Final Report for GNE13-070
Project Information
This project aimed to develop bioassays for screening candidate bacteria and was an attempt at biological control of blueberry anthracnose caused by Colletotrichum fioriniae but was later expanded to cranberry fruit, caused by numerous fungal pathogens. 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 and inefficient. The chemical fungicide regime necessary to control blueberry anthracnose is the driving force of the blueberry fruit rot control spray program in the northeast. In the cranberry system there is a complex of fungi associated with cranberry fruit rot, this study specifically evaluates Coleophoma empetri and Colletotrichum fructicola. Previous research at our laboratory (manuscript pending) presents a phenomenon of fungal disease cycle synchronization to host plant phenology via host water-soluble aqueous floral extracts in which the fungi either directly form disease enabling appressoria or other disease associated morphologies. It is of importance to note that adequate disease control is only accomplished via well-timed fungicide applications during the bloom period, in these crops and many others, indicating a possible target for biological control applications.
In this research bacterial screening methods have been identified as well as future directions for improved screening methods. Bacteria collected from New Jersey, Wisconsin and Massachusetts from cranberry fruit and grape flowers as well as two commercial biological controls, Serenade® Optimum WP and Double Nickel® LC, were screened for biological control components. Screening assays consisted of ability to inhibit the previously observed activity of floral extracts either by metabolizing active stimuli or the production of antifungal compounds within the floral extracts thereby reducing the ability of the pathogen to form disease-enabling structures in a suitable time frame, a form competitive exclusion. In order to observe this effect a “rescue” assay was utilize comparing disease structures stimulated by floral extracts that were either virgin or had experienced bacterial inoculation and subsequent bacterial removal. Bacteria were then screened for antibiosis properties, the ability to inhibit hyphal growth of pathogens on nutrient rich agar media plates. The final in-vitro testing was to determine the direct interaction effects of the candidate bacteria on established pathogen media cultures, hyphal killing, growth of bacteria and pathogen sporulation.
One bacterium collected from grape flowers digested blueberry floral extracts, reducing disease-enabling structures of C. fioriniae without antibiosis effects. Multiple isolates, notably from the cranberry system had strong antifungal effects comparable to commercial biological controls. Field bacterial inoculation trials were conducted on two blueberry cultivars and a very common commercial variety of cranberry.
These field trials were largely unsuccessful in disease suppression of fruit rot but gave valuable application and management information. Understanding the importance of bacterial populations and dynamics of them within the context of traditional disease management strategies is crucial to grower adoption and gradual transition as to minimize monetary risks.
Introduction:
Species of the Colletotrichum acutatum complex have been implicated in a number of costly anthracnose diseases throughout the region, country and world. This proposal focuses on blueberry anthracnose caused by Colletotrichum fioriniae, which is the driving force behind the fungicide spray regime of blueberries, and cranberry fruit rot representatives Colletotrichum fructicola and Coleophoma empetri. The major problem is that blueberry anthracnose and cranberry fruit rot cannot be adequately controlled without the use of chemistry-based fungicidal applications. This problem leads to many others for those who cultivate blueberries and cranberries in the region; increased health risk to the farmers, field workers, their families and ultimately the consumers; deterioration of soils, leaching of chemicals into water supplies and depletion of other natural resources like beneficial micro-organisms. This proposal aimed at exploiting observed responses of these pathogens to respective host plant floral extracts as a means of synchronization to host plant phenology when disease enabling structures (appressorium) are stimulated to develop then infect and causes latent infections that become evident at the time of harvest and during storage. In our research we have been exploring the idea of negation of these floral stimulants and targeting pathogen development at this time frame, as germinating conidia are extremely vulnerable to disease control measures. If the pathogen is able to form an appressorium an infection will ensue.
Many biological controls have been developed and utilized including Sonata® (Bacillus pumilus (QST 2808))1, Serenade® MAX (Bacillus subtilis (strain QST 713))1, TRICHODEX® (T. harzianum isolate T-39)2, Paenibacillus polymyxa (AB15)3 and many others. These biological controls’ modes of action include antibiosis / antifungal properties, activation of SAR and ISR (systemic required resistance and induced systemic resistance, respectively), antagonism and niche competition 1,2,3. The bacteria evaluated in this proposal are Paenibacillus spp. isolated from cranberry, two bacterial isolates cultured from grape cultivars Cabernet Franc and Traminette floral extracts and established biocontrols Serenade® Optimum (Bacillus subtilis strain QST 713) and Double Nickel® (Bacillus amyloliquefaciens strain D747). Serenade® MAX was initially intended for use because it has disease controlling efficacy data, which in some studies, show a higher level of anthracnose control compared to chemistry-based fungicides like Pristine, Abound and Bravo4 but was substituted for Serenade® Optimum WP because of availability, the active bacterium being the same for both. Serenade® Optimum’s mode of action is through the utilization of lipo-peptides, which puncture the pathogenic fungal mycelium. Paenibacillus spp. are gram-negative rod shaped bacteria, which acts as an antagonist and participates in antibiosis. Previously graduate students’ laboratory has demonstrated Paenibacillus spp.’s ability to reduce disease occurrence on C. fioriniae inoculated apples. Paenibacillus functions as an antagonist to C. fioriniae utilizing antibiosis / antifungal properties as well as possible niche competition. Many related bacteria in this class of microorganisms confer a range of antimicrobial properties. Data generated via the interactions of C. fioriniae with blueberry cv. Bluecrop floral-extracts on glass-coverslips, apple cuticles and blueberries demonstrates a statistical increase in germination rates and appressorium formation compared to water only controls. Very similar results are seen with Coleophoma empetri with cranberry floral-extracts. This increase in germination confers a window of vulnerably, which if utilized correctly could be a powerful tool in treating these diseases without such heavy reliance on chemistry-based fungicide/stats. It is known that development of young hyphal structures from new and dormant conidia are the vulnerable time period for the fungus5. If one can apply disease-controlling biocontrol agents prior to the formation of the appressoria during the elongation of hyphal structures, much higher disease-controlling efficacy can be achieved.
