Lab Tuesday: Electron Microscopy and Nematode Extraction

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Richard Cain
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Lab Tuesday: Electron Microscopy and Nematode Extraction Quiz for Viruses (pp 75-83) and Observation of Viral Movement in Plants (p.

Finish Koch s postulates (re-examine symptoms) Demonstration of electron microscopy Extract nematodes lab Observe movement of GFP-expressing virus under black

CELL-TO-CELL MOVEMENT Virus Life Cycle 2 Viral genome Genome uncoating, expression and replication Host ribosomes translate (express) viral genome Gene

1 Lab Tuesday: Electron Microscopy and Nematode Extraction Quiz for Viruses (pp 75-83) and Observation of Viral Movement in Plants (p ), and intro sections for Electron Microscopy (pp.21) and Nematode Extraction from Soils (p. 84 week #1). Finish Koch s postulates (re-examine symptoms) Demonstration of electron microscopy Extract nematodes lab Observe movement of GFP-expressing virus under black light Record data on from virus inoculations RNA genome of TMV: ~6,400 nucleotides, three genes, and three major functions RNA REPLICATION RNA ENCAPSIDATION CELL-TO-CELL MOVEMENT Virus Life Cycle 2 Viral genome Genome uncoating, expression and replication Host ribosomes translate (express) viral genome Gene Products: Replicase enzyme makes new copies of virus genome uncoating of virion translation replication Replicase (RNA polymerase) enzyme (gene product) New protein coat subunit (gene product) Cell to cell movement protein (gene product) 1

2 Replicase (RNA polymerase) enzyme Host ribosomes Cell to cell movement protein New protein coat subunits The replication cycle of Tobacco mosaic virus (TMV). TMV enters a wounded plant cell to begin the replication cycle [1]. As the cost protein (CP) molecules are stripped away from the RNA [2], host ribosomes begin to translate the two replicase-associated proteins. The replicase proteins (RP) are used to generate a negative-sense (- sense) RNA template from the virus RNA [3]. This - sense RNA is, in turn, used to generate both full-length positive-sense (+ sense) TMV RNA [4] and the + sense subgenomic RNAs (sgrnas) [5] that are used to express the movement protein (MP) and CP. The + sense TMV RNA is either encapsidated by the CP to form new TMV particles [6] or wrapped with MP [7] to allow it to move to an adjacent cell for another round of replication. Cell-to-cell movement of Tobacco mosaic virus (TMV). In this model, the movement protein (MP) binds to the viral RNA [1]. Host proteins and/or other virus-encoded proteins may be included in the MP-complex [2]. The MPcomplex then moves from cell-to-cell through the plasmodesmata [3]. When the complex is localized to a new cell, the MP (and any host proteins) are presumably released from the TMV RNA [4], allowing for translation of the genomic RNA to express the replicase proteins and to initiate a new round of replication [5], as shown in previous slide. Review of RNA silencing A wayward petunia leads to the discovery of modest little molecules with enormous medical and agricultural promise*. *Potential to eliminate virus diseases from agriculture *Potential to cure human afflictions 2

RNA silencing of African Cassava Mosaic viruses Hi Ken In fact, I'm in Uganda right now, working on the project. We have 100% incidence of cassava mosaic viruses - ACMV and EACMV-UG.

3 RNA silencing of African Cassava Mosaic viruses Hi Ken In fact, I'm in Uganda right now, working on the project. We have 100% incidence of cassava mosaic viruses - ACMV and EACMV-UG. EACMV-UG gives severe symptoms alone - yield loss can be up to 70%. ACMV is not so bad alone, but when the two are together, it is devastating. With control of EACMV-UG only by RNA silencing, we went from 4.0 ton/ha to 31 ton/ha. We had 100% ACMV in the plots, so our silenced cultivar took away the worst virus and the synergy. We should do better when once we have transgenic plants for both viruses. I believe the local adoption rate will be great, but right now we are keeping a low profile since we're evaluating silencing constructs for the two viruses separately in a model cultivar. The final product is a ways off. Principles of Plant Disease Control Review Disease management strategies revolve around three manageable factors: reduce the amount or efficacy of initial inoculum x o or amount of initial disease y o -- sanitation reduce the rate of disease development r change the time frame t Disease severity time Principles of Disease Control Exclusion: preventing the entrance of inoculum or establishment of a pathogen within an uninvaded area x o Pathogen Eradication: reducing, eliminating or destroying inoculum at the source. x o Therapy: curing diseased plants by use of chemicals (drugs), surgery or an altered environment (temperature, drainage). x o Host Host resistance: reducing efficiency of inoculum via host genetics x o, r, t Environment Protection: reducing the efficiency of inoculum r by interposing a barrier between host and the pathogen Avoidance: Avoiding disease by altering the environment r, t 3

4 Principles: Methods: Exclusion: today certification, quarantine Eradication: Fri wk 10 rotation, pasteurization, fumigation Therapy: today surgery, drug treatment, heat therapy Host resistance: R-genes, polygenic, pathogen-derived Protection: Wed wk 10 chemical paints, biological agents Avoidance: Wed wk 9 planting site, date, depth and maturity irrigation method, drainage Exclusion of pathogens Purpose: to prevent the introduction of a pathogen(s) into an area where susceptible plants will be grown (this area can be as small as a greenhouse or as large as a country) Goal is to produce susceptible plants in a pathogen-free environment Pathogens distributed with propagative parts are vulnerable to control by exclusionary procedures Methods to achieve pathogen exclusion legally enforced quarantines seed certification Distribution of pathogen-free propagation stock 4

Quarantine Governmental action to prevent spread of disease Quarantine legislative control of the propagation, culture or transport of plants or plant parts to

in Pennsylvania, Citrus canker in Florida Plant Quarantine Act of 1912 regulates importation of plants from foreign countries as well as trade of commodities (and

Citrus canker quarantine and eradication effort in southern Florida - 2005 Citrus canker on grapefruit Orange = diseased tree was found Quarantine area within county

5 Quarantine Governmental action to prevent spread of disease Quarantine legislative control of the propagation, culture or transport of plants or plant parts to prevent the spread of pests or pathogens Areas where alternate hosts susceptible to wheat stem rust were restricted Other examples: Sudden Oak Death in PNW, Plum Pox in Pennsylvania, Citrus canker in Florida Plant Quarantine Act of 1912 regulates importation of plants from foreign countries as well as trade of commodities (and consequently, trade disputes can result). Inter-state transport can be regulated similarly. Citrus canker quarantine and eradication effort in southern Florida Citrus canker on grapefruit Orange = diseased tree was found Quarantine area within county These areas are under quarantine. No citrus trees, fruits or plant parts may be moved from the quarantine areas. No citrus trees may be planted in the quarantine area. No potted citrus may be kept in a quarantine area unless in a registered nursery. 5

6 Citrus Canker: Taking action within a quarantined, residential area Questions about Quarantine? Disease Certification of Propagation Stocks and Exported Plant Material Governmental assurance of quality Seed and Propagation Stocks Certification tag on the stock (states) Exported produce Phytosanitary certificate with shipment (feds: USDA APHIS) 6

Disease Certification -- purpose is to ensure low disease Certification programs exist for nursery stock, ornamentals, seed potatoes, vegetable seeds, grass and cereal seeds, fruit trees, berries,

7 Disease Certification -- purpose is to ensure low disease Certification programs exist for nursery stock, ornamentals, seed potatoes, vegetable seeds, grass and cereal seeds, fruit trees, berries, mint, etc. Inspectors visually certify propagation fields and shipped plant parts. They do this by looking for symptoms of target diseases. For every crop that is certified, there is a list of diseases (also weed seeds) and the amount of each tolerated. The inspection reports are public records and are available to the buyer.. Maximum tolerances for diseases in seed potatoes Disease Tolerance (Gen III) mosaic 0.1% potato leaf roll 0.05% ring rot 0.0% Zero tolerance blackleg 0.1% Questions about Certification? Pathogen-free or pathogen-tested seed and propagation materials purpose is to ensure little to no pathogen resides in propagation stock Note: this is a more stringent goal than is usually the case with a disease certification program typically, pathogen-testing requires lab testing by government agency or increasingly, by a private company 7

Pathogen-free or pathogen-tested seed and propagation materials Often goes hand-in-hand with disease certification but may involve testing planting stock for the presence of pathogen(s) Obtaining

