Progress 06/26/13 to 02/13/18
Outputs
Progress Report Objectives (from AD-416): This project applies biotechnology to effectively mitigate the impact of pests and pathogens on priority floral crops and develops systems that can be manipulated to preserve select hardwood tree taxa for future genetic engineering applications. Genetic engineering of floral crops for pest and pathogen resistance contributes to sustainable production. There has been little research done on migratory nematodes such as Pratylenchus, and this project addresses the use of transgenes that may be effective in controlling Pratylenchus. Viruses are always a problem for propagated plants, particularly flower bulb crops that are propagated from the same bulb for many years. This project determines if an RNAi approach is effective for control of Cucumber mosaic virus (CMV), an economically important virus that affects numerous crops. In vitro manipulation of plants through genetic engineering allows for rapid clonal propagation, ploidy level manipulation, and preservation of germplasm and cryopreservation. Hardwood trees have been difficult to manipulate in vitro, and there are few reports of research done with North American hardwood tree taxa. This project will explore the possibilities of regenerating plants from select oak, elm, or maple taxa for future applications by using either embryogenesis or organogenesis and maintaining plants in vitro by micropropagation. Objective 1: Characterize the interaction between Pratylenchus and transgenic Easter lilies transformed with currently available anti- nematode genes for resistance response. (NP301; C1, PS 1B) Objective 2: Identify genes involved in the metabolic activities of Pratylenchus, using an RNAi approach, and determine if they are effective in enhancing resistance to Pratylenchus, using a soybean hairy root system. (NP301; C1, PS1B) Objective 3: Develop and evaluate lilies containing an antiviral gene for resistance to Cucumber mosaic virus. (NP301; C1, PS 1B) Objective 4: Develop a regeneration system from embryogenic callus for priority hardwood tree species in the tree improvement program at the U.S. National Arboretum. (NP301; C3, PS 3B) Approach (from AD-416): Genetic engineering will be used to introduce genes into Easter lily for nematode and cucumber mosaic virus resistance. Genes that will be tested for nematode resistance are two Bt genes and a cystatin genes. Two genes targeting nematode movement will be used in an RNAi approach against the root lesion nematode. Seeds and cuttings from select hardwood tree species will be cultured in vitro and used for micropropagation and induction of callus. Treatments will be used to determine if plants can be regenerated from the callus. This is the final report for the Project 8020-21000-058-00D which ended February 13, 2018. The new NP301 OSQR approved project 8020-21000-068-00D entitled �Biotechnology Applied to Ornamental Plants for Controlling Migratory Nematodes� began February 14, 2018. Progress was made on all four objectives during the five years of this project. Under Objective 1 the mas2 promoter was found to direct gene expression preferentially in roots of lilies making it a useful promoter when engineering lilies for root lesion nematode resistance. Lilies were transformed with the cystatin gene and found to be resistant to root lesion nematodes in vitro. Under Objective 2, a second approach to nematode resistance was to target nematode-specific parasitism genes for silencing. Gene sequences were obtained from root lesion nematodes, and 22 parasitism genes were identified. Eight metabolic genes and 7 parasitism genes of root lesion nematodes were targeted for silencing in hairy roots of soybean. Several of these targeted gene sequences reduced nematode reproduction, showing that they can be used as a new technology for controlling root lesion nematodes. Two parasitism genes have been selected for engineering lilies for nematode resistance. Lilies have also been engineered with a gene that targets Cucumber Mosaic Virus to prevent is replication (Objective 3) . Under Objective 4, regeneration and micropropagation systems were developed for a priority hardwood tree species, Chinese elm, at the U.S. National Arboretum. Micropropagation and regeneration of trees can contribute to the USNA breeding program because they can be used to maintain elite clones, and seeds of elms are not ideal for storing elm genotypes. Accomplishments 01 New technology for root lesion nematode resistance in plants. The root lesion nematode, Pratylenchus penetrans, ranks third among nematodes for the economic loss that it causes to crops. ARS researchers in Beltsville, Maryland, identified a sequence for a parasitism gene specific to nematodes and expressed this gene in soybean roots. The expression of this gene resulted in inhibited reproduction of root lesion nematodes. This technology can be applied to genetic improvement of plants that are susceptible to root lesion nematodes such as potatoes, soybeans, corn, fruits crops, and lilies. The technology can decrease the use of chemicals to control nematodes; because it is nematode-specific, it will not affect non-target organisms such as insects, birds, and humans.
