RejuAgro A as an antimicrobial for fire blight control of pome fruits and beyond

Apple (Malus domestica) is an important fruit commodity worldwide and is cultivated in nearly 100 countries on six continents. In 2020, the global apple yield reached 86.4 million tons with a planting area of 4.6 million hectares¹. In the United States, apples are the second most consumed fruit after bananas, with an estimated production of 5 million tons in the 2023–2024 crop year (Industry Outlook 2023, USApple). Global pear (Pyrus communis, P. pyrifolia) production in 2022 was 26.5 million tons, with China growing over half of the world’s supply². The United States ranked second with 0.58 million tons.

Fire blight is a disease that severely hampers apple and pear production and is prevalent in all growing regions of the United States as well as in Europe, Central Asia, the Middle East, New Zealand, South Korea, and China^3,4,5,6,7,8. Fire blight is caused by the bacterial pathogen Erwinia amylovora, and this disease not only leads to decreased yields but can also cause tree mortality, thereby severely impacting production⁹. The pathogen E. amylovora grows epiphytically on flowers before infecting the flower base^10,11,12. Consequently, the primary focus of fire blight management has involved limiting pathogen colonization through the application of antibiotics (in the U.S. and Asia) or copper (in Europe)^13,14. Streptomycin sulfate and oxytetracycline hydrochloride are the primary antibiotics deployed in the battle against fire blight. However, the significant importance of antibiotics in human medicine adds complexity to their application in plant agriculture. Concerns include increased risks of developing and spreading antibiotic resistance among bacteria, antibiotic residues in produce, and environmental issues like soil and water pollution, which may prompt future regulatory actions^14,15,16,17. Kasugamycin is a third antibiotic and is registered only for use in plant agriculture, where it has been used for fire blight management in the U.S. since 2014¹⁸. Disease control efficacy of the alternate bactericide copper is significantly lower than that of antibiotics, and copper use is also limited due to its potential to cause phytotoxicity, such as fruit russeting¹⁹. Streptomycin-resistant E. amylovora strains are present in almost all major pome fruit-producing regions in the U.S., including California, Michigan, Washington, and New York^{16,20,21,22,23,24}, and strains of E. amylovora with resistance to oxytetracycline were recently isolated in California²⁵. McManus²⁶ reported that streptomycin use for fire blight does not significantly affect bacterial community structure or increase resistance genes²⁶. However, the presence of streptomycin-resistant isolates in apple orchards raises concerns about the potential transfer of resistance genes to human pathogens²⁷. As highlighted in a recent review²⁸, resistance to streptomycin and other antibiotics has reduced their utility in many regions, prompting renewed attention to the risks of resistance development and emphasizing the need for effective alternatives.

In this research, we identify an antimicrobial compound, RejuAgro A (RAA), produced by the bacterium Pseudomonas soli 0617-T307, isolated from soil in Wisconsin, U.S. RAA demonstrates strong antimicrobial activities against all tested E. amylovora strains, including those resistant to streptomycin. Our field experiments show that RAA effectively reduced the incidence of fire blight in several trials to a level comparable to streptomycin and kasugamycin. Mechanistic investigations demonstrate that RAA functions as an inhibitor, disrupting RNA, DNA, and protein synthesis in target pathogens. The chemical structure of RAA and the gene cluster encoding RAA biosynthesis are characterized. We also determined that RAA is effective against a wide range of phytopathogenic bacteria and fungi, suggesting its potential application in the management of various plant diseases.

Identification of Pseudomonas soli 0617-T307

To discover antimicrobial compounds effective against E. amylovora, we isolated over 40,000 bacteria from a wide range of soil samples. These samples were collected from diverse natural settings throughout Wisconsin, encompassing forests, lake shores, and marshlands. A culture extract from each isolate was obtained using ethyl acetate and was tested for antibiosis activity against the E. amylovora strain Ea110. This effort led to the discovery of bacterial isolate 0617-T307, whose culture extract displayed strong inhibition of E. amylovora growth in vitro (Fig. 1a). In addition, co-culturing E. amylovora with isolate 0617-T307 resulted in significant inhibition of E. amylovora growth (Fig. 1b). Bacterium 0617-T307 was identified using a multifaceted approach. Firstly, based on multilocus sequence analysis utilizing the 16S rRNA, gyrB, rpoB, and rpoD genes, 0617-T307 was classified as a member of Pseudomonas soli²⁹. Secondly, a phylogenetic sequence analysis of the aforementioned genes was performed, showing that 0617-T307 and other P. soli strains formed a strongly supported monophyletic clade and were grouped under the P. putida group (Supplementary Fig. 1). Finally, whole genome sequencing revealed that 0617-T307 shared 88.3% coverage and 95.3% average nucleotide identity (ANI) with the P. soli type strain (Supplementary Table 1). These values are above the guidelines of 70% coverage and 95% ANI suggested for delineating prokaryotic species³⁰, confirming that 0617-T307 is a P. soli strain (P. soli 0617-T307 hereafter). P. soli 0617-T307 strain has been deposited in the American Type Culture Collection (ATCC) with the accession number PTA-126796.

Discovery of an antimicrobial compound produced by P. soli 0617-T307

To identify antimicrobial compounds produced by P. soli 0617-T307, the bacterial culture supernatant was extracted with ethyl acetate. The resulting crude extract was then separated using a silica gel column, yielding eight fraction groups (Fig. 2a). Among these, group 3, fraction F29-42, exhibited potent antimicrobial activity against E. amylovora. To further isolate and purify the active components in F38-40 (a narrowed-down fraction of F29-42), we employed preparative High-Performance Liquid Chromatography (HPLC) utilizing a C18 column (Fig. 2a). Antagonistic activity against E. amylovora strain Ea110 was evident (Fig. 2b).

The active compounds were further characterized using high-resolution mass spectrometry (HR-MS), which revealed a dominant compound with a molecular formula of C₇H₇NO₃S and a molecular weight of 185.2 Da (Fig. 2c). This purified compound inhibited the growth of E. amylovora in vitro (Fig. 2b). The structure of this compound was successfully determined using X-ray crystallography. The compound comprises 7 types of carbon groups, including three types of carbonyl, two types of tertiary carbons, and two types of methyl carbons (Fig. 2c). We have characterized this compound and named it RejuAgro A (RAA).

RAA displays potent antimicrobial efficacy against E. amylovora at a comparable level to streptomycin

To assess the antimicrobial potency of RAA, the minimum inhibitory concentration (MIC) of HPLC-purified RAA was determined for several E. amylovora strains. The MIC for the less virulent strain Ea1189 and the highly virulent strain Ea110^31,32 was determined to be 5 µg/mL, which is comparable with that of streptomycin (Table 1). Additionally, RAA was equally effective in inhibiting the growth of three streptomycin-resistant strains (CA11, DM1, and Ea88)²¹ with a MIC of 10 µg/mL (Table 1). These findings suggest that RAA is a highly effective antimicrobial that displays similar efficacy as streptomycin against E. amylovora.

We determined that RAA also exhibits high potency against a broad spectrum of other bacterial as well as fungal plant pathogens. Among the bacterial pathogens tested, RAA is particularly effective against Xanthomonas and Ralstonia species, with MICs comparable to or lower than those of streptomycin. For example, MIC values for the citrus canker pathogen X. citri pv. citri and the bacterial spot pathogen of tomato X. campestris pv. vesicatoria XV-16 were 5 and 2.5 µg/mL, respectively (Supplementary Data 1), whereas MICs for strains of the bacterial wilt pathogen R. solanacearum ranged from 3.1 to 6.3 µg/mL. The activity of RAA in suppressing the growth of other Gram-negative bacterial phytopathogens, including soft rot pathogens like Pectobacterium and Dickeya species, was also comparable to that of streptomycin. Moreover, RAA was highly potent against Gram-positive phytobacteria, with MIC values for the tomato canker pathogen Clavibacter michiganensis ranging from 1.6 to 12.5 µg/mL. RAA inhibited the growth of P. savastanoi pv. savastanoi and three P. syringae pathovars at concentrations of 10–40 µg/mL, which are higher than those for streptomycin (Supplementary Data 1). For Oomycota and fungal plant pathogens, strong inhibitory effects were observed for Phytophthora infestans and Venturia inaequalis at 40 and 80 µg/mL, respectively (Supplementary Data 1). Unlike streptomycin, RAA is a potent, broad-spectrum antimicrobial effective against both bacterial and fungal plant pathogens. Antibiotics are typically defined as substances produced by or derived from microorganisms that inhibit or kill bacteria, whereas antimicrobials encompass agents active against bacteria, fungi, and other pathogens. RAA is thus referred to as an antimicrobial to reflect its broader spectrum of activity. This dual action makes RAA a versatile option for managing a wide range of plant diseases.

