The occurrence of fire blight caused by Erwinia amylovora in apple and pear trees in Korea showed regularity in terms of newly infected orchards that were contaminated in the vicinity of already infected orchards. However, despite being a long distance from the infected orchards, fire blight made an unexpected appearance in many newly infected orchards. Therefore, the management of the spread of E. amylovora has been unsuccessful most likely due to its several modes of dissemination, including natural factors such as wind-driven rain (McManus and Jones, 1994) and honeybees (Choi et al., 2022a), and artificial factors such as the movement of workers and agricultural equipment (Choi et al., 2019; van der Zwet et al., 2012). Therefore, active management strategies must be developed, based on the current dispersal patterns in Korea, to prevent the spread of this pathogenic bacterium.

Commercial products including 12 antibiotics and 8 copper compounds, either as single antibiotics (streptomycin, oxytetracycline, oxolinic acid, and validamycin) and copper compounds (copper hydroxide, copper sulfate basic, tribasic copper sulfate, and copper oxychloride), or their mixtures have been registered by the Rural Development Administration of Korea (RDA) as active ingredients of commercial products for the fire blight control (Park et al., 2017). The use of antibiotics for plant protection has been either not permitted or prohibited in the European Union, United States, and Canada due to the increased risk of resistance problems and adverse environmental impacts (Fried et al., 2013; Mikiciński et al., 2020). However, Korea has permitted their use. The use of copper compounds is also limited because they only have a protective effect but not a curative effect, and also have a side effect of the formation of russet on the leaves and fruits of apples (Mikiciński et al., 2020). Thus, biological control agents (BCAs) developed using antagonistic bacteria effective against E. amylovora has garnered increasing attention as a potential resources of controlling the fire blight. To date, several BCAs, including the genera Pseudomonas: P. fluorescens A506 (Wilson and Lindow, 1993) and EPS62e (Cabrefiga et al., 2007), P. orientalis F9 (Zengerer et al., 2018), P. vancou-verensis L16, P. chlororaphis 3M, P. congelans 35M. P. protegens 59M (Mikiciński et al., 2020); the genera Pantoea: P. agglomerans E325 (Pusey, 2002) and P10c (Vanneste et al., 2002), P. vagans C9-1 (Ishimaru et al., 1988; Smits et al., 2010); the genera Bacillus: B. subtilis QST713 (Aldwinckle et al., 2002) and BD170 (Broggini et al., 2005). B. amyloliquefaciens LMR2 (Bahadou et al., 2018), MB40 (Shemshura et al., 2020), and FZB42 (Chen et al., 2009); Paenibacillus polymixa N179 (Fallahzadeh-Mamaghani et al., 2021); Lactobacillus plantarum (Roselló et al., 2013); Enterobacter sp. En113 and Serratia sp. Se111 (Sharifazizi et al., 2017); and the fungal BCA Aureobasidium pullulans (Kunz, 2006), have been reported to have potential use against fire blight worldwide. Among these, several BCAs have been commercialized as biopesticides, such as BlightBan A506 (P. fluorescens A506, USA), Bloomtime (P. agglomerans E325, USA), BlossomBless (P. agglomerans P10c, New Zealand), BlightBan C9-1 (P. vagans C9-1, USA), Serenade (B. subtilis QST713, USA), Biopro (B. subtilis BD170, Switzerland), and BlossomProtect (A. pulllans, Germany). In Korea, two biopesticides, Serenade (B. subtilis QST713, USA) and Serifel (B. amyloliquefaciens MBI600, USA) (Fravel, 2005), have recently been registered by the RDA for fire blight control.

However, the efficacy of biopesticides for fire blight control is controversial because their effects vary depending on the environmental conditions and disease severities in each orchard (Reininger et al., 2019). Therefore, in order to succeed in fire blight control using BCAs, it is necessary to intensive research on mechanisms of action and targets of BCAs will be required in Korea beside other countries. Biopesticides for fire blight control may be permitted in Korea if the novelty and biosafety of their microbial sources are proven. Recently, few studies have been conducted to develop domestic BCAs using antagonistic bacteria (Kim et al., 2022), lichen extracts (Lee et al., 2021), and bacteriophages (Park et al., 2018, 2022). Although these BCAs were developed domestically to en-sure originality, they came from a single source. To satisfy originality, in this study, we isolated antagonistic bacteria from different apple tissues, collected in Korea to satisfy originality, and tested their effectiveness in suppressing fire blight. Furthermore, cocktails of various antagonistic bacteria were investigated in order to maximize the effect of controlling fire blight.

