Fire blight disease, caused by the plant pathogenic bacteria Erwinia amylovora
, is one of the most economically important diseases in pome fruits [1
], limiting the production of apples in fruit growing regions of the eastern United States and many other apple and pear growing regions of the world [3
]. It is also a severe problem in pear orchards in the eastern United States, and because of that, the production acreage in this region is kept quite small—only 3200 acres worth $
10 million compared to 61,200 acres worth $
291 million along the West Coast [7
]. If not controlled in orchards, E. amylovora
infects blossoms, fruits, as well as vegetative parts such as shoots and wood in host trees [8
]. In severe infections, it can also infect rootstock crowns of fruit trees, eventually causing death [4
]. Major changes in the apple production industry in the last 20 years have resulted from the widespread adoption of new cultivars and high-density tree plantings in commercial orchards to reduce labor costs. Recent plantings of different susceptible apple cultivars and certain dwarf rootstocks have greatly increased the risk of fire blight disease in orchards [2
]. An incidence of only 10% fire blight disease in new high-density orchards with 4-year old trees is estimated to cause losses of up to USD 8400/hectare [10
]. In the recent past, just a single incidence of fire blight epidemic in apple orchards in Michigan resulted in the loss of 220,000 trees with an estimated economic loss of about USD 42 million [5
]. Fire blight causes heavy losses in other geographical regions as well. For example, the eradication of fire blight-infected dwarf pome orchards in Switzerland cost approximately CHF 50 million [6
]. In addition, fire blight incidences may cause quarantine issues resulting in export denial in some countries.
Effective management of the fire blight disease in commercial apple orchards requires integrated approaches. Most fire blight management strategies have focused on the reduction of inoculum in the orchard (e.g., early season sprays of copper bactericides or the pruning of cankers and infected tissues), and the use of antibiotic treatments to prevent infection during the blossom blight phase [2
]. Control of the blossom blight phase of the disease remains the critical component in fire blight management, and approximately 10,000 lb (~4,536 kg) of the active ingredient (a.i.) of streptomycin are applied prophylactically each year to apples and 7000 lb (~3175 kg) a.i. to pears in the US [13
]. Properly timed applications of this antibiotic during the bloom period in orchards can provide over 80% control of sensitive strains of the pathogen, but resistant strains have developed in most areas of western United States and Michigan that greatly reduce its effectiveness [14
]. Although another antibiotic, oxytetracycline, can be used in orchards for these pathogen strains [16
], it is partially effective against these strains and there are no other registered alternatives that provide a high level of control. If fruit growers fail to control blossom blight during the spring, no control strategies are available to control shoot blight and rootstock blight during the summer months. Use of streptomycin in organic apple production, while allowed, is strictly regulated. The continued use of large amounts of antibiotics for agricultural production has raised serious concerns about developing cross-resistance in human pathogenic bacteria [18
]. The need for different management tactics and alternative tools (such as the use of biological control methods or development of resistant varieties) for the management of fire blight, therefore, cannot be overstated.
Biological control of E. amylovora
is an important alternative to conventional antibiotics in managing fire blight disease [19
]. Several biocontrol products for fire blight control have been registered or are at an advanced stage of registration. These include different products based on Bacillus subtilis, Pseudomonas fluorescens
], as well as several strains of Pantoea agglomerans
that are at advanced stages of testing [22
]. Epiphytic bacterial biocontrol agents have not consistently provided high levels of fire blight control [23
]. In biological control, inhibition of E. amylovora
results from the pre-emptive and competitive colonization of the near-sterile environments of the nutrient-rich flower stigma of newly opened flowers. If environmental conditions are highly favorable for multiplication or if the initial inoculum is very high, pre-emptive colonization becomes the major factor responsible for disease control [25
]. Current applications of biological control-based products with airblast sprayers are unable to target the material applied directly onto the stigmatic surfaces and floral nectarines where initial pathogen build-up and subsequent host colonization begins [26
]. Of the biocontrol bacteria that do reach the flowers, most never reach the stigma because of flower location/orientation or because they were not open (i.e., flowering) at the time of product application. Targeted delivery directly to the stigma and nectaries would greatly reduce both the rates and costs of bacterial biological control agents as well as improve their efficacy.