Assessing the efficacy of RAA in controlling fire blight in field trials

To assess the effectiveness of RAA against fire blight under field conditions, trials were conducted over two seasons under various climates in California, Connecticut, Michigan, and New York, each utilizing locally adapted apple or pear cultivars and cultivation practices. In the California trial on pears in 2023, disease pressure was low with a natural incidence of 4.1% of blighted flower/fruitlet clusters. RAA at 20 ppm was similarly effective as kasugamycin (both 0.3% incidence). In 2024, under higher disease pressure, the disease was reduced from 35.2% in the untreated control to 12.4% incidence using RAA at 30 ppm (Table 2). In the apple trials, between 64% and 85% of untreated flowers exhibited symptoms of fire blight. In contrast, infection of flowers treated with the antibiotics streptomycin or kasugamycin at a standard rate of 100 ppm was effectively suppressed to 9 to 32.2%. RAA, when applied at 20 ppm or higher, consistently led to significant suppression of fire blight (7–33%; Table 2). For example, in four of the five trials (2022 CT, 2023 CT, 2023 NY, and 2023 CA), the efficacy of RAA at 20 ppm was similar to that of the antibiotic controls at 100 ppm. In the 2023 NY trial, RAA at 20 ppm (7% incidence) even surpassed the performance of streptomycin (18% incidence) in fire blight suppression. These results highlight the consistent disease suppression of RAA in different field locations with different environmental and growing conditions, suggesting that RAA is an alternative to antibiotics in fire blight management.

Identification of the RAA biosynthesis gene cluster in P. soli

To identify genes responsible for RAA biosynthesis, the genome of P. soli 0617-T307 was analyzed for gene clusters potentially involved in secondary metabolite biosynthesis using AntiSMASH³³. This analysis led to the identification of 10 putative secondary metabolite biosynthetic gene clusters (BGCs 1–10) (Supplementary Table 2). Among them, BGCs 3–8 and 10 are predicted to function in synthesizing previously characterized antimicrobials.

To pinpoint which BGCs are involved in RAA biosynthesis, mutations were generated in the core biosynthesis genes of BGC 1, BGC 2, BGC 7, BGC 8, and BGC 9 (Supplementary Fig. 2a). Results indicated that mutation of BGC 9 abolished RAA production. However, mutations in BGC 1, 7, and 8 did not affect RAA production, although the mutation in BGC 2 showed a slightly reduced production of RAA (Supplementary Fig. 2b). BGC 9 consists of 31 genes, including an operon that contains the core predicted biosynthesis gene and five additional genes. These six genes, designated as ras1-6, stand for RAA biosynthesis genes and encode hydroxymethylglutaryl-CoA synthase (encoded by the predicted core biosynthesis gene), enoyl-CoA hydratase, asparagine synthase, NAD(P)/FAD-dependent oxidoreductase, acyl-CoA dehydrogenase, and Paal family thioesterase, respectively (Fig. 3a). Next, to determine which ras genes are responsible for RAA biosynthesis, single deletion mutants were constructed. The mutation of ras1, ras3, ras4, and ras6 led to a complete abolishment of RAA production (Fig. 3b and Supplementary Fig. 3), suggesting that these genes are essential for RAA biosynthesis. Additionally, the cell-free culture supernatant extract from the ∆ras1 strain lacked inhibitory activity against E. amylovora strain Ea110 (Fig. 1a). Moreover, co-culturing Ea110 with ∆ras1 resulted in no inhibition of E. amylovora growth (Fig. 1b). Single deletion mutations of ras2 or ras5 resulted in partial reductions in RAA production, yet double mutation of ras2 and ras5 led to the complete abolishment of RAA synthesis (Fig. 3b). This observation suggests that Ras2 and Ras5 contribute to the biosynthesis of RAA with overlapping functions. It should be noted that the more substantial reduction in RAA production in ∆ras5, as opposed to ∆ras2, indicates the dominant role of Ras5 in RAA biosynthesis. Finally, the altered phenotypes in RAA biosynthesis observed in ras mutants could be restored through the complementation of selected genes (Fig. 3c and Supplementary Fig. 3), which confirms their roles in RAA biosynthesis.

The biosynthesis of RAA has two steps, with RAB being an intermediate

During the HPLC analysis of extraction fractions, we identified a compound from fraction F43-56 comprising two symmetrically independent structures, each resembling RAA (Fig. 2a, c). This compound, with a molecular formula of C₁₂H₈N₂O₆ and a molecular weight of 276.2 Da, was named RejuAgro B (RAB) due to its potential role as an intermediate in RAA biosynthesis. Unlike RAA, RAB did not inhibit the growth of E. amylovora (Fig. 2b and Supplementary Fig. 4).

To determine whether RAB is a potential intermediate during the biosynthesis of RAA, HPLC and LCMS analyses were conducted to determine whether the production of RAB is affected by the mutation of various ras genes. Our results showed that RAB was not detected in the ∆ras2∆ras5 double mutant but was detected in wild type, ∆ras2, and ∆ras5 strains (Fig. 4a, b), suggesting that Ras2 and Ras5 are both involved in RAB biosynthesis and their functions are likely redundant. Adding RAB to the culture medium did not affect RAA production in the WT but partially restored the reduced RAA production in ∆ras2 at 20 ppm, and in ∆ras5 and ∆ras2∆ras5 at both 5 ppm and 20 ppm (Fig. 4c–f). This confirms that Ras5 has a more dominant role in RAA biosynthesis than Ras2. RAB production was not detected in ∆ras1, ∆ras3, ∆ras4, and ∆ras6 (Fig. 4a and Supplementary Fig. 5), and supplementation of RAB did not restore RAA production in these strains (Supplementary Fig. 6).

The experimental evidence obtained from genetic deletion studies indicates that RAA biosynthesis proceeds through a coordinated enzymatic cascade involving the ras1–ras6 gene products, with RAB serving as an intermediate in the pathway. The proposed biosynthetic mechanism involves a sequential multi-enzyme process with defined functional roles: The initial steps likely involve Ras2 (enoyl-CoA hydratase) and Ras5 (acyl-CoA dehydrogenase), which catalyze the modification of acyl-CoA substrates to generate RAB as an intermediate. Subsequently, RAB undergoes further enzymatic processing through the downstream enzymes. Ras1 (hydroxymethylglutaryl-CoA synthase) likely facilitates structural modifications to the RAB scaffold, while Ras3 (asparagine synthase) may mediate additional nitrogen incorporation or structural rearrangements. Ras4 (NAD(P)/FAD-dependent oxidoreductase) appears to catalyze redox reactions necessary for pathway progression, and Ras6 (Paal family thioesterase) is predicted to perform the final hydrolytic step to release RAA or its immediate precursor (Fig. 4g).

Antimicrobial mechanism of RAA against E. amylovora

Macromolecular synthesis assays demonstrated that RAA at 2× MIC (20 μg/mL) simultaneously inhibits DNA, RNA, and protein synthesis in E. amylovora 1189, as evidenced by decreased incorporation of [³H]-thymidine, [³H]-uridine, and [³H]-leucine, respectively (Fig. 5a, c, e and Supplementary Fig. 7). This broad inhibitory profile resembles that of the natural compound allicin, suggesting similar mechanistic properties in thiol reactivity³⁴. Concurrent viability measurements revealed progressive bactericidal activity by reducing bacterial survival to below 50% within 60 min post-treatment (Fig. 5b, d, f).

To investigate potential thiol-reactive properties, glutathione (GSH) supplementation experiments were conducted. Results demonstrated dose-dependent protection against RAA-mediated growth inhibition, with 5 mM GSH significantly restoring bacterial viability to approximately 80% of untreated controls (Fig. 6). Importantly, GSH supplementation failed to mitigate the antimicrobial effects of conventional antibiotics (tetracycline, ciprofloxacin, rifampicin) at equivalent concentrations (Supplementary Fig. 8), indicating specificity in the protective effect.

Two-dimensional principal component analysis (PCA) confirmed that RAA possesses unique structural properties, placing it in a distinct chemical space compared to established classes of antibiotics (Supplementary Data 2 and Supplementary Fig. 9). Collectively, these findings suggest that the antimicrobial activity of RAA is distinct from that of conventional antibiotics.

