorate ivy vein clearing virus (IVCV). Ultrastructural aspects of CyRV infection in Datura stramonium and Nicotiana glutinosa were very like those described for the same virus in the past except for presence of longer virions which were often encountered in infected cells during this study.     

Comparison of some apple latent viruses

Apple latent viruses were eliminated from, the tips of apple shoots by exposure to a temperature of 36 °C for various periods. The length of treatment needed to eliminate a particular virus differed from plant to plant, but viruses were always inactivated in the same order: first chlorotic leaf spot, followed by stem pitting and finally Spy decline. Quince plants developed sooty ring-spot and bark necrosis when inoculated with buds from some heat-treated apple clones infected with Spy decline virus. Only chlorotic leaf spot virus was transmitted to herbaceous hosts by sap extracts from apple leaves, petals and fruits, and returned from herbaceous plants to apple. This virus, isolated from either apple or cherry, caused a dark green mottle on peach leaves.     

Effect of plant architecture and hail nets on temperature of codling moth habitats in apple orchards

Plant architecture of apple trees in commercial orchards was rapidly changed from traditional tall trees to dwarf trees to optimize yield and fruit quality. Additionally, hail nets are widely used to prevent yield loss by hail. These changes are expected to considerably influence the orchard microclimate and thus the developmental rates of pest insects in apple. However, these relationships have not yet been fully elucidated. The present study was conducted over the seasonal cycle to investigate the influence of plant architecture and hail nets on the habitat temperatures of the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), in apple, Malus domestica Borkh. (Rosaceae). Within the canopies, leaf area index (LAI) and global site factor (GSF) were quantified using hemispherical photography. Temperature was analysed for the main habitats of the different codling moth stages, i.e., air within the canopy, bark of tree stems, and apple fruit. In dwarf trees, LAI was lower, leading to a higher GSF than in tall trees. Hail nets did not influence LAI and GSF. Results for dwarf trees compare as follows with those for tall trees: Average air temperatures within the canopy were 0.7 °C higher during daytime, whereas 0.4 °C lower at night. Mean surface temperatures of bark were 0.9 °C higher on sunny and 0.4 °C on overcast days. Mean surface temperatures of apple fruits were 1.8–2.7 °C higher on sunny days, but 0.6 °C cooler on overcast days. The effect of hail nets was confined to a reduction of the air temperature within the canopy by approximately 0.2–0.8 °C. Bark and apple surface temperatures were not significantly affected. Based on the temperature differences in the habitats considered, the calculated development of the codling moth in dwarf trees was on average 3 days faster than in tall trees. The calculations imply a negligible effect of hail nets on codling moth development.     

Properties of a resistance-breaking strain of potato virus X

During indexing of a potato germplasm collection from Bolivia, a strain of potato virus X (PVX), X_HB, which failed to cause local lesions in inoculated leaves of Gomphrena globosa was found in 7% of the clones. X_HB was transmitted by inoculation of sap to 56 species from 11 families out of 64 species from 12 families tested. It was best propagated in Nicotiana glutinosa or N. debneyi; Montia perfolia and Petunia hybrida were useful as local lesion hosts. Inoculated leaves of G. globosa plants kept at 10°, 14°, 18°, 22°, or 26 °C after inoculation were always infected symptomlessly. X_HB caused a mild mosaic, systemic chlorotic blotching or symptomless infection in 16 wild potato species and eight Andean potato cultivars, systemic necrotic symptoms in clone A6 and cultivar Mi Peru, and bright yellow leaf markings in cultivar Renacimiento. It caused necrotic local lesions in inoculated leaves of British potato cultivars with the PVX hypersensitivity gene Nb but then invaded the plants systemically without causing further necrosis; with gene Nx systemic invasion occurred but no necrotic symptoms developed. These reactions resemble those of PVX strain group four. X_HB differed from other known strains of PVX in readily infecting PVX-immune clones 44/1016/10, G. 4298.69 and USDA 41956, cultivars Saphir and Saco, and Solanum acaule PI 230554. X_HB had slightly flexuous filamentous particles with a normal length of 516 nm. It was transmitted readily by plant contact and it partially protected G. globosa leaves from infection with X_CP, a group two strain of PVX. Sap from infected N. glutinosa was infective after dilution to 10^--⁶ but not 10^--⁷ after 10 min at 75° but not 80 °C and after 1 yr at 20 °C. X_HB was readily purified from infected N. debneyi leaves by precipitation with polyethylene glycol followed by differential centrifugation. Microprecipitin tests showed that X_HB and X_CP are closely related serologically.     

