Zhibo Hamborg
Forsker
Lenker
ResearchGateSammendrag
Potato (Solanum tuberosum L.) is one of the most important crops grown in Norway, and virus-free plants are required for commercial potato production and for preservation of potato germplasm. The present study evaluates three in vitro therapies – meristem culture, cryotherapy, and chemotherapy combined with thermotherapy – to eliminate viruses from eight historically valuable potato cultivars belonging to the Norwegian potato germplasm. Potato virus Y, potato virus M, potato virus X and potato virus S were present in eight selected old potato cultivars due to long-term conservation in open field. Double-antibody sandwich enzyme-linked immunological assay (DAS-ELISA) and biological indicators were the standard tests used to confirm virus infection in our study. Six virus-free plants from four potato cultivars were obtained after meristem culture, and no virus-free potato cultivars were obtained after cryotherapy. Virus-free frequency for eight different potato cultivars after combining chemotherapy with thermotherapy varied from 36.4% to 100%, with single virus elimination rates of between 74.2% and 92.9%. Chemotherapy compared with thermotherapy was the most effective of the three in vitro therapies used in this study. Highly sensitive small RNA high-throughput sequencing (HTS) was used to evaluate the virus status of potato virus-free materials after virus eradication, and no virus was found, which was consistent with the results of DAS-ELISA and biological indicators. Small RNA HTS has been reported for the first time to evaluate the virus status after virus elimination and to control virus-free potato nuclear stocks.
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Virus diseases have been a great threat to production of economically important crops. In practice, the use of virus-free planting material is an effective strategy to control viral diseases. Cryotherapy, developed based on cryopreservation, is a novel plant biotechnology tool for virus eradication. Comparing to the traditional meristem culture for virus elimination, cryotherapy resulted in high efficiency of pathogen eradication. In general, cryotherapy includes seven major steps: (1) introduction of infected plant materials into in vitro cultures, (2) shoot tip excision, (3) tolerance induction of explants to dehydration and subsequent freezing in liquid nitrogen (LN), (4) a short-time treatment of explants in LN, (5) warming and post-culture for regeneration, (6) re-establishment of regenerated plants in greenhouse conditions, and (7) virus indexing.
Forfattere
Zhibo Hamborg YeonKyeong Lee Carl Jonas Jorge Spetz Jihong Liu Clarke Qiaochun Wang Dag-Ragnar BlystadSammendrag
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Jana Fránová Jaroslava Pribylová Rostislav Zemek Jiunn Luh Tan Zhibo Hamborg Dag-Ragnar Blystad Ondrej Lenz Igor KoloniukSammendrag
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Jana Fránová Jaroslava Pribylová Rostislav Zemek Jiunn Luh Tan Zhibo Hamborg Dag-Ragnar Blystad Ondrej Lenz Igor KoloniukSammendrag
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Igor Koloniuk Jana Fránová Jaroslava Pribylová Tatiana Sarkisova Josef Špak Jiunn Luh Tan Rostislav Zemek Radek Cmejla Martina Rejlová Lucie Valentová Jiri Sedlák Jan Holub Jan Skalik Dag-Ragnar Blystad Bijaya Sapkota Zhibo HamborgSammendrag
Raspberry plants, valued for their fruits, are vulnerable to a range of viruses that adversely affect their yield and quality. Utilizing high-throughput sequencing (HTS), we identified a novel virus, tentatively named raspberry enamovirus 1 (RaEV1), in three distinct raspberry plants. This study provides a comprehensive characterization of RaEV1, focusing on its genomic structure, phylogeny, and possible transmission routes. Analysis of nearly complete genomes from 14 RaEV1 isolates highlighted regions of variance, particularly marked by indel events. The evidence from phylogenetic and sequence analyses supports the classification of RaEV1 as a distinct species within the Enamovirus genus. Among the 289 plant and 168 invertebrate samples analyzed, RaEV1 was detected in 10.4% and 0.4%, respectively. Most detections occurred in plants that were also infected with other common raspberry viruses. The virus was present in both commercial and wild raspberries, indicating the potential of wild plants to act as viral reservoirs. Experiments involving aphids as potential vectors demonstrated their ability to acquire RaEV1 but not to successfully transmit it to plants.