Impacts
(N/A)
Publications
- Vieira, P., Mayer, T., Eves-Van Den Akker, S., Howe, D., Zasada, I., Baum, T., Eisenback, J.D., Kamo, K.K. 2018. Identification of candidate effector genes of Pratylenchus penetrans. Molecular Plant Pathology.
- Lakshman, D.K., Kamo, K.K. 2018. First report of lily root rot caused by Thantephorus cucumeris AG 2-1 in the United States. Plant Disease.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report Objectives (from AD-416): This project applies biotechnology to effectively mitigate the impact of pests and pathogens on priority floral crops and develops systems that can be manipulated to preserve select hardwood tree taxa for future genetic engineering applications. Genetic engineering of floral crops for pest and pathogen resistance contributes to sustainable production. There has been little research done on migratory nematodes such as Pratylenchus, and this project addresses the use of transgenes that may be effective in controlling Pratylenchus. Viruses are always a problem for propagated plants, particularly flower bulb crops that are propagated from the same bulb for many years. This project determines if an RNAi approach is effective for control of Cucumber mosaic virus (CMV), an economically important virus that affects numerous crops. In vitro manipulation of plants through genetic engineering allows for rapid clonal propagation, ploidy level manipulation, and preservation of germplasm and cryopreservation. Hardwood trees have been difficult to manipulate in vitro, and there are few reports of research done with North American hardwood tree taxa. This project will explore the possibilities of regenerating plants from select oak, elm, or maple taxa for future applications by using either embryogenesis or organogenesis and maintaining plants in vitro by micropropagation. Objective 1: Characterize the interaction between Pratylenchus and transgenic Easter lilies transformed with currently available anti- nematode genes for resistance response. (NP301; C1, PS 1B) Objective 2: Identify genes involved in the metabolic activities of Pratylenchus, using an RNAi approach, and determine if they are effective in enhancing resistance to Pratylenchus, using a soybean hairy root system. (NP301; C1, PS1B) Objective 3: Develop and evaluate lilies containing an antiviral gene for resistance to Cucumber mosaic virus. (NP301; C1, PS 1B) Objective 4: Develop a regeneration system from embryogenic callus for priority hardwood tree species in the tree improvement program at the U.S. National Arboretum. (NP301; C3, PS 3B) Approach (from AD-416): Genetic engineering will be used to introduce genes into Easter lily for nematode and cucumber mosaic virus resistance. Genes that will be tested for nematode resistance are two Bt genes and a cystatin genes. Two genes targeting nematode movement will be used in an RNAi approach against the root lesion nematode. Seeds and cuttings from select hardwood tree species will be cultured in vitro and used for micropropagation and induction of callus. Treatments will be used to determine if plants can be regenerated from the callus. Lilies transformed with the rice cystatin gene showed resistance to root lesion nematodes in vitro, and bulbs of the transformed lilies were larger and weighed more than lilies lacking the rice cystatin gene. These results in vitro showed that the rice cystatin may be useful for nematode resistance if lilies with this gene show resistance to a high population of nematodes under field conditions. Successful genetic engineering for nematode resistance requires expression of the transgene in roots where root lesion nematodes feed. Expression of the GUS reporter gene under the control of either the CaMV 35S, mas2, rice RPC1, or rolD promoters was examined because a promoter that expresses at high levels in roots is needed for effective resistance to root lesion nematodes. Levels of GUS expression were highly expressed throughout roots with the CaMV 35S and mas2 promoters indicating the possibility of using the mas2 promoter for expression in roots of lilies. GUS expression was significantly lower with the rolD promoter, and expression was only in the stele of roots with the rice RPC1 promoter, so these two promoters are not useful when engineering lilies for root lesion resistance. In Objective 2, eight metabolic and seven parasitic genes were identified from a transcriptome of the root lesion nematode and used as dsRNA gene constructs to transform soybean hairy roots. Soybean hairy roots showed resistance to nematodes with several of these targeted genes using an RNAi approach. Under Objective 3 we developed lilies with a dsRNA antiviral gene for Cucumber mosaic virus and screened the lilies for resistance to the virus. Expression of the transgene was verified in lilies, and experiments to challenge the lily lines with virus are in progress. Under Objective 4, we found that meta-topolin is better for micropropagation and regeneration of Chinese elm trees from leaves and callus as compared to two other cytokinins tested (thidiazuron and benzyladenine). Both micropropagated and regenerated plants grown in the greenhouse appear phenotypically normal. Accomplishments 01 Improved production of Easter lilies. Easter lilies are a valuable commodity to the floral industry, but are increasingly difficult to grow because of their susceptibility to a variety of pathogens. Many of the pesticides used to control these pathogens have been banned, and others are expected to be removed from the market in the future, so growers are looking for methods to produce quality lily plants with decreased pesticide use. An ARS scientist in Beltsville, Maryland, created pathogen-free Easter lily plants in tissue culture and sent them to the Easter Lily Foundation in Brookings, Oregon, where they were grown for three seasons in the field. The pathogen-free lilies from tissue culture were much more vigorous than the controls. Growers are now planning to put lily bulbs through tissue culture in order to create pathogen-free planting stock to increase yield and quality of lily plants.
Impacts
(N/A)
Publications
- Vieira, P., Lakshman, D.K., Pandey, R., Slovin, J.P., Kamo, K.K. 2017. Symptom development in response to combined infection of in vitro grown Lilium longiflorum with the root lesion nematode Pratylenchus penetrans and soilborne fungi collected from diseased roots of field-grown lilies. Plant Disease. 101:1-8.
- Vieira, P., Kamo, K.K., Eisenback, J.D. 2017. Plant-mediated silencing of the fatty acid- and retinoid-binding Pp-far-1 gene can reduce development of the root lesion nematode, Pratylenchus penetrans. Plant Pathology. 1:11.
- Vieira, P., Mowery, J.D., Kilcrease, J., Eisenback, J.D., Kamo, K.K. 2017. Cytological changes of Easter lily (Lilium longiflorum) upon root lesion nematode (Pratylenchus penetrans) infection. Plant Pathology. 49:1-11.
- Wade, E.H., Kamo, K.K. 2016. Gladiolus diseases. Handbook on Florist Crop Diseases. 2-19.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): This project applies biotechnology to effectively mitigate the impact of pests and pathogens on priority floral crops and develops systems that can be manipulated to preserve select hardwood tree taxa for future genetic engineering applications. Genetic engineering of floral crops for pest and pathogen resistance contributes to sustainable production. There has been little research done on migratory nematodes such as Pratylenchus, and this project addresses the use of transgenes that may be effective in controlling Pratylenchus. Viruses are always a problem for propagated plants, particularly flower bulb crops that are propagated from the same bulb for many years. This project determines if an RNAi approach is effective for control of Cucumber mosaic virus (CMV), an economically important virus that affects numerous crops. In vitro manipulation of plants through genetic engineering allows for rapid clonal propagation, ploidy level manipulation, and preservation of germplasm and cryopreservation. Hardwood trees have been difficult to manipulate in vitro, and there are few reports of research done with North American hardwood tree taxa. This project will explore the possibilities of regenerating plants from select oak, elm, or maple taxa for future applications by using either embryogenesis or organogenesis and maintaining plants in vitro by micropropagation. Objective 1: Characterize the interaction between Pratylenchus and transgenic Easter lilies transformed with currently available anti- nematode genes for resistance response. (NP301; C1, PS 1B) Objective 2: Identify genes involved in the metabolic activities of Pratylenchus, using an RNAi approach, and determine if they are effective in enhancing resistance to Pratylenchus, using a soybean hairy root system. (NP301; C1, PS1B) Objective 3: Develop and evaluate lilies containing an antiviral gene for resistance to Cucumber mosaic virus. (NP301; C1, PS 1B) Objective 4: Develop a regeneration system from embryogenic callus for priority hardwood tree species in the tree improvement program at the U.S. National