The discovery of RAA offers potential perspectives and solutions for plant disease management and could significantly impact agricultural activities. The current widespread use of the same antibiotics to treat human, animal, and plant diseases could shrink the pool of effective treatment options, emphasizing the need for alternative management tools. To reduce resistance development to antibiotics that are used in human medicine, it is imperative that the plant agricultural sector gradually reduces its reliance on commonly used antibiotics such as oxytetracycline and streptomycin for the management of bacterial diseases. This strategic shift is essential for preserving the efficacy of these critical drugs for future generations, ensuring their continued effectiveness in human medicine. In light of this, RAA emerges as an alternative approach to managing plant diseases. It exhibits effective antimicrobial activity against bacterial, Oomycota, and fungal phytopathogens (Table 1 and Supplementary Data 1), and thus, has broad-spectrum efficacy. RAA also exhibits antimicrobial activity against bacterial strains of medical relevance, with MICs of 100, 250, 100, and 150 µg/mL against Escherichia coli O157:H7, Pseudomonas aeruginosa PAO1, Salmonella Enteritidis (strain 155350A, Carolina Biological Supply, USA), and Mycobacterium smegmatis (strain 155180A, Carolina Biological Supply), respectively. Given its multi-target antimicrobial mechanism, its occupation of a distinct chemical space apart from conventional antibiotics, and its resemblance to established thiol-reactive compounds used in human therapeutics (e.g., auranofin and allicin), RAA may hold promise beyond agriculture. This compound not only has the potential to provide a sustainable and effective solution for managing plant diseases but can also have a crucial role in preserving the effectiveness of conventional antibiotics for human health applications.

In this research, RAA demonstrated efficacy in managing fire blight in field trials, with the notable advantage of requiring a lower dosage compared to streptomycin. The molecular weight of RAA is 185.2 Da, which is lower than that of streptomycin with a molecular weight of 581.6 Da. The smaller molecular size potentially enhances the ability of RAA to penetrate plant tissues more effectively, offering an advantage in its distribution within the plant system for improved disease control. Physicochemical properties of compounds such as the pKa and the lipid-water partition coefficient (log Kow, a marker of polarity or membrane permeability) significantly influence their translocation through the cuticle when applied to flowers^35,36. RAA with a pKa of 7.76 and a log Kow of 1.5 likely has improved translocation ability compared to streptomycin (pKa of 10 and log Kow of −7.5). The higher lipophilicity and partial non-ionized state of RAA suggest better penetration through the waxy plant cuticle and more efficient movement across cell membranes. Additionally, the higher lipophilicity of RAA, likely resulting in superior penetration, cellular uptake, and adherence to plant surfaces, suggests potential for effective control of fire blight at lower doses.

The diminished effectiveness and agricultural dependence on conventional antibiotics have heightened concerns about environmental antibiotic resistance, posing risks to environmental and human health^14,17. Currently, available alternatives include copper products, hydrogen peroxide, peroxyacetic acid, sulfur, and essential oils, as well as biological control agents^37,38. However, these options are limited due to variable efficacy and the risk of phytotoxicity^4,39,40,41. The exclusion of streptomycin and other antibiotics from organic cultivation in the U.S. highlights the need for effective alternative treatments, steering organic producers toward biological control strategies. Biological control agents such as microorganisms antagonistic to E. amylovora can protect against infection^{42,43,44,45,46,47,48,49,50,51,52,53,54,55}, but the performance of biocontrol agents is inconsistent across U.S. regions, with generally better efficacy in the West compared to the East. For instance, BlightBan A506 significantly reduced fire blight by 40 to 80% in the Pacific Northwest over a 6-year period, whereas in the Eastern U.S., it only resulted in a 9.1% reduction³⁹. Bloomtime Biological E325 also displayed variable control efficacy across different states. These inconsistencies emphasize the challenges of biocontrol agents relative to traditional antibiotics³⁷. Blossom Protect, a commercial biological control product composed of two strains of the yeast Aureobasidium pullulans, has shown effectiveness in managing fire blight, particularly within organic production systems in both western and eastern apple orchards^38,56. However, our compatibility tests revealed that RAA at 20 and 30 ppm inhibits A. pullulans; therefore, compatibility with biological control agents for the management of blossom blight requires further investigation.

Over 3 consecutive years, field studies were conducted in Connecticut, Michigan, New York, and California, covering both the Western and Eastern U.S. These studies included a range of local apple and pear cultivars to provide a comprehensive assessment of RAA performance under diverse conditions. The incidence of fire blight in the Western U.S., exemplified by California, is highly variable depending on environmental conditions, especially temperature, in a particular season, and rattail bloom contributes to frequent disease outbreaks, making fire blight a serious annual disease problem. The Eastern U.S., with a humid environment, has conditions more consistently favorable for the spread of fire blight. The effectiveness of RAA in controlling fire blight across these different climates and pome fruit cultivars highlights its adaptability for good efficacy. In the 2023 New York field trial, RAA with an infection rate of 7% demonstrated superior effectiveness in suppressing fire blight, compared to 18% for streptomycin, despite being used at a significantly lower concentration (20 versus 100 ppm). This finding is particularly relevant considering that strains of streptomycin-resistant E. amylovora were consistently identified in apple-producing regions across New York²², underscoring the potential of RAA as a more effective alternative in areas where streptomycin is failing. We further tested whether citric acid can promote the efficacy of RAA in controlling apple fire blight. The 2024 field data in New York at Cornell AgriTech in Geneva showed similar efficacy in fire blight control with or without supplementation with citric acid (Supplementary Table 3). Additionally, our 2023 and 2024 field assays in New York evaluated RAA for controlling apple scab caused by the fungal pathogen V. inaequalis (Supplementary Tables 4 and 5). Due to the resistance of V. inaequalis to conventional fungicides, managing apple scab typically requires a combination of treatments. In our assays, RAA was applied as a major component of integrated management programs, and while RAA showed efficacy, all treatments included multiple components. Further field trials using RAA alone are necessary to assess its standalone efficacy.

We have identified the BGC of RAA by constructing insertion mutants according to the AntiSMASH prediction. In P. soli 0617-T307, BGC 9 was found to be essential for RAA synthesis, as deleting the ras1, ras3, ras4, or ras6 genes within the BGC completely abolished RAA production. Despite BGC 9 being categorized as a type III polyketide synthase (T3PKS) gene cluster, the specific functions of its constituent genes remain largely unexplored, complicating the current efforts to delineate the biosynthetic pathway of RAA. Nonetheless, the identification of another compound, RAB, provides clues about how RAA is being synthesized. RAB is composed of two linked RAA molecules but lacks the thiomethyl group (-SCH₃) of RAA. RAB is an essential intermediate for RAA synthesis because RAA production can be rescued when RAB is supplied in ∆ras2∆ras5. This suggests that the larger RAB molecule is first synthesized, followed by modifications by other enzymes in the RAA biosynthesis pathway. The complete abolishment of both RAA and RAB production observed in Δras1, Δras3, Δras4, and Δras6 mutants suggests that ras1, ras3, ras4, and ras6 might be involved in either RAB biosynthesis or the conversion of RAB to RAA. However, no RAA production in these mutants when supplied with exogenous RAB indicates that these genes are most likely not responsible for RAB biosynthesis, but rather play roles downstream in the pathway to convert RAB to RAA. The failure to rescue RAA production with RAB supplementation in mutants lacking these essential genes confirms the sequential nature of the biosynthetic pathway, wherein disruption of any downstream enzymatic step prevents final product formation. This proposed biosynthetic pathway represents a working model derived from genetic deletion studies and bioinformatic predictions; however, direct enzymatic characterization and identification of all pathway intermediates will be required to fully elucidate and validate the complete biosynthetic mechanism.

RAA demonstrates a multi-target inhibitory profile against E. amylovora, simultaneously suppressing RNA, DNA, and protein synthesis pathways. The observed protective effects of GSH against RAA’s antimicrobial activity suggest potential involvement of a thiol-reactive mechanism. This reactivity pattern parallels that of established natural antimicrobials, such as allicin from garlic (Allium sativum), which irreversibly modifies cysteine thiols via sulfenic acid intermediates, forming mixed disulfides and disrupting multiple enzymatic pathways in bacterial cells^34,57. Similarly, auranofin, a synthetic gold(I)-containing compound inspired by natural organogold chemistry, exerts antimicrobial effects by irreversibly binding to selenocysteine residues in thioredoxin reductase, a critical enzyme in bacterial redox homeostasis^58,59. These precedents highlight the regulatory viability of thiol-reactive antimicrobials, with auranofin approved for rheumatoid arthritis and repurposed for antimicrobial use⁵⁹, and allicin recognized as a food-safe antimicrobial (GRAS status)³⁴. While this study did not address resistance mechanisms to RAA, the possibility of tolerance development through spontaneous mutations remains a consideration. In related Pseudomonas species, resistance pathways such as efflux pumps and enzymatic detoxification could theoretically contribute to tolerance^60,61. Although GSH was shown to attenuate RAA’s antimicrobial activity, suggesting that resistance could theoretically arise through inactivation of its thiol-reactive group, the multi-target mode of action of RAA makes the development of stable resistance less likely compared to conventional antibiotics. RAA thus represents an agricultural antimicrobial with a mechanism rooted in natural product-based thiol reactivity⁶², offering potential regulatory advantages through established mechanistic precedents.