8 Pathogen-free or pathogen-tested seed and propagation materials Often goes hand-in-hand with disease certification but may involve testing planting stock for the presence of pathogen(s) Obtaining pathogen-free seed: Grow seed crop in area isolated from the pathogen or area where the climate is not suitable for the disease (dry climate) examples: bean seed in Treasure Valley, ID Obtaining pathogen-free clonal plant material: Use micro-propagation to cure infected plants and to (at least initially) mass produce pathogen-free plants examples: fruit crops, potatoes European Plant Protection Organization Programs in pathogen tested planting stock PM 4/1(1) Virus-free or virus-tested fruit trees and rootstocks PM 4/2(1) Pathogen-tested material of carnation PM 4/3(2) Pathogen-tested material of pelargonium PM 4/4(1) Pathogen-tested material of lily PM 4/5(1) Pathogen-tested material of narcissus PM 4/6(1) Pathogen-tested material of chrysanthemum PM 4/7(1) Nursery requirements - recommended requirements for participation in certification of fruit or ornamental crops PM 4/8(1) Pathogen-tested material of grapevine varieties and rootstocks PM 4/9(1) Pathogen-tested material of Ribes PM 4/10(1) Pathogen-tested material of Rubus PM 4/11(1) Pathogen-tested material of strawberry PM 4/12(1) Pathogen-tested citrus trees and rootstocks PM 4/16(1) Pathogen-tested material of hoppm 4/17(1) Pathogen-tested olive trees and rootstocks PM 4/18(1) Pathogen-tested material of Vaccinium spp. PM 4/19(1) Pathogen-tested material of begonia PM 4/20(1) Pathogen-tested material of New Guinea hybrids of impatiens PM 4/21(1) Pathogen-tested material of rose PM 4/26(1 )Pathogen-tested material of petunia Example of a pathogen tested potato seed program ELISA testing Once propagated from a pathogen-tested tissue culture, clonal plant material can exist in the program for up to 8 years. Annual visual inspection 8

9 Questions about Pathogen Tested? Micropropagation Therapy to obtain clean, mother plants of clonally propagated plant materials Typically, systemic, obligate pathogens are targeted e.g. viruses, phytoplasmas Micropropagation Goals: 1) Cure infected plants to obtain pathogen-free clonal stock plants 2) Maintain pathogen-free condition as number of plants is increased Procedures: 1. Thermotherapy (give the plant a fever) hold mother plant at high temperature, C, for 2-6 wk to slow viral spread into growing tips 2. Excise meristematic cells from growing tips and isolate in tissue culture 3. Index (i.e., test) for pathogens to determine if the new regenerated plantlet is pathogen free 4. Increase plantlets in pathogen-free environment, then retest 9

10 Example of micropropagation program 1 This plant may have received thermotherapy Excise meristem Produce callus in culture Regenerate plantlet Samples being prepared for ELISA or PCR 5 Index (test) for pathogens Grow plantlets (test tube babies) Micropropagation Program continued 6 Divide indexed plantlets 7 8 Root increased plantlets Plant in soil-based medium Micropropagation Program continued 9 10 Increased indexed plantlets in pathogen-free and insect-free greenhouse 11 Harvest propagation unit that will go to the field mini-tubers 10

Micropropagation Program continued 12 13 14 Start

potatoes, at this point, the indexed generation could

Annually inspected then winter- tested in Florida

managed fields Potential number of pathogen-free

compared to field-based visual inspection program

11 Micropropagation Program continued Start indexed plants for field production as transplants For potatoes, at this point, the indexed generation could be grown as certifiedseed for several years. Annually inspected then winter- tested in Florida Produce pathogen-tested stock in isolated, intensely managed fields Potential number of pathogen-free plants produced in potato micropropagation program compared to field-based visual inspection program where pathogen status is uncertain Total no. of tubers: 10 7,812,500 Field Tissue culture 11

Specific Certification Standards Micropropagated Virus-indexed Sweetpotato

Specific Certification Standards Micropropagated Virus-indexed Sweetpotato I. Explanation of General Standards as Applied to Sweetpotato A. The General Planting stocks (roots, plants & cuttings) Certification