Molecular diagnosis of apple virus and viroid pathogens from India

Apple is known to be susceptible to various virus and viroid pathogens. Symptomatic apple cultivars and rootstocks were collected and analyzed by ELISA and then through RT-PCR. The study reports the presence of Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV), the major apple viruses and Prunus necrotic ringspot virus (PNRSV), a minor apple virus, at the molecular level in India. Apple scar skin viroid (ASSVd) infection was also confirmed at the molecular level. Sporadic incidences of Tomato ringspot virus and Arabis mosaic virus infections were also detected by ELISA in nursery plants.     

Interrelations between respiration and dormancy in buds of three hardwood species with different chilling requirements for dormancy release

 Respiration in vegetative buds of mature Betula pendula, Alnus glutinosa and Prunus padus trees was measured monthly at 15°C from mid-October 1996 to natural outdoor budburst in April 1997. In B. pendula the effect of bud water content on respiration was also estimated (December–April) by artificial imbibition of buds for 24 h prior to measurement of respiration. For estimation of corresponding bud dormancy status, batches of twigs were forced at identical monthly intervals at 15°C in long days (24 h), and budburst recorded. In all species dormancy was deepest when the leaves were shed in October, and dormancy was first alleviated in P. padus followed by B. pendula and A. glutinosa. However, bud respiration capacity was not related to dormancy release as it decreased in all species from October to November and displayed no notable increase until February in P. padus, March in B. pendula and April in A. glutinosa, after completion of dormancy release. Rather, increase in respiration coincided with growth resumption prior to budburst. Artificial imbibition of B. pendula buds increased the water content by approximately 10% (FW) and induced a doubling of the respiration rate (December–February). Moreover, the seasonal variation in bud water content (October–April) explained 94% of the variation in respiration in B. pendula and P. padus, and 84% in A. glutinosa. These observations suggest an important role of water content for respiration. During a cold period from mid-December to mid-January with mean temperature of –9.7°C dormancy release was arrested in P. padus, and to some degree in A. glutinosa, whereas dormancy release progressed normally in B. pendula. This indicates species differences in lower critical temperatures for dormancy release. Received: 30 June 1997 / Acceped: 1 October 1997     

Cytopathology of Apple stem pitting virus in Nicotiana occidentalis L.

Apple stem pitting virus (ASPV) is a causal agent of stem pitting associated disease in pomes fruit trees. The present report focuses on a cytopathological effect of ASPV infection in a herbaceous host Nicotiana occidentalis ‘37B’. A leaf dip preparation shows predominantly basic virus particles and aggregated particles 800 nm and 3200 nm long respectively. The main cytopathological effect observed in ASPV infected N. occidentalis includes fibrous aggregates of virus particles (massive/or few), formation of membranous vesicles and proliferation of the endoplasmic reticulum.     

Phloem Unloading in Stem Parts Parasitized by Cuscuta: The Release of 14C and K+ to the Free Space at 0°C and 25°C

By washing out ¹⁴C-solutes or K⁺ from the free space of stem segments of Vicia faba L. and Urtica dioica L., efflux from stem tissues was measured. At 25°C the efflux of ¹⁴C or K⁺ from non-parasitized stem parts was found to be comparable with the pattern known for parenchyma cells. The efflux pattern from parasitized stem parts kept at 0°C was also a normal one, but at 25°C a more complicated pattern was found. After addition of metabolic inhibitors, however, the efflux pattern appeared to be comparable with the pattern found in 0°C experiments. Also, after washing stem segments for several hours in water at 0°C, Phloem unloading was shown to start unimpeded after transfer to 25°C. Stem parts parasitized by Cuscuta can be regarded as a model system for studying the fundamental processes underlying movement out of sieve tubes in strong sink regions.     

Purification and properties of sweet potato mild mottle, a white-fly borne virus from sweet potato(Ipomoea batatas) in East Africa

A virus obtained from sweet potatoes in Kenya, Uganda and Tanzania was transmitted by inoculation of sap and by whiteflies (Bemisia tabaci). It infected forty-five of 119 plant species in fourteen of thirty-six plant families. It was propagated in Nicotiana glutinosa and N. tabacum, in which diagnostic symptoms of vein clearing, leaf curling and distortion developed. Cheno-podium quinoa was a good local lesion host. Different seedling lines of sweet potato differed greatly in their susceptibility to infection and in symptoms produced; some developed leaf mottling and were stunted, some were symptomless, and some appeared immune. The virus was transmitted by dodder (Cuscuta campestris) but not by aphids, or through seed of Ipomoea nil or N. clevelandii. Sweet potato sap contained strong inhibitors of infection, and a low concentration of virus. Virus-free cuttings of sweet potato were obtained by thermotherapy (4–5 wk at 35 °C), or by meristem-tip culture. The virus remained infective in sap of N. tabacum after dilution to 10-³, or after 10 min at 55 °C (but not 60 °C), 3 but not 7 days at 18 °C, or 42 but not 49 days at 2 °C. Infectivity was abolished by sonication or u.v. irradiation, by 2% formaldehyde or 2% tri-sodium orthophosphate, and was greatly decreased by 20 % CHC1₃ or 20 % ether. Purified virus preparations were obtained from N. tabacum by clarifying phosphate buffer extracts with n-butanol, virus precipitation with polyethylene glycol, and differential centrifugation. The virus sedimented as one band in density gradients, and produced a single sedimenting boundary in analytical centrifugation (s°20, w = 1555)- It contained one polypeptide species of mol wt 37700, and preliminary digestion experiments suggested a single-stranded RNA. Antisera prepared against the virus reacted specifically in precipitin tube tests with titres of 1/16384, but no serological relationships could be found between the virus and fourteen viruses of the potato virus Y group. Electron micrographs showed straight, filamentous particles c. 950 nm long when mounted in MgCl_a, but 800–900 nra long in EDTA. The present cryptogram is: (R/i):*/*:E/E:S/Al. This virus is probably the same as Sheffield's virus B.     

Low soil temperatures induce water deficits in olive (Olea europaea) trees

Olive trees are often subjected to low temperatures during winter. To quantify the effects of low temperatures on the water relations of olive trees, we studied the responses to low soil temperatures on winter days of variable evaporative demand (ET₀) in 1-year-old potted olive (Oleo europaea L. cv. Picual) trees in 1996 and 1997. Low night (2.5 and 5.2°C) but ambient day soil temperatures (above 10°C) did not affect stomatal conductance (g_s), leaf (Ψ_leaf) and stem (Ψ_stem) water potentials. Soil temperature levels inducing water stress in olive trees were determined for winter days with ET₀ typical for southern Spain (ET₀= 1.5 ± 0.3 mm day^?1). Leaf and stem water potential decreased and root hydraulic resistance (r_root) increased when trees were exposed to night and day soil temperatures below 10°C. Stomatal conductance was not affected at soil temperatures between 6.4 and 10°C, but decreased at temperatures below 6.4°C. The soil temperature levels affecting the water uptake of olive trees remained relatively constant over the range of ET₀ of 1-2 mm day^?1 during winter and early spring months. However, the soil temperature influencing gs appeared to be more variable and was affected by ET₀. Olive tree recovery from low soil temperature stress depended on stress duration and severity and interacted with ET₀. Recovery of ψ started already during the stress period, probably induced by stomatal closure and high r_root, thus allowing tree rehydration overnight. Root hydraulic resistance contributed the major part of whole-tree hydraulic resistance in response to cold stress, accounting for 76 and 89% at 6.4 and 4.6°C, respectively; which indicates that r_root is the primary control of the water status in olive trees under low temperatures.