In summary, the introduction of RAA signifies a notable development in agriculture, introducing a fresh perspective on disease management. This advancement brings additional possibilities for improving crop protection and sustainability. Biochemical research and comprehensive field studies have demonstrated the efficacy of RAA in plant disease management. These findings suggest that RAA could be a valuable addition to the toolbox for managing diseases of specialty crops.

Microbial strains, plasmids, primers, and media

Microbial strains and plasmids used in this study are listed in Table 1, Supplementary Data 1 and 3. E. amylovora strains, P. soli, P. savastanoi pv. savastanoi, P. syringae strains, R. solanacearum, Dickeya and Pectobacterium strains, C. michiganensis strains, X. campestris strains, and X. arboricola pv. juglandis were grown in Luria Bertani (LB) broth (tryptone, 10 g/L; yeast extract, 5 g/L; NaCl, 10 g/L) at 28 °C. X. citri pv. citri strains were grown in NA (nutrient broth) medium (beef extract, 3 g/L; yeast extract, 1 g/L; polypeptone, 5 g/L; and sucrose, 10 g/L) at 28 °C. Escherichia coli O157:H7, P. aeruginosa PAO1, and Salmonella Enteritidis (strain 155350A, Carolina Biological Supply, USA) were grown in LB media at 37 °C. Mycobacterium smegmatis (strain 155180A, Carolina Biological Supply) was grown in LB supplemented with 0.15% (v/v) glycerol (Fisher Scientific, United Kingdom) and 0.10% (v/v) Tween-80 (Fisher Scientific, United Kingdom) at 37 °C⁶³. Fermentation of P. soli 0617-T307 was conducted in YM (yeast extract, 4 g/L and malt extract, 10 g/L) or YME medium (yeast extract, 4 g/L; glucose, 4 g/L; and malt extract, 10 g/L) at 16 °C. Venturia inaequalis was grown on PDA (potato dextrose agar: potato infusion (infusion from 200 g potatoes), 4 g/L; dextrose, 20 g/L; agar, 15 g/L), while P. infestans was grown on RYE medium (dry rye berries, 60 g/L and sucrose, 20 g/L) at room temperature (22 °C). Oligonucleotide primers used for cloning are listed in Supplementary Data 4.

Genome sequencing, assembly, and annotation

High molecular weight genomic DNA of P. soli 0617-T307 was extracted, and the quality of the obtained DNA was checked by spectrophotometry at the Next Generation Sequencing Core (UW-Madison, Madison, WI). Genome sequencing was conducted on the Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio) HiFi platforms. ONT facilitated the assembly of genomes with reads ranging from 50 to 120 kb in length. Consensus error corrections on the genomes and/or additional extrachromosomal elements were performed with PacBio reads at 8–14 kb size that were mapped against the assembly created from ONT reads. Genome sequencing resulted in a total of one single circular contig with a length in the 1+ MB range. For genome assembly and annotation, the polished contigs were compared against a BRC-curated subset of NCBI Prokaryotic RefSeq and GenBank accessions. This involved using a custom database of prokaryotic sequences constructed by UW-Madison, sourced from NCBI on January 27, 2020. The five best matches are sorted by a BLAST+ v2.8.0 bit score (blastn). A comparative analysis of the assembled contig and the highest-scoring NCBI match was made using MUMmer4⁶⁴. Each contig × NCBI reference MUM comparison was filtered, requiring an exact match length of at least 2 kb. The dotplot was generated with MUMmer4 by computing maximal exact matching, match clustering, and alignment extension between the contig and the single best-match NCBI sequence. The assembly features of the polished assembly were depicted by CIRCOS⁶⁵. Annotation of coding regions (genes on forward and reverse strands), including ORFs, was determined by PROKKA⁶⁶. The boundary was defined by the gene dnaA, a protein that activates the initiation of DNA replication in nearly all bacteria (the genes dnaN and gyrB are usually associated with dnaA)⁶⁷. A low error-rate assembly was generated after three error-type corrections. The complete genome was deposited in GenBank with accession number CP151184 (BioProject accession: PRJNA1094439).

Species identification

For species identification, the genome sequences of representative Pseudomonas species were obtained from the NCBI RefSeq database. The marker genes were parsed from the genome sequences and analyzed according to the guidelines established for Pseudomonas²⁹. The procedure for molecular phylogenetic analysis was based on the approach described previously⁶⁸. Briefly, the multiple sequence alignment was performed using MUSCLE v3.8.31, and the maximum likelihood phylogeny was inferred using PhyML v3.3.20180621. The genome-wide ANI was calculated using FastANI v1.1³⁰.

Construction of deletion, insertion, and complementation strains

Deletion mutants Δras1 to Δras6 were generated using a double cross-over gene knock-out method adapted from Huang and Wilks⁶⁹. Sequences flanking ras1 at 714 bp upstream and 910 bp downstream were amplified by PCR using primers XbaI-ras1-UF/ras1UR and ras1-UF/EcoRI-ras1-DR (Supplementary Data 4), respectively. The two fragments were fused by overlapping PCR and cloned into a suicide plasmid pEX18-Gm with restriction sites of XbaI and EcoRI. The construct was first transformed into E. coli S17-1 and conjugated with P. soli 0617-T307 on an LB agar plate at 28 °C. The cells on the plate were then rinsed off with 0.9% NaCl solution and spread on a selection plate with gentamicin (50 µg/mL) and carbenicillin (100 µg/mL) at 28 °C for 2 days. P. soli 0617-T307 is naturally resistant to carbenicillin, while S17-1 is not. The selection pressure of gentamicin forced the integration of the plasmid into the genome through homologous recombination (first homologous recombination) at the ras1 upstream location. The positive clone was selected and incubated on YM medium with 12% sucrose for the second homologous recombination that forced the excision of the plasmid sequence. Due to the high G-C content in the P. soli 0617-T307 genome, the upstream and downstream primers were designed for efficient PCR to lower the annealing temperature by covering 15 bp upstream and 17 bp downstream sequences of ras1. Deletions of other ras genes were carried out in the same manner, and the design for upstream and downstream fragments (bp) is listed in Supplementary Data 4 and Supplementary Table 6. The Δras2Δras5 double mutant was made by deleting ras5 from the chromosome of mutant Δras2. The deletion construct pEX18-GmR-ras5 was delivered to E. coli S17-1 and transferred to mutant Δras2 by bi-parental mating. The deletion of targeted genes in all mutants was confirmed by PCR and DNA sequencing. A detailed procedure for making insertion mutants for BGC 1, 2, 7, 8, and 9 is provided in Supplementary Method 1.

Complementation of mutants was done by cloning the gene back to its original location through homologous recombination. The sequences upstream and downstream of the target gene used in the knock-out method, along with the target gene sequence, were amplified by PCR using XbaI-target gene-UF and EcoRI-target gene-DR (Supplementary Data 4). The amplified sequence was cloned into the suicide vector pEX18-GmR with the restriction sites XbaI and EcoRI. Subsequent steps of homologous recombination in the deletion mutant strain were done as described in the knock-out method. The complementation of mutants was confirmed by PCR and DNA sequencing.

Field trials

Over 3 years, field trials were conducted in California, Connecticut, Michigan, and New York, utilizing local spray application tools on regional apple or pear cultivars. Trials employed a randomized complete block design with 3–11 replicate trees per treatment, depending on location. In all field assays, the surfactant Regulaid was added to RAA. Treatments were applied at critical bloom stages (70–100% bloom) using calibrated backpack or motorized sprayers. Trees were inoculated with E. amylovora strains (Ea110, Ea273, or MASHBO) at concentrations of 1 × 10⁶ CFU/mL during bloom. Untreated or water controls were applied as negative controls. Streptomycin or kasugamycin at a concentration of 100 ppm were applied as positive controls. RAA treatments were performed at concentration 5, 10, 20, and 30 ppm, respectively. Disease incidence was assessed 3–6 weeks post-inoculation by evaluating 50–300 flower clusters per tree, depending on tree age and size. Detailed protocols for each location and year are provided in Supplementary Method 2. The detailed protocol for the management of apple scab by RAA treatments is provided in Supplementary Method 2 as well. All percentage data were subjected to arcsine square root transformation prior to analysis. Data were assumed to be normally distributed and to have equal variation across groups. Multiple comparisons for significance were determined using one-way ANOVA followed by a two-sided Tukey–Kramer test, which adjusts for multiple comparisons while accommodating unequal group sizes. Data were analyzed by IBM SPSS Statistics (Version 31.0.0.0).

