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					                                     ICARDA - LITP 2011/2012
NURSERY INFORMATION


Nursery                                        Faba Bean International Chocolate Spot Nursery - 2012
Abbreviation                                   FBICSN-2012
Number of test entries                         29
Number of checks                               Two:          a) local check - not supplied
                                                             b) ILB 365 (Rebaya 40) susceptible check repeated
                                                              after every 2 test entries
Total number of entries                        31
Total number of plots/replication              46
Number of replicates                           2
Design                                         Randomized Complete Block
Recommended no. of rows/plot                   1
Recommended row length                         3m
Recommended row width                          50 cm (or as recommended for your area)

Total plot area                                1.5 m2
No. of seeds per plot                          15
Seed treatment                                 Fungicide: Vitavax 200
                                               Insecticide: Cygnus 500 EC (a.i. Dichlorophos)
Plot numbers                                   Rep 1: 45101-45146
                                               Rep 2: 45201-45246
IMPORTANT
1) Inoculate arificially, if possible. The recommendation for inoculation are:
        Time of artificial inoculation: 10-12 weeks after planting;
        Spore density: 500,000 spores/ml;
        Spray volume: 20 ml per plant; and
        Age of culture: 12 days.

For details of the inoculation technique please see the procedures for the creation of disease epiphytotics in
faba beans as given in the write up "Procedures for the creation of disease epiphytotics in Faba Beans ".
2) The trial should be planted in an area commonly receiving chocolate spot epiphytotics. Date of planting
should be decided to maximize disease incidence.
3) The packets numbered 45105 and 45212 have been left empty for you to include your susceptible local
cultivar as a check.
4) PLNO in Field Book is for recording plant number per harvested area of each plot.
5) DIS1 and DIS2 in field book are for recording disease scores at flowering and podding stages
                                     ICARDA - LITP 2011/2012
                             PROCEDURES FOR THE CREATION OF DISEASE
                                   EPIPHYTOTICS IN FABA BEANS

                                  CHOCOLATE SPOT (BOTRYTIS FABAE)


PROCEDURE NO. 1:

This procedure is suggested for areas where pathologists and laboratory facilities are available.

1.1. Preparation of faba bean-dextrose-agar (FDA)

1.1.1. Weigh out 200 gr. of faba bean seeds, place in 1.5 L-flask, add 1 L of water, then autoclave at 15 p.s.i.
       for 30 min.
1.1.2. Pass through a sieve, add 18 gr. Agar to the leachates and boil till dissolved.
1.1.3. Add 20 gr. dextrose, stir till dissolved and make up volume to 1 L by adding tap water.
1.1.4. Autoclave at 15 p.s.i. for 20 min., then distribute medium in petri dishes (normally 40 petri dishes to a
       liter).

1.2. Isolation of Botrytis fabae

1.2.1. Collect black sclerotia from a culture of B. fabae kept in the refrigerator from the previous season, or
       collect infected faba bean leaves or stems having the aggressive stage of chocolate spot (dark brown
       blackish lesions) from plants grown during current season.
1.2.2. Surface sterilize sclerotia or infected tissues in 0.5% sodium hypochlorite solution (10% clorox) for 1-2
       min., plate on FDA medium and incubate at room temperature (20-25 oC).
1.2.3. When black sclerotia germinate, or fungal colonies develop from plant pieces (4-6 days later), transfer
       to another FDA plate. This can be done by stabling inoculating needle into sterile medium, touching
       the spores and then streaking onto a plate.
1.2.4. Step 3 should be repeated until pure colonies are obtained.
1.2.5. Incubate at room temperature, for about one week, until spores have formed (exposing plates for
       alternating cycles of 12 hrs. Light, from 40 W fluorescent tube, and 12 hrs. Darkness enhances
       sporulation).

1.3. Propagation of B. fabae

1.3.1. Add 5-10 ml. of sterilized tap water to the surface of pure colonies in petri dishes.
1.3.2. Shake gently in a round motion until spores are released into the water.
1.3.3. Inoculate new plates by transferring this spore suspension from one plate to another. Inoculated
       plates must be shaken gently until their entire surface is covered with spore suspension.
1.3.4. Stack up plates on top of each other in groups, 10 plates each, and keep at room temperature for 3-4
       days (until little mycelial growth is seen on the surface of the plate).
1.3.5. Arrange plates side by side in one layer on a bench, and expose to alternating cycles of 12 hrs light,
       from 40 W fluorescent tube, and 12 hrs darkness.
1.3.6. After 3 days stop light treatment, then use cultures when they are 12 days old.
                                    ICARDA - LITP 2011/2012

