This project consisted of three activities. 1) A biocontrol yeast, developed by USDA-ARS (WRL-076) was tested to determine whether it could survive in the field long enough to serve as a control agent for Alternaria alternata. 2) The ability of the yeast to control A. alternata in the field was evaluated with a replicated test against a water control. Efficacy was evaluated from counts of damaged fruit clusters and marketable yield. The effect of the yeast on other nut micro-organisms was also evaluated. 3) Additional yeast strains were tested using a plate challenge test followed by scratch tests in the greenhouse.
Two years of sampling (in-husk nuts were evaluated) were conducted to determine whether the yeast would persist in the field for a sufficient time period to provide protection against A. alternata infection. Adequate levels of residual yeast remained on the nuts after 17 days throughout the growing season.
Commercial scale yield trials were conducted over 4 years. Significant A. alternata epiphytotics were observed in 2006 and 2009, but environmental conditions prevented A. alternata development in 2007 and 2008. The yeast produced about 14% higher marketable yield in 2006 and 2009 and no detectable difference in 2007 and 2008. However, these difference were not statistically significant due to high levels of uncontrollable variation. Nut cluster damage was also evaluated and treatment effects for this measure of A. alternata infection was highly significant (P=0.001), due to the large number of error df for this study. Cluster damage was also moderately correlated (r=0.47, P=0.002) with yield differences.
Two additional yeasts strains were selected for controlled greenhouse testing against both A. alternata and A. solani. A. alternata was evaluated on pistachio while A. solani was evaluated initially on tomato and later on potato using leaf scratch tests. High levels of uncontrolled variation were present and significant differences from water controls or from WRL-076 were not established.
The WRL-076 yeast was found to be an effective control measure on a waterborne bacterium, Burkholderia cepacia, a contaminant in the spray feedwater in 2007.
1. Evaluate biocontrol yeast that has been released by USDA-ARS.
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.
Objective 1 was the major focus of the project 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 Alternaria alternata at a field scale in an organic production environment where possible interaction effects from prior pesticide sprays are absent. Four years of data were collected for yield and fruit cluster survival. Additional strains were screened for ability to inhibit A. alternata on culture plates and the best 2 were selected for greenhouse tests with pistachio (A. aternata) and potato and tomato (A. solani). Additional data was collected to determine the effect of the field sprayed yeast on other micro-organisms found on the nuts.
This report provides a summary of several separate but related experiments and studies designed to determine whether a biocontrol yeast developed by the USDA-ARS at the Western Regional Research Lab at Albany CA could be used as a biocontrol agent to control Alternaria alternata on pistachio. The yeast, a Pichia anomala strain, designated as WRL-076 and patented by the USDA, was developed as a potential control agent for Aspergillus flavus on pistachio. The current project was carried out as a proof of concept under standard commercial field conditions. Yeast survival, efficacy at reducing damage, potential for improving yield were all evaluated. In addition greenhouse experiments were conducted to see if other similar yeasts could provide a greater level of protection and to evaluate the potential of applying biocontrol yeast for control of other Alternaria species on annual crop plants.
Survival of yeast in the field – Yeast levels in the field were monitored prior to and following spray treatments for the first 2 years of the project. 5-10 nut samples per treatment-replicate were collected and taken to the lab for analysis. Nuts with husks were washed and an aliquot of the wash was spiral plated on a selective medium and counted. Initial spray concentrations of 5 x 107 CFU were applied at five dates during the growing season. CFU concentration and application dates were selected based on our experience with prior trials from 2003 to 2005.
