Steen Lykke Nielsen

Varroa-Virus Interaction in Collapsing Honey Bee Colonies

Varroa mites and viruses are the currently the high-profile suspects in collapsing bee colonies. Therefore, seasonal variation in varroa load and viruses (Acute-Kashmir-Israeli complex (AKI) and Deformed Wing Virus (DWV)) were monitored in a year-long study. We investigated the viral titres in honey bees and varroa mites from 23 colonies (15 apiaries) under three treatment conditions: Organic acids (11 colonies), pyrethroid (9 colonies) and untreated (3 colonies). Approximately 200 bees were sampled every month from April 2011 to October 2011, and April 2012. The 200 bees were split to 10 subsamples of 20 bees and analysed separately, which allows us to determine the prevalence of virus-infected bees. The treatment efficacy was often low for both treatments. In colonies where varroa treatment reduced the mite load, colonies overwintered successfully, allowing the mites and viruses to be carried over with the bees into the next season. In general, AKI and DWV titres did not show any notable response to the treatment and steadily increased over the season from April to October. In the untreated control group, titres increased most dramatically. Viral copies were correlated to number of varroa mites. Most colonies that collapsed over the winter had significantly higher AKI and DWV titres in October compared to survivors. Only treated colonies survived the winter. We discuss our results in relation to the varroa-virus model developed by Stephen Martin.

Incidence of acute bee paralysis virus, black queen cell virus, chronic bee paralysis virus, deformed wing virus, Kashmir bee virus and sacbrood virus in honey bees (Apis mellifera) in Denmark

Samples of adult honey bees from apiaries with unusually high winter mortality and brood from hives with symptoms of disease were tested for presence of acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Kashmir bee virus (KBV) and sacbrood virus (SBV) by RT-PCR. All six viruses were detected, but the frequencies varied significantly: SBV was detected in 78 apiaries, DWV in 55, ABPV in 11, CBPV in 4, BQCV in 1 and KBV in 1. This is the first record of KBV in Denmark. A large majority of the bee samples were infected with one or more viruses. Single, dual and triple infections were observed. Nucleotide sequences of the PCR products from each virus were determined and found to be 98–99% identical to GenBank accessions except CBPV, which was only 88–90% identical to known CBPV sequences.ZusammenfassungNeuere Untersuchungen zur Verbreitung von Bienenviren unter Verwendung molekulargenetischer Diagnosemethoden wurden in Europa lediglich in Frankreich und Deutschland durchgeführt. Um auch für Dänemark einen detaillierten Überblick zu erhalten, wurden adulte Honigbienen von Bienenständen mit außergewöhnlich hoher Wintermortalität sowie Brutwaben von Bienenvölkern mit Krankheitssymptomen mittels RT-PCR auf folgende Viruserkrankungen hin untersucht: Akute-Bienenparalyse-Virus (ABPV), Schwarze-Königinnenzellen-Virus (BQCV), Kaschmir-Bienen-Virus (KBV), Chronische-Bienenparalyse-Virus (CBPV), Verkrüppelte-Flügel-Virus (DWV) und Sackbrut-Virus (SBV). Die verwendeten Primer und RT-PCR-Verfahren wurden der Literatur entnommen. Alle Proben wurden auf das Vorkommen aller sechs Bienenviren hin analysiert. Um die PCR-Produkte eindeutig zu identifizieren, wurden repräsentative Proben sequenziert und die Sequenzen gegen die GenBank-Datenbank geblasted.Insgesamt wurden 96 Bienenstände auf das Vorkommen der sechs Bienenviren hin untersucht. Alle sechs Viren wurden nachgewiesen, allerdings in sehr unterschiedlicher Häufigkeit. SBV wurde in 78, DWV in 55, ABPV in 11, CBPV in 4 und BQCV sowie KBV in einem Bienenstand festgestellt. Die Mehrzahl der Bienenproben war von mehr als einem Bienenvirus befallen, wobei Einzel-, Doppel- und Tripelbefall vorkamen. Die Nukleotidsequenzen der PCR-Produkte aller Viren waren zu 98–99% identisch mit den GenBank-Sequenzen mit Ausnahme von CBPV, der lediglich eine Übereinstimmung von 88–90% zu den bekannten CBPV-Sequenzen aufwies. Die Häufigkeit der sechs Bienenviren in Dänemark unterscheidet sich erheblich von den Ergebnissen aus anderen europäischen Ländern. Im Gegensatz zu diesen Ländern war SBV am meisten verbreitet, gefolgt von DWV. Außerdem war die Häufigkeit von ABPV, BQCV und CBPV deutlich geringer als sonst beschrieben. KBV wurde erstmals in Dänemark nachgewiesen und erweitert die bisherigen Funde von KBV aus fünf anderen europäischen Ländern.

