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TWV/46/31

ORIGINAL: English

DATE: May 15, 2012

INTERNATIONAL UNION FOR THE PROTECTION OF NEW VARIETIES OF PLANTS

Geneva

Technical working party for VEGETABLES

Forty-Sixth Session
near the city of Venlo, Netherlands, June 11 to 15, 2012

PARTIAL REVISION OF THE TEST GUIDELINES FOR LETTUCE


(DOCUMENT TG/13/10 Rev.)

Document prepared by experts from Japan


1 The Technical Working Party for Vegetables (TWV), at its forty-fifth session, held in Monterey, California, United States of America, from July 25 to 29, 2011 agreed to propose a partial revision of the Test Guidelines for Lettuce concerning resistance to Fusarium and to lettuce big-vein disease.
2 It is proposed to make a partial revision of the Test Guidelines for Lettuce (document TG/13/10 Rev.) in order to include:


  1. Resistance to Fusarium oxysporum f.sp. lactucae Race 1 and 2

  2. Resistance to lettuce big-vein disease (Olpidium brassicae carrying Mirafiori Lettuce Big-Vein Virus(MLBVV) and/or Lettuce Big-Vein associated Virus)

3 Annex I to this document contains the proposed partial revisions of the Test Guidelines for Lettuce.


[Annex I follows]

PROPOSED PARTIAL REVISION OF THE TEST GUIDELINES FOR LETTUCE

(DOCUMENT TG/13/11)




new 42.

(+)


MS/VG

Resistance to Fusarium oxysporum f.sp. lactucae
















42.1

MS/VG

-Race 1
















QL




susceptible










Patriot , Salinas

1







resistant










Costa Rica No.4 Romasol’

9

42.2



MS/VG

-Race 2
















QL




susceptible










Patriot

1







resistant










Banchu red fire

9

43.

(+)


MS/VG

Resistance to lettuce big-vein disease (Olpidium brassicae carrying Mirafiori Lettuce Virus and/or Lettuce Big-Vein Associated Virus)
















QN




susceptible










Cisco

3







moderately resistant










Logic

5







highly resistant










Antore 411

7


Ad 42. Resistance to Fusarium oxysporum f.sp. lactucae (Fol)







* 1. Pathogen

Fusarium oxysporum f.sp. lactucae

2. Quarantine status

EPPO alert list

* 3. Host species

Lactuca sativa L.

* 4. Source of inoculum

NIAS Genebank,

INRAN, Naktuinbouw, GEVES



* 5. Isolate

Fol : 1 and Fol : 2

6. Establishment isolate identity

Use microscope and inoculation to lettuce susceptible standard

7. Establishment pathogenicity

Use lettuce susceptible standard

8. Multiplication inoculum




8.1 Multiplication medium

Inoculation by sowing on contaminated soil: Wheat bran-soil medium.

Inoculation by soaking seedlings: on synthetic liquid medium (e.g. Potatoes Dextrose Broth)



8.2 Multiplication variety




8.3 Plant stage at inoculation

See 10.3

8.4 Inoculation medium




8.5 Inoculation method

See 10.4

8.6 Harvest of inoculum

Inoculation by sowing on contaminated soil: 7-10 day-old culture

Inoculation by soaking seedlings: 15 days



8.7 Check of harvested inoculum




8.8 Shelf life/viability inoculum




9. Format of the test




* 9.1 Number of plants per genotype

20 plants

* 9.2 Number of replicates




* 9.3 Control varieties




Susceptible

Patriot

Resistant to Fol:1

Costa Rica No.4, Romasol

Resistant to Fol:2

Banchu red fire

* 9.4 Test design

Include control varieties

9.5 Test facility

Greenhouse or climate room

9.6 Temperature

20-28 oC

9.7 Light

Under natural day length

9.8 Season




9.9 Special measures




10. Inoculation




10.1 Preparation inoculum

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



10.2 Quantification inoculum

Inoculation by sowing on contaminated soil: soil: culture =20: 1

Inoculation by soaking seedlings: spores are harvested and adjusted to 107 sp/mL



*10.3 Plant stage at inoculation

Inoculation by sowing on contaminated soil: seeds stimulated to emerge

Inoculation by soaking seedlings: cotyledons



*10.4 Inoculation method

Two methods can be use for inoculation :

by sowing seeds to contaminated soil or by soaking seedlings



10.5 First observation

After 7- 10 days from inoculation

10.6 Second observation

14 days from inoculation

*10.7 Final observations

20-25 days after inoculation (sowing or soaking)