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Tomat er følsom for mange virus og viroider, men det har likevel ikke vært vanlig å finne virus og viroider i norske tomatveksthus de siste ti-årene. Det har nå dukket opp et nytt virus i tomat i Europa som vekker stor bekymring. Det er tomatbrunflekkvirus (tomato brown rugose fruit virus, ToBRFV). Mattilsynet gjennomførte derfor i 2021 og 2022 et kartleggingsprogram for ToBRFV i samarbeid med NIBIO. I 2021, ble det samlet inn 394 prøver (som representerte 3940 individuelle planter) fra i alt 17 gartnerier, hovedsakelig fra Rogaland. Det ble i 2022 samlet inn 150 prøver (som representerte 3000 individuelle planter) fra i alt 7 småplante tomatgartnerier. Det ble ikke påvist ToBRFV i noen av prøvene. Det ble imidlertid funnet ToBRFV i en prøve som ble sendt inn til Planteklinikken fra et mindre gartneri i Vestfold på forsommeren 2021.
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Xiaoyan Ma Junhua Bao Jinwei Li Xi Cheng Muhammad Mobeen Tahir Meizi Liu Xian Lu Min-Rui Wang Zhibo Hamborg Dong ZhangSammendrag
Apple stem grooving virus (ASGV) is one of the most widespread and asymptomatic main viruses, that restricts the production of apples worldwide. Establishment of rapid, simple, and effective early detection methods of apple virus is important. In this study, we established and optimized a one-step reverse transcription - recombinase polymerase amplification (RT-RPA) method, using the target-specific primers of ASGV coat protein gene sequence, and M-MLV reverse transcriptase. This method could be completed within 30 min at 40 °C, followed by a visual detection of the results within 5 min by using lateral flow dipstick (LFD). The specificity results showed that only samples infected with ASGV showed a test line, while no test line appeared in the ASGV-negative samples. In addition, when crude extract of leaves was used, the whole detection could be completed within 1 h, which was shortened by 4 to 6 times compared with the RT-PCR method. The detection made on more field samples showed that the RT-RPA-LFD method is of high stability and reliability for ASGV diagnosis, with a great potential in the rapid on-site detection of plant viruses.
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The estimated global production of raspberry from year 2016 to 2020 averaged 846,515 tons. The most common cultivated Rubus spp. is European red raspberry (Rubus idaeus L. subsp. idaeus). Often cultivated for its high nutritional value, the red raspberry (Rubus idaeus) is susceptible to multiple viruses that lead to yield loss. These viruses are transmitted through different mechanisms, of which one is invertebrate vectors. Aphids and nematodes are known to be vectors of specific raspberry viruses. However, there are still other potential raspberry virus vectors that are not well-studied. This review aimed to provide an overview of studies related to this topic. All the known invertebrates feeding on raspberry were summarized. Eight species of aphids and seven species of plant-parasitic nematodes were the only proven raspberry virus vectors. In addition, the eriophyid mite, Phyllocoptes gracilis, has been suggested as the natural vector of raspberry leaf blotch virus based on the current available evidence. Interactions between vector and non-vector herbivore may promote the spread of raspberry viruses. As a conclusion, there are still multiple aspects of this topic that require further studies to get a better understanding of the interactions among the viral pathogens, invertebrate vectors, and non-vectors in the raspberry agroecosystem. Eventually, this will assist in development of better pest management strategies.
Forfattere
Jiunn Luh Tan Nina Trandem Rostislav Zemek Zhibo Hamborg Bijaya Sapotka Dag-Ragnar Blystad Jana FránováSammendrag
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Plant virus eradication is a prerequisite for the use of virus-free propagules for sustainable crop production. In contrast, virus preservation is required for all types of applied and basic research of viruses. Shoot tip cryopreservation can act as a double-edged strategy, facilitating either virus eradication or virus preservation in cryoderived plants. Here, we tested the efficacies of shoot tip cryopreservation for virus eradication and preservation in shallot (Allium cepa var. aggregatum). In vitro stock shallot shoots infected with onion yellow dwarf virus (OYDV) and shallot latent virus were thermotreated for 0, 2, and 4 weeks at a constant temperature of 36℃ before shoot tip cryopreservation. Results showed that viruses were preserved in recovered shoots when thermotherapy was not applied. Although thermotherapy lowered the regrowth levels of cryotreated shoot tips, the efficiency of virus eradication increased from 5% to 54%. Immunolocalization of OYDV and histological observation of cryotreated shoot tips showed the high frequency of virus preservation was due to the viral invasion of cells close to the apical meristem and the high proportion of cells surviving. Four weeks of thermotherapy drastically decreased the distribution of OYDV, as well as the percentage of surviving cells within the shoot tips, thereby promoting virus eradication. Virus-free plants obtained from combining thermotherapy with cryotherapy showed significantly improved vegetative growth and bulb production. The present study reports how thermotherapy can act as a trigger to facilitate either the safe preservation of Allium viruses or the production of virus-free shallot plants.
Forfattere
Min-Rui Wang Wenlu Bi Mukund R. Shukla Li Ren Zhibo Hamborg Dag-Ragnar Blystad Praveen K. Saxena Qiao-Chun WangSammendrag
Cryopreservation is considered an ideal strategy for the long-term preservation of plant genetic resources. Significant progress was achieved over the past several decades, resulting in the successful cryopreservation of the genetic resources of diverse plant species. Cryopreservation procedures often employ in vitro culture techniques and require the precise control of several steps, such as the excision of explants, preculture, osmo- and cryoprotection, dehydration, freeze-thaw cycle, unloading, and post-culture for the recovery of plants. These processes create a stressful environment and cause reactive oxygen species (ROS)-induced oxidative stress, which is detrimental to the growth and regeneration of tissues and plants from cryopreserved tissues. ROS-induced oxidative stresses were documented to induce (epi)genetic and somatic variations. Therefore, the development of true-to-type regenerants of the source germplasm is of primary concern in the application of plant cryopreservation technology. The present article provides a comprehensive assessment of epigenetic and genetic integrity, metabolic stability, and field performance of cryopreserved plants developed in the past decade. Potential areas and the directions of future research in plant cryopreservation are also proposed.
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Min-Rui Wang Zhibo Hamborg Rune Slimestad Abdelhameed Elameen Dag-Ragnar Blystad Sissel Haugslien Gry Skjeseth Qiao‑Chun WangSammendrag
Shallot (Allium cepa var. aggregatum), a small bulb onion, is widely grown in the world. We previously reported a droplet-vitrification for cryopreservation of in vitro-grown shoot tips of shallot genotype ‘10603’. The present study further evaluated rooting, vegetative growth, bulb production and contents of biochemical compounds, as well as genetic stability in cryo-derived plants. The results showed no significant differences in rooting, vegetative growth, bulb production and contents of soluble sugars and flavonols between the cryo- and in vitro-derived plants. Analyses of ISSR and AFLP markers did not detect any polymorphic bands in the cryo-derived plants. These results indicate rooting and vegetative growth ability, biochemical compounds and genetic stability were maintained in cryo-derived plants. The present study provides experimental evidences that support the use of cryopreservation method for long-term preservation of genetic resources of shallots and other Allium species.
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The present study described a combining thermotherapy with meristem culture for improved eradication of onion yellow dwarf virus (OYDV) and shallot latent virus (SLV) from co‐infected in vitro‐cultured shallot shoots. In vitro‐cultured shoots infected with OYDV and SLV were thermo‐treated at a constant temperature of 36°C for 0, 2 and 4 weeks, and then meristems (0.5 mm) containing 1–2 leaf primordia were excised and cultured for shoot regrowth. Meristem culture without thermotherapy produced much higher levels of survival (100%) and shoot regrowth (55%) than those (62% survival and 32% shoot regrowth) produced by the procedure combining 4 weeks of thermotherapy with meristem culture. However, much higher virus‐free frequencies (70% for OYSV, 80% for SLV and 50% for both viruses) were obtained in the latter than those (10% for OYSV, 15% for SLV and 10% for both viruses) obtained in the former. Histological and subcellular studies showed that thermotherapy imposed stress or damage to the cells of meristems, thus resulting in reduced meristem survival and shoot regrowth. Studies on virus location revealed considerable alternations of virus distribution patterns in the thermo‐treated meristems. The results of histological and subcellular studies and analysis of virus distribution pattern added valuable experimental data in the combining thermotherapy with meristem culture for virus eradication. These data provided explanations as to why combining thermotherapy with meristem culture improved the eradication of OYDV and SLV from the virus‐infected in vitro shallot shoots.
