Infected Raspberry Research Articles

Efficiency of eradication of Raspberry bushy dwarf virus from infected raspberry (Rubus idaeus) by in vitro chemotherapy, thermotherapy and cryotherapy and their combinations

Raspberry bushy dwarf virus (RBDV) is one of the few horizontally transmitted plant viruses and is difficult to eradicate from infected raspberry (Rubus idaeus) by conventional methods because of its ability to infect meristematic cells in the apical dome. This virus is present where ever raspberry is grown and its adverse effects on quality and yield of the crop has necessitated development of methods to eradicate RBDV from infected stock plants. We applied ribavirin treatment (30 mg/L for 2 weeks—chemotherapy), thermotherapy (24 °C for 8 h in dark and 39 °C for 16 h with light for 2 weeks), cryotherapy (droplet vitrification of shoot tips) and their different combinations to assess the efficiency of virus eradication from an infected raspberry genotype. Virus testing was done on plantlets in tissue culture after 3–4 months following plant regeneration and repeated in half the population of virus-free plants in the greenhouse, 1 year after the initial test. Results showed that ribavirin treatment severely affected plant regeneration, resulting in low efficiency of virus eradication (10.6% regenerated plantlets free of RBDV out of all explants treated). When it was combined with thermotherapy, plant regeneration was significantly improved, resulting in higher efficiency of virus eradication (44.4%). When the shoot tips subjected to these two treatments were cryopreserved and regenerated, virus eradication efficiency further improved to 58.5%. The control of this treatment where plant material was not treated in liquid nitrogen produced the highest efficiency of virus eradication (78.8%), partly because of better plant regeneration. Thermotherapy and cryotherapy when applied alone failed to remove the virus from infected material; however, when combined, 48.6% efficiency of virus eradication was recorded. Inclusion of in vitro thermotherapy in combination with chemotherapy or cryotherapy or both is important to improve efficiency of RBDV eradication from infected raspberry. Nevertheless this virus can be eradicated by different approaches, and our results are useful for planning virus eradication programmes based on the efficiency of eradication. It is important to test the virus status after greenhouse acclimation, as virus may be suppressed and not completely eliminated in some treatments at tissue culture stage, as shown in ribavirin treatment in our research. Eradication of Raspberry bushy dwarf virus from infected red raspberry is possible by chemotherapy, but the efficiency can be significantly improved when combined with thermotherapy or cryotherapy, or both.

Behavioral evidence for contextual olfactory-mediated avoidance of the ubiquitous phytopathogen Botrytis cinerea by Drosophila suzukii.

Herbivorous insects may benefit from avoiding the smell produced by phytopathogens infecting plant host tissue if the infected tissue reduces insect fitness. However, in many cases the same species of phytopathogen can also infect host plant tissues that do not directly affect herbivore fitness. Thus, insects may benefit from differentiating between pathogen odors emanating from food and nonfood tissues. This is based on the hypothesis that unnecessarily staying attentive to pathogen odor from nonfood tissue may incur opportunity costs associated with not responding to other important survival functions. In this study adults of Drosophila suzukii Matsumura, an invasive larval frugivore, showed reduced attraction to the odor of raspberry fruit, a food tissue, when infected with Botrytis cinerea Pers., a ubiquitous phytopathogen, in favor of odors of uninfected raspberry fruit. Moreover, D. suzukii oviposited fewer eggs on infected raspberry fruit relative to uninfected raspberry fruit. Larval survival and adult size after eclosion were significantly reduced when reared on B. cinerea-infected raspberry relative to uninfected fruit. Interestingly, when the behavioral choice experiment was repeated using Botrytis-infected vs. -uninfected strawberry leaves, a nonfood tissue, in combination with fresh raspberry fruit, odor from B. cinerea-infected leaves did not reduce D. suzukii attraction to raspberries relative to raspberries with uninfected leaves. These behavioral results illustrate the important role context can play in odor-mediated interactions between insects, plants and microbes. We discuss implications of our findings for developing a repellent that can be useful for the management of D. suzukii.

Elimination of Phytoplasmas in Rubus Mother Plants by Tissue Culture Coupled with Heat Therapy.

Phytoplasmas are plant-pathogenic bacteria that cause a disease in Rubus species which is referred to as Rubus stunt. As phytoplasmas can be spread by vegetative propagation and latency periods of Rubus stunt can be up to one year, the use of pathogen-free Rubus propagation material in plant nurseries is important in order to stop the spread of this disease. Even though heat therapy has been commonly applied against viruses in many plants, its potential for phytoplasma eradication has been much less explored. Here, the efficacy of heat therapy with subsequent tissue culture to eliminate phytoplasmas from infected raspberry and blackberry plants is evaluated. Heat therapy was performed on 25 phytoplasma-infected raspberry and 33 infected blackberry plants, out of which 100 raspberry and 65 blackberry plants were regenerated via subsequent tissue culture. All plants were negative for the presence of phytoplasma DNA by qPCR at the end of cultivation periods of 481 to 565 days for the treated raspberry plants and 231 to 337 days for the treated blackberry plants. These results show the suitability of heat therapy combined with tissue culture as a routine tool to ensure the presence of phytoplasma-free Rubus mother plants in nurseries.

EliminatingRaspberry bushy dwarf virus(RBDV) from infected raspberry tissue cultures with ribavirin

Eliminating<i>Raspberry bushy dwarf virus</i>(RBDV) from infected raspberry tissue cultures with ribavirin

Penetration of pollen tubes with accumulated Raspberry bushy dwarf virus into stigmas is involved in initial infection of maternal tissue and horizontal transmission

Torenia fournieri (Torenia) plants were infected with Raspberry bushy dwarf virus (RBDV) by pollination with RBDV-infected raspberry pollen grains. The infected raspberry pollen grains germinated on Torenia stigmas, and then the pollen tubes penetrated into the stigma, even though the pollen tubes were arrested in the styles. In whole-mount in situ hybridization of germinating infected raspberry pollen grains, RBDV accumulated in the tips of the pollen tubes. Tissue blot hybridization of Torenia plants pollinated with infected raspberry pollen grains revealed that the first virus infection site leading to systemic infection is the stigma. When infected raspberry pollen grains that had lost germination capacity were pollinated on Torenia stigmas, RBDV could not infect the stigmas, and no horizontal transmission occurred. These results indicate that penetration of pollen tubes with accumulated RBDV into stigmas is essential in causing the first viral infection in the stigma to lead to systemic infection.

Changes in phenolic content induced by infection with Didymella applanata and Leptosphaeria coniothyrium, the causal agents of raspberry spur and cane blight

The phenolic profile of healthy and infected raspberry canes was investigated in three raspberry cultivars: Autumn Bliss, Himbo Top and Polka. The content of total phenols and tannins was determined using spectrophotometric methods, whilst individual phenolic acids, flavan‐3‐ols, ellagic acid derivatives and glycosides of quercetin were analysed using HPLC/MS analysis. The content of secondary metabolites varied considerably among the analysed raspberry cultivars. Moreover, Didymella applanata and Leptosphaeria coniothyrium infection significantly altered the metabolism of phenolic compounds. Flavanols represented the greatest share of all identified phenolics in raspberry canes (90%), followed by glycosides of quercetin (6%), derivatives of ellagic acid (3%) and traces of hydroxycinnamic acid derivatives. Spur and cane blight diseases caused an increase of flavan‐3‐ols and tannins but the levels of hydroxycinnamic acid derivatives, conjugates of ellagic acid and quercetin glycosides were significantly reduced. Cultivars Himbo Top and Polka contained higher levels of hydroxycinnamic acid and ellagic acid derivatives in healthy and infected canes compared to cv. Autumn Bliss. Cultivar Polka also contained the highest level of flavanols and tannins. However, despite high levels of flavanols and total phenols measured in cv. Polka, the canes were highly diseased following infection with D. applanata and L. coniothyrium. The results of the study provide evidence that the level of phenolic compounds in the canes could be causally linked to the differences in disease susceptibility.

ALASKAN RIBES L. AND RUBUS L. PLANT SPECIES SURVEYED FOR VIRUSES

Alaska’s domesticated and native Ribes and Rubus genera have virtually gone unchecked for pathogen detections. Cultivated Ribes species are predominantly found in home gardens and landscape areas along highways and in cities. In 2008, while surveying native plants for diseases in North Central Alaska near the town of North Pole, mottled leaves were readily visible on wild raspberries (Rubus strigosus L.) growing in agricultural berms and on recent clearings next to forests. In 2009 and 2010, other sites close to the original sites were observed to have similar symptoms. Initial protein extracts from partially purified preparations revealed a putative coat protein about 30 kDa. Mechanical transmission occurred in Nicotiana benthamiana and Chenopodium quinoa. Virion RNA preparations contained two dominant bands about 5.9 kb and 1.9 kb. Based on these biological parameters and similarities with Raspberry bushy dwarf virus, leaf sap and purified virion from infected raspberry and N. benthamiana were assayed for RBDV by ELISA and western blot. Leaf samples were also tested specifically for RBDV by RT-PCR. Negative results indicated that RBDV was not the causal agent involved in the native raspberry disease. Commercial raspberries growing near infected wild plants did not have symptoms and did not test positive for viruses. Another virus was discovered and detected from domesticated black and red currants (Ribes nigrum L. and R. rubrum L.) in 2008 and 2010 on the Kenai Peninsula, Alaska. Diseased plants usually contained leaves with vein-clearing that developed into large spots. Field collected leaf samples tested positive for vitivirus using a RT-PCR assay adapted for vitivirus detection. Direct sequencing of the ca. 200 bp PCR product resulted in nucleotides that were most similar to species in the genus Vitivirus when analyzed in BLAST. This is the first time that viruses have been detected in currants and raspberries from Alaska and the first report of a vitivirus from Ribes spp.