Fermentation and compound extraction

P. soli 0617-T307 was grown in 500 mL of YME medium at 28 °C for 24 h. Subsequently, the seed culture was inoculated into a 20-L fermenter (BioFlo IV, New Brunswick Scientific Co., NJ) containing 12 L of YME medium. The fermentation proceeded at 16 °C for 24 h. The agitation speed and the airflow rate were 200 rpm and 2 L/min, respectively.

Bacterial metabolites were extracted with ethyl acetate. The organic layer was separated and dried using sodium sulfate and rotary-evaporated at 35 °C. Metabolites were then resuspended in 20 mL of methanol, and the methanol was evaporated in a fume hood. This resulted in 2.9 g of crude extract per 12-L culture.

Compound separation and antimicrobial activity identification

The crude extract was dissolved in acetone and mixed with silica gel, which was loaded onto a silica gel column (φ3.0 × 20 cm) on a flash chromatography system (Yamazen AI-580) equipped with a UV detector. The sample was eluted with 280 mL of each of the following solvents in order with increasing polarity: 100% hexane, 75% hexane/25% ethyl acetate, 50% hexane/50% ethyl acetate, 25% hexane/75% ethyl acetate, 100% ethyl acetate, 50% ethyl acetate/50% acetone, 100% acetone, and 100% methanol at a flow rate of 20 mL/min. The eluate was monitored at UV 254 nm, fractions were collected in time-dependent mode at 20 mL/tube, and 114 fractions/tubes (F1-114) were generated.

The fractions were used in antimicrobial plate assays. Aliquots of 1 mL of each fraction were first vacuum evaporated using a vacuum concentrator (Eppendorf, Enfield, CT) and re-dissolved in 50 µL DMSO. To test the antimicrobial activity, overnight cultures of E. amylovora were diluted 1:100 with water (~10⁸ CFU/mL), spread onto LB agar plates, and 2 µL of each re-dissolved fraction was added equidistantly. DMSO alone was used as a negative control. The plates were then incubated at 28 °C for 24 h, and the presence or absence of inhibition zones was observed.

The flash chromatographic fractions containing RAA (F38-40) and RAB (F50-54) were subjected to prep-HPLC purification on an Agilent C18 column (2.12 × 25 cm, 3.5 μm) with mobile phase A: Water with 0.1% formic acid, and mobile phase B: Methanol with 0.1% formic acid. The flow rate was 8.0 mL/min. The eluate was monitored at 254 nm using a DAD detector. For RAA, the gradient program 40–100% B in 19 min was used. RAA was eluted at Rt 17.5 min. For RAB, the gradient program 20 to 60% B in 10 min was used. RAB was eluted at Rt 10.5 min.

RAA and RAB characterization

RAA and RAB crystals were obtained through slow evaporation of their respective chloroform solutions at room temperature. The X-ray diffraction analysis was performed at the Marquette University Diffraction Facility using an Oxford Diffraction SuperNova diffractometer equipped with Cu(Kα) radiation (λ = 1.54184 Å) at 100 K. The diffraction images were processed and scaled with CrysAlisPro software. Using the Olex2 graphical interface, structures were solved by direct methods (ShelXS-2023/1) and refined by full-matrix least-squares methods against F² (ShelXL-2025/1). Detailed crystallographic procedures and refinement data are provided in Supplementary Method 3 and Supplementary Table 7 and Supplementary Figs. 10 and 11. HR-MS was performed to confirm the molecular formulas determined by X-ray crystallography. Detailed HR-MS method and data are provided in Supplementary Method 4 and Supplementary Fig. 12.

RAA: C₇H₇NO₃S, M.W. 185.20. HR-MS (ESI⁻) m/z: [M−H]⁻ calculated for C₇H₆NO₃S⁻ 184.0068, found 184.0070 ± 0.0003 (n = 8). Crystal data: monoclinic, space group P2₁/n, a = 5.30391(6) Å, b = 13.97822(13) Å, c = 10.74471(13) Å, β = 101.5883(12)°, V = 780.367(15) ų, Z = 4, R = 0.0253.

RAB: C₁₂H₈N₂O₆, M.W. 276.20. HR-MS (ESI⁻) m/z: [M−H]⁻ calculated for C₁₂H₇N₂O₆⁻ 275.0304, found 275.0308 ± 0.0001 (n = 5). Crystal data: triclinic, space group P-1, a = 7.0528(3) Å, b = 11.7911(5) Å, c = 14.6888(6) Å, α = 72.249(4)°, β = 79.265(3)°, γ = 86.633(3)°, V = 1143.02(8) ų, Z = 4, R = 0.0413.

Crystallographic data for the structures have been deposited in the Cambridge Crystallographic Data Centre under accession codes CCDC 2503436 (RAA) and CCDC 2503437 (RAB).

HPLC analytical methods

Analytical HPLC was done using an Agilent 1260 Infinity II system (Agilent, Santa Clara, CA). For the analysis of RAA (Method A), a PHENOMENEX 00B-4018-E0 3 µm, 50 × 4.6 mm column was used to achieve separation. Detection occurred at 406 nm with a retention time of 2.5 min. The mobile phase consisted of 10% acetonitrile (ACN) and 90% water + 0.1% formic acid. The flow rate was set to 0.6 mL/min, and the autosampler was configured to inject 10 μL aliquots of each sample. The standard curve of HPLC-purified RAA was used to determine RAA concentrations.

For HPLC analysis of RAB (Method B), a Phenomenex® Luna® Phenyl Hexyl HPLC Column 3 µm 150 × 4.6 mm 00F-4256-E0 was used. Detection occurred at 254 nm with a retention time of 9.8 min. The mobile phase consisted of 10% acetonitrile (ACN) and 90% water + 0.1% formic acid. The flow rate was set to 0.4 mL/min, and the autosampler was configured to inject 10 μL aliquots of each sample. The standard curve of HPLC-purified RAB was used to determine RAB concentrations.

LC-MS analysis

LC-MS analysis was performed using a Shimadzu LC-MS-2020 system (Shimadzu, Japan). Chromatographic separation was achieved on a Phenomenex Luna Phenyl Hexyl column (150 × 4.6 mm, 3 µm) maintained at 40 °C. The mobile phases consisted of 0.1% formic acid in water (A) and acetonitrile (B). The flow rate was set at 0.4 mL/min with a gradient elution program: 10% B (0–1.0 min), 10–90% B (1.0–10.0 min), 90% B (10.0–14.9 min), 90-10% B (14.9–15.0 min), and 10% B (15.0-20.0 min). The injection volume was 5 μL, and the total run time was 20 min. Mass spectrometric detection was carried out using combined electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in negative mode. The desolvation line was heated to 250 °C, and the heat block was at 400 °C. Nebulizing gas flow was 1.3 L/min, and drying gas flow was 10.0 L/min. The mass scan range was m/z 100–1000.

For sample preparation, 5 mL of bacterial culture was extracted with an equal volume (5 mL) of ethyl acetate. The organic phase was collected and air-dried at room temperature. The dried residue was dissolved in 500 µL of methanol. A total of 14 strains were analyzed, including WT, ∆ras1, ∆ras2, ∆ras3, ∆ras4, ∆ras5, ∆ras6, ∆ras2∆ras5, CΔras1, CΔras2, CΔras3, CΔras4, CΔras5, and CΔras6. HPLC-purified RAA and RAB were analyzed as positive standards. Methanol was used as a blank control for all LC-MS analyses. Data acquisition and analysis were performed by using LabSolutions software (Version 5.123; Shimadzu, Japan). RAA and RAB were identified in bacterial culture extracts based on their retention times and mass-to-charge ratio (m/z) values by comparison with HPLC-purified positive standards. RAA was detected at a retention time of 9.8 min with m/z 184 [M−H]⁻, and RAB was detected at a retention time of 7.8 min with m/z 275 [M−H]⁻. The identities were confirmed by matching retention times and m/z values with the positive standards. All samples and controls were analyzed under identical conditions. Three biological replicates were prepared for each strain, and each sample was subjected to three independent LC-MS analyses.