Forfattere
Min-Rui Wang Zhibo Hamborg Sissel Haugslien Astrid Sivertsen Morten Rasmussen Qiaochun Wang Dag-Ragnar BlystadSammendrag
Shallot (Allium cepa var. aggregatum) is an important vegetable crop belonging to the genus Allium. The present study attempted to develop an efficient droplet-vitrification cryopreservation method for shallot ‘10603’ shoot tips. In vitro stock shoots were maintained on Murashige and Skoog (1962) medium (MS) supplemented with 30 g L-1 sucrose, 0.5 mg L-1 BAP, 0.1 mg L-1 NAA and 8 g L-1 agar (pH=5.8). Shoot tips (2.0-3.0 mm in length) were excised from 4-week-old stock shoots and stepwise precultured with increased sucrose concentrations from 0.3 to 0.5 M, each concentration for 1 day. The precultured shoot tips were then loaded for 20 min with a solution composed of 2 M glycerol and 0.5 M sucrose, before exposure to PVS3 for 3 h at room temperature. Dehydrated shoot tips were transferred onto aluminum foils (2×0.8 cm), prior to direct immersion into liquid nitrogen (LN) for cryostorage. For thawing, frozen aluminum foils were moved from LN and immediately transferred into unloading solution composed of liquid MS containing 1.2 M sucrose. After incubation at room temperature for 20 min, shoot tips were post-cultured on solidified MS medium containing 0.3 M sucrose for 2 days and then transferred onto a recovery medium for shoot regrowth. With this procedure, 94% shoot tips survived, and 58% shoot tips regenerated into shoots following cryopreservation.
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Min-Rui Wang Zhibo Hamborg Jiří Zámečník Alois Bilavčík Dag-Ragnar Blystad Sissel Haugslien Qiao-Chun WangSammendrag
The present study described a droplet-vitrification cryopreservation for shoot tips of shallot (Allium cepa var. aggregatum), a small bulb onion. Shoot tips taken from in vitro stock shoots were precultured with 0.3 M and 0.5 M of sucrose, with 1 day for each concentration. Precultured shoot tips were treated with a loading solution containing 2 M glycerol and 0.6 M sucrose for 20 min and then exposed to plant vitrification solution 3 (PVS3) at 24 °C for 3 h of dehydration. Following exposure to PVS3, shoot tips were moved onto 5.0 μl PVS3 droplets on aluminum foil strips, followed by direct immersion into liquid nitrogen for 1 h. Frozen shoot tips were thawed by incubation in liquid MS medium containing 1.2 m sucrose for 20 min at room temperature, and then post-thaw cultured for shoot regrowth. Exposure of the shoot tips to PVS3 produced shoot regrowth (58%). Differential scanning calorimetry (DSC) detected 1.8% of freezable water in the shoot tips that had been dehydrated by PVS2, and no freezable water in those by PVS3 treatment. Exposure to PVS3 provided a broader safe temperature range (− 196 °C to − 88 °C), compared to that (− 196 °C to − 116 °C) of PVS2, for cryopreserved samples. Histological observations found that PVS3 dehydration allowed many cells in the apical dome and in the leaf primordia to survive following freezing in LN, while PVS2 dehydration resulted in much fewer surviving cells in the apical dome. The droplet-vitrification cryopreservation produced 56%, 72% and 32% shoot regrowth in cryopreserved shoot tips taken from in vitro shoots, adventitious buds regenerated from stem discs and field-grown bulbs, respectively. Advantages and disadvantages of the use of different source explants for cryopreservation were discussed. The droplet-vitrification cryopreservation produced 45% and 70% shoot regrowth in the additional two shallot genotypes ‘Kverve’ and ‘Lunteviga’. The results obtained in this study provide technical supports for setting-up cryo-bankings of genetic resources of shallots and other Allium species.