Seed transmission of Pepino mosaic virus in tomato

In this manuscript we provide evidence for the seed transmission of Pepino mosaic virus (PepMV) in tomato. Fruit was harvested from a tomato crop artificially infected with both European and CH2 genotypes of PepMV and more than 100,000 seeds were extracted and cleaned using an enzymatic treatment without disinfection. Infection assays using indicator plants confirmed the presence of viable virus on the seeds. Seeds were distributed to ten different laboratories in three separate batches, where they were germinated and the young plants tested by ELISA. In total over 87,000 plants were tested and 23 positives detected, indicating an overall transmission rate of 0.026%. However, the observed seed transmission rates varied from 0.005% to 0.057%, depending on the seed batch used. Results clearly showed that PepMV can be transmitted from seeds contaminated with virus to seedlings, highlighting the risk of using seeds from PepMV-infected plants and the potential for seed transmission to contribute to the further spread of PepMV.

Alternative hosts for potato mop-top virus, genus Pomovirus and its vectorSpongospora subterranea f.sp. subterranea

SummarySeventeen weed species common in the potato fields in Denmark were grown in a hydroponic system infested with viruliferous zoospores ofSpongospora subterranea f.sp.subterranea carrying potato mop-top virus (PMTV). The plants were examined for infection with PMTV andS.s.s. using DAS-ELISA and based on visible symptoms. Only two weed species were found to be infected with PMTV,Chenopodium album andSolanum nigrum, whereas 13 became infected withS.s.s. C. album was infected with PMTV by mechanical inoculation were the infection became systemically and the leaves showed necrotic spots.S. nigrum became local infected with PMTV by mechanical inoculation. By inoculation with viruliferousS.s.s. the roots ofS. nigrum became infected but in three weeks PMTV had not spread into the top of the plants.Nicotiana benthamiana was used as a control susceptible toS.s.s. and PMTV.

Development and inter-laboratory evaluation of real-time PCR assays for the detection of pospiviroids.

Assays based on real-time PCR (TaqMan) that can detect a number of viroids in the genus Pospiviroid have been developed and evaluated. The assays are designed for detecting viroids from tomato leaf material but detection from other solanaceous hosts of these viroids has been confirmed. These methods have been validated by nine laboratories and comprise a reliable set of assays for the detection of CEVd, TASVd, CLVd and a generic assay which will detect the six viroids of concern to European tomato growers: PSTVd, TCDVd, CEVd, CLVd, TASVd and CSVd.

Molecular identification of a new begomovirus associated with mosaic disease of Jatropha curcas L. in Nigeria.

We report the complete nucleotide sequence of DNA-A of a begomovirus naturally infecting Jatropha curcas L. in Nigeria. Symptoms observed on infected plants were severe mosaic, mottling and blistering of leaves. The virus, which we provisionally name “jatropha mosaic Nigeria virus” (JMNV), has a monopartite genome of 2,779 to 2,789 nucleotides. Pairwise comparisons of DNA-A sequences showed that JMNV had maximum nucleotide sequence identity (72%) with a strain of tomato yellow leaf curl virus. Since there are widespread infections of jatropha in Nigeria showing similar symptoms as those investigated in the present study, JMNV may represent a significant threat to a promising bioenergy crop.

Effects of sewage sludge stabilization on fertilizer value and greenhouse gas emissions after soil application