Arboretum. (NP301; C3, PS 3B) Approach (from AD-416): Genetic engineering will be used to introduce genes into Easter lily for nematode and cucumber mosaic virus resistance. Genes that will be tested for nematode resistance are two Bt genes and a cystatin genes. Two genes targeting nematode movement will be used in an RNAi approach against the root lesion nematode. Seeds and cuttings from select hardwood tree species will be cultured in vitro and used for micropropagation and induction of callus. Treatments will be used to determine if plants can be regenerated from the callus. The level of beta-glucuronidase (GUS) expression was determined for lilies transformed with the uidA gene under control of either the CaMV 35S, mas2, or rolD promoters. Levels of GUS expression were comparable for roots with either the CaMV 35S or mas2 promoters, and levels were generally very low for roots with the rolD promoter. GUS expression was high in leaves with the CaMV 35S promoter and low with the mas2 and rolD promoters. Hairy roots of soybean have been transformed with eight nematode metabolic genes, seven parasitism-related genes, and currently five putative effector genes. These plants were confirmed to contain the transgenes by PCR, and levels of expression determined by qPCR. Hairy roots with the highest levels of expression are being multiplied for future challenge with the root lesion nematode, P. penetrans. Lilies transformed with dsRNA of the replicase gene have been multiplied and planted in soil for future challenge. Expression of the transgene was determined using qPCR and PCR of the intron. Plants of Ulmus parvifolia, the Chinese elm, that resulted from regeneration or in vitro multiplication have been established in the greenhouse. The acclimatization rate was high (96-100%). Accomplishments 01 Characterization of four promoters for expression in roots of lilies. Successful genetic engineering of lilies for resistance to root lesion nematodes requires a promoter that directs high levels of expression in roots. It is desirable that the promoter direct expression specifically in roots rather than other plant organs. ARS scientists in Beltsville, MD compared expression in roots of lilies using either the CaMV 35S, rolD, rice root3, or mas2 promoters and determined that levels of expression were comparable with the CaMV 35S and mas2 promoters. Expression was low with the rolD promoter. Histological staining showed expression throughout the root with the CaMV 35S, rolD, and mas2 promoters, but expression was limited to the stele with the rice root3 promoter. The CaMV 35S promoter directed high levels of expression in leaves as compared to the low levels from the mas2 and rolD promoter. The mas2 promoter has been identified as a promoter that directs expression of gene expression primarily in roots rather than shoots making it potentially useful for genetic engineering lilies or other crops for resistance to the root lesion nematode that feeds on roots.
Impacts
(N/A)
Publications
- Kamo, K.K., Lakshman, D.K., Pandey, R., Guaragna, M.A., Okubara, P.A., Rajasekaran, K., Cary, J.W., Jordan, R.L. 2015. Resistance to Fusarium oxysporum f. sp. gladioli in transgenic Gladiolus plants expressing either a bacterial chloroperoxidase or fungal chitinase genes. Plant Cell Tissue And Organ Culture. 124:541.
- Vieira, P., Eves-Van Den Akker, S., Verma, R., Wantoch, S., Eisenback, J.D. , Kamo, K.K. 2015. Characterization of the Pratylenchus penetrans transcriptome including data mining of putative nematode genes involved in plant parasitism. PLoS One. 10(12):e0144674. doi: 10.1371/journal.pone. 0144674.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): This project applies biotechnology to effectively mitigate the impact of pests and pathogens on priority floral crops and develops systems that can be manipulated to preserve select hardwood tree taxa for future genetic engineering applications. Genetic engineering of floral crops for pest and pathogen resistance contributes to sustainable production. There has been little research done on migratory nematodes such as Pratylenchus, and this project addresses the use of transgenes that may be effective in controlling Pratylenchus. Viruses are always a problem for propagated plants, particularly flower bulb crops that are propagated from the same bulb for many years. This project determines if an RNAi approach is effective for control of Cucumber mosaic virus (CMV), an economically important virus that affects numerous crops. In vitro manipulation of plants through genetic engineering