11. Observations




*11.1 Method

Visual and/or counting number of plants with symptom

*11.2 Observation scale

Inoculation by sowing on contaminated soil:




Symptoms: stunting, wilting, dead plant




As reference calculate of Disease Severity Index (DSI) and Disease Incidence(DI)




0: healthy




1: slightly stunting, growing reduction




2: severely stunting




3: die




DSI = (0A + 1B + 2C + 3D) / (A + B + C + D)




*A ~ D: number of plants of each category




DI = (0A + 1B + 2C + 3D) *100/ ((A + B + C + D)*3)




Inoculation by soaking seedlings:




Symptoms: growth reduction and brown vessels above cotyledons, dead plant

*11.3 Validation of test

Analysis of results should be calibrated with results of controls

11.4 Off-types




*12. Interpretation of data

Inoculation by sowing on contaminated soil:




Susceptible: severely stunting, wilting, dead plant

(DSI :Relative evaluation to DSI of example variety) (Race1:DI Value is higher than 10%)







Resistant: no stunting, no wilting

(DSI :Relative evaluation to DSI of example variety), (Race1:DI Value is lower than 10%)






Inoculation by soaking seedlings:




susceptible: growth reduction and brown vessels above cotyledons, dead plant




Resistant: no growth reduction and no brown vessels above cotyledons

13. Critical control points:

Avoid rotting seeds

Plant stage at inoculation: inoculation of 30 day old plants can change the results from susceptible to resistant



Race 1 and 2:




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


Only Race 1:

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



Naktuinbouw, Sotaweg 22, 2370 AA Roelofarendsveen. THE NETHERLANDS

Email: info@Naktuinbouw.nl



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















Ad 43. Resistance to lettuce big-vein disease










* 1. Pathogen

Olpidium brassicae carrying Mirafiori Lettuce Big-Vein Virus and/or Lettuce Big-Vein Associated Virus

2. Quarantine status




* 3. Host species

Lactuca sativa L.

* 4. Source of inoculum

`

* 5. Isolate

Non-resistance breaking

6. Establishment isolate identity

Use lettuce susceptible standard

7. Establishment pathogenicity

Use lettuce susceptible standard

8. Multiplication inoculum




8.1 Multiplication medium

Living plant

8.2 Multiplication variety

’Cisco’ or other susceptible varieties

8.3 Plant stage at inoculation

See 10.3

8.4 Inoculation medium

-

8.5 Inoculation method

See 10.4

8.6 Harvest of inoculum

Root systems are collected without soil

8.7 Check of harvested inoculum

Visual check for symptom of leaves or ELISA

8.8 Shelf life/viability inoculum

One day

9. Format of the test




* 9.1 Number of plants per genotype

20 plants

* 9.2 Number of replicates




* 9.3 Control varieties




Susceptible

‘Cisco’

Moderately resistant

‘Logic’

Resistant

‘Antore 411’

* 9.4 Test design

Include control varieties

9.5 Test facility

Greenhouse or climate room

9.6 Temperature

Not over 20 oC

9.7 Light

Natural day length

9.8 Season

-

9.9 Special measures

-

10. Inoculation




10.1 Preparation inoculum

Homogenized one g of roots from plants show severe symptom with 60ml distilled water

10.2 Quantification inoculum

10 ml per plant

*10.3 Plant stage at inoculation

Three to four true leaves are emerged

*10.4 Inoculation method

Root suspensions are poured onto the base of young plants

10.5 First observation

30 – 40 days after inoculation (‘Cisco’ begins to show the symptom)

10.6 Second observation

15 days after first observation

*10.7 Final observations

60 – 70 days after inoculation (30 days after first observation)

11. Observations




*11.1 Method




*11.2 Observation scale

Counting the number of plants with symptom

*11.3 Validation of test

on standards







11.4 Off-types

Resistant varieties may have a few plants with symptom

*12. Interpretation of data




[3] About 50 % of plants show the symptom at second observation. Almost plants show the symptom at the end of test.

[5] Few plants show the symptom at second observation. About 50 % of plants show the symptom at the end of test.

[7] Almost plants show no or slightly mild symptom until the end of test.

Transition of rate of plants with severe symptom is shown in the glaph.



13. Critical control points:

Avoid rotting roots; high temperature causes masking of symptom.

Literature references




・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)



[End of Annex and of document]





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