Application of sewage sludge on agricultural land becomes more and more common in many parts of the world in order to recycle the nutrients from the sludge. A range of sewage sludge stabilization techniques are available to make the sludge more stable prior to storage, transportation, and application. These stabilization techniques include dewatering, drying, anaerobic digestion, composting, and reed bed sludge treatment. However, very few studies have investigated the effect of these techniques after the sludge has been applied to agricultural land. The objective of the current study was therefore to investigate the effect of sewage sludge stabilization techniques on the C and N mineralization and gaseous emissions from soil. A soil incubation was conducted to determine the rate of C and N mineralization and N2O and CH4 emissions of sewage sludge stabilized using different techniques. Unstabilized sludge released up to 90% of their C content as CO2, part of which could be caused by release of CO2 from carbonates. Compared with this, sludge stabilization including anaerobic digestion and drying resulted in a reduction of the C mineralization rate of about 40%. Liming reduced C mineralization with around 29%, while treatment in a reed bed system reduced it by 74%. The current study thus clearly demonstrated that stabilization techniques resulted in sludge that was more stable once they were applied to agricultural land. Stabilization also reduced the N immobilization phase, potentially improving the value of the sludge as a fertilizer. Emissions of CH4 were also reduced through sludge stabilization and mainly occurred after application of easily degradable sludge types, which is likely to have enhanced the creation of anaerobic microsites. The stabilization processes also decreased emissions of N2O. The results for both CH4 and N2O indicate that the stabilization tends to reduce the chance of developing conditions where these gases could be produced.

Influence of post harvest temperature treatments, storage period and harvest date on development of spraing caused by tobacco rattle virus and potato mop-top virus

SummaryThe frequency of PMTV-spraing and TRV-spraing increased during storage in several cultivars. The only exception was cv. Saturna, where a decrease was observed in 1992. A spraing inducing treatment of one week at 18 °C followed by one week at 8 °C immediately after an early harvest increased the frequency and provided a rough estimate of the development of spraing in untreated tubers during storage. Under most circumstances wound healing at 25 °C compared with 15 °C resulted in a lower frequency of spraing during the storage period. The date of harvest had an important influence on development of spraing during storage and on the effect of post harvest temperature treatments. Therefore, physiological processes that induce or erase spraing are strongly related to tuber maturity. The mechanisms controlling development of symptoms seemingly are identical for the two spraing inducing vira.

Efficacy of mineral oil combined with insecticides for the control of aphid virus vectors to reduce potato virus Y infections in seed potatoes (Solanum tuberosum)

Abstract Aphids are major vectors of plant viruses. Potato virus Y (PVY) is the most important aphid-transmitted virus affecting potato crops in Denmark. Because of a changed seed potato growing strategy, the seed potato area in Denmark is changing from regions with a low average temperature to regions with a higher average temperature. This means that the aphids may infest the potato crops earlier and the population development of the aphids may be faster, and consequently PVY may more easily become epidemic in seed potato crops. With a view to reducing the spread of PVY a 3-year experiment was carried out with a combination of mineral oil and insecticides. In 2005 and 2007 when a very high number of aphids were present, nearly all plants were infected with PVY. In 2006 with a lower number of aphids a smaller proportion of the plants were infected, and a tendency to a lower PVY incidence in mineral-oil treated plots was found, but more than the 8% threshold value. Even in plots where systemic neonicotinoids were applied and very few aphids were recorded, no significant reduction in infestation level of PVY was found. The present experiment shows that mineral oil and insecticides applied to potato crops each week for a 6-week period as protection against aphid transmission of PVY did not significantly reduce the level of PVY infestations in potatoes.

Patterns of viral infection in honey bee queens

The well-being of a colony and replenishment of the workers depends on a healthy queen. Diseases in queens are seldom reported, and our knowledge on viral infection in queens is limited. In this study, 86 honey bee queens were collected from beekeepers in Denmark. All queens were tested separately by two real-time PCRs: one for the presence of deformed wing virus (DWV), and one that would detect sequences of acute bee-paralysis virus, Kashmir bee virus and Israeli acute paralysis virus (AKI complex). Worker bees accompanying the queen were also analysed. The queens could be divided into three groups based on the level of infection in their head, thorax, ovary, intestines and spermatheca. Four queens exhibited egg-laying deficiency, but visually all queens appeared healthy. Viral infection was generally at a low level in terms of AKI copy numbers, with 134/430 tissues (31 %) showing the presence of viral infection ranging from 101 to 105 copies. For DWV, 361/340 tissues (84 %) showed presence of viral infection (DWV copies ranging from 102 to 1012), with 50 tissues showing viral titres >107 copies. For both AKI and DWV, the thorax was the most frequently infected tissue and the ovaries were the least frequently infected. Relative to total mass, the spermatheca showed significantly higher DWV titres than the other tissues. The ovaries had the lowest titre of DWV. No significant differences were found among tissues for AKI. A subsample of 14 queens yielded positive results for the presence of negative-sense RNA strands, thus demonstrating active virus replication in all tissues.