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Potato (Solanum tuberosum L.) is one of the most important crops grown in Norway, and virus-free plants are required for commercial potato production and for preservation of potato germplasm. The present study evaluates three in vitro therapies – meristem culture, cryotherapy, and chemotherapy combined with thermotherapy – to eliminate viruses from eight historically valuable potato cultivars belonging to the Norwegian potato germplasm. Potato virus Y, potato virus M, potato virus X and potato virus S were present in eight selected old potato cultivars due to long-term conservation in open field. Double-antibody sandwich enzyme-linked immunological assay (DAS-ELISA) and biological indicators were the standard tests used to confirm virus infection in our study. Six virus-free plants from four potato cultivars were obtained after meristem culture, and no virus-free potato cultivars were obtained after cryotherapy. Virus-free frequency for eight different potato cultivars after combining chemotherapy with thermotherapy varied from 36.4% to 100%, with single virus elimination rates of between 74.2% and 92.9%. Chemotherapy compared with thermotherapy was the most effective of the three in vitro therapies used in this study. Highly sensitive small RNA high-throughput sequencing (HTS) was used to evaluate the virus status of potato virus-free materials after virus eradication, and no virus was found, which was consistent with the results of DAS-ELISA and biological indicators. Small RNA HTS has been reported for the first time to evaluate the virus status after virus elimination and to control virus-free potato nuclear stocks.
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Zhibo Hamborg Minrui Wang Sissel Haugslien Astrid Sivertsen Morten Rasmussen Qiaochun* Wang Dag-Ragnar BlystadSammendrag
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Virus diseases have been a great threat to production of economically important crops. In practice, the use of virus-free planting material is an effective strategy to control viral diseases. Cryotherapy, developed based on cryopreservation, is a novel plant biotechnology tool for virus eradication. Comparing to the traditional meristem culture for virus elimination, cryotherapy resulted in high efficiency of pathogen eradication. In general, cryotherapy includes seven major steps: (1) introduction of infected plant materials into in vitro cultures, (2) shoot tip excision, (3) tolerance induction of explants to dehydration and subsequent freezing in liquid nitrogen (LN), (4) a short-time treatment of explants in LN, (5) warming and post-culture for regeneration, (6) re-establishment of regenerated plants in greenhouse conditions, and (7) virus indexing.
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Zhibo HamborgSammendrag
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Zhibo HamborgSammendrag
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Viral diseases (a biotic stress) and salinity (an abiotic stress) have been/are the two major constraints for sustainable development of the world’s agricultural production including potato. Crops grown in field are often exposed simultaneously to abiotic and biotic stress, and responses of plants to co-stress by two or more factors may differ from those to each of the multiple stresses. Using in vitro cultures, we demonstrated that virus infection (singly and in combination) or salt, and co-stress by virus infection (singly and in combination) and salt significantly reduced growth and microtuber production, and caused severely oxidative cell damage determined by levels of O2·− and methane dicarboxylic aldehyde, and H2O2 localization in situ. Alterations in physiological metabolism by increasing total soluble sugar and free proline, and by decreasing chlorophyll content are responses of potato plantlets to virus infection (singly and in combination) or salt stress and co-stress by virus infection (singly and in combination) and salt. Oxidative cell damage and reduced chlorophyll content caused by virus and/or salt are believed to be responsible for the reduced growth, eventually resulting in decreased tuber yield. Results reported here would help us to better understand possible mechanism of reduced tuber yield by virus infection and/or salt stress.