allows for rapid clonal propagation, ploidy level manipulation, and preservation of germplasm and cryopreservation. Hardwood trees have been difficult to manipulate in vitro, and there are few reports of research done with North American hardwood tree taxa. This project will explore the possibilities of regenerating plants from select oak, elm, or maple taxa for future applications by using either embryogenesis or organogenesis and maintaining plants in vitro by micropropagation. Objective 1: Characterize the interaction between Pratylenchus and transgenic Easter lilies transformed with currently available anti- nematode genes for resistance response. (NP301; C1, PS 1B) Objective 2: Identify genes involved in the metabolic activities of Pratylenchus, using an RNAi approach, and determine if they are effective in enhancing resistance to Pratylenchus, using a soybean hairy root system. (NP301; C1, PS1B) Objective 3: Develop and evaluate lilies containing an antiviral gene for resistance to Cucumber mosaic virus. (NP301; C1, PS 1B) Objective 4: Develop a regeneration system from embryogenic callus for priority hardwood tree species in the tree improvement program at the U.S. National Arboretum. (NP301; C3, PS 3B) Approach (from AD-416): Genetic engineering will be used to introduce genes into Easter lily for nematode and cucumber mosaic virus resistance. Genes that will be tested for nematode resistance are two Bt genes and a cystatin genes. Two genes targeting nematode movement will be used in an RNAi approach against the root lesion nematode. Seeds and cuttings from select hardwood tree species will be cultured in vitro and used for micropropagation and induction of callus. Treatments will be used to determine if plants can be regenerated from the callus. The soybean hairy root system was used to test the effectiveness of two cystatin genes (OCI and OCII) and two Bt genes in conferring resistance to the root lesion nematode (Pratylenchus). These four genes were not as effective as the modified rice cystatin gene that has a deletion. Additional biological replicates have been examined for the transcriptome analysis of Pratylenchus infection of soybean. This provides information about the metabolic pathways and genes that are up- or down-regulated during infection of soybean roots by Pratylenchus. Several Pratylenchus genes were identified and are being subcloned for transformation of soybean hairy roots. An RNAi strategy will be evaluated for its effectiveness in decreasing infection of soybean roots by nematodes. The GUS gene was subcloned under either the rolD, mas2, or rice root3 promoters and used to transform lilies. The three promoters resulted in expression primarily in the roots, although there was some expression in the shoots. These promoters should be useful when transforming lilies with anti-nematode genes because the nematode infects roots. Lilies have been transformed with a dsRNA construct that contains a portion of the replicase gene from a lily isolate of Cucumber mosaic virus with the intent of creating virus-resistant lilies. Currently these plants are being multiplied for future challenge with Cucumber mosaic virus. A system for micropropagation and regeneration of Ulmus parvifolia has been defined. Meta-topolin added to Driver Kuniyaki Walnut medium resulted in regeneration of plants from leaves. Micropropagation required either benzylaminopurine or meta-topolin. Accomplishments 01 Transcriptome analysis of Pratylenchus penetrans, the root lesion nematode. Worldwide crop losses due to plant-parasitic nematodes have been estimated at $118 billion annually, with root lesion nematodes, Pratylenchus spp., ranking third in terms of economic loss. The molecular mechanisms of pathogenicity of root lesion nematodes are poorly understood, and this may be attributed to the limited knowledge of the functional analysis of their genes. ARS researchers in Beltsville, Maryland, have completed an overall analysis of the transcriptome of P. penetrans. A subset of genes, potential effector genes, has been identified and will be the subject for future research. Understanding the interactions between P. penetrans and its host plant at the molecular level will provide a basis for controlling this nematode species.
Impacts
(N/A)
Publications
- Kamo, K.K., Lakshman, D.K., Bauchan, G.R., Rajasekaran, K., Cary, J.W., Jaynes, J. 2015. Expression of a synthetic antimicrobial peptide, D4E1, in Gladiolus plants for resistance to Fusarium oxysporum f. sp. gladioli. Plant Cell Tissue And Organ Culture. 121:459-467.