Yield comparisons – Yields were evaluated in a commercial pistachio orchard near Madera CA. This orchard was selected because the grower maintains an organic production system and therefore was not spraying chemical fungicides to control fungal pathogens. In addition, this was a location that was known to have an Alternaria problem and the grower has a history of working with UC Extension for testing pest solutions that can be used with his organic production system. Five blocks of 10 tree replicates for both water control and yeast treated trees were used. Four or five sprays were applied throughout the season at 15-20 day intervals, beginning when conditions were judged to be favorable for A. alternata development and continuing through harvest (Fig 1). Commercial bin harvesting was used to evaluate yield. Fruit were collected in standard bins and weighed (Fig. 2). An appx. 5-10 kg. sample was taken for each treatment-replicate and weighed. Samples were husked, floated to remove blanks and damaged nuts, dried and re-weighed. These sample were used to adjust bin weights to commercial dry weight yields.
Nut and cluster damage – Disease impact was also evaluated by counting the number of damaged vs. intact fruit clusters on each tree in the above described experiment about 3-7 days prior to harvest. Damaged clusters are rendered unharvestable after infection spreads from an initial site on a nut husk and moves into the rachis. Once the rachis dies, all of the fruits in the cluster dry and cannot be harvested. They are not removed by shaking. 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. Clusters were scored as alive vs. dead. Dead clusters were scored if the cluster rachis and most nuts were brown or black. (Figs. 3 and 4.) Data was collected on a per tree basis and transformed using the square root transformation for ANOVA.
Other micro-organisms – During year 2 we decided to see what other pathogens might be present on treated and control nuts sampled from the field. 10 nuts per treatment-replicate were collected and placed on agar medium. After several days, fungal and yeast colonies were isolated and cultured. DNA was isolated and amplified for rDNA sequencing and characterization.
In year 2 we observed a bacterial contaminant in the feedwater used to dilute the yeast. We determined that the contaminant was Burholderia cepacia, a human water borne pathogen from the well water source (determined from rRNA sequencing). Very significant differences in the amount of contaminant were found on the nuts that were sampled for the previously described yeast survival experiment.
Test of additional yeast strains – Yeast strains, isolated from pistachio orchards, were provided by Dr. Hua of the USDA-ARS Western regional lab. These isolates were challenged with several biotypes of A. alternata, isolated from pistachio orchards as part of a prior study of Alternaria biotype diversity in Dr. Parfitt’s lab. The best 2 yeast isolates were retained and used for whole plant testing in the greenhouse. We tested these cultures against both A. alternata and a tomato biotype of A. solani (obtained from the Amer. Type Culture Collection). Initially, we used tomato as the test plants, but found these to be unsuitable for accurate scoring due to difficulties associated with insect control and the tendency of tomato to have considerable natural leaf senecence on older leaves in the greenhouse. Subsequently, both potato and seedling pistachio were used for these tests and proved to be much more easily scoreable. Initial experiments were conducted by spraying both pathogen and and yeast inocula on the subject plants. Unfortunately we could not obtain controllable infection with this method and subsequently a leaf scratch test protocol was adopted. Two leaves from each plant were selected for inoculation, scratched and inoculum applied (Fig. 5). 4 to 5 plants per replicate were inoculated with yeast strains or water control and a randomized block design applied (Fig. 6).
- Fig. 1. Field spraying of yeast and water control in Madera pistachio orchard.
- Fig. 5. Scratch test protocol for inoculation of plants in the greenhouse.
- Fig. 6. Greenhouse test of 3 yeast strains (including WRL-076) and water control
- Fig. 2. Plot harvesting under commercial conditions with standard equipment.
- Fig. 3. A. alternaria damaged nut cluster
- Fig. 4. Normal nut cluster for comparison
Survival of yeast in the field – Yeast levels in the 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.
Yield comparisons – Significant differences between (yeast) treatment and (water) controls were not found. Year by treatment effects were also not significant. Only year effects were significant, which was expected since during 2 years (2007 and 2008) no disease was present in the field while in 2006 and 2009, strong disease development occurred. Despite the absence of statistical significance, mean yield differences (higher yield for the yeast treatments) of approximately 14% were observed for 2006 and 2009. A major limitation to the experiment was the need to use commercial harvesting equipment and variation associated with bin weight (tare) differences from the commercial bins (unfilled bins varied by as much as 7 kg.).
