Objective and Rationale:

Determine if the bacterial isolates 5.40 (Pseudomonas putida) and 1.2 (Bacillus subtilis) have efficacy in controlling losses in greenhouse grown strawberries (cv. Pajaro) due to grey mold caused by Botrytis cinerea. Both isolates have shown effectiveness in inhibiting growth of vegetative mycelia of B. cinerea in vitro, and also in inhibiting conidia , germination in vitro. The logical extension of this is to determine of these organisms can be used as a foliar spray for control of grey mold in plantae. The efficacy of these putative control agents will be compared to that of just a foliar application of PDB in order to discern between effects of the biocontrol organisms and any possible, media effects. Efficacy will also be compared to Rovral 50 WP (Ioprodione; Rhone-Polenc), the fungicide of choice for the control of Botrytis fruit mold of strawberry because of the ability of applying Rovral up to one day before harvest. Another treatment will be a negative control block in which the plants do not receive any foliar treatment application.

Methods:

1. Growth of strawberries:

   Strawberry plants (cv. Pajaro) were supplied as rooted crowns by Dr. Beth Crandall (BHN Research, Watsonville, CA). Plants were potted into 6" plastic pots (1 plant/pot) filled with Black Gold 100% organic soil (Black Gold Inc., Hubbard, OR). Plants were watered and fertilized regularly (Peters 20:20:20) and grown under and 18 hour light:6 hour dark cycle.

2. Treatments:

   1. Foliar applications
      
      1. Rovral 50 WP at 1.5 lb/100 gallons water.
      
      
      2. Pseudomonas putida (5x10-8 CFU/ml).
      
      
      3. Bacillus subtilis (5x10-8 CFU/ml).
      
      
      4. PDB (Potato dextrose broth, full-strength, Difco).
      
      
      5. No treatment.

   2. Growth of biological control agents: Single colonies of P. putida and B. subtilis were used to inoculate two, 2 liter flasks containing PDB (1 L/flask). Flasks were placed in a rotary shaker and grown for 48 hours at 28^oC, 225 rpm. Cultures were then assayed for growth concentration by spectrophotometer (A600nm) and adjusted with PDB to realize a final cell density of approximately 5x10-8 cells/ml.
   3. Timing, frequency and intensity of foliar spray applications:
   
   
   1. All treatments 1-4 were applied 5 times during the test #1 - 10% flower bloom. #2-5, at 2 week intervals, the 5th occurring 1 day before the first harvest.
   
   
   2. All treatments were applied by spraying plants with a hand sprayer that contained various treatm6nts. Plants were sprayed to run-off, approximately 75 ml/plant.
   
   

3. Artificial greenhouse inoculation of strawberry plants with B. cinerea:

   1. Intensity: Plants were applied by spraying with s 4x10-3 conidia/ml suspension. Leaves, flowers and fruit were sprayed to run-off with the conidia suspension (approximately 75 ml/plant).
   
   
   2. Frequency: Plants were sprayed three times with a 4x10-3 conidia/ml suspension.
   
   
   3. Timing: 1st spray = day inflorescence is observed
      2nd spray = 2 weeks after 1st spray
      3rd spray = 2 weeks after 2nd spray

4. Harvesting:

   All ripe berries were harvested from all plants, once weekly, for three consecutive weeks.

5. Sample size:

   1. All treatments applied to plants
   
   
   2. All blocks of 5 plants replicated 4 times
   
   
   3. 20 plants/treatment x 5 treatments = 100 plants.
   
   

6. Parameters for evaluation:

   1. Upon each harvest the following parameters were evaluated, for each block of 5 plants (control, Rovral, Bacillus, Pseudomonas), with four replicas of 5 plants for each treatment (replications A,B,C,D):
      
      1. # Healthy fruits (g)
      
      
      2. Weight of healthy fruits (g)
      
      
      3. Weight of diseased fruits (g)
      
      
      4. % Total weight of harvest that are diseased fruits.
      
      

   2. After the evaluation of the above parameters upon harvest, the healthy fruit was placed in plastic trays (so that no fruit touched another fruit), The tray was then covered with plastic wrap and stored at 4^oC for 7 days. This 4^oC, 95% RH treatment was done to mimic storage conditions in the wholesale/retail distribution chain. The development of Botrytis grey mold during storage was then evaluated to determine if any treatments were effective at reducing post harvest losses due to grey mold. After 7 days storage at 4^oC, 95% RH the healthy fruit was evaluated for the following parameters:
   
   
   1. Weight of healthy fruit (g)
   
   
   2. Weight of diseased fruit (g)
   
   
   3. % Total weight of harvest that are diseased fruit
   
   

Results and Conclusions:

1. Pseudomonas and Bacillus were as effective as Rovral in controlling losses due to grey mold (% total harvest that is diseased fruit) for the first 2 of 3 harvests. Efficacy of the biocontrol agents was severely diminished by the 3rd harvest. It may be possible to overcome this loss by another application of the biocontrol agents during the harvest cycle, for example; between the first and second harvests or between the second and third harvests.
2. Yield of healthy fruit:


1. Bacillus and Pseudomonas treatment means were never significantly less than control or PDB treatments


2. Bacillus treatment mean was never significantly less than Rovral treatment.


3. Pseudomonas vs. Rovral was equivalent at the first harvest and significantly better at the second and third harvest.