3 Annex I to this document contains the proposed partial revisions of the Test Guidelines for Lettuce.
Ad 42. Resistance to Fusarium oxysporum f.sp. lactucae (Fol)
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* 1. Pathogen
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Fusarium oxysporum f.sp. lactucae
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2. Quarantine status
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EPPO alert list
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* 3. Host species
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Lactuca sativa L.
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* 4. Source of inoculum
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NIAS Genebank,
INRAN, Naktuinbouw, GEVES
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* 5. Isolate
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Fol : 1 and Fol : 2
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6. Establishment isolate identity
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Use microscope and inoculation to lettuce susceptible standard
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7. Establishment pathogenicity
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Use lettuce susceptible standard
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8. Multiplication inoculum
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8.1 Multiplication medium
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Inoculation by sowing on contaminated soil: Wheat bran-soil medium.
Inoculation by soaking seedlings: on synthetic liquid medium (e.g. Potatoes Dextrose Broth)
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8.2 Multiplication variety
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8.3 Plant stage at inoculation
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See 10.3
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8.4 Inoculation medium
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8.5 Inoculation method
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See 10.4
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8.6 Harvest of inoculum
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Inoculation by sowing on contaminated soil: 7-10 day-old culture
Inoculation by soaking seedlings: 15 days
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8.7 Check of harvested inoculum
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8.8 Shelf life/viability inoculum
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9. Format of the test
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* 9.1 Number of plants per genotype
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20 plants
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* 9.2 Number of replicates
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* 9.3 Control varieties
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Susceptible
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Patriot
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Resistant to Fol:1
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Costa Rica No.4, Romasol
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Resistant to Fol:2
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Banchu red fire
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* 9.4 Test design
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Include control varieties
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9.5 Test facility
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Greenhouse or climate room
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9.6 Temperature
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20-28 oC
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9.7 Light
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Under natural day length
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9.8 Season
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9.9 Special measures
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10. Inoculation
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10.1 Preparation inoculum
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Inoculation by sowing on contaminated soil: Wheat bran-soil medium culture are mixed with sterilized soil
Inoculation by soaking seedlings: soaking of roots and of hypocotyls axis for 5 to 15 min in the inoculums suspension and transplantation of inoculated plantlets in soil
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10.2 Quantification inoculum
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Inoculation by sowing on contaminated soil: soil: culture =20: 1
Inoculation by soaking seedlings: spores are harvested and adjusted to 107 sp/mL
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*10.3 Plant stage at inoculation
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Inoculation by sowing on contaminated soil: seeds stimulated to emerge
Inoculation by soaking seedlings: cotyledons
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*10.4 Inoculation method
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Two methods can be use for inoculation :
by sowing seeds to contaminated soil or by soaking seedlings
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10.5 First observation
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After 7- 10 days from inoculation
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10.6 Second observation
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14 days from inoculation
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*10.7 Final observations
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20-25 days after inoculation (sowing or soaking)
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11. Observations
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*11.1 Method
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Visual and/or counting number of plants with symptom
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*11.2 Observation scale
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Inoculation by sowing on contaminated soil:
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Symptoms: stunting, wilting, dead plant
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As reference calculate of Disease Severity Index (DSI) and Disease Incidence(DI)
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0: healthy
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1: slightly stunting, growing reduction
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2: severely stunting
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3: die
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DSI = (0A + 1B + 2C + 3D) / (A + B + C + D)
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*A ~ D: number of plants of each category
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DI = (0A + 1B + 2C + 3D) *100/ ((A + B + C + D)*3)
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Inoculation by soaking seedlings:
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Symptoms: growth reduction and brown vessels above cotyledons, dead plant
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*11.3 Validation of test
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Analysis of results should be calibrated with results of controls
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11.4 Off-types
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*12. Interpretation of data
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Inoculation by sowing on contaminated soil:
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Susceptible: severely stunting, wilting, dead plant
(DSI :Relative evaluation to DSI of example variety) (Race1:DI Value is higher than 10%)
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Resistant: no stunting, no wilting
(DSI :Relative evaluation to DSI of example variety), (Race1:DI Value is lower than 10%)
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Inoculation by soaking seedlings:
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susceptible: growth reduction and brown vessels above cotyledons, dead plant
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Resistant: no growth reduction and no brown vessels above cotyledons
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13. Critical control points:
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Avoid rotting seeds
