Alternaria Control Using Biocontrol Yeast in Organic Pistachio Production Systems

Alternaria Control Using Biocontrol Yeast in Organic Pistachio Production Systems

Summary

Pichia anomala strain WRL-076 was tested for control of Alternaria alternata and Botryosphaeria dothidea in pistachio. Field application of WRL-076 yeast produced significantly reduced cluster loss in 2005-2006 and significantly greater 12% harvestable yield in 2006. Little damage from disease was observed in 2007, and the treatment yield was 6% greater than the control, not significantly different. The yeast controlled a waterborne bacterial contaminant that was found in the spray water midseason in 2006 and may be useful as a control measure against human pathogenic bacterial contaminants in the field.

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).

   Test additional biocontrol yeast strains that have shown particular efficacy against multiple A. alternaria strains found on pistachio.

   Test promising strains from objective 2 in the greenhouse and field.

Our initial objective is 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.
We will continue to screen additional strains of naturally occurring yeast for efficacy against multiple A. alternata biotypes. Initial tests will be done as in-vitro tests, followed by greenhouse tests in 2007. If successful control is shown in greenhouse tests, small scale field tests will be conducted in 2008

Accomplishments/Milestones

A field experiment was conducted in an organic grower’s pistachio orchard near Madera CA during the summers of 2006 and 2007. Five replicates each of control and yeast treated trees (10 trees/replicate) were arranged in two rows to facilitate spraying and machine harvest in a randomized block design. Five preharvest sprays of 5 x 107 cfu/ml WRL-76 yeast were applied to the treatments with water applied to the controls at 17-day intervals, beginning July 11 and extending to harvest in 2006. Four sprays were applied in 2007 which had a significantly shorter growing season. A postharvest spray was applied in 2006 with the idea that it may provide prophylactic effect for the following year. The postharvest spray was not used in 2007 due to the low level of A. alternaria present in both controls and treatments.

Five nuts/tree, 10 trees/rep x treatment were collected and counted in both years to evaluate yeast survival and test for yeast on the controls before and after spraying. The before spray evaluation represented the residual yeast levels from the prior spray while the after spray evaluations provided an estimate of the amount of yeast applied. As expected, the yeast levels declined to approximately 3- 8% of the sprayed yeast after 17 days (3d – 5th sprays), representing an approximately 10x reduction in inoculum from spray application to just prior to the following application. However, significant levels of yeast were maintained in the treated plots throughout the season. Based on these results, at least one fewer spray could be applied in the future. Further, we did not observe any beneficial value to the post-harvest spray (see discussion of Alternaria control).

Control of Alternaria alternata on the pistachio leaves was evaluated using a whole tree visual score at three dates (9/14/06, 9/21/06, and 11/8/06) during the season. Evaluation was initiated when significant infection was observed until the end of the season, shortly before leaf drop. Scoring was from 0=no lesions to 5=whole tree defoliation. Very little Alternaria was seen until about 2 weeks from harvest. After harvest, infection levels increased rapidly. Significant differences between treatments and controls for leaf infection were not visually obvious, on any of the dates. However, subsequent variance analysis of the data on 2/21/06 (the middle date) showed significant differences (P=0.02) between the yeast treatment and water control. Leaves were also scored once in 2007, but very little A. alternata was observed on either treatments or controls.

Cluster counts were made twice prior to harvest in 2006, with approximately 100-150 clusters/tree evaluated in each year and once in 2007. 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. The dead clusters were well established by the beginning of August and levels did not increase significantly thereafter. Because of the relatively early manifestation of this behavior and visual evaluation of initial nut infection (prior to spread through the cluster), Dr. Holtz was of the opinion that Botryosphaeria dothidea was a more important agent of cluster death than Alternaria alternata. In any event, the results of the yeast spray were very significant (P=0.002 for treatment, P=0.2 for assay date, and P=0.00 for block). The number of dead clusters was 9.5% less in the yeast treated trees. These results closely track the yield results below. These results are consistent with the results that we saw in the preliminary trial with four trees per treatment in 2005, except that the overall cluster damage level was much less (15% for the water control in 2006 vs. 49% in 2005, and 10% for the yeast treated vs. 23% in 2005). Differences in overall levels may be due to the larger sample size used in 2006 (50 trees vs. 4 trees) or to seasonal variables. Differences in 2007 were not significant (P=0.2) due to the low level of A. alternata present in the orchard. The number of dead clusters was only 0.6 % less in the yeast treated trees only 3% in the control trees.