Nut and cluster damage – The distinctions between live and dead clusters were easy to score. Year, treatment, and year by treatment interaction effects were all highly significant. Higher levels of dead clusters (1-live clusters) were observed in 2006 and 2009. During the low disease years (2007 and 2008) only 3-4% of fruit clusters were lost, while in 2006, losses were 13% and in 2009 losses were 30% (combined yeast and water treatments), clearly showing the difference in disease incidence across years. The difference between yeast and control was 5% in 2006 and 9% for 2009. Expressed as a percentage of control, the yeast treatment produced 9.8 % more live cluster for those 2 years. Percent live clusters were also compared with harvested yield (adjusted marketable dry wt.) on a treatment by year basis. The correlation was r = 0.48 with a P value of 0.002. Therefore, evaluation of % live clusters may be used as a rough estimate of harvest yield losses, and is potentially a more accurate measure of loss since degrees of freedom are larger and errors associated with commercial harvesting are not present. In addition, cluster counts can be made earlier in the season. We found that the % damaged clusters increased as the season progressed but that trends observed earlier, continued to be seen at the end of the season.
Other pathogens – Figures 7 and 8 show the results from visual evaluation of nuts that were collected and evaluated for the occurance of surface pathogens on nuts compromised by insect or mechanical damage (early split nuts) in 2007. This analysis was a followup to prior work in the USDA-ARS lab on the occurance of Aspergillus flavus and the potential control of A. flavus with the biocontrol yeast. Both figures show the amounts of non-yeast micro-organisms found on both yeast and water treated nuts collected from the field (a selective medium was used to suppress yeast growth on the plates). Yeast treated nuts in the field had more nuts with low (1-2) levels of contamination while water treated nuts had more nuts in the high contamination categories (4-5). This was a very small pilot study and would have to be much expanded to draw significant conclusions. A variety of micro-organisms were found on the water control nuts, while most of the micro-organisms sampled from the yeast treated nuts were the bio-control yeast (Table 4). These experiments suggest that the yeast is active in the field and may suppress levels of other pathogens.
An interesting and very significant observation was made during the 2006 season. We found that the feed water that was used during the second and third sprays was contaminated with a water borne bacterium Burholderia cepacia. However, as seen in figure 9, bacterial levels on the pistachio nuts were highly reduced on the 3d spray date (3.5 times less) and were completely absent on the 4th spray date, despite even higher levels of bacteria on the nuts for the water control. These observations, combined with the observations on early split and damaged nuts, suggest that this yeast may be a highly effective control agent for other bacterial contaminants of agricultural crops, such as E. coli or Salmonella sp.
Laboratory and greenhouse tests of additional yeast strains – From the 2008 annual report: 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 reported in the 2008 annual report follows.
3/20/08: This experiment was a scratch test conducted on potato – A. solani was applied first by scratch inoculation followed 1 day later by yeast sprays. No significant differences were observed for any of the yeast treatments or the water control. Overall infection rates were modest (2 to 3).
6/2/08: This was a leaf scratch test on potato with A. solani – Both yeast and fungus were sprayed either at the same time or A. solani sprayed 3 days before yeast. The fungus application treatment (applied before or at the same time) were not significantly different. 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. Both of the other yeast produced lower rates of infection than WRL-076, but were similar to water (score of appx. 2).
9/18/08: The experiment was a scratch test on pistachio with A. alternata – A. alternata was applied 3 days before yeast treatments to leaf scratches. Water controls had similar levels of disease development as the A. alternata treatments for all of the applied yeast treatments. Significant differences were not seen among the yeast treatments.
12/8/08: Pistachio leaf scratch experiment with 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, but a high level of infection was obtained for the water control. All of the yeasts gave some measure of control (significantly better than no yeast), but all of the yeasts produced similar results. None of the yeast strains were effective for controlling A. solani.