Plant stage at inoculation: inoculation of 30 day old plants can change the results from susceptible to resistant
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Race 1 and 2:
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NIAS: National Institute of Agrobiological Sciences
2-1-2, Kannondai, Tsukuba, Ibaraki,305-8602, Japan
Tel: +81-29(838)7406, fax: +81-29(838)7408, E-mail: genebank@nias.affrc.go.jp
http://www.gene.affrc.go.jp/about_en.php
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Only Race 1:
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INRAN: National Research Institute for Food and Nutrition
Loc. Corno d’Oro SS 18, km 77.70 – 84091 Battipaglia (SA) ITALY
Tel: +39 0828 309484, fax +39 0828 302382, E-mail: r.bravi@ense.it
http://www.ense.it
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Naktuinbouw, Sotaweg 22, 2370 AA Roelofarendsveen. THE NETHERLANDS
Email: info@Naktuinbouw.nl
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GEVES : Groupe d’Etude et de contrôle des Variétés Et des Semences
Rue Georges Morel, BP 90 024, 49071 Beaucouzé Cedex, France
Valerie.GRIMAULT@geves.fr
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Ad 43. Resistance to lettuce big-vein disease
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* 1. Pathogen
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Olpidium brassicae carrying Mirafiori Lettuce Big-Vein Virus and/or Lettuce Big-Vein Associated Virus
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2. Quarantine status
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* 3. Host species
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Lactuca sativa L.
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* 4. Source of inoculum
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`
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* 5. Isolate
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Non-resistance breaking
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6. Establishment isolate identity
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Use lettuce susceptible standard
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7. Establishment pathogenicity
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Use lettuce susceptible standard
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8. Multiplication inoculum
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8.1 Multiplication medium
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Living plant
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8.2 Multiplication variety
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’Cisco’ or other susceptible varieties
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8.3 Plant stage at inoculation
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See 10.3
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8.4 Inoculation medium
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-
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8.5 Inoculation method
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See 10.4
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8.6 Harvest of inoculum
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Root systems are collected without soil
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8.7 Check of harvested inoculum
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Visual check for symptom of leaves or ELISA
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8.8 Shelf life/viability inoculum
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One day
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9. Format of the test
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* 9.1 Number of plants per genotype
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20 plants
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* 9.2 Number of replicates
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* 9.3 Control varieties
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Susceptible
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‘Cisco’
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Moderately resistant
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‘Logic’
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Resistant
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‘Antore 411’
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* 9.4 Test design
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Include control varieties
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9.5 Test facility
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Greenhouse or climate room
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9.6 Temperature
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Not over 20 oC
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9.7 Light
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Natural day length
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9.8 Season
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-
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9.9 Special measures
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-
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10. Inoculation
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10.1 Preparation inoculum
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Homogenized one g of roots from plants show severe symptom with 60ml distilled water
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10.2 Quantification inoculum
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10 ml per plant
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*10.3 Plant stage at inoculation
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Three to four true leaves are emerged
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*10.4 Inoculation method
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Root suspensions are poured onto the base of young plants
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10.5 First observation
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30 – 40 days after inoculation (‘Cisco’ begins to show the symptom)
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10.6 Second observation
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15 days after first observation
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*10.7 Final observations
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60 – 70 days after inoculation (30 days after first observation)
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11. Observations
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*11.1 Method
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*11.2 Observation scale
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Counting the number of plants with symptom
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*11.3 Validation of test
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on standards
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11.4 Off-types
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Resistant varieties may have a few plants with symptom
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*12. Interpretation of data
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[3] About 50 % of plants show the symptom at second observation. Almost plants show the symptom at the end of test.
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[5] Few plants show the symptom at second observation. About 50 % of plants show the symptom at the end of test.
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[7] Almost plants show no or slightly mild symptom until the end of test.
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Transition of rate of plants with severe symptom is shown in the glaph.
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13. Critical control points:
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Avoid rotting roots; high temperature causes masking of symptom.
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Literature references
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･Y. Kawazu, et. Al. A transgenic Lettuce line with resistance to both Lettuce Big-Vein associated virus and Mirafiori Lettuce Virus (J. Amer. Soc. Hort. Sci. 131(6):760-763.2006
･Shoji KOBAYASHI, Shinji NISHIGUTI, Tetsuji OSIMOTO and Masanobu KATO. Searching for Resistant Varieties to Lettuce Big Vein Disease (Bull. Hyogo Pre. Tech. Cent. Agr. Forest. Fish. (Agricuture)53, 17-23 (2005)
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