2012 - Washington Red Raspberry Commission

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acutely toxic products. Alternative control strategies are very limited: rotations are not a feasible option in perennial crops, and resistant cultivars, which could be one of the most economic and sustainable control tactics, are not available. Therefore, the urgent development of new control methods is necessary to prevent significant yield losses due to P. penetrans infestations in the future. Current strategies to develop alternative control tactics against P. penetrans in raspberries are aimed at testing new synthetic nematicides and cover crops/green manure. The research proposed here will complement ongoing efforts to control root-lesion nematodes in raspberries as part of an integrated pest management program. Plant-parasitic nematodes release secretions produced in their salivary glands into host plants during infection. These secretions degrade plant cell walls, thereby enabling nematode invasion and interfere with the normal functions of host plant physiology. Plant-parasitic nematodes depend on these secretions for their survival and their ability to establish themselves as parasites, which makes these secretions central to their infection strategy and an attractive target for new control methods. Recent studies have shown that disabling the nematode genes (effector genes) that produce these secretions can result in dramatically increased resistance of the host plant (Huang et al. 2006, Sindhu et al. 2009). This can either be achieved through biotechnology or traditional breeding. Whereas significant progress has been made in identifying and disabling effector genes in cyst and root-knot nematodes, information about similar genes in root-lesion nematodes is very limited. We propose to exploit a weak link in the infection strategy of P. penetrans, namely its dependency on effector genes to develop new control strategies against root-lesion nematodes. In order to use nematode effector genes as new control targets, they need to be identified, which is the goal of this project. No similar research is conducted in Oregon or Idaho. Relationship to WRRC Research Priorities: This project will improve our understanding about root-lesion nematodes and can lead to the development of new control strategies. This project directly addresses two #1 WRRC priorities: i) understanding soil ecology and soil borne pathogens and ii) alternatives to control soil pathogens and nematodes. Objectives: 1) Sequence P. penetrans transcriptome 2) Identify P. penetrans secretion genes and effector gene candidates Procedures: 1) Sequence P. penetrans transcriptome. We will follow a three-step process to find P. penetrans effector genes: i) sequence all P. penetrans genes (i.e., its transcriptome), ii) separate genes that produce secretions from other genes and iii) screen for effector genes among secretion-producing genes. 039
We will collect large quantities of P. penetrans and clean the nematodes from contaminating plant material and other nematode species. Root-lesion nematodes will be stored in an ultra-low freezer at -80 ºC. Once a sufficient amount of nematode material has been obtained, we will isolate mRNA, which represents the message of genes. To create a collection of all P. penetrans genes, we will sequence all genes using high-throughput sequencing technologies. Once sequence data has been obtained, we will process it to remove contaminating sequences from plants and other organisms. The more active a given gene is, the more sequences we will obtain for that particular gene. We will sort all sequence data and generate a profile for all genes that will show us a range from the most to the least active nematode genes. 2) Identify P. penetrans secretion genes and effector gene candidates. To separate P. penetrans genes that produce secretions from other genes, we will search for genes that contain a signal peptide-coding region using SignalP software (Bendtsen et al. 2004). The signal peptide region is a hallmark of genes that produce secretions and is therefore present in all effector genes. Once we have narrowed down the total gene library to those genes that produce secretions, we will screen for the most active effector genes. To this end, we will use a technique, in situ hybridization, which employs a color reaction specific to individual gene sequences in question. By using a microscope, we will be able to determine whether the produced pigment is present in P. penetrans salivary gland cells. This would indicate that the respective gene is active at that location and therefore a potential effector gene (de Boer et al. 1998). We will focus on those effector gene candidates that have the most sequence data and are therefore most active, which will facilitate further screening steps. Simultaneously, we will compare P. penetrans genes with genes in other root-lesion nematode species to find putative effectors. We anticipate that addressing objective 2 will take up the majority of this funding year. Anticipated Benefits and Information Transfer: The identification of P. penetrans effector genes is important, because once their identities are known, effector genes can be used as control targets. Blocking their functions would result in increased P. penetrans resistance. The research proposed here is important and benefits <strong>Washington</strong>’s raspberry growers, because it aids in developing new nematode control methods that reduce the use of costly nematicides and that provide an alternative control tactic as part of an integrated pest management program. Results will be disseminated through presentations at grower meetings and scientific congresses, and through publications in trade magazines and scientific journals. References: Bendtsen, J.D., Nielsen, H., von Heijne, G. and Brunak, S. 2004. Improved prediction of signal peptides: SignalP 3.0. Journal of Molecular Biology 340: 783-795. de Boer, J.M., Yan, Y., Smant, G., Davis, E.L. and Baum, T.J. 1998. In-situ hybridization to messenger RNA in Heterodera glycines. Journal of Nematology 30: 309-312. Gao, B., Allen, R., Maier, T., Davis, E.L., Baum, T.J. and Hussey, R.S. 2003. The parasitome of the phytonematode Heterodera glycines. Molecular Plant-Microbe Interactions 16: 720-726. 040
Page 1 and 2: 2013 Research Proposals and 2012 Re
Page 3 and 4: Table of Contents Primary Investiga
Page 5 and 6: 2012 WASHINGTON RED RASPBERRY COMMI
Page 7 and 8: ut the effect was short lived (Pink
Page 9 and 10: Privѐ, J., J.A. Sullivan, and J.T.
Page 11 and 12: Corbin, A Benedict, C Cogger, C Bar
Page 15 and 16: Budget: Indirect or overhead costs
Page 17 and 18: Justification and Background: In 20
Page 21 and 22: 2012 PROGRESS REPORT Title: Red Ras
Page 23 and 24: than most other varieties but are s
Page 25 and 26: Table 1. Yield and fruit weight of
Page 27 and 28: Table 3. Harvest data for hand-harv
Page 29 and 30: Table 5. Susceptibility (S) and res
Page 31 and 32: identifying new, unbroken sources o
Page 35 and 36: tested rigorously over a period of
Page 37 and 38: Other funding sources: All amounts
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Page 43 and 44: Current & Pending Support NAME (Lis
Page 45 and 46: Appendices Table RY1. Red raspberry
Page 47 and 48: Table RY3. Mean yield and berry siz
Page 49 and 50: Table RY5. Mean yield and berry siz
Page 51 and 52: 8 Table RY8. Ripening season for pr
Page 53 and 54: - Cultivars for the Pacific Northwe
Page 55 and 56: Current & Pending Support Chad Finn
Page 57 and 58: Washington Red Raspberry Commission
Page 59 and 60: Title of project: Support of SCRI P
Page 61 and 62: We will develop markers that will b
Page 63 and 64: the operations amenable to and desi
Page 67 and 68: Current & Pending Support-Tarara In
Page 69 and 70: Table 1. Primocane injury and weed
Page 71 and 72: Importantly, floricane injury and b
Page 75 and 76: density. The roots and shoots are s
Page 77 and 78: Name (List PI #1 first) Moore, P.P.
Page 79 and 80: Publications/Presentations: July 18
Page 81 and 82: Table 2. 2011 and 2012 harvest of 2
Page 83 and 84: PROJECT: 13C-3755-5641 TITLE: Red R
Page 85 and 86: PROPOSED BUDGET: Funds from the Nor
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• Maximum number of plants per se
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Project Title: Fungicide Resistance
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society’s/the environment’s bes
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aged, contact/ingestion residues fo
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Table 3. Clay color weevil topical
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Project No.: New Title: Management
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growers in the Lynden area that hav
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Application Date: November 13, 2012
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particular inoculum levels are asso
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References: Berkeley, G. H. and Jac
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2013 WASHINGTON RED RASPBERRY COMMI
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technical bulletin states “For pe
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Budget: 2013 2014 Salaries $0 $0 Ad
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2012 - Washington Red Raspberry Commission

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