Overall this project was a first step at evaluating a new approach to biological control of blueberry and cranberry fruit rot targeting the stimulatory effects of host floral extracts and hindrance of this stimulation.
- Margaret Tuttle McGrath. Efficacy of Various Biological and Microbial Fungicides - Does That Really Work?. Department of Plant Pathology and Plant-Microbe Biology, Cornell University Bulletin.
- Freeman, S., Minz, D., Kolesnik, I., Barbul, O., Zveibil, A., Maymon, M., Nitzani, Y., Kirshner, B., Rav-David, D., Bilu, A., Dag, A., Shafir, S. and Elad, Y. 2004. Tricoderma biocontrol of Colletotrichum acutatum and Botrytis cinerea and survival of in strawberry. European J. of Plant Pathology 110:361-370.
- Lamsal, K., Kim, S., Kim, Y., Lee, Y. 2012. Application of Rhizobacteria for Plant Growth Promotion Effect and Biocontrol of Anthracnose Caused by Colletotrichum acutatum on Pepper. Mycobiology 40(4):244-251.
- Schilder, A., Gillet, J. and Sysak, R. Evaluation of fungicides for control of anthranose fruit rot in ‘Rubel’ blueberries. 2005. F&N Test Vol 61:SMF026.
- Engle, J. S., Lipps, P. E., Graham, T. L., and Boehm, M. J. 2004. Effects of choline, betaine, and wheat floral extracts on growth of Fusarium graminearum. Plant Dis. 88:175-180.
- Develop bacterial bioassay: A multitude of new assays have been developed for this research consisting of a utilization assay, an antibiosis assay and a direct contact plate method. A screening method, which evaluates the bacterium’s ability to utilize 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. Assays developed are practical and open the door to more specific bioassays and screening procedures. Specific floral chemicals utilized by the pathogens are currently being investigated and will become more specific targets for biocontrol screening procedures.
- 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 biological control efficacy of disease occurance evaluated. Field trials were largely unsuccessful but the reasons for which, due to design, where ambiguous. Creation of field ready biocontrols from this study are still very much in their infancy and more work is needed before large-scale acceptance and use is implemented.
- Compare commercial chemical fungicides to commercially available biological controls to new (this study) biological controls: Crucial to this portion a New Jersey Pesticide Applicators License was obtained prior to chemical applications in the categories of CORE, 1A and 10. Two blueberry cultivars were subjected to control applications each with 2 randomized complete block design experiments, early variety Duke and mid-season variety Bluecrop. The cranberry variety selected for control applications was the common commercial variety Stevens. 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 WP(Bayer) and Double Nickel® LC (Certis) were evaluated. Serenade® Optimum WP was as effective as newly created biological control applications in some blueberry experiments of this individual study. In our cranberry field trial neither were effective. The results obtained are from a single season application and may not reflect the actual efficacy of the above-mentioned biological controls. 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.
- Microscopy: Microscopy of all phenological stages of both crop systems was not possible as the sheer volume of work required was prohibitive.
- 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 evaluated for fruit rot at 7-20 days post picking and then again 14-30 days post pick depending on the cultivar. Described later in much more detail.
Cooperators
Research
Floral Extractions: Flower extracts from blueberry cultivar Bluecrop, grape varieties Traminette and Cabernet franc were used in this research and all prepared the same way by gently grinding flowers using mortar and pestle combined with sterile distilled water (SDW) (1:9 flower wt/vol). The resulting pulp was strained through 4 layers of sterile cheesecloth and the filtrate centrifuged for 10 min at 7,000 rpm. The supernatant was then vacuum filtered through Whatman No.1 filter paper (Maidenstone, UK).
Bacteria sources: Two groups of bacteria were investigated in this research, those isolated from grape flowers at the peak of bloom and those isolated from sound cranberry fruit. Grape flower isolates referred to as GB4 and GB6 were isolated from Traminette and Cabernet-franc grape varieties, respectively, were collected at the Rutgers RAREC in Bridgeton, New Jersey. Sound cranberry fruit isolates referred to as CB2, CB3, CB4, CB5, CB6. CB2 and CB3 were isolated from a grower sample in Massachusetts; CB4 was isolated from a grower sample in Wisconsin; CB5 and CB6 were isolated from 2013 cranberry fruit rot culturing performed annually at our laboratory, the Phillip E. Marucci Center for Blueberry and Cranberry Research and Extension in Chatsworth, New Jersey.
Bacterial isolation: Grape isolates were collected from water-soluble floral extracts made using each varieties flowers. Extract was initially intended for use in comparative studies with other floral extracts from the area, but the floral extract was full of bacteria, which caused the pathogen Colletotrichum fioriniae to not form appressoria in a coverslip assay. An aliquot from both varieties of grape were plated onto nutrient agar plates and the most numerous bacterial colonies were isolated and further plated to single colony forming units (CFU). Two isolates were picked, primarily due to their vigorous growth, a valuable component to a prospective biological control bacteria application. Cranberry bacterial isolates were all initially isolated in the same fashion regardless of location of origin. Fruit collected from cranberry bogs at or near the time of harvest are then surface sterilized in a 10% bleach solution for five minutes and then left to dry. Once dried, cranberries are cut into halves and each half is placed on nutrient rich agar and left to incubate until growth appears. Bacteria of interest are observed at the later time of growth inspection, as they either grow from completely sound fruit or are growing near a pathogen in which a zone of inhibition is observed, both valuable biocontrol components.
Bacteria and pathogen maintenance and storage: Bacteria stored on nutrient agar plates in the dark at 25?C or on nutrient agar slants at 4?C for longer-term storage. Pathogens Colletotrichum fioriniae (previously named C. acutatum) and Colletotrichum fructicola (previously named C. gloeosporioides) were maintained on clarified V8® agar without calcium carbonate, to induce high-density sporulation. Pathogen Coleophoma empetri was maintained on water agar amended with rhododendron leaves to give substrate for active sporulation.
Density determinations: Bacterial suspensions were calculated using optical density, using a Beckman DU® 530 Spectrophotometer at 580nm with a target working concentration of 108 colony forming units (CFU) per ml of SDW. A standard curve for each bacteria used was generated and graphically established with R2 values ranging from 0.93– 0.99, checked via dilution plating. Pathogen conidial suspension were calculated and adjusted using a hemocytometer with a target working concentration of 105 conidia per ml of SDW.
Utilization of floral extracts: (Figure 1 a, b) Blueberry cultivar Bluecrop floral extracts, extracted as described above, were subjected to 1X108 bacteria per ml for 24h in the dark at 25?C. All bacteria designated for use in the blueberry spray trials were used independently. Bacteria and floral extracts were at equal volumes, which allowed for incorporation into subsequent bioassays.
Removal of bacteria from floral extract: (Figure 1 b) Once floral extracts were subjected to 24h bacterial inoculation, the bacteria were then removed. In order to determine the most effective mode of bacterial removal a representative bacteria, grape bacteria #4 (GB4), was inoculated as described above and various modes of removal or sterilization were performed including autoclave to kill bacteria, sterile filtration using a 0.22µm nylon filter, and combination of the two methods and then utilized in a glass coverslip assay. The ratio of this pre-sterilized portion is 1-part bacteria in water and 1-part floral extract, once sterilized using filter described above this portion can be considered a 2-volume water portion. Sterilized samples were divided in half and one half of samples received virgin blueberry floral extract. Treatments for the coverslip assay all contained 1 X105 conidia per ml sterile deionized water (SDW) of 7d old Colletotrichum fioriniae conidia and are paired for individual floral extract controls; Filter sterilized bacteria + blueberry cv Bluecrop floral extract (uGB4 F BCFE) and filter sterilized blueberry cv Bluecrop floral extract (F BCFE); Autoclave sterilized bacteria + blueberry cv Bluecrop floral extract (uGB4 A BCFE) and autoclave sterilized blueberry cv Bluecrop floral extract (A BCFE); Filtered then autoclave sterilized bacteria + blueberry cv Bluecrop floral extract (uGB4 F+A BCFE) and filtered then autoclave sterilized blueberry cv Bluecrop floral extract (F+A BCFE). An internal control design also included a positive control 1 X105 conidia per ml SDW of 7d old Colletotrichum fioriniae + blueberry cv Bluecrop floral extract + SDW (BCFE) and a negative control of 1 X105 conidia per ml SDW of 7d old Colletotrichum fioriniae + 2 SDW (Water). Treatments were then run in a glass coverslip assay modified from Leandro et al. (2003) in which equal volumes of SDW, conidia and floral extracts or portions considered on of those volumes are compared to all other treatments and a negative control consisting of 2 volumes SDW and 1 volume conidia. Individual treatments are applied to the surface of a 22mm X 22mm glass coverslip at a volume of 40µl. One coverslip per replicate of treatment per time point; for this experiment there were three replicates per treatment and a single time point of 24h postinoculation. At the specified time point 15µl of lactophenol cotton blue was added which stops all biological activity, considered a vital stain. At this time the coverslip was gently inverted onto a glass slide and number appressoria per 16 fields counted at 24h postinoculation at 400X magnification. (Fig. 1 d)
Rescue of floral stimulation: (Figure 1 c,d) Result from removal of bacteria from flower extract assay demonstrated that sterile filtration using above described filter alone was the appropriate means of bacteria removal. This assay investigated all bacteria used for the blueberry spray trials and the ability of those bacteria to utilize and hinder the stimulatory effects of blueberry cv Bluecrop floral extract. Bacteria used as described above in bacterial utilization methods included grape bacteria #4 and #6 (GB4 and GB6) and cranberry bacteria #6 (CB6) in blueberry cultivar Bluecrop floral extract. The ratio of this pre-sterilized portion is 1-part bacteria in water and 1-part floral extract, once sterilized using filter described above this portion can be considered a 2-volume water portion. Sterilized samples were divided in half and one half of samples received virgin blueberry floral extract (VFE). The half receiving virgin floral extract have the ratio of 1 volume virgin floral extract + 1 volume conidia + 1 volume sterilized bacteria + floral extract which is considered a water volume. The addition of virgin floral extracts is considered a rescue of stimulatory activity due to floral extract. The half that did not receive virgin floral extract is now considered 2 volumes sterilized bacteria + floral extract which is considered 2 volumes water, equivalent to the standard negative control of 2 volumes water and 1 volume conidia. Treatments for the coverslip assay all contained 1 X105 conidia per ml sterile deionized water (SDW) of 7d old Colletotrichum fioriniae conidia and are paired for individual floral extract controls; Filter sterilized GB4 utilized blueberry cv Bluecrop floral extract (uGB4) and filter sterilized GB4 utilized blueberry cv Bluecrop floral extract plus 1 volume virgin blueberry cv Bluecrop floral extract (uGB4+VFE); Filter sterilized GB6 utilized blueberry cv Bluecrop floral extract (uGB6) and filter sterilized GB6 utilized blueberry cv Bluecrop floral extract plus 1 volume virgin blueberry cv Bluecrop floral extract (uGB6+VFE); Filter sterilized CB6 utilized blueberry cv Bluecrop floral extract (uCB6) and filter sterilized CB6 utilized blueberry cv Bluecrop floral extract plus 1 volume virgin blueberry cv Bluecrop floral extract (uCB6+VFE). An internal control design also included a positive control 1 X105 conidia per ml SDW of 7d old Colletotrichum fioriniae + blueberry cv Bluecrop floral extract + SDW (BCFE) and a negative control of 1 X105 conidia per ml SDW of 7d old Colletotrichum fioriniae + 2SDW (Water). A randomized complete block design coverslip assay was then preformed as above and number appressoria per 16 fields counted at 24h postinoculation at 400X magnification.
Antibiosis in-plate: (Figure 2) All bacteria used in both blueberry and cranberry spray trials and pathogens Colletotrichum fioriniae, Colletotrichum fructicola and Coleophoma empetri were used in this antibiosis assay and maintained as described above. A paper print out of 6 adjoining 0.5” by 1” grids were attached to the back of 3 nutrient agar or potato dextrose agar plates per combination of pathogen and bacteria. In the center of the second and fifth grids a 0.25” by 0.25” grid indicated the location of pathogen or bacterial inoculation site. The two 0.5” grids in the center represented the area of interaction and the two 0.5” grids on the perimeter represented non-influenced areas. Once all plates where set up, 1w old 0.25” by 0.25” actively growing hyphal plugs of pathogen were placed into the second grid and a high-density streak of bacteria was placed into the 0.25” by 0.25” of the fifth grid on nutrient agar plates for C. fioriniae or potato dextrose agar for C. empetri. Once inoculated, plates incubated at 25?C in the dark and where evaluated for up to 1w and pictorial observations collected. Combinations are representative of spray trials; C. fioriniae + one of following bacteria: GB4, GB6, CB6; C. fructicola + one of the following bacteria: CB2, CB3, CB4, CB5; C. empetri + one of the following bacteria: CB2, CB3, CB4, CB5.
Antibiosis direct contact: (Figure 3) Pathogen C. fioriniae was plug inoculated onto and grown for 2w on clarified V8® agar plates and allowed to grow dense mycelium. At 2w 40µl drops of each of the following bacteria: GB4, GB6, CB6 and Bacillus subtilis QST713 (Serenade Optimum ®) was added directly onto the surface of the mycelium in a randomized complete four block design, blocking for possible variation between plates. Once inoculated plates were placed into 25?C incubator in the dark. Plates were observed and photographed at 1w post inoculation. Pathogen C. empetri was plug inoculated onto and grown for 1w on potato dextrose plates and allowed to grow dense mycelium, very slow growing. At 1w 40µl drops of each of the following bacteria: CB2, CB3, CB4, CB5 and Bacillus subtilis strain QST713 (Serenade® Optimum WP) and Bacillus amyloliquefaciens strain D747 (Double Nickel® LC) was added directly onto the surface of the mycelium in a randomized complete four block design, blocking for possible variation between plates. Once inoculated plates were placed into 25?C incubator in the dark. Plates were observed and photographed at 5d and 7d post inoculation.
Field bacterial inoculum preparation: Bacteria used in field inoculations were prepared by scraping the surface of nutrient agar plates containing 3d old bacterial cultures into SDW and quantified using optical density at 580nm, adjusting to 108 CFU per ml SDW using previously established standard curve and later checked using serial dilution plating. Once bacterial solution was at specified density, the label for Bacillus subtilis QST713 (Serenade Optimum ®) was followed to an estimated 20oz per acre rate (high-rate), which is roughly 2.5 X 106 CFU per ml of water at an application volume of 50 gallons per acre.
Field applications: All applications began at early bloom and followed Rutgers Commercial Blueberry and Cranberry Pest Control Recommendations for each crop. Blueberry and cranberry spray trials received different fungicide regimes. Blueberry: Two independent experiments for each of the two blueberry cultivars evaluated, Duke (DK1 and DK2) and Bluecrop (BC1 and BC2), a total of 4 experiments received treatments described below in a randomized complete block design for each experiment, total of 7 treatments in each of 10 blocks, applications were made at 7-10d intervals to individual bushes within these blocks using a CO2 pressurized spray canister set to deliver an application rate of 50 gallons per acre. Chemistry based fungicides: standard recommendation (high-rate) of Abound Flowable® (Azoxystrobin: methyl (E)-2-{2-[6-(2-cyanophenoxy)pyrimidin-4-yloxy]phenyl}-3-methoxyacrylate) at 15 fluid ounces per acre and Ziram 76DF® (United Phosphorous, Inc.) (Zinc dimethyldithiocarbamate @ 76%, zinc expressed as metallic @ 16.25%) at 4 pounds per acre (via a 24(C) NJ, label exemption for blueberries in New Jersey). Commercial biological control: Serenade Optimum® was applied at the labeled high-rate of 20 ounces per acre. Experimental: biological controls from this study were prepared as described above and applied following the commercial biological control’s label. Bacteria applied were grape bacteria # 4 (GB4), grape bacteria #6 (GB6) and cranberry bacteria #6 (CB6) and a combination treatment consisting of equal volumes of each of the above experimental bacteria (Combo). A non-treated control was also utilized in these trials. Cranberry: One experiment received treatments described below in a randomized complete block design, total of 8 treatments in each of 8 blocks, applications were made at 7-10d intervals to 4’ by 4’ plots within these blocks using a CO2 pressurized spray canister set to deliver an application rate of 130 gallons per acre, which allows for feasible application conductance. Chemistry based fungicides: standard recommendation (high-rate) of Indar 2F® (Dow AgroSciences LLC) (fenbuconazole: a-[2-(4-chlorophenyl)ethyl]-aphenyl-1H-1,2,4-triazole-1-propanenitrile) at 15 fluid ounces per acre which is in a combination spray with Abound Flowable® (Syngenta) (Azoxystrobin: methyl (E)-2-{2-[6-(2-cyanophenoxy)pyrimidin-4-yloxy]phenyl}-3-methoxyacrylate) at 15.2 fluid ounces per acre and Bravo Weather Stick® (Syngenta) (Chlorothalonil:2,4,5,6-tetrachloroisophthalonitrile) at 5.5 pints per acre. Commercial biological controls: Serenade® Optimum WP was applied at the labeled high-rate of 20 ounces per acre and Double Nickel® LC was applied at the labeled high-rate of 6 quarts per acre. Experimental: biological controls from this study were prepared as described above and applied following the labeled high rate instructions of Serenade® Optimum WP. Bacteria applied were cranberry bacteria # 2-5 (CB2, CB3 CB4, CB5). A non-treated control was also utilized in these trials.
Immature disease occurrence ratings: Blueberry immature fruit disease occurrence methods: 10 clusters of fruit survey collected from each bush (replicate) from every treatment were incubated, not touching other clusters reducing berry to berry spread, in incubation chambers and misted with SDW at the start of the incubation period. For a total of 7 treatments X 10 replicates equaling 70 incubation chambers per cultivar, for a total of 140 chambers. 1 experiment of each blueberry cultivar Duke and Bluecrop were visually observed for disease signs 2w postinoculation. Cranberry immature fruit disease occurrence methods: 20 uprights from each plot (4’X4’) of the randomized complete block design were evaluated for disease occurrence 2 months prior to harvest. The design included 8 treatments within 8 blocks for a total of 64 plots each having 20 analysis points, which were summed for analysis. Upright analysis included number of fruit and subsequent number of diseased fruit. Cranberry fruit rot is caused by a complex of fungi so the causal agent did not need to be specified to deem the fruit as rotten. Data was analyzed in a one-way ANOVA with comparisons made using a Student Newman Keuls post-hoc test with an alpha set to 0.05, utilizing a randomized complete block design for both crops.
Blueberry fruit harvesting and storage trials: Fruit was collected by block and starting treatment was denoted randomly. Each treated bush was totally stripped of mature fruit and was then placed into one of four pint containers with a moistened paper towel. Pint containers were for 2 room temperature (RT) analyses and 2 cooler (C) analyses at time points dependent on each cultivars’ sensitivity to blueberry anthracnose. Blueberry harvest was dependent on maturation stage of the two cultivars. Blueberry cultivar Duke was harvested at peak maturation on June 24-25, DK1 and DK2 respectively. Blueberry cultivar Bluecrop was harvested on July 8-10, BC1 and BC2. Duke storage analysis time points: 20d and 30d cooler, 7d and 14d room temperature. Bluecrop storage analysis time points: 10d and 20d cooler, 7d and 14d room temperature. Fruit evaluated at these time points were accessed for total number of fruit and total number of fruit with obvious C. fioriniae signs like orange conidial discharge. These experiments were analyzed using a one-way ANOVA with comparisons made using a Fisher’s Least Significant Difference post-hoc test with an alpha set to 0.05, utilizing a randomized complete block design for both cultivars. Each time point for each cultivar was displayed and analyzed separately.
Cranberry fruit harvest and fruit rot analysis: Fruit was collected by block and starting treatment was denoted randomly. Within each 4’X4’ block a 3’X3’ grid was centrally placed and all fruit collected. Once fruit collected, the total number of fruit and total number of diseased fruit was recorded and the percentage of sound/healthy fruit was displayed. Data was analyzed in a one-way ANOVA with comparisons made using a Student Newman Keuls post-hoc test with an alpha set to 0.05, utilizing a randomized complete block design.
Removal of bacteria from floral extracts: (Figure 4 a) Bacteria that were inoculated into blueberry cultivar Bluecrop virgin floral extracts (BCFE) and let incubate, thereby utilizing the floral extracts for 24h had to be removed/sterilized in order to not confound subsequent experiments and needed to provide a high degree of separation between bacteria treated extracts and virgin extracts that both received the same sterilization methods i.e. sterile filtration (F), autoclaving (A) and sterile filtration and then autoclaving (F+A). Grape bacteria # 4 (GB4) was used in this assay as previous research had demonstrated that its effect on C. fioriniae’s ability to form appressoria was likely not due to inhibitory compounds. Each sterilization method was paired with GB4 treated floral extract (uGB4) and a virgin Bluecrop floral extract (BCFE) that received one of the sterilization methods above. Number of appressoria were then collected as described above. The internal positive and negative controls (BCFE) and (Water), respectively had the highest degree of separation at 7.13 appressoria per field difference followed by GB4 utilized BCFE (uGB4 F BCFE) and (F BCFE) with a delta of 4.6 appressoria per field. Autoclaving had a delta of 1.37 and filtration then autoclaving had a delta of 0.91. A one-way ANOVA was performed on raw summed data of counted appressoria (14 d.f, LSD=29.001, with a blocking p-value of 0.1239 and treatment p-value of 0.000). A Student Newman Keuls post–hoc test was performed with an alpha set to 0.05 and the treatments were scored as follows: uGB4 F BCFE (b), F BCFE (a), uGB4 A BCFE (b), A BCFE (b), uGB4 F+A BCFE (b), F+A BCFE (b), BCFE (a), water (b). The result of this section lead to future bacterial removal being performed using 0.22µm nylon filter.
Rescue of floral stimulation: (Figure 4 b) Grape bacteria #4 and #6 (GB4 and GB6) and cranberry bacteria # 6 (CB6) were used in this assay along with virgin blueberry cultivar Bluecrop floral extract (VFE) and a water treatment, each treatment contained 1X105 C. fioriniae conidia per ml as described above. A one-way ANOVA was performed on raw summed data of counted appressoria (14 d.f, LSD=18.229, with a blocking p-value of 0.227 and treatment p-value of 0.000). A Student Newman Keuls post–hoc test was performed with an alpha set to 0.05 and the treatments were scored as follows: uGB4 (b), uGB4+VFE (a), uGB6 (b), uGB6+VFE (b), uCB6 (b), uCB6+VFE (b), BCFE (a), water (b). The activity or ability of C. fioriniae to form appressoria could not be returned with the addition of virgin floral extracts, indicating the presence of possible inhibitory compounds produced by GB6 and CB6. However activity was returned in the uGB4+VFE treatment indicating that the observed effect was more likely to be due to the ability of the bacteria to utilize and thereby negate the stimulatory effects on appressoria induction. Microcyclic conidiation, where one conidium gives rise to several others without building a traditional conidial bearing structure, was observed and not different from BCFE positive control, in all treatments except water, which is consistent with our findings involving floral extracts. Data not displayed, a one-way ANOVA was performed on raw summed data of counted conidia (14 d.f, with a blocking p-value of 0.996 and treatment p-value of 0.0015). A Student Newman Keuls post–hoc test was performed with an alpha set to 0.05 and the treatments were scored as follows: uGB4 (a), uGB4+VFE (a), uGB6 (a), uGB6+VFE (a), uCB6 (a), uCB6+VFE (a), BCFE (a), water (b). The results of this assay lead our group to the understanding that there were two separate routes of bacterial hindrance of appressorium formation; antibiosis and competition for resources/exclusion.
Antibiosis in-plate: (Figure 2) This assay was observation based, looking for areas of inhibition due to interactions between pathogens of interest and the bacteria used in their respective field trials. Center two grids indicate area of interaction, P was where pathogen plug was placed and B is where high-density bacteria were streaked (Fig. 2 a). Commercial biocontrols Bacillus subtilis strain QST713 (Serenade® Optimum WP) and Bacillus amyloliquefaciens strain D747 (Double Nickel® LC) 5d postinoculation verses Coleophoma empetri (Ce11.3) and Colletotrichum fiorniniae (Ca10), (figure 2 b1 and b2) respectively. Bacillus amyloliquefaciens strain D747 (Double Nickel® LC) verses Ce11.3 (Fig. 2 b3). All commercial biological controls showed very strong antifungal properties in this assay, with growth of the fungi away from the zone of inhibition. Colletotrichum fructicola (PMAP182) verses bacteria used in cranberry spray trial at 5d postinoculation, (Fig. 2 c1): verses cranberry bacteria #2 (CB2), (Fig. 2 c2): verses CB3, (Fig. c3): verses CB4 and (Fig. 2 c4): verses CB5. All bacteria produced limited inhibition with the exception of CB5 that caused the pathogen to form a linear line of hyphae indicating the zone of inhibition had been reached. Coleophoma empetri (Ce11.3) verses bacteria used in cranberry spray trial at 5d postinoculation, (Fig. 2 d1): verses cranberry bacteria #2 (CB2), (Fig. 2 d2): verses CB3, (Fig. 2 d3): verses CB4 and (Fig. 2 d4): verses CB5. Cranberry bacteria # 2 and #5 had the most pronounced inhibitory effects, where the hyphae grew away from the zone of inhibition and in some cases there was a distinct change in mycelial morphology from uniform to sparsely filamentous. Colletotrichum fiorniniae (Ca10) verses bacteria used in blueberry spray trial at 7d postinoculation, (Fig. 2 e1): verses grape bacteria #4 (GB4), (Fig. 2 e2): verses GB6 and (Fig. 2 e3): verses CB6. Almost all bacteria used in this assay showed some degree of antibiosis except for grape bacteria #4, GB4 did not hinder the growth of C. fioriniae. Grape bacteria # 6 interactions showed a slight inhibitory response but cranberry bacteria #6 had the highest level of inhibition comparable to that of the commercial biological controls. In response to CB6 C. fioriniae was stopped half way through the first inhibition zone grid and began to melanize a perceived defense response, and actively grew in the opposing direction.
Antibiosis direct contact: (Figure 3) This assay was designed to observe the effects of direct bacterial inoculation on established pathogen cultures. (Fig. 3 a) Two pathogens were used; Colletotrichum fioriniae as the blueberry anthracnose causal agent and Coleophoma empetri as a representative of a cranberry fruit rot pathogen inoculated with bacteria that were used in respective field trials. (Fig. 3 b1, b2) All observations were made at 7d postinoculation. Observations included both what the bacteria did while a top the pathogen mycelium as well as the fungal response. Bacterial responses on C. fioriniae mycelium included hyphal discoloration and likely destruction as with cranberry bacteria #6 (CB6) (Fig. 3 c3) and most notably Bacillus subtilis strain QST713 (Serenade® Optimum). (Fig. 3 c4, c5). One very important observation was that when inoculated with grape bacteria #4 and #6 the pathogen began to sporulate. This is an important aspect to a screening method as induction of sporulation may be counter to disease control even if the sporulation is a defensive response. Observations made of Coleophoma empetri responses to bacteria were quite different. A notable observation was the change in growth morphology of both the inoculation site and the morphology of extending hyphal growth once inoculated. In the whole plate picture, figure 3 b2, the inoculation sites are obvious with sunken areas and the mycelium extending from them has gone from a very defined slow growing border to one that is more sparsely growing mycelium and appears to be growing away from the bacteria. (Fig. 3 d6) Bacteria inoculated often began to replicate on the surface of C. empetri as in the case of cranberry bacteria # 4 (Fig. 3 d1, d2) and Bacillus subtilis strain QST713 (Serenade® Optimum) (Fig. 3 d3, d4). It was interesting as to our understanding there was no induction of sporulation of C. empetri in any of the bacterial inoculations. This screening method will be a valuable tool in the future as this step in a screening procedure may give insight into possible problem areas and morphological indicators of response to bacterial biological control agents.
Immature disease occurrence ratings: (Figure 5) In order to evaluate the progression of disease in the field when using both experimental and commercial biological controls, fruit pre-harvest and immature fruit were evaluated in cranberry and blueberry respectively. Cranberry: Cranberry fruit 2 months prior to harvest, August data collection with an October pick date. Result from this section indicated that the biological controls (all types) were not having a substantial effect on disease suppression as no treatment other than the standard recommended chemical fungicides described above were statistically different than water in percent sound/healthy fruit. (Fig. 5 a) A one-way ANOVA was performed on percent sound/healthy fruit utilizing a randomized complete block design (49 d.f, LSD=10.349, with a blocking p-value of 0.0054 and treatment p-value of 0.000). There was slight variation from block to block but the magnitude of rotten fruit made this point trivial. The result of this analysis was a foreshadowing of limited disease suppression expected later at the time of harvest. Blueberry: Clusters from each treatment per block per cultivar per 1 experiment of that cultivar were collected and analyzed for disease occurrence once incubated as described above. Blueberry cultivar Duke analysis of blueberry anthracnose occurrence on detached clusters; a one-way ANOVA was performed on percent C. fioriniae infected fruit per treatment of each block utilizing a randomized complete block design (54 d.f, LSD=1.488, with a blocking p-value of 0.4576 and treatment p-value of 0.000). A Student Newman Keuls post–hoc test was performed with an alpha set to 0.05 and the treatments were scored as follows (uppercase letters in figure 5 b): STD RECS (C), Serenade® Optimum (B), grape bacteria # 4 (GB4) (AB), GB6 (AB), cranberry bacteria #6 (CB6) (A), combination of equal parts GB4, GB6, CB6 (Combo) (C), and an untreated control (UTC) (AB). The standard chemical fungicides had the highest level of disease suppression as was expected followed by Serenade® Optimum and interestingly by the combination of bacteria. It is possible that at this early stage of maturation a synergistic effect, using both inhibitory and floral extract utilizing bacteria lead to the result observed with the combination treatment in the blueberry cultivar Duke. Blueberry cultivar Bluecrop analysis of blueberry anthracnose occurrence on detached clusters; a one-way ANOVA was performed on percent C. fioriniae infected fruit per treatment of each block utilizing a randomized complete block design (54 d.f, LSD=1.533, with a blocking p-value of 0.1192 and treatment p-value of 0.001). A Student Newman Keuls post–hoc test was performed with an alpha set to 0.05 and the treatments were scored as follows: (lowercase letters in figure 5 b): STD RECS (C), Serenade® Optimum (a), GB4 (a), GB6 (ab), CB6 (ab), combination of equal parts GB4, GB6, CB6 (Combo) (bc), and an untreated control (UTC) (ab). As expected the standard chemical fungicides had the highest level of disease suppression again with this very susceptible blueberry cultivar. All other treatments were not dramatically different from the untreated control but a similar trend was observed in the combo treatment like that of the blueberry cultivar Duke. Again these results were a foreshadowing of limited disease suppression expected later at the time of harvest.
Rot analysis: (Figure 5c and Figure 6) Cranberry: Fruit was collected by block and starting treatment was denoted randomly. Within each 4’X4’ block a 3’X3’ grid was centrally placed and all fruit collected. Once fruit collected, the total number of fruit and total number of diseased fruit was recorded and the percentage of sound/healthy fruit was displayed. Data was analyzed in a one-way ANOVA was performed on percent healthy sound/healthy fruit utilizing a randomized complete block design (49 d.f, LSD=4.87, with a blocking p-value of 0.0825 and treatment p-value of 0.000). There was slight variation from block to block but the magnitude of rotten fruit made this point trivial. Only the standard chemical fungicides provided disease suppression. Blueberry: Fruit was collected by block and starting treatment was denoted randomly. Each treated bush was totally stripped of mature fruit and was then placed into one of four pint containers with a moistened paper towel. Pint containers were for 2 room temperature (RT) analyses and 2 cooler (C) analyses at time points dependent on each cultivars’ sensitivity to blueberry anthracnose. Blueberry harvest was dependent on maturation stage of the two cultivars. Blueberry cultivar Duke was harvested at peak maturation on June 24-25, DK1 and DK2 respectively. Blueberry cultivar Bluecrop was harvested on July 8-10, BC1 and BC2. Duke storage analysis time points: 20d and 30d cooler, 7d and 14d room temperature. Bluecrop storage analysis time points: 10d and 20d cooler, 7d and 14d room temperature. Fruit evaluated at these time points were accessed for total number of fruit and total number of fruit with obvious C. fioriniae signs like orange conidial discharge.
A one-way ANOVA was performed on percent C. fioriniae fruit infected per treatment of each block of each independent experiment at each of the individual storage time points utilizing a randomized complete block design. A Fisher’s Least Significant Difference post–hoc test was performed with an alpha set to 0.05 and the treatments were scored (lowercase letters in parenthesis) with lowest alpha numeric score equaling the highest percent rotten blueberry fruit (a = highest percent rot). Duke experiment 1 (DK1) 20d cooler (C) (DK1 20d C) (41 d.f, LSD=4.478, with a blocking p-value of 0.5174 and treatment p-value of 0.5174): STD RECS (a), Serenade® Optimum (a), GB4 (ab), GB6 (a), CB6 (a), combination of equal parts GB4, GB6, CB6 (Combo) (ab), and an untreated control (UTC) (ab). Duke experiment 2 (DK2) 20d cooler (C) (DK2 20d C) (42 d.f, LSD=5.95, with a blocking p-value of 0.2573 and treatment p-value of 0.0003): STD RECS (c), Serenade® Optimum (ab), GB4 (a), GB6 (a), CB6 (ab), combination of equal parts GB4, GB6, CB6 (Combo) (bc), and an untreated control (UTC) (ab). Duke experiment 1 (DK1) 30d cooler (C) (DK1 30d C) (42 d.f, LSD=10.648, with a blocking p-value of 0.4405 and treatment p-value of 0.4218): STD RECS (b), Serenade® Optimum (ab), GB4 (ab), GB6 (ab), CB6 (ab), combination of equal parts GB4, GB6, CB6 (Combo) (ab), and an untreated control (UTC) (a). Duke experiment 2 (DK2) 30d cooler (C) (DK2 30d C) (42 d.f, LSD=8.822, with a blocking p-value of 0.9435 and treatment p-value of 0.0357): STD RECS (b), Serenade® Optimum (a), GB4 (a), GB6 (a), CB6 (a), combination of equal parts GB4, GB6, CB6 (Combo) (a), and an untreated control (UTC) (a). Duke experiment 1 (DK1) 7d room temperature (RT) (DK1 7d RT) (43 d.f, LSD=18.09, with a blocking p-value of 0.2808 and treatment p-value of 0.0011): STD RECS (C), Serenade® Optimum (ab), GB4 (a), GB6 (ab), CB6 (ab), combination of equal parts GB4, GB6, CB6 (Combo) (ab), and an untreated control (UTC) (b). Duke experiment 2 (DK2) 7d room temperature (RT) (DK2 7d RT) (42 d.f, LSD=11.744, with a blocking p-value of 0.4642 and treatment p-value of 0.000): STD RECS (d), Serenade® Optimum (bc), GB4 (bc), GB6 (b), CB6 (b), combination of equal parts GB4, GB6, CB6 (Combo) (c), and an untreated control (UTC) (a). Duke experiment 1 (DK1) 14d room temperature (RT) (DK1 14d RT) (41 d.f, LSD=34.4, with a blocking p-value of 0.8998 and treatment p-value of 0.1997): STD RECS (ab), Serenade® Optimum (b), GB4 (ab), GB6 (b), CB6 (b), combination of equal parts GB4, GB6, CB6 (Combo) (a), and an untreated control (UTC) (ab). Duke experiment 2 (DK2) 14d room temperature (RT) (DK2 14d RT) (42 d.f, LSD=21.908, with a blocking p-value of 0.7019 and treatment p-value of 0.4490): STD RECS (b), Serenade® Optimum (b), GB4 (a), GB6 (ab), CB6 (ab), combination of equal parts GB4, GB6, CB6 (Combo) (ab), and an untreated control (UTC) (a). Bluecrop experiment 1 (BC1) 10d cooler (C) (BC1 10d C) (38 d.f, with a blocking p-value of 0.5174 and treatment p-value of 0.2751): STD RECS (b), Serenade® Optimum (b), GB4 (b), GB6 (a), CB6 (b), combination of equal parts GB4, GB6, CB6 (Combo) (b), and an untreated control (UTC) (b). Bluecrop experiment 2 (BC2) 10d cooler (C) (BC2 10d C) (50 d.f, LSD=6.00, with a blocking p-value of 0.7567 and treatment p-value of 0.000): STD RECS (d), Serenade® Optimum (c), GB4 (c), GB6 (a), CB6 (bc), combination of equal parts GB4, GB6, CB6 (Combo) (ab), and an untreated control (UTC) (ab). Bluecrop experiment 1 (BC1) 20d cooler (C) (BC1 20d C) (39 d.f, LSD=5.28, with a blocking p-value of 0.2968 and treatment p-value of 0.2628): STD RECS (b), Serenade® Optimum (ab), GB4 (ab), GB6 (ab), CB6 (a), combination of equal parts GB4, GB6, CB6 (Combo) (ab), and an untreated control (UTC) (b). Bluecrop experiment 2 (BC2) 20d cooler (C) (BC2 20d C) (49 d.f, LSD=7.743, with a blocking p-value of 0.5066 and treatment p-value