Alternaria Control Using Biocontrol Yeast in Organic Pistachio Production Systems

Alternaria Control Using Biocontrol Yeast in Organic Pistachio Production Systems

Summary

The application of Pichia anomala yeast strain WRL-076 for the potential control of Alternaria alternata was continued in 2008. A pistachio plot located near Madera CA was used for the test. Treatment and control trees and replicates were the same as used in the fall of 2006 and 2007. Yeast was applied 4 times during the growing season at a concentration of 5 x 10(7) CFU/l. 2008 was a dry year and very little A. alternata was observed in the plot, similar to 2007, but a marked contrast to the 2005 and 2006 growning seasons. Cluster counts were taken prior to harvest. No significant difference in the percentage of damaged clusters were observed between water control and treated replicates. No significant yield differences were observed as well. We believe that these results are due to the absence of any significant A. alternaria in the plot. (A. alternaria infection severity is a function of seasonal environment and to a lesser extent, cropping level. 2008 was an ‘off’ low production year and A. alternata was expected to be less severe that for an ‘on’ year.)

Objectives/Performance Targets

1.Evaluate biocontrol yeast that has been released by USDA-ARS (presently going through registration as a control measure for Aspergillus flavus).
2. Test additional biocontrol yeast strains that have shown particular efficacy against multiple A. alternaria strains found on pistachio.
3. Test promising strains from objective 2 in the greenhouse and field.

Our central objective has been to test the efficacy of a specific strain (WRL-076) of Pichia anomala that was isolated from the natural environment (a pistachio orchard) for control of A. alternata at a field scale in an organic production environment where possible interaction effects from prior pesticide sprays are absent. Additional strains of naturally occurring yeast (provided by Dr. Hua) were screened for efficacy against A. alternata on pistachio and A. solani (tomato and potato).

If funds remain at the end of the project period, we plan to ask for a ‘no cost’ extension to evaluate this treatment for a 4th year.

1.Evaluate biocontrol yeast that has been released by USDA-ARS (presently going through registration as a control measure for Aspergillus flavus).
2. Test additional biocontrol yeast strains that have shown particular efficacy against multiple A. alternaria strains found on pistachio.
3. Test promising strains from objective 2 in the greenhouse and field.

Our central objective has been to test the efficacy of a specific strain (WRL-076) of Pichia anomala that was isolated from the natural environment (a pistachio orchard) for control of A. alternata at a field scale in an organic production environment where possible interaction effects from prior pesticide sprays are absent. Additional strains of naturally occurring yeast (provided by Dr. Hua) were screened for efficacy against A. alternata on pistachio and A. solani (tomato and potato).

If funds remain at the end of the project period, we plan to ask for a ‘no cost’ extension to evaluate this treatment for a 4th year.

Accomplishments/Milestones

Survival of yeast in the field –
Yeast levels in field were monitored just prior to harvest. WRL-076 yeast at reduced concentrations was still present on sampled nuts, showing that the yeast was active during the growing season and especially during the period needed to protect the nuts from A. alternata.

Alternaria alternata control –
Control of Alternaria alternata on the pistachio leaves was evaluated with counts of damaged (dead) fruit clusters and undamaged clusters, as has been done during the previous 3 years. Quatitative evaluations of leaf symptoms were not taken since no leaf symptoms were observed in 2008. Very little Alternaria was seen in 2008, even at harvest.

Nut and cluster damage –
Cluster counts were made about a week prior to harvest in 2008. Clusters were scored as alive vs. dead. Dead clusters were scored if the cluster rachis and most nuts were brown or black. Once the rachis turns black where it attaches to the branch, all of the nuts will turn brown with associated husk adhesion to the nuts. The distinction between live and dead clusters was easy to score. No significant differences between the yeast treated trees and the water control trees were observed in 2008. Very few dead clusters were observed for either treatment. 5% and 4% dead clusters were observed for the water control and the yeast sprayed treatments respectively, for a 1% difference.

Yield comparisons –
As in prior years, yield differences for the yeast treated vs. water controlled trees paralleled the results that we observed for the cluster counts. No significant differences in yield between the treated and control trees were seen. The yeast treated trees produced 8% less yield (P=0.29) than the water control trees. This was departure from our prior observations, in which the yeast treated trees produced significantly more yield. Three reasons for the observation are possible. 1) Differences were not significant due to a low level of A. alternata in the orchard. 2) 2007 was a high yield year and since this crop alternately bears, the trees with the best yields in 2007 consequently tend to have the worst yield in 2008. Therefore the better yielding blocks in 2007 (yeast treated) became the poorer yielding blocks in 2008. 3) It appears that mite damage was greater in the yeast treated plots than in the water treated controls (Parfitt, personal observation). It is possible that the yeast treatments provided a more favorable environment for the proliferation of mites.