Antimicrobial assay

To assess the growth inhibition of E. amylovora strains by P. soli, 100 µL of P. soli and 10 µL of E. amylovora containing a pML123 plasmid⁷⁰ at an optical density of 590 nm (OD₅₉₀ = 2.0) were co-cultivated in 10 mL of YM medium in 150-mL Erlenmeyer flasks at 28 °C with shaking at 220 rpm. The population of E. amylovora was quantified after 18 h on plates supplied with Kanamycin 50 μg/mL by colony-forming unit assay.

The antimicrobial assay was conducted following the CLSI Antimicrobial Susceptibility Testing Standards, as outlined in CLSI document M07-A10 issued in January 2015. In brief, overnight bacterial cultures were diluted to an optical density at 590 nm (OD₅₉₀) of 0.01 using the appropriate media and then aliquoted into 96-well plates with 200 µL per well. RAA or streptomycin was added to each well to final concentrations of 40, 20, 10, 5, 2.5, 1.25, 0.62, 0.31, 0.15, or 0.07 µg/mL, respectively. Control wells were supplemented with either water (for streptomycin) or DMSO (for RAA). The plates were then incubated at 28 °C without agitation. To evaluate the antimicrobial activity of RAA on bacterial strains of medical relevance, overnight cultures of E. coli O157:H7, P. aeruginosa PAO1, and S. enteritidis (strain 155350 A) were diluted 1:1000 into fresh LB medium to an initial optical density (OD₅₉₀) of 0.01. M. smegmatis (strain 155180A) was similarly diluted 1:1000 into LB medium supplemented with 0.15% (v/v) glycerol and 0.10% (v/v) Tween-80 (OD₅₉₀ = 0.01). Aliquots of 2 mL culture were dispensed into sterile tubes, and RAA was added to final concentrations of 100, 150, or 250 µg/mL. Control tubes received methanol alone. Cultures were incubated at 37 °C with shaking at 200 rpm for 24 h. MIC values, defined as the lowest concentration of the compound that resulted in no bacterial growth after 24 h, were determined.

V. inaequalis was cultivated on PDA agar in the dark at room temperature (22 °C). A suspension containing conidia and mycelia in 0.01 M PBS was prepared, and 10 µL aliquots were applied to each RAA-amended PDA plate. Plates containing 0.4% DMSO served as a negative control, while those with 1000 µg/mL CuSO₄ were used as a positive control. Plates were incubated at room temperature in the dark, and the diameter of each V. inaequalis colony was measured after 14 days. MIC values were determined after 7 days. P. infestans strains were cultured on RYE plates at room temperature. Mycelia were washed with 0.01 M PBS, centrifuged for 20 min at 2465 × g, and resuspended in sterile distilled water. Ten microliters of this suspension were placed onto each of three equal-sized sections of each RAA-amended plate. RYE plates containing 0.4% DMSO served as the negative control. Plates were incubated at room temperature in the dark, diameters of P. infestans colonies were measured after 4 days, and MIC values were determined.

Macromolecular synthesis assay

The macromolecular synthesis assay was performed using E. amylovora strain Ea1189 to assess the effects of RejuAgro A on DNA, RNA, and protein synthesis. Cultures grown to log phase in M9 minimal media were labeled with [³H]-thymidine, [³H]-uridine, or [³H]-leucine, then treated with test compounds. Radioactive incorporation into macromolecules was measured using trichloroacetic acid precipitation and liquid scintillation counting, with cell viability determined by plate counting⁷¹. Ea1189 was cultured in M9 minimal media (Na₂HPO₄, 6 g/L; KH₂PO₄, 2 g/L; NaCl, 0.5 g/L; NH₄Cl, 1 g/L; MgSO₄, 0.24 g/L; nicotinic acid, 0.2 g/L; glucose, 9.01 g/L) at 28 °C with shaking at 200 rpm. Growth was monitored by measuring optical density at 600 nm (OD₆₀₀) hourly after inoculation with 1% (v/v) of an overnight culture. The MICs of RAA, tetracycline, ciprofloxacin, and rifampicin against Ea1189 were determined using the same conditions after 24 h of incubation.

An overnight culture of Ea1189 was grown in M9 medium at 28 °C with shaking (200 rpm) to an OD₆₀₀ of 0.3–0.4 (log phase). The culture was then diluted 1:100 (v/v) into fresh M9 medium and spiked with either 4.16 nM [³H]-thymidine (VT 155, ViTrax, California), 17.85 nM [³H]-uridine (VT 158, ViTrax, California), or 9.7 nM [³H]-leucine (NET460250, Revvity, Massachusetts) with specific activity 60 Ci/mmol, 14 Ci/mmol, and 103 Ci/mmol respectively at time zero. After 60 min, samples were treated with inhibitors or with 10 µM of unlabeled precursors (thymidine or uridine), or 50 µM of unlabeled leucine, or without (untreated). All samples treated with [³H]-leucine were supplemented with 10 µM unlabeled leucine to dilute the specific activity before samples were spiked with tritiated leucine. Because protein is the largest single growth product, undiluted [³H]-leucine uptake exhausted the external pool too rapidly. At time zero and other time points, 1 mL samples were collected for two measurements: (1) Total radioactivity: Samples were directly mixed with 10 mL Hydro-Fluor organic LSC cocktail. (2) Incorporated radioactivity: Samples were mixed with 100 µL of 100% trichloroacetic acid (TCA) and incubated overnight at 4 °C. The TCA-precipitated macromolecular material was then washed with 5% TCA and resuspended in 1 mL of Hydro-Fluor organic LSC cocktail. Radioactivity was quantified using a liquid scintillation analyzer (Tri-Carb 2900 TR, Perkin Elmer), with each sample counted for 20 min. Disintegrations per minute (DPM) per milliliter were normalized using the external standard [³H]-quench curve function from the counter. Cell viability for each sample was determined by plating appropriate dilutions on LB agar and incubating overnight. All experiments were performed in biological triplicate and repeated independently twice.

Effect of glutathione on RejuAgro A antimicrobial activity

E. amylovora 1189 was cultured overnight and diluted 1:100 (v/v) into fresh M9 medium containing RejuAgro A at 2× MIC and supplemented with reduced glutathione at final concentrations of 0 mM, 0.25 mM, 0.5 mM, or 5 mM. Cell viability for each sample was determined by plating appropriate dilutions on LB agar after 24 h of incubation. All experiments were performed in biological triplicate and repeated independently twice.

Principal component analysis (PCA) of compounds’ chemical properties

PCA in two dimensions was performed to analyze and visualize the structural similarity between RAA and the selected antibiotics. Analysis was conducted using the ChemBio Server 2.0 (Biomedical Research Foundation, Academy of Athens). A detailed protocol for compound selection for PCA is provided in Supplementary Method 5.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

1. FAO & WFP. The State of Food Security and Nutrition in the World 2021. https://openknowledge.fao.org/server/api/core/bitstreams/522c9fe3-0fe2-47ea-8aac-f85bb6507776/content (2021).

2. USApple. Industry Outlook 2023. https://usaa.memberclicks.net/assets/2023/Outlook2023/USAPPLE-OutlookReport-2023.pdf (2023).

3. Sun, W. et al. Current situation of fire blight in China. Phytopathology 113, 2143–2151 (2023).

   Article  PubMed  Google Scholar 

4. van der Zwet, T., Orolaza-Halbrendt, N. & Zeller, W. Fire Blight: History, Biology, and Management (American Phytopathology Society, 2012).

5. Denning, W. On the decay of apple trees. N. Y. Soc. Promot. Agric. Arts Manuf. Trans. 2, 219–222 (1794).

   Google Scholar 

6. Soukainen, M., Santala, J. & Tegel, J. First report of Erwinia amylovora, the causal agent of fire blight, on pear in Finland. Plant Dis. 99, 1033 (2015).

   Article  Google Scholar 

7. Myung, I.-S. et al. Fire blight of apple, caused by Erwinia amylovora, a new disease in Korea. Plant Dis. 100, 1774 (2016).

   Article  Google Scholar 

8. Rhouma, A., Helali, F., Chettaoui, M., Hajjouji, M. & Hajlaoui, M. First report of fire blight caused by Erwinia amylovora on pear in Tunisia. Plant Dis. 98, 158 (2014).

   Article  CAS  PubMed  Google Scholar 

9. Kharadi, R. R. et al. Genetic dissection of the Erwinia amylovora disease cycle. Annu. Rev. Phytopathol. 59, 191–212 (2021).