1.4. Artificial inoculation

1.4.1. Add 10-15 ml. of distilled water to the surface of 12-day-old cultures, and rub gently employing a small
       brush, until spores are released.
1.4.2. Dilute with distilled water until a spore suspension containing 400,000 to 500,000 spores/ml is
       obtained.
1.4.3. Use this spore suspension to inoculate 10-12 week-old plants in the field after sunset, employing 10-15
       ml. of the spore suspension per plant. A kanap-sack sprayer can be used to apply the inoculum.
1.4.4. Cover inoculated nurseries immediately with polythene sheets, supported by metal frames, to provide
       adequate humidity and a water film on leaf surface. Uncover nurseries, spray with water, and cover
       again every other day to maintain high humidity.
1.4.5. After 6 days of incubation, uncover plants. Disease readings can be recorded 2-3 weeks after
       inoculation. Polythene sheets should be removed on sunny days and placed back after sunset. Under
       continuous rainy humid weather conditions and, as far as leaf surface remains wet, there is no need to
       cover the nursery with polythene sheets.


PROCEDURE NO. 2:

This is a simpler procedure, suggested only for a small scale artificial inoculations in areas where laboratory
facilities are not available. It consists of the following steps:

2.1.   Collect chocolate spot-infected leaves, flower parts, and stems having the aggressive stage.
       (Defoliated infected leaves from the field constitute a good source of inoculum.)
2.2.   Rinse infected plant parts in water, place in pans (moist chambers) then cover with plastic sheets to
       maintain high humidity.
2.3.   Keep at room temperature for 3-5 days until spores have formed.
2.4.   Place sporulating plant parts on top of a sieve and wash vigorously with distilled water, while collecting
       leachates containing spores in a container.
2.5.   Count spores in the leachates if possible, and use for direct artificial inoculation in the field as
       mentioned above.
                              ICARDA - LITP 2011/2012


                                General Information About Trial


Date of planting: ____________________________________________________________

Date of Inoculation: _________________________________________________________

Time of Inoculation: _________________________________________________________

Age of Inoculation: _________________________________________________________

Spore Density: ____________________________________________________________

Spray Volume: ____________________________________________________________

Date of First Symptoms: ____________________________________________________

Most Promising Lines: ______________________________________________________
                                   ICARDA - LITP 2011/2012

