Biological Control of Blueberry Anthracnose and Cranberry Fruit Rot: Exploiting Fungal Responses to Blueberry and Cranberry Bloom in Biocontrol Treatments

Biological Control of Blueberry Anthracnose and Cranberry Fruit Rot: Exploiting Fungal Responses to Blueberry and Cranberry Bloom in Biocontrol Treatments

Summary

This project was initially intended to evaluating biological control of blueberry anthracnose but was expanded to cranberry fruit rot as well. The purpose of this study was to address the issue that adequate fruit rot control without the use of chemical fungicides on blueberry and cranberry crops is currently impractical. In the blueberry system the driving fruit rotting pathogen is Colletotrichum fioriniae, previously known as C. acutatum. In the cranberry system there is a complex of fungi associated with cranberry fruit rot, this study specifically evaluates Coleophoma empetri and cranberry isolates of Colletotrichum acutatum and Colletotrichum fructicola. Previous research presents a phenomenon of fungal disease cycle synchronization to host plant phenology via floral extracts in which the fungi either directly form disease enabling appressoria or other disease associated morphologies in vitro and via detached fruit inoculation studies. It is of importance to note that disease control is only accomplished via well-timed fungicide applications during the bloom period, indicating a possible target for biological control applications. So far bacterial screening, digestion assays, plate inhibition assays, field inoculations, fruit analysis, fruit storage assays and comparisons to established, commercial biological controls (Serenade Optimum and Double Nickel) have been conducted. Bacteria have been screened for the ability to do one or more of the following: digestion of floral extracts, thereby reducing the ability of the pathogen to form disease enabling structures in a suitable time frame, a form competitive exclusion; produce antifungal compounds in plate assays, a from of antibiosis; direct digestion and or cell death of the pathogen by bacterial biological controls, direct interactions. One bacterium collected from grape flowers digested blueberry floral extracts reducing disease-enabling structures of C. fioriniae. Multiple isolates, notably from the cranberry system had strong antifungal effects comparable to commercial biological controls. In field bacterial inoculation trials were conducted on two blueberry cultivars and a very common commercial variety of cranberry. The field trials were largely unsuccessful in the prevention of fruit rot but gave valuable application and management information.

Objectives/Performance Targets

1. 1. Develop bacterial bioassay: A multitude of new assays have been developed for this research, the group consists of a digestion assay, an antibiosis assay and a direct contact plate method. A screening method, which evaluates the bacterium’s ability to digest and or inactivate the effects of floral extracts, has been designed and implemented. Antibiosis assay consist of dual inoculation of bacteria and pathogen on nutrient agar and area of inhibition observed. Direct contact assay evaluates the ability to kill or directly impede growth of an established population of the pathogen.

1. 1. Develop new biological control solutions: Bacterial isolates from Cabernet Franc grape flowers, Traminette grape flowers, cranberry bacterial isolates from New Jersey, Massachusetts and Wisconsin were all applied to blueberry and cranberry plantings and used in bioassays.

1. 1. Compare commercial chemical fungicides to commercially available biological controls to new (this study) biological controls: As is crucial to this portion a New Jersey Pesticide Applicators License was obtained. As stated in the introduction, it is extremely difficult to control fruit rot in New Jersey and much of the northeast without the use of chemical fungicides. Applications of the standard chemical fungicides had by far the highest levels of disease control and suppression. Commercially available biological controls Serenade Optimum (Bayer) and Double Nickel (Certis) were evaluated. Serenade Optimum was as effective as newly created biological control applications in some blueberry experiments. In the cranberry field trial neither were effective. Comparisons of in-plate assays were interesting as some of the cranberry bacterial isolates were as effective as established biological controls in the inhibition of fungal growth.

1. 1. Microscopy: Microscopy of digestion assays has been performed in great detail. Previously it was outlined in the proposal that evaluations would be made at all fruit developmental points, this did not happen as the sheer volume of work required for this project was prohibitive.

1. 1. Fruit rot analysis and storage tests: fruit from both crops field trials were collected during the their respective fruit bearing season. Blueberry fruit were collected then sampled for fruit rot at 7-10 days post pick and then again 14-20 days post pick depending on the cultivar. Cooler storage tests were performed for 1-month post picking.

1. 1. Outreach: To date I have disseminated this information (whole or in part) at the National American Phytopathological Society meeting in Minnesota and the Northeast Division of American Phytopathological Society in New Hampshire. I will be speaking at upcoming twilight meetings for both blueberry and cranberry growers. In January I will be speaking with the American Cranberry Growers Association (ACGA).

Accomplishments/Milestones

To date:

• • Obtained a New Jersey Pesticide Applicators License for CORE, 1A and 10.

• • Hiring and management of field season hires.

• • Determined optical density curves for calibration of application concentrations of bacterial biological controls.

• • Flagged out two 7 TRT by 10 Block experiments for each blueberry cultivar; Duke (early season) and Bluecrop (main season commercial variety).

• • Flagged out 1 8 TRT by 8 Block experiment in an established cranberry cultivar Stevens (commercial variety) bog. Plots were 4’ by 4’.

• • Performance of bacterial digestion of blueberry floral extract via biological control bacteria assays. Assay included the pathogen fioriniae.

• • Performance of bacterial digestion of cranberry floral extract via biological control bacteria assays. Assay included the pathogen empetri.