   Article  CAS  PubMed  Google Scholar 

10. Slack, S. M. et al. In-orchard population dynamics of Erwinia amylovora on apple flower stigmas. Phytopathology 112, 1214–1225 (2022).

    Article  PubMed  Google Scholar 

11. Pusey, P. & Curry, E. Temperature and pomaceous flower age related to colonization by Erwinia amylovora and antagonists. Phytopathology 94, 901–911 (2004).

    Article  CAS  PubMed  Google Scholar 

12. Malnoy, M. et al. Fire blight: applied genomic insights of the pathogen and host. Annu. Rev. Phytopathol. 50, 475–494 (2012).

    Article  CAS  PubMed  Google Scholar 

13. McManus, P. S., Stockwell, V. O., Sundin, G. W. & Jones, A. L. Antibiotic use in plant agriculture. Annu. Rev. Phytopathol. 40, 443–465 (2002).

    Article  CAS  PubMed  Google Scholar 

14. Sundin, G. W., Castiblanco, L. F., Yuan, X., Zeng, Q. & Yang, C. H. Bacterial disease management: challenges, experience, innovation and future prospects: challenges in bacterial molecular plant pathology. Mol. Plant Pathol. 17, 1506–1518 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

15. Rezzonico, F. et al. Burning questions for fire blight research: I. Genomics and evolution of Erwinia amylovora and analyses of host-pathogen interactions. J. Plant Pathol. 106, 797–810 (2024).

16. Sundin, G. W. & Wang, N. Antibiotic resistance in plant-pathogenic bacteria. Annu. Rev. Phytopathol. 56, 161–180 (2018).

    Article  CAS  PubMed  Google Scholar 

17. Sundin, G. & Bender, C. Dissemination of the strA–strB streptomycin-resistance genes among commensal and pathogenic bacteria from humans, animals, and plants. Mol. Ecol. 5, 133–143 (1996).

    Article  CAS  PubMed  Google Scholar 

18. Adaskaveg, J., Förster, H. & Wade, M. Effectiveness of kasugamycin against Erwinia amylovora and its potential use for managing fire blight of pear. Plant Dis. 95, 448–454 (2011).

    Article  CAS  PubMed  Google Scholar 

19. Aldwinckle, H. S., Bhaskara Reddy, M. V. & Norelli, J. L. Evaluation of control of fire blight infection of apple blossoms and shoots with SAR inducers, biological agents, a growth regulator, copper compounds, and other materials. Acta Hortic. 590, 325–331 (2002).

    Article  CAS  Google Scholar 

20. Norelli, J., Holleran, H., Johnson, W., Robinson, T. & Aldwinckle, H. Resistance of Geneva and other apple rootstocks to Erwinia amylovora. Plant Dis. 87, 26–32 (2003).

    Article  CAS  PubMed  Google Scholar 

21. McGhee, G. C. et al. Genetic analysis of streptomycin-resistant (Sm^R) strains of Erwinia amylovora suggests that dissemination of two genotypes is responsible for the current distribution of Sm^R E. amylovora in Michigan. Phytopathology 101, 182–191 (2011).

    Article  PubMed  Google Scholar 

22. Tancos, K. & Cox, K. Exploring diversity and origins of streptomycin-resistant Erwinia amylovora isolates in New York through CRISPR spacer arrays. Plant Dis. 100, 1307–1313 (2016).

    Article  CAS  PubMed  Google Scholar 

23. Chiou, C. & Jones, A. Nucleotide sequence analysis of a transposon (Tn5393) carrying streptomycin resistance genes in Erwinia amylovora and other gram-negative bacteria. J. Bacteriol. 175, 732–740 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

24. Förster, H., McGhee, G. C., Sundin, G. W. & Adaskaveg, J. E. Characterization of streptomycin resistance in isolates of Erwinia amylovora in California. Phytopathology 105, 1302–1310 (2015).

    Article  PubMed  Google Scholar 

25. Sundin, G. W. et al. A novel IncX plasmid mediates high-level oxytetracycline and streptomycin resistance in Erwinia amylovora from commercial pear orchards in California. Phytopathology 12, 2165–2173 (2023).

    Article  Google Scholar 

26. McManus, P. S. Does a drop in the bucket make a splash? Assessing the impact of antibiotic use on plants. Curr. Opin. Microbiol. 19, 76–82 (2014).

    Article  PubMed  Google Scholar 

27. Ham, H., Oh, G. R., Lee, Y. H. & Lee, Y. H. Comparison of resistance acquisition and mechanisms in Erwinia amylovora against agrochemicals used for fire blight control. Plant Pathol. J. 40, 525–536 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

28. Zeng, Q. et al. Burning questions for fire blight research. II. Critical next steps in disease management and in host resistance breeding of apple and pear. J. Plant Pathol. 106, 811–822 (2024).

    Article  Google Scholar 

29. García-Valdés, E. & Lalucat, J. Pseudomonas: Molecular and Applied Biology 1–23 (Springer, 2016).

30. Konstantinidis, K. T. Sequence-discrete species for prokaryotes and other microbes: a historical perspective and pending issues. mLife 2, 341–349 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

31. Cabrefiga, J. & Montesinos, E. Analysis of aggressiveness of Erwinia amylovora using disease-dose and time relationships. Phytopathology 95, 1430–1437 (2005).

    Article  PubMed  Google Scholar 

32. Puławska, J. & Sobiczewski, P. Phenotypic and genetic diversity of Erwinia amylovora: the causal agent of fire blight. Trees 26, 3–12 (2012).

    Article  Google Scholar 

33. Blin, K. et al. antiSMASH 7.0: new and improved predictions for detection, regulation, chemical structures and visualisation. Nucleic Acids Res. 51, W46–W50 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

34. Borlinghaus, J., Albrecht, F., Gruhlke, M. C. H., Nwachukwu, I. D. & Slusarenko, A. J. Allicin: Chemistry and biological properties. Molecules 19, 12591–12618 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

35. DeBoer, G. & Satchivi, N. Retention, Uptake, and Translocation of Agrochemicals in Plants 75–93 (ACS Publications, 2014).

36. Kleier, D. A. Phloem mobility of xenobiotics: I. Mathematical model unifying the weak acid and intermediate permeability theories. Plant Physiol. 86, 803–810 (1988).

    Article  CAS  PubMed Central  Google Scholar 

37. Johnson, K. & Stockwell, V. Management of fire blight: a case study in microbial ecology. Annu. Rev. Phytopathol. 36, 227–248 (1998).

    Article  CAS  PubMed  Google Scholar 

38. Johnson, K. B., Temple, T. N., Kc, A. & Elkins, R. B. Refinement of nonantibiotic spray programs for fire blight control in organic pome fruit. Plant Dis. 106, 623–633 (2022).

    Article  CAS  PubMed  Google Scholar 

39. Sundin, G. W., Werner, N. A., Yoder, K. S. & Aldwinckle, H. S. Field evaluation of biological control of fire blight in the eastern United States. Plant Dis. 93, 386–394 (2009).

    Article  CAS  PubMed  Google Scholar 

40. Winkler, A., Athoo, T. & Knoche, M. Russeting of fruits: etiology and management. Horticulturae 8, 231 (2022).

    Article  Google Scholar 

41. Sugar, D., Powers, K. A. & Basile, S. R. Mancozeb and Kaolin applications can reduce russet of ‘Comice’ pear. HortTechnology 15, 272–275 (2005).

    Article  CAS  Google Scholar 

42. Pusey, P. L., Stockwell, V. O. & Mazzola, M. Epiphytic bacteria and yeasts on apple blossoms and their potential as antagonists of Erwinia amylovora. Phytopathology 99, 571–581 (2009).

    Article  PubMed  Google Scholar 

43. Stockwell, V., Johnson, K., Sugar, D. & Loper, J. Mechanistically compatible mixtures of bacterial antagonists improve biological control of fire blight of pear. Phytopathology 101, 113–123 (2011).

    Article  CAS  PubMed  Google Scholar 

44. Freimoser, F. M., Rueda-Mejia, M. P., Tilocca, B. & Migheli, Q. Biocontrol yeasts: mechanisms and applications. World J. Microbiol. Biotechnol. 35, 154 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

45. Gayder, S., Parcey, M., Castle, A. J. & Svircev, A. M. Host range of bacteriophages against a world-wide collection of Erwinia amylovora determined using a quantitative PCR assay. Viruses 11, 910 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

46. Gayder, S., Parcey, M., Nesbitt, D., Castle, A. J. & Svircev, A. M. Population dynamics between Erwinia amylovora, Pantoea agglomerans and bacteriophages: exploiting synergy and competition to improve phage cocktail efficacy. Microorganisms 8, 1449 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

47. Gildemacher, P. et al. Can phyllosphere yeasts explain the effect of scab fungicides on russeting of Elstar apples? Eur. J. Plant Pathol. 110, 929–937 (2004).