Apple growers heavily rely on bees and other flower-visiting arthropods for pollination [27
]. The characteristics that make bees excellent pollinators also make them appropriate carriers of organically approved disease biocontrol formulations, which they can deposit directly on the stigma while foraging on open flowers. This strategy has met some success using honey bees (Apis mellifera
) in various crops [25
]. Many factors such as synchrony of life cycle with pome fruit phenology, crop specificity, and greater efficacy in pollinating flowers make solitary bees (e.g., the Japanese orchard bee, Osmia cornifrons
) more efficient vectors of biocontrol products than honey bees [30
]. Osmia cornifrons
is commercially available as a tree fruit pollinator, and fruit growers deploy them for pollination services in their orchards. Due to parasitic mites and several other stressors, the population of honey bees has been declining in different regions [32
]. In the recent past, such declines have greatly increased the cost of honey bee rentals to fruit growers. Alternative pollinators such as O. cornifrons
and the native Blue Orchard bee, O. lignaria
, are increasingly being used as replacements or supplements to honey bees in many crops [27
The main challenge while using these solitary bees as vectors of biocontrol agents has been to devise a strategy to pack the body of the individual bees with sufficient amounts of the biocontrol agent before they exit their nesting structures. In the case of colony bees such as honey bees, this difficulty was overcome by placing the biocontrol agent in a dispenser box attached to the standard bee hive [29
]. However, a different strategy is required for the solitary bees, which, by definition, do not live in large colonies like honey bees or bumble bees. Until recently, such a strategy had not been available, but research from Italy on the European Orchard bee, O. cornuta
, suggests that it is possible to devise a dispenser that would facilitate the delivery of biocontrol agents using other solitary bees such as the Japanese orchard bees [40
]. In this context, the objectives of the current study are to (1) design a vector dispenser suitable for the Japanese orchard bee (O. cornifrons
), (2) determine if the O. cornifrons
bees using the dispenser can vector the biocontrol product, and (3) determine the ability of O. cornifrons
bees to secondarily transmit biocontrol bacteria between flowers.
2. Materials and Methods
This study consisted of three experiments on bee vectoring of a biological control agent for fire blight management. Experimental procedures for each of these studies are summarized below.
2.1. Designing and Optimizing Nest Dispenser System for Osmia cornifrons
We designed and tested a nest dispenser suitable for the O. cornifrons
bees following a model described by Maccagnani et al. [41
] for the European orchard bee, O. cornuta
. The design is of a simple wooden structure (H,D,W: 26, 20.5, 17.5 cm) consisting of an exit ramp (made of transparent plastic) with a shallow station at the base of the ramp designed to hold the biocontrol product in a fine powder or granular form (Figure 1
). We scaled up this dispenser to accommodate at least 250 female O. cornifrons
, which are considered adequate for pollination of one acre of apples. In the dispenser system, we modified the upper exit ramp to allow for quick loading of the biocontrol product into the grooves with a small spoon. A previous similar design for a mason bee species allowed 20% of the bees to exit via the entrance holes [41
], thus reducing their effectiveness as active carriers of the antagonistic bacteria. The new dispenser design was modified to increase the efficiency of using the exit slot, by covering the screened front to allow light to enter only from above, attracting the bees upward to the exit ramp. We also separated the exit slot from the entrance tubes by fitting the entrance tubes with a screen flap directing the bees upward but restricting their movement downward, all of which greatly reduced the use of the entrance tubes by exiting bees.
Based on our experience from different trails with the O. cornifrons bees, we color-coded the entrance holes. Bees that have been shown the color-coded entrance tubes “remember” the entrance holes of their nest at the time of their initial orientation flight, so that upon returning, they fly directly to a specific entrance hole. After constructing the dispenser system, we tested the proper use of entrances and exits in the nesting dispenser by observing all bees entering and exiting the dispenser over a 15-min period and repeated the observations three times. The observations were made with the Serenade® MAX (B. subtilis strain QST 713) formulation (Bayer CropScience, St. Louis, MO) containing a minimum of 7.3 x 109 cfu/g placed in the exit grooves of the nest dispenser. The percentage of bees utilizing the exits and entrances correctly of the dispenser nest was calculated for each trial.