Neither of the yeasts tested as alternatives to WRL-076 performed significantly better or worse than WRL-076 in any of the experiments.. For some of the experiments, the water control was equivalent to the yeast treatments. We also did not develop a desirable level of infection in the control treatments.
- Fig. 8. Occurrence of micro-organism contamination on early split nuts.
- Fig. 9. Suppression of Burkholderia cepacia contamination on yeast treated nuts.
- Table 1. Yeast Survival in Pistachio Orchard in Madera 2006-2007
- Table 2. ANOVA for dry weight, in shell, split nut, marketable yield.
- Table 3. ANOVA and mean values for % live nut clusters for 4 years. Transformed values were used for ANOVA and untransformed values for means.
- Table 4. Some of the additional pathogens found on both yeast treated and water control damaged nuts and their identies as determined by rbcl sequence and genome match.
- Fig. 7. Occurrence of micro-organism contamination on insect damaged nuts.
We demonstrated that the biocontrol yeast WRL-076 could survive in the field on pistachio nuts for more than two weeks and that no more than 4 sprays were needed to provide overlapping coverage of the crop with yeast during the period when A. alternata was likely to be the greatest problem. The yeast was demonstrated to significantly reduce the number of damaged fruit clusters during years of high Alternaria infection. Impacts on yield were not as clearly defined. While increased yields from the yeast treatments were seen in the high Alternaria infection seasons, these differences were not statistically significant. Therefore, while further research is recommended, we cannot recommend the application of WRL-067 yeast as a control measure for A. alternata at the present time.
A very significant result from the field studies was the observation that the WRL-076 yeast (and probably other yeasts as well) have the potential for being an effective control agent against field bacterial contaminants of crops in the field. This is an area that should be explored much more thoroughly. It is possible that this yeast could be used to provide non-chemical control of E. coli in leafy vegetables or Salmonella in almonds.
One of the difficulties with commercial scale field experiments is variable disease incidence from season to season. One solution would be application of pathogen inoculum in the field. This is not an option that cooperators (or regulatory agencies) are likely to endorse. An alternative to field testing is the use of controlled environment testing. Our experience with greenhouse tests of other yeast strains compared to the WRL-076, showed that control of variation may be even more problematic in greenhouses. Better testing protocols need to be developed.
Educational & Outreach Activities
Parfitt, D., S.S.T. Hua, W. Gee, S.B. Ly, A. Almehdi, H. Chan, M. Braga, T. Martin-Duvall, B. Holtz. 2007. Control of pistachio fungal pathogens with biocontrol yeast. Hort. Sci. 42(4):991 (Abst.)
Although we could not prove statistical significance for the yeast application treatment (see above), during the 2 years that we had significant Alternaria infection in the orchard, we observed an approximately 14% improvement in yield during the two years with high A. alternata infection. At $3/lb, this produced an extra $857/acre. During the 2 years without A. alternata infection the average yield difference was 0.4%.
This product (yeast WRL-076) cannot be applied commercially until it is registered. A commercial company has obtained a license for the product and may register it in the future. Because, we have not conclusively proven it’s efficacy with respect to yield improvement, from a standard statistical approach (the ultimate test), growers are not likely to apply it until additional research shows it’s value. Additionally, A. alternata is not a problem in all years, so growers will have to know their location and the spring/early summer environmental conditions that are likely to promote Alternaria in their orchards.
Areas needing additional study
Prior to commercial adoption of this product, additional field testing is needed to establish statistical efficacy for yield. This will require a larger scale experiment with multiple locations, more replication, strong A. alternaria incidence, and better control of harvest conditions (especially the ability to carefully control the taring of the boxes used to collect the crop). A more effective greenhouse protocol for screening of alternative yeast strains is needed, especially a protocol that can produce a strong uniform disease response on the greenhouse grown plants.
The yeast was shown to have some efficacy against other types of field micro-organisms and could be an effective control for bacterial pathogens or weakly pathogenic fungi. Yeast antagonists may be less likely to be effective against highly pathogenic fungi, however.