of 0.0419): STD RECS (b), Serenade® Optimum (a), GB4 (a), GB6 (a), CB6 (ab), combination of equal parts GB4, GB6, CB6 (Combo) (a), and an untreated control (UTC) (a). Bluecrop experiment 1 (BC1) 7d room temperature (RT) (BC1 7d RT) (41 d.f, LSD=16.362, with a blocking p-value of 0.1349 and treatment p-value of 0.000): STD RECS (C), Serenade® Optimum (ab), GB4 (ab), GB6 (b), CB6 (ab), combination of equal parts GB4, GB6, CB6 (Combo) (ab), and an untreated control (UTC) (a). Bluecrop experiment 2 (BC2) 7d room temperature (RT) (BC2 7d RT) (49 d.f, LSD=17.854, with a blocking p-value of 0.4365 and treatment p-value of 0.0015): STD RECS (d), Serenade® Optimum (a), GB4 (ab), GB6 (cd), CB6 (bc), combination of equal parts GB4, GB6, CB6 (Combo) (bc), and an untreated control (UTC) (abc). Bluecrop experiment 1 (BC1) 14d room temperature (RT) (DK1 14d RT) and Bluecrop experiment 2 (BC2) 14d room temperature (RT) (DK2 14d RT) had 100% fruit rot in all treatments and therefore no variation among treatments.
The results from this section were not a surprise after the immature fruit analysis; the standard chemical based fungicide had the highest level of disease suppression for much of the storage times of this experiment. General trends for the other applications are not obvious and are not consistent from experiment 1 to experiment 2 from each of the two blueberry cultivars. Storage in cooler temperatures dramatically improved keeping quality in our experiments. Our results are from 1 season, replicated in two blueberry cultivars at one farm location.
Although the field trials were largely unsuccessful many insights came from our data collection, observations and experimentation. The development of screening assays that look for hindrance of floral extract stimulation with or without inhibitory effects could be an extremely useful and novel tool for use in future research. Another key screening type looks for effects on established populations and keeps both useful and possible problematic responses in mind like the induction of sporulation of the pathogen as a defense response. The field trails in this research have not lead to any real impact as of yet but have demonstrated the extreme challenges associated with growing blueberries and cranberries in the northeast, particularly in New Jersey without the use of traditional fungicides. In the areas needing more research section this will be discussed in more detail. The most important part of this research was step towards exploiting floral stimulants, understanding the biology of pathosystems and taking steps towards using biological associations to benefit sustainable agriculture in means that are more environmentally and socially sound.
Education & Outreach Activities and Participation Summary
Participation Summary:
To date the results of this work have been disseminated (whole or in part) at the 2014 National American Phytopathological Society meeting in Minnesota and the 2014 Northeast Division of American Phytopathological Society in New Hampshire. This research was presented along with other cranberry floral extract research at the fall 2014 meeting of the American Cranberry Growers Association in New Jersey. Although all contacts were established, an instructional video that was described in the initial proposal of this funding was not performed due to the need for further research prior to grower recommendations from this avenue of research. This research is part of continuing investigation by a PhD student and committee pertaining to pathogen stimulation and synchronization to host plant phenology via bloom and how best to utilize obtained information and implement real-world strides towards sustainability for the growers of these crops.
Project Outcomes
Farmer Adoption
Although field trails were largely unsuccessful, growers were genuinely interested in the idea of incorporation of biocontrol bacteria into their crops, a control measure that has the potential to regenerate season after season. Interest was generated in the screening procedures and bioassays as these assays can be used to screen not only biological controls but can also be used in active chemistries, the predominate control mechanism in blueberry and cranberry crops in the northeast, especially New Jersey.
Areas needing additional study
Modifications of bioassays targeting specific compounds that are responsible for the stimulatory effects previously described in Colletotrichum fioriniae and Coleophoma empetri’s appressorial and microcyclic conidiation responses to host plant floral extracts will have the most impact on real world biological control agents and or combination regimes. Characterizations of these compounds are currently part of graduate students dissertation research and will be reported on. Chemical profiling of experimental biological controls’ inhibitory compounds will also be useful for future research whether it be chemically or organically based. The failure of the field trials was likely due to many factors but two are of the most important; inoculum preparation and application as well as understanding how these bacteria are able to colonize the host plants. Inoculum production with the highest number and most vigorous strains of bacteria will aid in establishment on host plants. Establishment trails are currently being examined and will be reported on in subsequent manuscripts. Understanding the importance of bacterial populations and dynamics of them within the context of traditional disease management strategies is crucial to grower adoption and gradual transition as to minimize monetary risks.