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High-throughput sequencing for plant virus diagnosis in Norway
Forfattere
Jing-Wei Li Min-Rui Wang Hai-Yan Chen Lei Zhao Zhen-Hua Cui Zhibo Hamborg Dag-Ragnar Blystad Qiao-Chun WangSammendrag
Availability of and easy access to diverse plant viruses and viroids is a prerequisite in applied and basic studies related to viruses and viroids. Long-term preservation of viruses and viroids is difficult. A protocol was described for long-term preservation of potato leafroll virus (PLRV), potato virus S (PVS), and potato spindle tuber viroid (PSTVd) in cryopreserved shoot tips of potato cv. Zihuabai. Shoot regrowth levels following cryopreservation were higher in 1.5 mm-shoot tips (58–60%) than in 0.5-mm-ones (30–38%). All shoots recovered from 0.5-mm-shoot tips were PVS- and PSTVd-preserved, but none of them were PLRVpreserved. Cryopreservation of 1.5-mm-shoot tips resulted in 35% and 100% of PLRV- and PVS- and PSTVd-preserved shoots. Studies on cell survival patterns and virus localization provided explanations to the varying PLRV-preservation frequencies produced by cryopreservation of the two sizes of shoot tips. Although micropropagation efficiencies were low after 12 weeks of subculture following cryopreservation, similar efficiencies were obtained after 16 weeks of subculture in pathogen-preserved shoots recovered from cryopreservation, compared with the diseased in vitro stock shoots (the control). Pathogen concentrations in the three pathogens-preserved shoots analyzed by qRT-PCR were similar to those in micropropagated shoots. The three pathogens cryopreserved in shoot tips were readily transmitted by grafting and mechanical inoculation to potato plants. PLRV, PVS, and PSTVd represent a diverse range of plant viruses and viroid in terms of taxonomy and infectious ability. Therefore, shoot tip cryopreservation opens a new avenue for long-term preservation of the virus and viroid.
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Zhibo HamborgSammendrag
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Zhibo Hamborg YeonKyeong Lee Carl Jonas Jorge Spetz Jihong Liu Clarke Astrid Sivertsen Gry Skjeseth Sissel Haugslien Qiaochun* Wang Dag-Ragnar BlystadSammendrag
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Zhibo HamborgSammendrag
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Zhibo Hamborg YeonKyeong Lee Astrid Sivertsen Gry Skjeseth Sissel Haugslien Jihong Liu Clarke Qiao-Chun Wang Dag-Ragnar BlystadSammendrag
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Zhibo HamborgSammendrag
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Zhibo Hamborg Gry Skjeseth Abdelhameed Elameen Sissel Haugslien Astrid Sivertsen Jihong Liu Clarke Qiao-Chun Wang Dag-Ragnar BlystadSammendrag
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Zhibo Hamborg YeonKyeong Lee Carl Jonas Jorge Spetz Jihong Liu Clarke Qiaochun Wang Dag-Ragnar BlystadSammendrag
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Bjørnar Bjelland Katrine Loen Thomsen Peter van der Ende Zhibo Hamborg Dag-Ragnar BlystadSammendrag
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Dag-Ragnar Blystad Zhibo Hamborg Kari Ørstad Eva Borowski Sissel Haugslien Tor Munthe Åsmund AsdalSammendrag
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Zhibo Hamborg Sissel Haugslien Jihong Liu Clarke Carl Jonas Jorge Spetz Dag-Ragnar Blystad Qing Wang YeonKyeong Lee Astrid H Sivertsen Gry SkjesethSammendrag
Chrysanthemum stunt viroid (CSVd) was first reported in US in the 1940s and is widespread in the world wherever chrysanthemum is grown. Cryotherapy of shoot tips, a new biotechnology developed in the recent years, is a novel application of plant cryopreservation techniques that allows pathogen eradication at a high frequency. Existing studies have proven that this technique can efficiently eradicate pathogens such as virus, phytoplasma and bacterium. However, up to now, there has been no report on viroid eradication. In the present study, we attempted to establish a droplet vitrification cryotherapy method for Argyranthemum and to apply it to eradicate CSVd. Results obtained so far demonstrated that cryotherapy of shoot tips alone failed to eradicate CSVd from the infected shoot tips of Argyranthemum maderense ‘Yellow Empire’. Using in situ hybridization of CSVd and histological analysis, we found that CSVd can invade meristematic cells and at the same time, these cells were able to survive following cryotherapy. These findings explained why cryotherapy of shoot tips alone could not be efficient enough to eradicate CSVd from the diseased materials. Further studies combining cold treatment with cryotherapy are under investigation for CSVd eradication.
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Biao Wang Jing-Wei Li Zhibo Hamborg Ren-Rui Wang Yan-Li Ma Dag-Ragnar Blystad E.R. Joachim Keller Qiao-Chun WangSammendrag
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Zhibo Hamborg Sissel Haugslien Y Lee Astrid H Sivertsen Gry Skjeseth Jihong Liu Clarke Carl Jonas Jorge Spetz QC Wang Dag-Ragnar BlystadSammendrag
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