Partial sequence and RT‐PCR diagnostic test for the plant rhabdovirus Raspberry vein chlorosis virus

A partial (c. 3 kb) nucleotide sequence of the genome of Raspberry vein chlorosis virus (RVCV) has been obtained, confirming that it is a plant rhabdovirus. Sequence data were obtained using two approaches, conventional reverse transcriptase polymerase chain reaction (RT‐PCR) of single‐strand RNA using degenerate primers designed from rhabdovirus L gene sequences, and mass (454) sequencing of randomly‐amplified double‐stranded RNA. Amplification using the degenerate primers of RNA extracted from virus‐infected raspberry plants was unsuccessful, whereas amplification of rhabdovirus sequences from RNA extracts of the RVCV vector aphid, Aphis idaei, was successful. From these sequences, RVCV‐specific primers were designed which could be used to direct amplification of fragments of the viral genome both from infected aphids and from infected raspberry plants. Phylogenetic analysis of available rhabdovirus sequences shows that RVCV is most closely related to Strawberry crinkle virus.

Application of the MaryBlytTM version 4.3 to the prediction of fire blight in raspberry

Fire blight of apple (Malus ×domestica) and pear (Pyrus communis), caused by Erwinia amylovora, is a well-known disease and has been extensively researched. Fire blight of raspberry (Rubus idaeus), caused by a distinct strain of E. amylovora is less well known but appears to be similar in its behavior. The purpose of this study was to determine if the MaryBlytTM version 4.3 forecasting system for predicting fire blight in apples and pears could be applied to the prediction of fire blight in raspberry. Seven years of weather data and field observations from a naturally infected plot of Kentville raspberry selection ‘K81-6’ were entered into the MaryBlyt version 4.3 forecasting system. This system predicted flower infection events and the subsequent appearance of symptoms within 0-3 days of the observed symptoms for 2000-2006. In 4 of the 7 years, the appearance of systemically infected raspberry cane laterals was within ≤3 days of the MaryBlyt version 4.3 prediction for canker blight symptoms in apple when a modified degree-day threshold specifically for raspberry was used. Also, changing the degree-day base temperature from 12.7 °C to 4.0 °C reduced the error of prediction, but the error for three of the years was still unacceptably high. This study also showed that insect-vectored infections of primocanes cannot be successfully modeled by adaptations of the MaryBlyt version 4.3 program. However, a simple degree-day model independent of MaryBlyt was developed to predict flowering.

Homobotcinolide: a biologically active natural homolog of botcinolide from Botrytis cinerea.

A novel natural product exhibiting biological activity was isolated from a strain of Botrytis cinerea that had infected raspberry fruit (Rubus ideaus). Liquid fermentation and bioassay-directed fractionation of the organism yielded a compound with molecular formula C22H38O8 that is trivially named homobotcinolide. It significantly inhibited etiolated wheat coleoptile growth. Greenhouse-grown bean, corn, and tobacco plants were also affected by exogenous application of homobotcinolide, severe chlorosis and necrosis being exhibited in corn. The compound is a polyhydroxylated nonalactone esterified with 4-hydroxy-2-decenoic acid.

Diagnostics of NEPO Viruses in Raspberries

The diseases caused by viruses of the NEPO group constitute a potential threat for the raspberry plantations. The NEPO group is related to the raspberry monocultures and to the isolation of nematodes from the Longidorus and Xiphinema genera from the rhyzosphere of infected raspberry plants. The application of the sensitive ELISA method for virus diagnostics guarantees necessary for the production of planting material free from NEPO viruses.