2.2. Osmia cornifrons Vectoring of Biological Control Product Using the Dispenser
In this experiment, the amount of the biological control product collected by O. cornifrons
bees exiting the dispenser and the proportion of the product that was deposited on individual flowers following legitimate visits was documented at the deployment rate of 250 bees/acre (25+ nest tubes) recommended for pollination. The same biological control product formulation used in Experiment 1 was used for this experiment. To determine the amount of the biocontrol product collected by an individual insect, bees exiting the dispensers were captured and placed in glass vials containing 2 mL of sterile 0.1 M potassium phosphate buffer (KPB) amended with 0.1 mL of Tween-20 surfactant per liter. The vials were shaken vigorously for 30 s, followed by incubation in a sonicating bath for another 30 s to dislodge the bacteria adhered to the body of the vectors. The wash buffer was subjected to serial dilution and plated either on nutrient yeast extract dextrose agar (NYDA) medium for B. subtilis
]. All the plates were then incubated at 28 ˚C and the total number of colony-forming units (CFUs) were determined within three days of plating. Bees exiting dispensers in which no biocontrol product was placed were used as controls. Eight bees were assayed for the Serenade treatment in each of the three experimental runs. We also collected data on primary transmission. For this purpose, flowers (n = 8, from each experimental trial) were collected from potted crabapple trees (n = 6) immediately after being visited by the O. cornifrons
bees carrying B. subtilis
based biological control product and placed singly into vials (replicates) for assaying the quantity of B. subtilis
by dilution plating as described above. The amount of Bacillus subtilis
(in terms of CFUs) carried by the O. cornifrons
bees exiting the dispenser nest was quantified in each trial.
2.3. Determining the Ability of O. cornifrons to Secondarily Transmit Biocontrol Bacteria between Flowers.
This experiment was conducted in two parts to examine the secondary transmission of B. subtilis based biological control product (Serenade®) by O. cornifrons bees. In order to isolate both the potted crabapple trees from wild bee visitation and to concentrate the test O. cornifrons from the nest dispensers on them, the experiment was conducted very early in the season (~3 weeks prior to normal bloom period). Early bloom development in potted crabapple trees and early emergence of overwintering O. cornifrons bees were facilitated by warming in the greenhouse and laboratory, respectively.
In the first part of the experiment, we measured primary transmission using potted crabapple trees and treated O. cornifrons nest dispensers. Crabapple trees were used rather than apples for use in the greenhouse because of the much heavier flower production on small potted trees. The potted crabapples trees (at 25–50% bloom, trial 1: n = 10, trial 2: n = 4) and the treated O. cornifrons nest dispensers (n = 2) were placed in an isolated grass area where there were no crops in bloom at the time. Consequently, we were able to isolate O. cornifrons bees on the only food source (i.e., crabapple flowers) around and observed no other bee species foraging on the crabapple trees at that time. Flowers in potted trees were closely observed and were harvested and placed in vials containing 5 mL of KPB solution immediately after O. cornifrons visitation. The experiment was repeated twice, and the flowers (n = 120) visited by treated O. cornifrons bees were collected and processed for counting CFUs of B. subtilis as per the protocol described in Experiment 2.
In the second part of this experiment, we measured the secondary transmission, and potted crabapple trees (i.e., primary exposure trees) that had been directly exposed to O. cornifrons bees carrying Serenade were immediately moved about 5 miles (~8 km) to an isolated location in a wooded area. All unopened blossoms on these trees were removed prior to transport so that only flowers that had been exposed to O. cornifrons with the Serenade product were left for the secondary transmission trial. Another set of potted crabapple trees at 25–50% bloom, which had never been exposed to bees or Serenade, were moved from the greenhouse and placed within 2 m of the primary exposure trees. The experiment was repeated twice (Trial 1 and 2) with a total of 10 potted trees of each treatment in the first replicate and 4 trees in the second. After 24 h of exposure to the new (untreated) O. cornifrons nests, flowers were collected at random from the previously unexposed trees and processed following the dilution and agar plating protocol of Experiment 2. A total of 120 flowers were collected, serially diluted 4 times and 480 plates were incubated for counting CFUs. Standard errors and means of each trial were calculated. Flowers (n = 5) collected from unexposed trees (n = 4) from the greenhouse prior to moving them to the grower field site were used as controls to make sure there was no prior contamination of the trees with the biological control product used in this experiment.