In Korea, fire blight was first observed in 2015, has since remained a devastating disease in apple and pear orchards because the infected trees or orchards with >5% disease severity must be buried to reduce inoculum levels and prevent further spread of the disease. Moreover, it is not easy to erad-icate fire blight and it is likely to spread to more orchards, resulting in severe situation (Choi et al., 2022b). Commercial products, including 12 antibiotics and 8 copper compounds, either in single form or as mixtures have been registered by the RDA as commercial control agents against fire blight. The copper and antibiotic products are recommended for treating orchards in the winter and blooming seasons, respectively. However, these products are not preferred by cultivators owing to their low efficacy, at least in field conditions, their side effects such as inadequate fertilization and abscission of immature fruits, and public concerns over their use. Thus, BCAs should be considered as alternative control agents against fire blight in Korea. The increasing use of BCAs every year is evident in the total year end sale. For example, the annual sales of Serenade from 2014 to 2018 increased by 331, 338, 35, 792, and 1,561 million Korean Won, respectively. In 2021, the annual sales of Serenade were almost two-fold higher than that in 2018, and it is predicted to further expand in the future (personal communication with Dr. Oh, K. H. in Farmhannong Ltd.).

Bacillus spp. have the benefits ease of growth, low toxicity to plants, and production of long-living spores (Whipps and McQuiken, 2009). The five BCAs selected and identified as Bacillus spp. in this study may also have the aforementioned benefits. The strains KPB 15 and 39 were identified as B. amyloliquefaciens and B. subtilis, respectively (Fig. 2), corresponding to previously identified BCAs against fire blight (Aldwinckle et al., 2002; Bahadou et al., 2018; Broggini et al., 2005; Chen et al., 2009; Shemshura et al., 2020). In addition, KPB 21, 25, and 31, identified as B. stratosphericus, B. altitudinis, and B. safensis, respectively, demonstrate antagonism against E. amylovora in this study. To date, these three Bacillus species have been reported as plant biocontrol agents. For example, B. stratosphericus, which was isolated from zinc-contaminat-ed mine soils, showed inhibition against five plant pathogenic bacteria, Burkholderia glumae, Xanthomonas oryzae pv. oryzae, Pectobacterium carotovorum, Pseudomonas syringae, and Ralstonia solanacearum by catalyzing siderophore production in vitro (Durairaj et al., 2017). In addition, B. altitudinis and B. safensis have been suggested as potential plant growth promoting rhizobacteria and suppression of fungal diseases (Sunar et al., 2015), and can be used as biopesticides to control rice blast and oomycetes plant pathogens (Bibi et al., 2012; Rong et al., 2020), respectively. Among the isolated BCAs, the antagonism test showed that KPB 39 exhibited antagonism against KPB 21, 25, and 31, indicating B. subtilis as the dominant species in the soil and that several commercial biopesticides containing B. subtilis should not be mixed with other biopesticides.

The biocontrol efficacy of the five selected BCAs varied in both immature apple fruits and seedlings, but their abilities to reduce disease were more consistent in seedlings than that in immature fruits (Fig. 3A, B). In a previous report, several Bacillus species were identified as endophytes (Bibi et al., 2012), suggesting that the five selected BCAs may also be endophytes as they occupy the inner tissues of seedlings with more ease than in immature fruits. However, in the present study, we did not evaluate whether these BCAs are endophytes, and did not identify the active ingredients related to their control effects against fire blight. Therefore, future studies should analyze the active ingredients such as antibiotics, enzymes, and peptides, via genetic analyses to elucidate the mechanism underlying their antagonism against E. amylovora. Nevertheless, KPB 25 and 31, individually and in a cocktail, showed the greatest reduction in lesion size on immature fruits and consistent effect in seedlings with no antagonism against the other four BCAs, and were finally chosen as novel single and mixed cocktail BCAs. These strains show promising action against E. amylovora and can be substitutes to imported BCAs, such as Serenade and Serifel.