            Faba Bean International Chocolate Spot Nursery - 2012

 Entry No. Entry Name            Pedigree                                     Origin   FAO Status*
     1         FLIP11-029        F6/HBP / S0D / 2005 (Asc. x Bot.) /292 - 1   ICARDA       U
     2         FLIP11-030        F6/HBP / S0C / 2005 (Cold x Bot.) /185 - 1   ICARDA       U
     3         FLIP11-031        F6/HBP / S0C / 2005 (Cold x Bot.) /185 - 2   ICARDA       U
     4         FLIP11-032        F6/HBP / S0C / 2005 (Cold x Bot.) /196 - 4   ICARDA       U
     5         FLIP11-033        F6/HBP / S0C / 2005 (Cold x Bot.) /200 - 1   ICARDA       U
     6         FLIP11-034        F6/HBP / S0C / 2005 (Cold x Bot.) /200 - 2   ICARDA       U
     7         FLIP11-035        F6/HBP / S0C / 2005 (Cold x Bot.) /200 - 3   ICARDA       U
     8         FLIP11-036        F6/HBP / S0C / 2005 (Cold x Bot.) /206 - 4   ICARDA       U
     9         FLIP11-037        F6/HBP / S0C / 2005 (Cold x Bot.) /206 - 6   ICARDA       U
     10        FLIP11-038        F6/HBP / S0C / 2005 (Cold x Bot.) /206 - 1   ICARDA       U
     11        FLIP11-039        F6/HBP / S0C / 2005 (Cold x Bot.) /207 - 3   ICARDA       U
     12        FLIP11-040        F6/HBP / S0C / 2005 (Cold x Bot.) /207 - 5   ICARDA       U
     13        FLIP11-041        F6/HBP / S0C / 2005 (Cold x Bot.) /207 - 1   ICARDA       U
     14        FLIP11-042        F6/HBP / S0C / 2005 (Cold x Bot.) /207 - 2   ICARDA       U
     15        FLIP11-043        F6/HBP / S0D / 2005 (Asc. x Bot.) /306 - 3   ICARDA       U
     16        FLIP11-044        F6/HBP / S0D / 2005 (Asc. x Bot.) /319 - 2   ICARDA       U
     17        FLIP11-045        F6/HBP / S0C / 2005 (Cold x Bot.) /208 - 1   ICARDA       U
     18        FLIP11-046        F6/HBP / S0C / 2005 (Cold x Bot.) /210 - 1   ICARDA       U
     19        FLIP11-047        F6/HBP / S0C / 2005 (Cold x Bot.) /212 - 1   ICARDA       U
     20        FLIP11-048        F6/HBP / S0C / 2005 (Cold x Bot.) /212 - 2   ICARDA       U
     21        FLIP11-049        F6/HBP / S0C / 2005 (Cold x Bot.) /212 - 3   ICARDA       U
     22        FLIP11-050        F6/HBP / S0C / 2005 (Cold x Bot.) /212 - 4   ICARDA       U
     23        FLIP11-051        F6/HBP / S0C / 2005 (Cold x Bot.) /213 - 2   ICARDA       U
     24        FLIP11-052        F6/HBP / S0C / 2005 (Cold x Bot.) /214 - 1   ICARDA       U
     25        FLIP11-053        F6/HBP / S0C / 2005 (Cold x Bot.) /217 - 1   ICARDA       U
     26        FLIP11-054        F6/HBP / S0C / 2005 (Cold x Bot.) /259 - 2   ICARDA       U
     27        FLIP11-055        F6/HBP / S0C / 2005 (Cold x Bot.) /261 - 1   ICARDA       U
     28        FLIP11-056        F6/HBP / S0D / 2005 (Asc. x Bot.) /292 - 1   ICARDA       U
     29        FLIP11-057        F6/HBP / S0D / 2005 (Asc. x Bot.) /292 - 2   ICARDA       U
     30        Local check                 -                                  -            -
     31        Rebaya 40
               (Susceptible check)         ILB 365                            Egypt        D
* D = Designated, U = Undesignated
                               ICARDA - LITP 2011/2012
Randomization for Faba Bean International Chocolate Spot Nursery - 2012
Randomized Complete Block
                                 Plot receiving entry
                      Entry    Rep 1                Rep 2
                       1       45114                45223
                       2       45133                45236
                       3       45103                45220
                       4       45132                45209
                       5       45121                45206
                       6       45126                45233
                       7       45109                45229
                       8       45117                45230
                       9       45130                45226
                       10      45129                45244
                       11      45118                45238
                       12      45144                45203
                       13      45106                45232
                       14      45102                45214
                       15      45135                45205
                       16      45112                45235
                       17      45111                45221
                       18      45123                45245
                       19      45139                45202
                       20      45124                45215
                       21      45142                45211
                       22      45108                45241
                       23      45145                45218
                       24      45141                45227
                       25      45120                45224
                       26      45127                45242
                       27      45138                45208
                       28      45136                45239
                       29      45115                45217
                       30      45105                45212
                       31      45101                45201
                       31      45104                45204
                       31      45107                45207
                       31      45110                45210
                       31      45113                45213
                       31      45116                45216
                       31      45119                45219
                       31      45122                45222
                       31      45125                45225
                       31      45128                45228
                       31      45131                45231
                       31      45134                45234
                       31      45137                45237
                       31      45140                45240
                       31      45143                45243
                       31      45146                45246
                            ICARDA - LITP 2011/2012
Trial: FBICSN-2012
Replication: 1
 Plot Entry   DFLR   DMAT   PTHT    YLD    PLNO       DIS1   DIS2   REM
45101 31
45102 14
45103   3
45104 31
45105 30
45106 13
45107 31
45108 22
45109   7
45110 31
45111   17
45112   16
45113   31
45114    1
45115   29
45116   31
45117    8
45118   11
45119   31
45120   25
45121    5
45122   31
45123   18
45124   20
45125   31
45126    6
45127   26
45128   31
45129   10
45130    9
45131   31
45132    4
45133    2
45134   31
45135   15
45136   28
45137   31
45138   27
45139   19
45140   31
45141   24
45142   21
45143   31
45144   12
45145   23
             ICARDA - LITP 2011/2012
45146   31
                               ICARDA - LITP 2011/2012
Trial: FBICSN-2012
Replication: 2
 Plot Entry      DFLR   DMAT   PTHT    YLD    PLNO       DIS1   DIS2   REM
45201 31
45202 19
45203 12
45204 31
45205 15
45206   5
45207 31
45208 27
45209   4
45210 31
45211   21
45212   30
45213   31
45214   14
45215   20
45216   31
45217   29
45218   23
45219   31
45220    3
45221   17
45222   31
45223    1
45224   25
45225   31
45226    9
45227   24
45228   31
45229    7
45230    8
45231   31
45232   13
45233    6
45234   31
45235   16
45236    2
45237   31
45238   11
45239   28
45240   31
45241   22
45242   26
45243   31
45244   10
45245   18
             ICARDA - LITP 2011/2012
45246   31

				
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