The effect of cane management techniques on raspberry yellow rust (Phragmidium rubi‐idaei)

SUMMARYThe effects of various cane removal treatments on the epidemiology of Phragmidium rubi‐idaei were studied in four experiments in infected raspberry plantations in Wales and Scotland. In 1981, removal of 15 cm high young canes either by cutting or by spraying dinoseb‐in‐oil reduced infection of a second flush of canes (replacement canes) compared to that of the first flush of canes on untreated plots of cv. Mailing Delight in Wales and of cv. Glen Clova in Scotland. In 1982, removal of 20 cm high young canes reduced infection of cv. Mailing Delight in Wales but in Scotland only removal of 60 cm high canes by cutting reduced infection of replacement canes. Removal of all fruiting canes and old cane stubs reduced rust infection of young canes, but removal of successive flushes of replacement canes did not reduce infection of lateral shoots on the fruiting canes.Dinoseb‐in‐oil applied to the bases of fruiting canes before emergence of young canes had no effect on infection of young canes in Wales or Scotland. The production of basidiospores was inhibited in vitro by dinoseb‐in‐oil at concentrations over 1.0 μg a.i./ml.

Spread of raspberry bushy dwarf virus by pollination, its association with crumbly fruit, and problems of control

SUMMARYRaspberry bushy dwarf virus (RBDV) was transmitted to raspberry seed both through the pollen and through the ovule and it infected plants pollinated with infected pollen. It did not infect plants prevented from flowering, and transmission through pollen seems to be the only method of spread in the field; in the proximity of infectors, most plants became infected during the first two or three flowering seasons. Plants containing RBDV showed no obvious symptoms, but healthy or infected flowers pollinated with infected pollen produced ‘crumbly’ fruit, containing a high proportion of aborted drupelets. RBDV was difficult to eliminate from infected raspberry by heat therapy. Raspberry cultivars that fail to become infected naturally were also immune to infection by grafting. Use of immune cultivars offers the only method of control and, because infected plants may produce crumbly fruit, future cultivars should if possible possess immunity to RBDV.

Some properties of an inhibitor of virus infection from leaves of raspberry.

SUMMARY: Raspberry leaves contain a substance which prevents the infection of plants by viruses when it is mixed with the inoculum. It is lost on dialysis, precipitated by nicotine or added protein, unaffected by boiling or freezing, and is probably a phenolic tanning agent. Extracts from peach or apple leaves contain less of such substances than do those from leaves of plum, cherry, raspberry or strawberry. The degree to which virus infection is inhibited by tanning substances from raspberry or other sources depends on the virus and not on the species of test plant. Raspberry tannin and tannic acid combine irreversibly with some viruses but with others the combination is readily reversed by dilution or increase in pH value. With potato ring necrosis, tobacco mosaic and tobacco necrosis viruses, there seems to be a fixed ratio between the amount of virus and the amount of tannin needed to decrease infectivity by a fixed proportion. The infectivity of extracts made from raspberry leaves containing raspberry ring spot or beet ring spot viruses varied with the concentration of tannin present and the pH value. The amount of virus sedimented by centrifuging such extracts at low speed decreased with increase of pH value. Extracts made with 2-5 % nicotine in water were usually more infective than those made with alumina or phosphate buffer (pH 8), and much more infective than those made with water. Both viruses are precipitated by acetone or ammonium sulphate from extracts of infected raspberry leaves made with nicotine or alumina; beet ring spot virus was detected serologically in such preparations from infected raspberry leaves.

RASPBERRY YELLOW DWARF, A SOIL‐BORNE VIRUS

An apparently undescribed virus, provisionally named raspberry yellow dwarf virus (RYDV), was isolated from naturally infected raspberry, strawberry, blackberry and several weed species by mechanical inoculation of sap to Chenopodium amaranticolor. The severe disease it caused in Malling Exploit raspberry usually occurred patchily in otherwise normal plantations: these patches increased in size from year to year. RYDV was differentiated from raspberry ringspot and tomato black ring viruses by the symptoms produced in C. amaranticolor, tobacco and Petunia hybrida. RYDV lost infectivity when sap was heated for 10 min. at 61° C., diluted 10‐5or kept for 15 days at 18° C. RYDV was precipitated without inactivation by acetone and by ammonium sulphate.Isolates of RYDV from different plants and localities, and of different virulence, were identified by plant‐protection and serological tests. Such tests gave no evidence that RYDV was related to raspberry ringspot, tobacco ringspot, tomato black ring or cucumber mosaic viruses.Raspberry and sugar‐beet plants became systemically infected with RYDV when grown under glass in soil from a field where the disease had occurred in raspberry plants, and where the virus persisted in the soil for 3 years after the raspberry plants were removed. RYDV seems to be widely disseminated in England but recently introduced and rare in eastern Scotland.Like raspberry ringspot and tomato black ring viruses, RYDV causes symptoms of the ringspot type in tobacco, has a wide natural and experimental host range, is soil‐borne and of local importance. Such features seem characteristic of ringspot viruses as a group.