Laboratory and greenhouse tests of additional yeast strains –
Two potential biocontrol yeast strains in addition to WRL-076 were selected for greenhouse testing from our petri plate tests in 2006-2007. These tests were begun near the end of 2007 and continued through 2008 using various treatment combinations. A. alternata from a prior taxonomic study on pistachio and A. solani obtained from ATTC were used for the greenhouse tests. Tomatoes (2007), potatoes, and seedling pistachios were used as the test plants. Randomized complete block experiments were set up, using water controls for both pathogen application and yeast application. Pathogen treatments without yeast and yeast without pathogens were also used as controls. Tomatoes proved to be unsuitable because of difficulty with hand inoculation and scoring of damage. A summary of the experiments follows.
3/20/08: Potato experiment: A. alternata was applied first by scratch inoculation followed 1 day later by yeast sprays. Both selected yeast strains gave improved control, compared with
6/17/08: Potato experiment. Both yeast and fungus were sprayed either at the same time or A. solani sprayed 3 days before yeast. The fungus application treatment did not make any difference to the results. However, there were significant differences among the fungus control, water, and yeast treatments. The fungus control had very low levels of infection, while all of the yeast treatments resulted in much higher scores.
9/18/08: Pistachio experiment – A. alternata. A. alternata was applied 3 days before yeast treatments to leaf scratches. Water controls consistently gave more disease readings than the A. alternaria treatments for all of the applied yeast treatments. Significant differences were not seen among the yeast treatments.
12/8/08: Pistachio experiment – A. alternata. Both yeasts and A. alternata were applied directly to scratches on the pistachio leaves. Results were inconclusive. Both treatment with fungus and control (nothing) gave similar results for all of the yeast treatments. All of the yeasts gave some measure of control (significantly better than no yeast), with the new strains providing somewhat better control than WRL-076.
None of the yeast strains were effective for controlling A. solani.
Good results were obtained with seedling pistachios. Neither of the new yeast strains produced better control of the fungi than did the WRL-076 strain or water.

Impacts and Contributions/Outcomes

Control of Alternaria alternata on pistachio – Significant differences in yield were not obtained in 2008 either for percentages of damaged clusters or for yield differences. This was probably the result of very low disease incidence in the orchard during 2008. Levels of disease were lower than observed in 2007 and much lower than in 2006 with consequent reduction in differences between control and yeast spray treatments. Our experience in 2008, points out one of the difficulties with commercial scale field experiments, eg. variable disease incidence from season to season. One solution would be application of pathogen inoculum in the field. However, this was not an acceptable option for our grower/cooperator.

Pistachio yield – The results for 2008 suggest that where significant A. alternata disease incidence is low or absent, biocontrol yeast did not contribute to improved productivity and may in yet unknown ways have a negative impact. Note: These results are also influenced by the tendency of pistachio to exhibit an alternate bearing pattern. Therefore, the final analysis after 3-4 years should provide more insight into the overall benefits of applying biocontrol yeast.

Greenhouse tests of additional yeast biotypes – The two additional yeast strains, selected for likely efficacy from prior petri plate tests, were similar to the the WRL-076 strain that was used in the field. None of the strains provided high level of protection against A. solani. A major problem was plant-to-plant variation in response to the different applied treatments, which prevented detection of statistical differences. We were especially interesting in the potential of these potential biocontrol yeasts to control Alternaria solani, which could have significant implications for control of that pathogen on tomato and potato. Tomato was found to be a poor subject for these tests due to cultural problems when grown in the greenhouse (a lot of leaf material and high levels of natural leaf senescence made scoring difficult). Potatoes, however, were good source materials for the experiments, easily grown and managed with easily scorable leaf surfaces. Seedling pistachio plants were also easily grown and scored, but could only be used twice (early and late summer).

Summary of impacts and outcomes – The results of the field study in 2008 did not provide additional support for the application of the biocontrol yeast, a different result than obtained in prior years, and a reflection of the low level of disease in the orchard. Greenhouse testing of additonal yeast strains were not successful because of high levels of uncontrollable experimental variation within treatments.

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