Not surprisingly, yield differences for the yeast treated vs. water controlled trees paralleled the results that we observed for the cluster counts. An overall yield improvement of 12.3% was observed for the yeast treatment. This was statistically significant (P=0.05). As noted above, there was only a low level of A. alternata in the orchard during 2007. Nevertheless, a yield increase of 7.8% (P=0.23) was observed for the yeast treatment. Differences were not significant due to the reduced level of A. alternata in the orchard, much higher mean yields (this was an on year for the orchard) and the need to collect each sample in two harvest bins rather than one due to the higher yield which increased measurement error.

Samples from the third spray in 2006 were found to be contaminated with a bacterial agent. Analysis of the 16S RNA sequence and BLAST matching identified the bacterium as Burkholderia cepacia. The source of this contaminant was found to be the well water used to mix the control and yeast sprays. The bacterium disappeared from the feedwater toward the end of the season in 2006 and was not found in 2007. Other than potentially interfering with the experiment, this event provided evidence that the yeast treatment effectively controlled a water borne bacterium. The significance of this observation is that this bacterium, which has also been shown to be a weak human pathogen, was eliminated by the yeast treatment and that the yeast may have broader application as a biocontrol agent. Our end of season results for A. alternata suggested that the Burkholderia did not seem to reduce the incidence of A. alternata near the end of the season when symptoms from A. alternata are usually observed. The Burkholderia cepacia was not found in the field or water after the fourth spray, either.

The literature on insect damage as a source of Aspergillus flavus contamination in pistachio has been mixed. However, several researchers have suggested that insect damaged nuts are much more likely to host A. flavus. The orchard being used for the present experiment had a significant level of nut damage from leaf footed bug feeding in June 2006 and to an even greater extent in 2007. This is different than the insect damage typically associated with A. flavus contamination, which is from Naval Orangeworm, and is associated with colonization of early split nuts. However, we decided to investigate whether the nuts attacked by leaf footed bugs (nuts that subsequently died and were not included in the harvest stream) had significant levels of and whether those levels were affected by the yeast spray. A total of 250 nuts were collected for each treatment (5 plates per treatment x replicate, 10 nuts per plate). Three plates of green healthy nuts, one plate of brown damaged nuts, and one plate of insect damaged nuts were collected per treatment x replicate. Higher levels of A.flavus were seen in both healthy and insect damaged nuts from the water control than from the yeast treated nuts. It is interested to note that the levels of A. flavus observed at this late season sampling were the highest observed at any point during the season and higher than seen from the sampling at spray dates. 26S RNA analysis of other organisms found on the nuts from this study were done and are reported in attached materials.

We have run some preliminary tests of 7 additional potential biocontrol yeasts using the A. alternata and B. dothidea biotypes from the Madera grower fields as challenges. We used streak tests and also did mixed culture tests similar to those used in 2004. Two selections that showed high levels of A. alternata control from the plate tests were tested against water controls and the WRL-076 yeast selection in 2007. Both pistachio and tomato were used as test plants. Tomatoes proved to be unsuitable because of difficulty with hand inoculation and scoring of damage. Good results were obtained with seedling pistachios. Significant differences in levels of infection were observed for both Alternaria alternata and Botryosphaeria dothidea with the different yeast treatments. Both of the new yeast strains produced better control of the fungi than did the WRL-076 strain, suggesting that even more effective yeast treatments may be possible in the future. An additional greenhouse test is being conducted with potato to test its application as a test plant and to confirm the results of the previous experiment.

Impacts and Contributions/Outcomes

The results from 2005 and 2006 showed that the Pichia anomala WRL-076 yeast treatment could significantly reduce the loss of fruit clusters when A. alternata was present.

Results from 2006 showed a 12.3% yield improvement for the yeast treated trees. In 2007, a yield improvement of 7.8% was observed, even with a low level of A. alternata.
This is a 42 kg/ha yield improvement in 2006 and a 50 kg/ha improvement in 2007, or 229 lb/ac. in 2006 and 272 lb/ac. in 2007. At $3 acre for organic pistachios in 2006 this translates into an additional profit of approximately $686/acre for the grower (less the cost of the spray treatments which have not been estimated).

The results from this part of the study suggest that the yeast spray treatments may also provide effective control of other pistachio pathogens such as Botryosphaeria dothidea and Aspergillus flavus. A. flavus is the agent responsible for aflatoxin contamination in pistachio, so control of this fungus should help to limit alflatoxin contamination. The control of the Burkholderia cepacia contaminant in the spray feedwater suggests that the use of preharvest field application of biocontrol yeast could have applications for improving food safety in some annual crops.

The results of the field study in 2006 continue to support our prior experiences with biocontrol yeast, and in particular with WRL-076, as an alternative to chemical pesticide for the control of fungal pathogens in pistachio. The yield comparisons suggest that application of this material will result in substantial improvements in revenue for the grower. Biocontrol yeast application may have other applications in agricultural systems for disease control beyond the specific system being testing in this study.

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