- Kamo, K.K., Lakshman, D.K., Rathore, K. 2014. Disease resistance: Molecular mechanisms and biotechnological applications. Plant Science. 228:1-2.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report Objectives (from AD-416): This project applies biotechnology to effectively mitigate the impact of pests and pathogens on priority floral crops and develops systems that can be manipulated to preserve select hardwood tree taxa for future genetic engineering applications. Genetic engineering of floral crops for pest and pathogen resistance contributes to sustainable production. There has been little research done on migratory nematodes such as Pratylenchus, and this project addresses the use of transgenes that may be effective in controlling Pratylenchus. Viruses are always a problem for propagated plants, particularly flower bulb crops that are propagated from the same bulb for many years. This project determines if an RNAi approach is effective for control of Cucumber mosaic virus (CMV), an economically important virus that affects numerous crops. In vitro manipulation of plants through genetic engineering allows for rapid clonal propagation, ploidy level manipulation, and preservation of germplasm and cryopreservation. Hardwood trees have been difficult to manipulate in vitro, and there are few reports of research done with North American hardwood tree taxa. This project will explore the possibilities of regenerating plants from select oak, elm, or maple taxa for future applications by using either embryogenesis or organogenesis and maintaining plants in vitro by micropropagation. Objective 1: Characterize the interaction between Pratylenchus and transgenic Easter lilies transformed with currently available anti- nematode genes for resistance response. (NP301; C1, PS 1B) Objective 2: Identify genes involved in the metabolic activities of Pratylenchus, using an RNAi approach, and determine if they are effective in enhancing resistance to Pratylenchus, using a soybean hairy root system. (NP301; C1, PS1B) Objective 3: Develop and evaluate lilies containing an antiviral gene for resistance to Cucumber mosaic virus. (NP301; C1, PS 1B) Objective 4: Develop a regeneration system from embryogenic callus for priority hardwood tree species in the tree improvement program at the U.S. National Arboretum. (NP301; C3, PS 3B) Approach (from AD-416): Genetic engineering will be used to introduce genes into Easter lily for nematode and cucumber mosaic virus resistance. Genes that will be tested for nematode resistance are two Bt genes and a cystatin genes. Two genes targeting nematode movement will be used in an RNAi approach against the root lesion nematode. Seeds and cuttings from select hardwood tree species will be cultured in vitro and used for micropropagation and induction of callus. Treatments will be used to determine if plants can be regenerated from the callus. In FY2014, the project had significant progress in the areas of evaluation of transgene expression and evaluation. Lilies and soybean hairy roots transformed with a cystatin, a gene that confers resistance to nematode feeding, showed a reduction in the number of nematodes. Transgenic lilies also showed an increase in plant weight. This correlation shows that we should be able to use the much faster soybean hairy root system as a model for testing anti-nematode activity of genes before spending the long time needed to transform lilies. Anti-nematode genes that appear to confer resistance in the soybean hairy root system will be used to transform lilies. Following a transcriptome analysis of Pratylenchus (nematode) infection with soybean, several genes have been selected for testing in soybean. These studies could lead to the identification of an effective gene that can be used to confer nematode resistance in lilies. Cucumber mosaic virus (CMV) affects lilies, and a portion of the CMV replicase gene has been subcloned and used to transform lilies. If successful, this gene may result in lilies that are resistant to CMV. Ulmus parvifolia, elm, has been established in vitro and optimal micropropagation parameters are being determined. This will lead to a method to multiply select clones or taxa of these elms for future conservation or distribution. Accomplishments 01 Transgenic nematode resistant lilies created. Lilies are an important floral crop sold as cutflowers, pot plants, or grown in gardens. Attack by the root lesion nematode is a serious problem when growing lilies in the field, as it leads to reduced growth and increased susceptibility to attack by fungal pathogens. There are no commercially important lilies resistant to nematodes that can be used in breeding. ARS researchers in Beltsville, MD have developed lily plants that contain cystatin, an anti-nematode gene. Lily plants with the cystatin gene were challenged with nematodes in culture, and there was a decrease in the number of nematodes. These plants are now being grown under field conditions in the Pacific Northwest where root lesion nematodes are a major problem.
Impacts
(N/A)
Publications
- Kamo, K.K. 2014. Transgene expression of lilies grown in the greenhouse and outdoors. Scientia Horticulturae. 167:158-163.
- Ozel, C.A., Kamo, K.K. 2014. Agrobacterium-mediated transformation of Easter lily (Lilium longiflorum cv. Nellie White). Acta Horticulturae. 1002:231-236.
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