    Article  CAS  Google Scholar 

48. Heidenreich, M. M., Corral-Garcia, M., Momol, E. & Burr, T. Russet of apple fruit caused by Aureobasidium pullulans and Rhodotorula glutinis. Plant Dis. 81, 337–342 (1997).

    Article  PubMed  Google Scholar 

49. Ippolito, A., El Ghaouth, A., Wilson, C. L. & Wisniewski, M. Control of postharvest decay of apple fruit by Aureobasidium pullulans and induction of defense responses. Postharvest Biol. Technol. 19, 265–272 (2000).

    Article  CAS  Google Scholar 

50. Kunz, S. Development of “Blossom-Protect” – a yeast preparation for the reduction of blossom infections by fire blight. In Ecofruit—11th International Conference on Cultivation Technique and Phytopathological Problems in Organic Fruit-growing (ed. Boos, M.) 108–112 (Fördergemeinschaft Ökologischer Obstbau e.V. (FÖKO), 2004).

51. Leibinger, W., Breuker, B., Hahn, M. & Mendgen, K. Control of postharvest pathogens and colonization of the apple surface by antagonistic microorganisms in the field. Phytopathology 87, 1103–1110 (1997).

    Article  CAS  PubMed  Google Scholar 

52. Slack, S. M., Outwater, C. A., Grieshop, M. J. & Sundin, G. W. Evaluation of a contact sterilant as a niche-clearing method to enhance the colonization of apple flowers and efficacy of Aureobasidium pullulans in the biological control of fire blight. Biol. Control 139, 104073 (2019).

    Article  CAS  Google Scholar 

53. Schnabel, E. L. & Jones, A. L. Isolation and characterization of five Erwinia amylovora bacteriophages and assessment of phage resistance in strains of Erwinia amylovora. Appl. Environ. Microbiol. 67, 59–64 (2001).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

54. Spadaro, D. & Droby, S. Development of biocontrol products for postharvest diseases of fruit: The importance of elucidating the mechanisms of action of yeast antagonists. Trends Food Sci. Technol. 47, 39–49 (2016).

    Article  CAS  Google Scholar 

55. Thompson, D. W., Casjens, S. R., Sharma, R. & Grose, J. H. Genomic comparison of 60 completely sequenced bacteriophages that infect Erwinia and/or Pantoea bacteria. Virology 535, 59–73 (2019).

    Article  CAS  PubMed  Google Scholar 

56. Johnson, K. B. & Temple, T. N. Evaluation of strategies for fire blight control in organic pome fruit without antibiotics. Plant Dis. 97, 402–409 (2013).

    Article  PubMed  Google Scholar 

57. Borlinghaus, J. et al. Allicin, the odor of freshly crushed garlic: a review of recent progress in understanding Allicin’s effects on cells. Molecules 26, 1505 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

58. Harbut, M. B. et al. Auranofin exerts broad-spectrum bactericidal activities. Proc. Natl. Acad. Sci. USA 112, 4453–4458 (2015).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

59. Liu, Y. et al. Repurposing of the gold drug auranofin and a review of its derivatives as antibacterial therapeutics. Drug Discov. Today 27, 1961–1973 (2022).

    Article  CAS  PubMed  Google Scholar 

60. Pang, Z., Raudonis, R., Glick, B. R., Lin, T. J. & Cheng, Z. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies. Biotechnol. Adv. 37, 177–192 (2019).

    Article  CAS  PubMed  Google Scholar 

61. Zhang, Y. H. Substrate channeling and enzyme complexes for biotechnological applications. Biotechnol. Adv. 29, 715–725 (2011).

    Article  CAS  PubMed  Google Scholar 

62. Tian, C. et al. Multiplexed thiol reactivity profiling for target discovery of electrophilic natural products. Cell Chem. Biol. 16, 1416–1427.e5 (2017).

    Article  Google Scholar 

63. Riaz, M. S. et al. Dissecting the mechanism of intracellular Mycobacterium smegmatis growth inhibition by platelet activating factor C-16. Front. Microbiol. 10, 1046 (2020).

    Article  Google Scholar 

64. Marçais, G. et al. MUMmer4: A fast and versatile genome alignment system. PLoS Com. Biol. 14, e1005944 (2018).

    Article  Google Scholar 

65. Krzywinski, M. et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–1645 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

66. Seemann, T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30, 2068–2069 (2014).

    Article  CAS  PubMed  Google Scholar 

67. Gil, R., Silva, F. J., Peretó, J. & Moya, A. Determination of the core of a minimal bacterial gene set. Microbiol. Mol. Biol. Rev. 68, 518–537 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

68. Chung, W. C., Chen, L. L., Lo, W. S., Lin, C. P. & Kuo, C. H. Comparative analysis of the peanut witches’-broom phytoplasma genome reveals horizontal transfer of potential mobile units and effectors. PLoS ONE 8, e62770 (2013).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

69. Huang, W. & Wilks, A. A rapid seamless method for gene knockout in Pseudomonas aeruginosa. BMC Microbiol. 17, 199 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

70. Labes, M., Pühler, A. & Simon, R. A new family of RSF1010-derived expression and lac-fusion broad-host-range vectors for gram-negative bacteria. Gene 89, 37–46 (1990).

    Article  CAS  PubMed  Google Scholar 

71. Cunningham, M. L., Kwan, B. P., Nelson, K. J., Bensen, D. C. & Shaw, K. J. Distinguishing on-target versus off-target activity in early antibacterial drug discovery using a macromolecular synthesis assay. J. Biomol. Screen. 18, 1018–1026 (2013).

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

1. T3 Bioscience, Milwaukee, WI, USA

   Jian Huang & Xianyang Liu

2. Department of Biological Sciences, University of Wisconsin–Milwaukee, Milwaukee, WI, USA

   Ton Nu Bao Vy Huyen, Shreyashi Mitra, Manda Yu & Ching-Hong Yang

3. Department of Plant Pathology and Ecology, The Connecticut Agricultural Experiment Station, New Haven, CT, USA

   Quan Zeng

4. Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, MI, USA

   George W. Sundin

5. Section of Plant Pathology and Plant-Microbe Biology, School of Integrative Plant Science, Cornell AgriTech, Cornell University, Geneva, NY, USA

   Kerik D. Cox

6. Department of Microbiology and Plant Pathology, University of California, Riverside, CA, USA

   Helga Förster & James E. Adaskaveg

7. Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, ROC

   Chih-Horng Kuo

8. Department of Plant Pathology, Entomology and Microbiology, Iowa State University, Ames, IA, USA

   Xiaochen Yuan

9. School of Freshwater Sciences, University of Wisconsin–Milwaukee, Milwaukee, WI, USA

   Russell L. Cuhel

Contributions

J.H. conducted molecular analyses of genes involved in RejuAgro A production in Pseudomonas soli, designed field assays, investigated the mode of action of RejuAgro A, and wrote the initial draft of the manuscript. T.N.B.V.H. conducted molecular analyses of genes involved in RejuAgro A production, contributed to the mode of action studies, and assisted in manuscript writing. X.Y.L. isolated and identified RejuAgro A and determined its minimum inhibitory concentration (MIC). S.M. conducted MIC assays and contributed to manuscript preparation. M.Y. performed MIC assays and assisted in manuscript writing. Q.Z., G.W.S., K.D.C., H.F., J.E.A., and X.Y. conducted independent field assays on apple and pear fire blight, as well as apple scab, and assisted in manuscript writing. C.K. analyzed and classified Pseudomonas soli and assisted in writing the classification section of the manuscript. R.L.C. assisted with macromolecular synthesis assays of RejuAgro A. C.-H.Y. conceived and supervised the project, designed experiments, and wrote and finalized the manuscript.

Corresponding author

Correspondence to Ching-Hong Yang.

Competing interests

C.-H.Y. is the Chief Scientific Officer of T3 BioScience, which supported aspects of this project. J.H. is a senior scientist at T3 BioScience and participated in conducting this research. The strain under accession number PTA-126796 (P. soli 0617-T307) is associated with a patent application (Application Number: 17063540). The co-inventors are C.-H.Y. and X.L. The remaining authors declare no competing interests.

Peer review information

Nature Communications thanks Antonet Svircev and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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