• • Sprayer calibrations and beginning of blueberry spray regimes for new (this study) biological controls, commercially recognized biological controls and chemical fungicides.

• • 1^st round of bacterial inhibition assays in culture.

• • CO2 high pressure sprayer calibration for use on cranberry bogs and beginning of blueberry spray regimes for new (this study) biological controls, commercially recognized biological controls and chemical fungicides.

• • Bacterial interaction (only investigating possible interaction between bacteria in culture) assays.

• • Bacterial inhibition assays for both blueberry and cranberry spray regime bacteria and recognized commercial biological controls against fioriniae, C. empetri and C. acutatum (isolate from cranberry).

• • Collection of ripe blueberry fruit. Disease severity and storage assays performed.

• • Disease phenology of cranberry spray trial conducted every two weeks during most of the fruiting season.    

• • Collection of ripe cranberry fruit (dry harvest). Disease severity assay performed.

• • Direct contact inhibition assay performed in which bacterial suspensions were added atop established pathogen cultures.

Current:

• • Bacteria producing highest antifungal response in culture (cranberry isolates CB2, CB5and CB6) are currently being evaluated for transferability of activity in culture media preparations against selected pathogens.

• • Descriptive characteristics of fungal response in digestion assays. Interesting modifications in fungal morphology in response to the addition and subsequent removal of bacteria may provide clues as to the actual biological role of these bacteria in these systems.

• • Bacterial digestion of cranberry floral extracts using other common cranberry pathogens like Phyllosticta vaccinii and Colletotrichum fructicola.

Future:

• • Isolation of fungal compounds responsible for inhibition of fungal pathogen growth in culture and subsequent application of this knowledge in down stream assays.

• • Establishment and sub-culturing from field inoculations on both blueberry and cranberry to establish if the bacteria are able to colonize the plants of importance. This will be an extremely valuable piece of information as it dictates how these biological controls will fit into a management program.

• • Screening for more bacteria like GB4 (no inhibition but actively digest and inactivates the stimuli of blueberry floral extracts on fioriniae).

• • Identification of bacteria of interest.

Accomplishments:

• • OD calibration curves for bacterial concentrations.

• • Bacteria isolate from grape (GB4) shows digestive activity and inactivation of blueberry floral extracts.

• • Bacterial isolates from cranberry (CB2, CB5, CB6) show fungal inhibition properties.

• • Experience of; managing employees, fungicide application, biological control application and experimental designs.

• • Creation of new assays and screening approaches.

• • Some (this study) biological control in blueberry cultivar Duke comparable to commercially available biological controls.

Expectations, problems and surprises:

I started this project with the expectations of having a number of new products available for ongoing investigation and practical application in both blueberry and cranberry crop systems. I expected that the field trials would be a good indicator of whether or not these biological controls would be a viable means of fruit rot control. I expected to be able to make a educational video and disseminate more information by this point in time, but I ran into a number of problems/experiences this year that taught me a great deal about running a grant, managing people and making/keeping a cohesive research project in high efficiency. The first problem I experienced was the sheer volume of work that needed to be done prior to bloom with very limited personnel, but through determination and many extra hours the spray program was ready for blueberry bloom and subsequently cranberry bloom about 1.5 months later. Calibration of optical densities also proved challenging, as some bacteria did not initially fully suspend in water and much troubleshooting was required to establish this critical tool for subsequent uses. As the season progressed cranberry disease phenology was conducted regularly and I had a larger team of people whom contributed a great deal to this area. Application days made me extremely aware of a number of things most importantly being the care needed to stay safe. Fungicides are dangerous, toxic and problematic. This alone in my personal thoughts about agriculture is a driving force to figure out a better, safer, way to control fungal diseases. Collection of blueberry and cranberry fruit and rot analysis revealed that the spray applications were not very effective but the reasons for this were ambiguous. I should have and will next time pay more attention to the establishment of the biological controls on the host plants as this is crucial to furthering possible management strategies. I was naïve in understanding the amount of work required to do the blueberry field experiments and rot analysis, I had upwards of 6 people helping me for weeks on end and still the work was mounting, but through hard work and many extra hours the analyses were completed.

Impacts and Contributions/Outcomes

Although the field trials were largely unsuccessful a great deal of information was obtained. Screening procedures, which demonstrated grape bacteria # 4 (GB4) as an agent of floral digestion and inactivation and cranberry bacteria #2,5,6 (CB2, CB5, CB6) as bacteria, which produce antifungal compounds, was a great success. This research has given new bacteria to evaluate for biological control and characteristics of which to look in future screenings. When these bacteria are better understood they will be valuable as tools for fruit rot control in blueberry and cranberry crops and hopefully in other systems that are affected by similar fungal pathogens. This research and hopeful outcomes may bring the possibly of organic IPM programs to areas of the northeast which currently have no other options than chemical fungicides. Understanding the establishment of these biological controls on host plants is crucial as it may lead to a system that naturally, in a sustainable fashion, rejuvenates bacterial protection against fungal pathogens. Personally the unsavory experiences I have had over the last growing season in regards to using chemical fungicides has shown me that growers would appreciate safer alternatives if the efficacy was up to the par they are currently accustomed to. Growers realize that science can and will make this a reality via understanding the biology and interactions of the host, pathogen and microbial community.                 

Collaborators: