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

Domain: biology
Published: 2026-03-11
Editorial outcome: major_revision

Abstract
Fire blight, caused by Erwinia amylovora, severely impacts global apple and pear production. Current control measures rely heavily on conventional antibiotics like streptomycin, oxytetracycline, and kasugamycin, which raise concerns regarding resistance development and environmental impacts. This research introduces RejuAgro A (RAA), an antimicrobial produced by Pseudomonas soli 0617-T307, showing potent activity against E. amylovora, including streptomycin-resistant strains. RAA demonstrates efficacy comparable to streptomycin in field trials, effectively reducing fire blight incidence. Studies on the antimicrobial mechanism reveal that RAA inhibits RNA, DNA, and protein synthesis, distinguishing from that of conventional antibiotics. Furthermore, RAA displays broad-spectrum activity against diverse plant bacterial and fungal pathogens. The RAA biosynthesis gene cluster in P. soli is identified, revealing key genes essential for its production. RAA presents an alternative to traditional antibiotics, potentially enhancing sustainable apple and pear production and addressing antibiotic resistance concerns.

Introduction
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, USA apple). 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 China3–8. 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 base10–12. 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 actions14–17. 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 York16,20–24, 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, Reju Agro 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.

Results
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, lakeshores, 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 name 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 Ea11031,32 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 wider 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 BGC1, BGC2, BGC7, BGC8, and BGC9 (Supplementary Fig. 2a). Results indicated that mutation of BGC9 abolished RAA production. However, mutations in BGC1, 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 Δras1 strain lacked inhibitory activity against E. amylovora strain Ea110 (Fig. 1a). Moreover, co-culturing Ea110 with Δras1 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 Δras5, as opposed to Δras2, 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 Δras2Δras5 double mutant but was detected in wild type, Δras2, and Δras5 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 Δras2 at 20 ppm, and in Δras5 and Δras2Δras5 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 Δras1, Δras3, Δras4, and Δras6 (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-enzymatic 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.

Methods
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 dot plot was generated with MUMmer 4 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 crossover 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 resolved 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) Å³, 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) Å³, 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, Δras1, Δras2, Δras3, Δras4, Δras5, Δras6, Δras2Δras5, 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 155350A) 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 ChemBioServer 2.0 (Biomedical Research Foundation, Academy of Athens). A detailed protocol for compound selection for PCA is provided in Supplementary Method 5.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript describes identification of a novel antimicrobial compound “RAA” showing potent activity against Erwinia amylovora, which causes the fire blight disease in apple and pear plants. “RAA” displayed potent antimicrobial efficacy against E. amylovora at a comparable level to streptomycin, which is the primary antibiotics deployed in the battle against fire blight. The efficacy of RAA in controlling fire blight in greenhouse and field trials was likely good. “RAA” displayed broad-spectrum activity against diverse plant bacterial and fungal pathogens. The biosynthetic gene cluster for “RAA” was identified in the “RAA”-producing Pseudomonas soli 0617-T307. However, enough experimental data are not available to gain insight into the biosynthetic pathway of “RAA”. There is no mention of any molecular targets of \"RAA\".\nMinor comments.\nPage 5, line 3 and Page 8, line 17-18. “data not shown” is not accepted. So, the authors should provide the corresponding data here.", "reviewer_2": "The manuscript entitled “RejuAgro A: A novel antimicrobial for fire blight control of pome fruits and beyond” by Huang, et al. describes the discovery of an antimicrobial compound isolated from Pseudomonas soli 0617-T307. The scope of the manuscript is broad and describes the isolation of the bacterium from soils, detection of inhibition of the bacterial plant pathogen Erwinia amylovora in culture. The authors then used a genomics-guided approach to determine of the genes, including one that encodes for a Type III polyketide synthetase involved in biosynthesis of a compound that inhibits growth of E. amylovora. The structure of the compound, abbreviated as RAA, and its likely precursor RAB was determined using several analytical methods. The genome research and genetic approach to identify the region associated with RAA biosynthesis appears to be sound, but I did not examine the primer sequences.\nThe authors demonstrate that RAA inhibits growth of the fire blight pathogen in in-vitro assays, and can reduce the incidence of fire blight on crabapple, apple or pear trees in greenhouse assays and field trials to levels comparable to streptomycin or kasugamycin, two antibiotics that are permitted for fire blight management in North America. In culture-based assays, the authors also found that RAA inhibited growth to varying degrees of several genera of plant pathogenic bacteria, a plant pathogenic oomycete, and an ascomycete plant pathogen. Please state if these assays were repeated. The capacity of RRA to inhibit these pathogens in planta was not investigated, but these assays demonstrate broad-spectrum activity in culture. The efficacy of RAA in planta appears to be statistically similar to streptomycin or kasugamycin in most field trials. I recommend adding in line 394 that Regulaid and citric acid was added to RAA preparations only in this field trial conducted in Michigan. In the Source Data spreadsheet, Table 3_NY 2023, the RAA treatments are labeled RAA10 B and 20 B. What does the ‘B’ mean?\nThe crabapple efficacy data presented in Table 2 also were provided in the Source Data spreadsheet, but the data for the two other replicate experiments conducted on crabapple trees in the greenhouse were not presented in the Source Data spreadsheet. Did the repeated experiments yield similar results? As is, RAA reduced the incidence of fire blight only 18 to 37% and streptomycin provided only 22% disease control relative to the check, which seems low for a streptomycin-sensitive pathogen strain. Are these values correct and complete for the crabapple trials?\nThe authors frequently refer to RAA as an antimicrobial, not an antibiotic, but they do not define the difference. Do they refer to RAA as an antimicrobial because it also inhibits some fungi or to make it clear that they consider RAA an ‘alternative to antibiotics’?\nThe driver of this research is to provide another material for fire blight management. Many of the hazards described for the use of two clinical antibiotics streptomycin and oxytetracycline and the non-clinical antibiotic kasugamycin in the abstract, lines 53, and 63 are not supported by the literature. The review article by McManus (http://dx.doi.org/10.1016/j.mib.2014.05.013) that addresses lack of effects related to antibiotic use on the environment and lack of residues on plants/produce should be cited.\nRegarding use of RAA as an antimicrobial pesticide, do the authors have information on the potential for spontaneous mutation resulting in tolerance? Are there genes in P. soli or other pseudomonads involved in tolerance, detoxification, or self-resistance? Could such genes be acquired by the target organisms?\nThe authors demonstrated that eukaryotic fungi are sensitive to RAA. Is there evidence that other eukaryotes, such as humans, fish, amphibians, mycorrhizae or beneficial insects and pollinators, such as honeybees are insensitive to RAA? This type of information would be essential for consideration for registration as a crop protection product.\nFinally, Blossom Protect, composed of two strains of the yeast Aureobasidium pullulans is a very effective commercial biological control product for fire blight, especially in organic production systems. This product should be mentioned (line 260) as an alternative to antibiotics that have been effective in western and eastern apple orchards (see DOI: 10.1094/PHYTO-12-22-0452-R; https://doi.org/10.1094/PDIS-07-12-0638-RE; https://doi.org/10.1094/PDIS-07-21-1405-RE). Have the authors tested the sensitivity of these A. pullulans strains to RAA? This would be important to growers that may want to integrate RAA with other methods for fire blight management.\nOverall, the manuscript describes the discovery of a novel antimicrobial isolated from a soil-borne pseudomonad. The potential for the material for crop protection applications is intriguing.", "reviewer_3": "This paper relates the discovery of an antimicrobial molecule, called RejuAgroA (RAA) that is as efficient as streptomycin against the Fire Blight pathogen Erwinia amylovora. This finding is interesting because E. amylovora is a treat to apple and pear production and streptomycin resistant strains are frequent due to the extensive use of this latter antibiotic to fight the disease.\nOur main comments are the following\n1. RAA been tested and found effective against a large number of plant pathogens. However, since E. amylovora belongs to the Enterobacteral order that also includes numerous Human bacterial pathogens, we think it should also be tested against human pathogens such as pathogenic E. coli, Yersinia, Shigella, and Salmonella strains. In addition, the authors justify the need for new molecules for use in agriculture as the extensive use of antibiotics threatens the long-term viability of these treatments. Tests with RAA on multi-resistant enterobacteria would have been appreciated and would have strengthened the rationale.\n2. How “new” is the discovered molecule RAA? Although it structure is well defined, we think it should be compared to the already available antibiotic class (beta-lactam, sulfonamide, fluoroquinone, tetracyclin, aminoglycoside, macrolide…..). This is not discussed and this should be clarified. Is it really new? or is it a variation close to other known antibiotics. From the knowledge of its structure and of the function of the genes involved in its synthesis could you (or not) speculate about its mode of action? Could IA be a help on that topic?\n3. The size of the gene cluster involved in RAA production should be clarified. In “Figure 3”, 6 genes putatively organized in an operon structure are depicted but in “extended data Figure 2”, describing the BGC9, 10 genes are depicted and the 6 last genes organized in an operon structure are not of the same size as in Figure 3. A better description of each gene of the operon/BGC9 cluster is required. Also, the number of genes involved in the biosynthesis of RAA should be clarified. If the first 4 genes are part of this biosynthetic cluster, mutants should be constructed.\n4. RAA is efficient in vitro against a large number of plant pathogens but its efficiency has only been tested in planta against fire blight. We think it would strengthen the article if the efficiency of RAA was also tested against a fungal pathogen.\n5. Protection assays against E. amylovora were performed by spraying plants with RAA just before and after inoculation, whether in greenhouse or in field trials. This procedure suggests that RAA is not stable, however, authors suggest in the discussion section that the high lipophilicity of RAA likely contributes to better persistence on flower and leaf surface. You should provide data regarding the stability of the RAA.\nMinor comments\nPage 2: please, provide the https links for\nFAO and WFP 2021\nIndustry outlook 2023, US apple\nFAOSTAT\nPage 3: You wrote “RAA effectively reduced the incidence of fire blight in some trials”\nThis sentence is surprising since RAA is as effective as streptomycin in all the presented trials (Table 2 and 3). Does it mean that some trials were not presented in the results?\nPage 4:\nMLSA analysis should be based on Pseudomonas core genome since the analysis was performed with sequenced strains.\nANI value is not sufficient to assign a strain to a species, you should also indicate the coverage.\nPage 6 and Table 2:\nIt is surprising that RAA effects in greenhouse tests are not dose dependent as in field trials. Could you comment.\nPage 11:\nPlease provide references or composition for LB medium and potato dextrose agar\nPage 12:\nAlthough you have a complete genome it seems from your explanation that you blasted on NCBI with contigs. Why? not clear to us.\nConcerning species identification, you should provide a MLSA tree based on core genome and not only the ANI but also the coverage. A 100% ANI with a 5% coverage does not classify you strain within the P. soli species.\nPage 14 and 15:\nWe imagine that the treatments applied to the field trials are the same as the one performed during the greenhouse assays but it is just a guess. Could you clarify this point.\nThe strains used for trials assays in Michigan, New York and Connecticut are different and in California the setting involves natural infections. We are concerned that the known strains may not have the same virulence. Could you perform a comparative plant test with all the strains used.\nPage 18\nCould you explain on which basis/which medium P. soli in counter selected when you co-cultivate E. amylovora and P.soli and then quantify E. amylovora. May be there is no counter selection and it is just on a visual basis? If so, provide a picture of the two types of colonies in supplemental data.\nL507 : please correct OD5900 to OD590\nPage 27: Extended Table 1\npv. pruni\npv. juglandis\nP. parmentieri and P. atrosepticum are not producing soft rot on multiple crops. These two species have been isolated only from potato.\nSubspecies do not longer exist within the Pectobacterium genus. Change to P. brasilisense and P. carotovorum\nTable page 28\nGive a number to this table\nWhy streptomycin was not tested on Phytophtora and Venturia?\nPage 29: extended data Table3\nThe P. soli species has been described as producing a secondary metabolites called Xantholysin (see doi: 10.1371/journal.pone.0062946 and 10.1016/j.syapm.2014.07.003 ) but this compound is not indicated on the table, could you clarified.\nPage 29: extended data Table 3\nThe blast results are clear enough to indicate these functions on the main text and on Fig3. From these functions, you could probably speculate the possible biosynthetic pathway(s).\nPage 38 Fig2\nOn the panel Fig2c, could you add the test with the purified RejuAgroB\nPage 40 Fig3\nGive details about the genes of the cluster\nPage 44 Extended Data Fig1\nChange the MLSA tree for a core genes tree\nRemove the part B that is not informative but indicate in the main text the size of the P. soli genome.\nAdd a table where you compare the ANI and the coverage of all the P. soli strains with that of the outgroup P. entomophila.\nPage 46 Extended data Fig2\nThe BGC9 cluster is not the same as in Fig3. Please clarify.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to the reviewer:\nWe appreciate the reviewer’s insightful comments on the biosynthetic pathway of RAA. To address the concern regarding the biosynthetic pathway, we have incorporated genetic deletion studies and an analysis of the functional roles of each encoding gene, providing a detailed understanding of the sequential enzymatic cascade involved in RAA biosynthesis. These findings are now presented in the Results and Discussion sections.\nAdditionally, we appreciate the reviewer’s comments regarding the molecular target of RAA and have incorporated additional experimental data to elucidate its antimicrobial mechanism. To address this, we conducted macromolecular synthesis assays, principal component analysis (PCA), and cell growth assays to evaluate the effect of glutathione (GSH) on RAA’s antimicrobial activity. RAA demonstrates a multi-target inhibitory profile against Erwinia amylovora, simultaneously suppressing RNA, DNA, and protein synthesis pathways. Moreover, the observed protective effects of GSH against RAA’s activity suggest the involvement of a thiol-reactive mechanism. These findings have been incorporated into the Results, Discussion, and Methods sections.\nResult section:\nPage 9:\n“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 a critical intermediate in the pathway. The proposed biosynthetic mechanism involves a sequential multi-enzyme process with defined functional roles: Initially, Ras2 (enoyl-CoA hydratase) and Ras5 (acyl-CoA dehydrogenase) catalyze the modification of acyl-CoA substrates, generating intermediates essential for subsequent reactions. These modified intermediates are then processed by Ras1 (hydroxymethylglutaryl-CoA enzyme), which facilitates the assembly of the core molecular scaffold. Subsequently, Ras3 (asparagine synthase) mediates the incorporation of nitrogen through an asparagine-dependent mechanism, enabling the formation of the NH-group that is structurally critical for RAB. Ras4 (NAD(P)/FAD-dependent oxidoreductase) appears to maintain redox homeostasis during the biosynthetic process, potentially stabilizing reactive intermediates through controlled electron transfer reactions. The terminal enzymatic step involves Ras6 (PaaI family thioesterase), which catalyzes the hydrolysis of thioester bonds, resulting in the release of either RAB or a closely related precursor that undergoes further modification to yield RAA (Fig. 4g).”\nDiscussion section:\nPage 13:\n“Notably, the complete abolishment of RAA production observed in Δras1, Δras3, Δras4, and Δras6 mutants suggests a high degree of interdependence among these enzymatic components. This \"all-or-none\" phenotype provides evidence that these enzymes might function as a tightly integrated enzymatic complex, wherein the disruption of any individual component results in the collapse of the entire biosynthetic pathway. This observation aligns with established models of secondary metabolite biosynthesis, where sequential enzymatic steps often exhibit strict substrate channeling and functional coupling.“\nResult section:\nPage 9:\nAntimicrobial mechanism of RAA against E. amylovora\nMacromolecular 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. 1). This broad inhibitory profile resembles that of the natural compound allicin, suggesting similar mechanistic properties in thiol reactivity 38. Concurrent viability measurements revealed progressive bactericidal activity by reducing bacterial survival to below 50% within 120 minutes post-treatment (Fig. 5b,d,f).\nTo 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. 2), indicating specificity in the protective effect.\nTwo-dimensional principal components analysis (PCA) confirmed that RAA possesses unique structural properties, placing it in a distinct chemical space compared to established classes of antibiotic (Supplementary Table 6 and Supplementary Fig. 3).\nCollectively, these findings suggest that the antimicrobial activity of RAA is distinct from that of conventional antibiotics.\nDiscussion section:\nPage 13:\nRAA 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 38, 64. 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 65, 66. These precedents highlight the regulatory viability of thiol-reactive antimicrobials, with auranofin approved for rheumatoid arthritis and repurposed for antimicrobial use 66, and allicin recognized as a food-safe antimicrobial (GRAS status) 38. The multi-target nature of these compounds offers significant resistance management advantages, as mutations across multiple pathways would be required for resistance development. RAA thus represents a promising agricultural antimicrobial with a mechanism rooted in natural product-based thiol reactivity 67, offering potential regulatory advantages through established mechanistic precedents.\nMethods section:\nPage 22:\nMacromolecular synthesis assay\nThe macromolecular synthesis assay was adapted with modifications from previously described protocol 75. E. amylovora 1189 (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 (OD₆₀₀) hourly after inoculation with 1% (v/v) of an overnight culture. The minimum inhibitory concentrations (MICs) of RAA, tetracycline, ciprofloxacin, and rifampicin against Ea1189 were determined using the same conditions after 24 hours of incubation.\nAn 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 4.16nM [³H]-thymidine, 17.85nM [³H]-uridine, or 9.7nM [³H]-leucine with specific activity 60 Ci/mmol, 14 Ci/mmol, and 103 Ci/mmol accordingly at time zero. After 60 minutes, samples were treated with inhibitors or with 10uM of unlabeled precursors (thymidine or uridine), or 50uM of unlabeled leucine, or without (untreated). All samples treated with [³H]-leucine was supplemented with 10uM unlabeled leucine to dilute the specific activity before samples were spiked with tritiated leucine.\nBecause protein is the largest single growth product, undiluted 3H-leucine uptake exhausted the external pool too rapidly. At time zero and other time points, 1mL 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 minutes. 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.\nEffect of glutathione on RejuAgro A antimicrobial activity\nE. amylovora 1189 was cultured overnight as previously described and diluted 1:100 (v/v) into fresh M9 medium containing RejuAgro A at 2x 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 hours of incubation. All experiments were performed in biological triplicate and repeated independently twice.\nPrincipal Component Analysis (PCA) of compounds’ chemical properties\nPrincipal component analysis in two dimensions (PCA-2D) 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).\nMinor comments.\nPage 5, line 3 and Page 8, line 17-18. “data not shown” is not accepted. So, the authors should provide the corresponding data here.\nResponse to the reviewer:\nFor Page 5, Line 3, and Page 8, Lines 17-18, where “data not shown” was previously noted, we have updated the manuscript to include the relevant experimental data. Specifically, we now provide data demonstrating that supplementation of RAB did not restore RAA production in the ∆ras1, ∆ras3, ∆ras4, and ∆ras6 mutant strains.\nReviewer #2 (Remarks to the Author):\nThe manuscript entitled “RejuAgro A: A novel antimicrobial for fire blight control of pome fruits and beyond” by Huang, et al. describes the discovery of an antimicrobial compound isolated from Pseudomonas soli 0617-T307. The scope of the manuscript is broad and describes the isolation of the bacterium from soils, detection of inhibition of the bacterial plant pathogen Erwinia amylovora in culture. The authors then used a genomics-guided approach to determine of the genes, including one that encodes for a Type III polyketide synthetase involved in biosynthesis of a compound that inhibits growth of E. amylovora. The structure of the compound, abbreviated as RAA, and its likely precursor RAB was determined using several analytical methods. The genome research and genetic approach to identify the region associated with RAA biosynthesis appears to be sound, but I did not examine the primer sequences.\nThe authors demonstrate that RAA inhibits growth of the fire blight pathogen in in-vitro assays, and can reduce the incidence of fire blight on crabapple, apple or pear trees in greenhouse assays and field trials to levels comparable to streptomycin or kasugamycin, two antibiotics that are permitted for fire blight management in North America. In culture-based assays, the authors also found that RAA inhibited growth to varying degrees of several genera of plant pathogenic bacteria, a plant pathogenic oomycete, and an ascomycete plant pathogen. Please state if these assays were repeated. The capacity of RRA to inhibit these pathogens in planta was not investigated, but these assays demonstrate broad-spectrum activity in culture.\nResponse to the reviewer:\nThe antimicrobial assays described in the manuscript were independently repeated in triplicate to ensure the reliability and reproducibility of the results. The MIC value of streptomycin against Xanthomonas axonopodis pv. citri was determined to be 5 µg/mL, and this correction has been made in the revised manuscript. Additionally, we have incorporated data from field trials conducted in 2023 and 2024 in New York to evaluate the efficacy of RAA against apple scab, caused by the fungal pathogen Venturia inaequalis. In these trials, RAA was applied as part of an integrated disease management strategy, which included pre-bloom protectant fungicides and targeted applications of RAA during high-risk infection periods identified by disease forecasting models (newa.cornell.edu). The consistent efficacy of RAA across both years supports its potential as a valuable tool in the integrated management of fungal diseases (see Supplementary Tables 4 and 5).\nThe efficacy of RAA in planta appears to be statistically similar to streptomycin or kasugamycin in most field trials. I recommend adding in line 394 that Regulaid and citric acid was added to RAA preparations only in this field trial conducted in Michigan. In the Source Data spreadsheet, Table 3_NY 2023, the RAA treatments are labeled RAA10 B and 20 B. What does the ‘B’ mean?\nResponse to the reviewer:\nPage 17: A sentence was added to the Materials and Methods section under the field assay portion: ” In all field assays, the surfactant Regulaid was added to RAA.”\nPage 17: The information regarding citric acid for B has been added to the text as: “In this assay, citric acid was incorporated into RAA at a concentration of 0.12% (W/V) before spraying.”\nPage 12: In the discussion section, the following information was added: “We further tested whether citric acid can promote the efficacy of RAA on controlling apple fire blight, the 2024 field data in New York at Cornell AgriTech in Geneva show similar efficacy on fire blight control with or without supplemented with citric acid (Supplementary Table 3).”\nSupplementary Table 3| Evaluation of RAA for fire blight control on 19-year-old ‘Idared’ trees in 2024 at Cornell University, AgriTech, Geneva, NY\nIncidence of Incidence of\nblossom blight shoot blight\nTreatment and concentration Timing* (%)** (%)**\nUntreated NA 77.6  4.4 a 22.4  3.5 a\nStreptomycin (100 μg/ml) 1,2 11.7  4.5 b 0.0  0.0 b\nRAA (10 µg/ml) with citric acid 0.12% (W/V) 1,2 31.3  17.8 b 0.0  0.0 b\nRAA (10 µg/ml) 1,2 27.8  8.7 b 0.0  0.0 b\nRAA (20 µg/ml) with citric acid 0.12% (W/V) 1,2 20.0  6.9 b 0.5  0.5 b\nRAA (20 µg/ml) 1,2 26.0  9.3 b 0.0  0.0 b\nRAA (30 µg/ml) with citric acid 0.12% (W/V) 1,2 9.0  3.3 b 0.0  0.0 b\nRAA (30 µg/ml) 1,2 8.0  4.1 b 0.0  0.0 b\n*Treatment timings were: 7 May- 80% bloom (application 1); 8 May-100% bloom/petal fall (application 2), 2024.\n**All values represent the means and standard errors of 4 replicate trees. Values within columns followed by the same letter are not significantly different (P < 0.05) according to the LSMEANS procedure in SAS 9.4 with an adjustment for Tukey’s HSD to control for family-wise error.\nThe crabapple efficacy data presented in Table 2 also were provided in the Source Data spreadsheet, but the data for the two other replicate experiments conducted on crabapple trees in the greenhouse were not presented in the Source Data spreadsheet. Did the repeated experiments yield similar results? As is, RAA reduced the incidence of fire blight only 18 to 37% and streptomycin provided only 22% disease control relative to the check, which seems low for a streptomycin-sensitive pathogen strain. Are these values correct and complete for the crabapple trials?\nResponse to the reviewer:\nThe data from the two additional replicate experiments on crabapple trees (Malus 'Snowdrift') are now included in the Source Data spreadsheet for clarity. This cultivar is highly susceptible to Erwinia amylovora, and unlike apple flowers, its fragile petals present additional challenges. In replicated greenhouse assays, we observed consistent efficacy results; however, they did not reach the control levels achieved with commercial apple cultivars under field conditions. These values are accurate and reflect the difficulty of achieving higher efficacy in greenhouse conditions with this specific crabapple cultivar.\nThe authors frequently refer to RAA as an antimicrobial, not an antibiotic, but they do not define the difference. Do they refer to RAA as an antimicrobial because it also inhibits some fungi or to make it clear that they consider RAA an ‘alternative to antibiotics’?\nResponse to the reviewer:\nWe refer to RAA as an antimicrobial to highlight its broad-spectrum activity against both bacterial and fungal pathogens, distinguishing it from conventional antibiotics typically used for bacterial targets in plant agriculture. This terminology also emphasizes the potential of RAA as an alternative to traditional antibiotics, aligning with sustainable agricultural practices. To address this, the following sentences were added to the results section (Page 6). “Unlike streptomycin, RAA is a potent, broad-spectrum antimicrobial with efficacy against both bacterial and fungal plant pathogens. This dual activity makes RAA a versatile and promising option for controlling a wide range of plant diseases.”\nThe driver of this research is to provide another material for fire blight management. Many of the hazards described for the use of two clinical antibiotics streptomycin and oxytetracycline and the non-clinical antibiotic kasugamycin in the abstract, lines 53, and 63 are not supported by the literature. The review article by McManus (http://dx.doi.org/10.1016/j.mib.2014.05.013) that addresses lack of effects related to antibiotic use on the environment and lack of residues on plants/produce should be cited.\nResponse to the reviewer:\nMcManus reference was added with the below modification (Page 3) “McManus (2014) reported that streptomycin use for fire blight does not significantly affect bacterial community structure or increase resistance genes (McManus 2014). However, the presence of streptomycin-resistant isolates in apple orchards raises concerns about the potential transfer of resistance genes to human pathogens.”\nRegarding use of RAA as an antimicrobial pesticide, do the authors have information on the potential for spontaneous mutation resulting in tolerance? Are there genes in P. soli or other pseudomonads involved in tolerance, detoxification, or self-resistance? Could such genes be acquired by the target organisms?\nResponse to the reviewer:\nWhile we have not yet conducted specific studies on the potential for spontaneous mutation conferring tolerance to RAA, as it is not the primary focus of this work, it is a recognized concern with many antimicrobial agents. In related pseudomonads, resistance mechanisms such as efflux pumps or enzymatic detoxification pathways have been observed in other contexts and could theoretically contribute to tolerance or self-resistance. We have added sentences addressing these considerations in the discussion section below (page 13). “While this study did not address resistance mechanisms to RAA, the possibility of tolerance development through spontaneous mutations remains a consideration. In related pseudomonads, resistance pathways such as efflux pumps and enzymatic detoxification could theoretically contribute to tolerance. ”\nThe authors demonstrated that eukaryotic fungi are sensitive to RAA. Is there evidence that other eukaryotes, such as humans, fish, amphibians, mycorrhizae or beneficial insects and pollinators, such as honeybees are insensitive to RAA? This type of information would be essential for consideration for registration as a crop protection product.\nResponse to the reviewer:\nWe have conducted extensive safety testing of RAA in collaboration with the U.S. Environmental Protection Agency (EPA) and the IR-4 Project, utilizing EPA-certified Good Manufacturing Practice (GMP) laboratories. Acute oral and inhalation toxicity studies in rats classified RAA as Category IV or 'not classified,' indicating the lowest level of acute toxicity. For pollinator safety, acute contact toxicity tests on honeybees demonstrated that RAA is relatively non-toxic, posing minimal risk. RAA was also shown to be non-mutagenic in a bacterial reverse mutation assay using Salmonella typhimurium. Additional ecotoxicological evaluations confirmed that RAA does not cause acute immobilization in Daphnia magna, acute toxicity in rainbow trout, or oral toxicity in Japanese quail. To ensure comprehensive safety data for EPA registration, sub-acute and sub-chronic oral toxicity studies are currently underway. Importantly, RAA has already passed prenatal developmental toxicity testing in rats, further supporting its safety profile across a broad range of eukaryotic organisms critical to ecological health.\nFinally, Blossom Protect, composed of two strains of the yeast Aureobasidium pullulans is a very effective commercial biological control product for fire blight, especially in organic production systems. This product should be mentioned (line 260) as an alternative to antibiotics that have been effective in western and eastern apple orchards (see DOI: 10.1094/PHYTO-12-22-0452-R; https://doi.org/10.1094/PDIS-07-12-0638-RE; https://doi.org/10.1094/PDIS-07-21-1405-RE). Have the authors tested the sensitivity of these A. pullulans strains to RAA? This would be important to growers that may want to integrate RAA with other methods for fire blight management.\nResponse to the reviewer:\nThe below sentences were added to the discussion section with the reference cited (DOI: 10.1094/PHYTO-12-22-0452-R) on page 11.\n“Blossom Protect, a commercial biological control product composed of two strains of the yeast Aureobasidium pullulans, has shown effective in managing fire blight, particularly within organic production systems in both western and eastern apple orchards.”\nAs suggested by the reviewer, we tested the compatibility of RAA with Aureobasidium pullulans. Our results indicated that RAA, at concentrations of 20 and 30 ppm, inhibits the growth of A. pullulans. Therefore, growers aiming to integrate both RAA and A. pullulans into their fire blight management programs should apply these treatments at separate times.\nOverall, the manuscript describes the discovery of a novel antimicrobial isolated from a soil-borne pseudomonad. The potential for the material for crop protection applications is intriguing.\nResponse to the reviewer:\nThank you for your positive feedback.\nReviewer #3 (Remarks to the Author):\nThis paper relates the discovery of an antimicrobial molecule, called RejuAgroA (RAA) that is as efficient as streptomycin against the Fire Blight pathogen Erwinia amylovora. This finding is interesting because E. amylovora is a treat to apple and pear production and streptomycin resistant strains are frequent due to the extensive use of this latter antibiotic to fight the disease.\nOur main comments are the following\n1. RAA been tested and found effective against a large number of plant pathogens. However, since E. amylovora belongs to the Enterobacteral order that also includes numerous Human bacterial pathogens, we think it should also be tested against human pathogens such as pathogenic E. coli, Yersinia, Shigella, and Salmonella strains. In addition, the authors justify the need for new molecules for use in agriculture as the extensive use of antibiotics threatens the long-term viability of these treatments.\nResponse to the reviewer:\nOur findings demonstrated that RAA is effective against E. amylovora strains from orchards that are resistant to streptomycin (Table 1), the primary antibiotic used in the field for fire blight control, highlighting its distinct mode of action. This differentiation is crucial, as it positions RAA as a viable agricultural alternative to streptomycin, reducing reliance on this important medical antibiotic for crop disease management. RAA is intended solely for agricultural applications, not for use against human-associated bacteria.\n2. How “new” is the discovered molecule RAA? Although it structure is well defined, we think it should be compared to the already available antibiotic class (beta-lactam, sulfonamide, fluoroquinone, tetracyclin, aminoglycoside, macrolide…..). This is not discussed and this should be clarified. Is it really new? or is it a variation close to other known antibiotics. From the knowledge of its structure and of the function of the genes involved in its synthesis could you (or not) speculate about its mode of action? Could IA be a help on that topic?\nResponse to the reviewer:\nWe appreciate the reviewer’s comments regarding the novelty of RejuAgro A (RAA) and its relationship to existing antibiotic classes. In response, we performed principal component analysis (PCA) comparing the chemical features of RAA to those of known antibiotic classes. As presented in the Results, Discussion, and Methods sections (Supplementary Table 6 and Supplementary Fig. 3), the analysis reveals that RAA occupies a distinct chemical space, supporting its classification as a novel antimicrobial compound rather than a structural variant of established antibiotics.\nTo further investigate RAA’s mode of action, we conducted macromolecular synthesis assays and cell growth assays to evaluate the effect of glutathione (GSH) on its antimicrobial activity. RAA exhibits a multi-target inhibitory profile against Erwinia amylovora, simultaneously suppressing RNA, DNA, and protein synthesis. Notably, the protective effect of GSH against RAA activity suggests the involvement of a thiol-reactive mechanism. These findings have been incorporated into the Results, Discussion, and Methods sections\nResult section:\nPage 9:\nAntimicrobial mechanism of RAA against E. amylovora\nMacromolecular 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. 1). This broad inhibitory profile resembles that of the natural compound allicin, suggesting similar mechanistic properties in thiol reactivity 38. Concurrent viability measurements revealed progressive bactericidal activity by reducing bacterial survival to below 50% within 120 minutes post-treatment (Fig. 5b,d,f).\nTo 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. 2), indicating specificity in the protective effect.\nTwo-dimensional principal components analysis (PCA) confirmed that RAA possesses unique structural properties, placing it in a distinct chemical space compared to established classes of antibiotic (Supplementary Table 6 and Supplementary Fig. 3).\nCollectively, these findings suggest that the antimicrobial activity of RAA is distinct from that of conventional antibiotics.\nDiscussion section:\nPage13:\nRAA 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 38, 64. 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 65, 66. These precedents highlight the regulatory viability of thiol-reactive antimicrobials, with auranofin approved for rheumatoid arthritis and repurposed for antimicrobial use 66, and allicin recognized as a food-safe antimicrobial (GRAS status) 38. The multi-target nature of these compounds offers significant resistance management advantages, as mutations across multiple pathways would be required for resistance development. RAA thus represents a promising agricultural antimicrobial with a mechanism rooted in natural product-based thiol reactivity 67, offering potential regulatory advantages through established mechanistic precedents.\nMethods section:\nPage 22:\nMacromolecular synthesis assay\nThe macromolecular synthesis assay was adapted with modifications from previously described protocol 75. E. amylovora 1189 (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 (OD₆₀₀) hourly after inoculation with 1% (v/v) of an overnight culture. The minimum inhibitory concentrations (MICs) of RAA, tetracycline, ciprofloxacin, and rifampicin against Ea1189 were determined using the same conditions after 24 hours of incubation.\nAn 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 4.16nM [³H]-thymidine, 17.85nM [³H]-uridine, or 9.7nM [³H]-leucine with specific activity 60 Ci/mmol, 14 Ci/mmol, and 103 Ci/mmol accordingly at time zero. After 60 minutes, samples were treated with inhibitors or with 10uM of unlabeled precursors (thymidine or uridine), or 50uM of unlabeled leucine, or without (untreated). All samples treated with [³H]-leucine was supplemented with 10uM unlabeled leucine to dilute the specific activity before samples were spiked with tritiated leucine.\nBecause protein is the largest single growth product, undiluted 3H-leucine uptake exhausted the external pool too rapidly. At time zero and other time points, 1mL 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 minutes. 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.\nEffect of glutathione on RejuAgro A antimicrobial activity\nE. amylovora 1189 was cultured overnight as previously described and diluted 1:100 (v/v) into fresh M9 medium containing RejuAgro A at 2x 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 hours of incubation. All experiments were performed in biological triplicate and repeated independently twice.\nPrincipal Component Analysis (PCA) of compounds’ chemical properties\nPrincipal component analysis in two dimensions (PCA-2D) 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).\n3. The size of the gene cluster involved in RAA production should be clarified. In “Figure 3”, 6 genes putatively organized in an operon structure are depicted but in “extended data Figure 2”, describing the BGC9, 10 genes are depicted and the 6 last genes organized in an operon structure are not of the same size as in Figure 3. A better description of each gene of the operon/BGC9 cluster is required. Also, the number of genes involved in the biosynthesis of RAA should be clarified. If the first 4 genes are part of this biosynthetic cluster, mutants should be constructed.\nResponse to the reviewer:\nThe biosynthetic gene cluster BGC 9 comprises a total of 31 genes, with 6 genes designated as core biosynthetic elements as illustrated in Fig. 3a. We generated Extended Data Figure 2a by directly incorporating the predicted gene cluster output from antiSMASH analysis, wherein the core biosynthetic genes are distinctly highlighted in dark red. This computational prediction guided our targeted insertional mutagenesis strategy focusing on these essential biosynthetic components.\nWithin BGC 9, a single core biosynthetic gene is classified as a type III polyketide synthase (T3PKS), which operates in conjunction with five additional genes organized in a functional operon. Notably, four of the six genes constituting this operon are categorized as biosynthetic genes. We hypothesized that this operon plays a crucial role in the primary biosynthetic pathway responsible for RAA production. To validate this hypothesis, we systematically introduced mutations into each of these genes independently. Our experimental results conclusively demonstrated that these genes are indispensable for RAA biosynthesis. The functional characterization of these genes and the proposed biosynthetic pathway for RAA production have been incorporated into the main text and revised Fig. 4g, which implies the sequential enzymatic transformations and intermediate compounds involved in this process.\n.\n4. RAA is efficient in vitro against a large number of plant pathogens but its efficiency has only been tested in planta against fire blight. We think it would strengthen the article if the efficiency of RAA was also tested against a fungal pathogen.\nResponse to the reviewer:\nWe thank the reviewer for the comment. We have added to the manuscript that two years of field trials, conducted in 2023 and 2024 to evaluate RAA for controlling apple scab caused by Venturia inaequalis, were performed by Dr. Kerik Cox at Cornell University. RAA was implemented as part of standard management containing pre-bloom applications of protectant fungicides with applications of RAA being made in the high-risk period for apple scab infection as predicted by disease forecasting models (newa.cornell.edu). This is mentioned in the discussion section and data is included in the supplementary table as below (page 12). “Additionally, our 2023 and 2024 field assays in New York demonstrated that RAA effectively controlled apple scab caused by the fungal pathogen V. inaequalis. RAA was implemented as part of standard management containing pre-bloom applications of protectant fungicides and applications of RAA being made in the high-risk period for apple scab infection as predicted by disease forecasting models (newa.cornell.edu). RAA’s consistent efficacy over both years highlights its potential role in integrated fungal disease management (see Supplementary Tables 4 and 5)\nSupplementary Table 4| Assessment of Apple Scab Management in 21-Year-Old 'Gala' Trees on B.9 Rootstocks at Cornell AgriTech, Geneva, NY (2023)\nIncidence of\napple scab Incidence of\nTreatment programs (materials listed in amt./Ha) Timing* lesions on apple scab on\nterminal leaves fruit (%)**\n(%)**\nUntreated na. 7.3  2.1 a 11.4  2.1 a\nKocide 3000 6.7 kg 1\nCaptec 4.7L + Manzate Max 5.7L 2, 4-5 0.3  0.3 b 2.0  1.4 b\nRejuAgro A 20 µg/mL 3, 6-8\nKocide 3000 6.7 kg 1\nCaptec 4.7L + Manzate Max 5.7L 2, 4-5 0.0  0.0 b 0.5  0.5 b\nRejuAgro A 40 µg/mL 3, 6-8\nKocide 3000 6.7 kg 1\nCaptec 4.7L + Manzate Max 5.7L 2,3,5,7\nAprovia 366 mL 4 0.3  0.3 b 0.0  0.0 b\nFlint Extra 219 mL 6\nCevya 366 mL 8\nKocide 3000 6.7 kg 1\nCaptec 4.7L + Manzate Max 5.7L 2\nDouble Nickel 4.7L 3,5,7\n0.3  0.3 b 0.0  0.0 b\nAprovia 366 mL 4\nFlint Extra 219 mL 6\nCevya 366 mL 8\n*Applications timing were 6 Apr-green tip (application 1); 13 Apr-half-inch green (application 2); 20 Apr-tight cluster (application 3); 25 April-pink (application 4); 4 May-bloom (application 5); 9 May-petal fall (application 6); 18 May-1st cover (application 7); 24 May-2nd cover (application 8).\n**All values are disease incidence and the means and standard errors of at least 10 sets of cluster leaves or 10 fruit collections across four replicate trees. Values within columns followed by the same letter are not significantly different (P < 0.05) according to LSMEANS procedure in SAS 9.4 with an adjustment for Tukey’s HSD to control for family-wise error.\nSupplementary Table 5| Assessment of Apple Scab Management in 21-Year-Old 'Gala' Trees on B.9 Rootstocks at Cornell AgriTech, Geneva, NY (2024).\nIncidence of apple\nIncidence of apple scab\nTreatment programs (amt./A) Timing* scab lesions on\non fruit (%)**\nterminal leaves (%)**\nUntreated na. 22.5  2.3 a 54.0  3.7 a\nKocide 3000 6 lb 1\nCaptec 2 qt+ Manzate Max 2.4 qts 2, 4, 5 8.8  2.2 b 23.0  3.1 b\nRejuAgro A 20 µg/mL 3, 6-8\nKocide 3000 6 lb 1\nCaptec 2 qt+ Manzate Max 2.4 qts 2, 4, 5 5.9  2.2 b 17.5  5.0 b\nRejuAgro A 40 µg/mL 3, 6-8\nKocide 3000 6 lb 1\nCaptec 4L 2 qt + Manzate Max 2.4 qt 2,3,5,7\nAprovia 5.0 fl oz 4 8.3  1.5 b 20.0  4.1 b\nFlint Extra 3 fl oz 6\nCevya 5 fl oz 8\nKocide 3000 6 lb 1\nCaptec 4L 2 qt + Manzate Max 2.4 qt 2\nDouble Nickel 2 Qts 3,5,7\n10.6  3.8 b 20.0  4.1 b\nAprovia 5.0 fl oz 4\nFlint Extra 3 fl oz 6\nCevya 5 fl oz 8\n*Applications timing were 6 Apr-green tip (application 1); 13 Apr-half-inch green (application 2); 19 Apr-tight cluster (application 3); 25 April-pink (application 4); 3 May-bloom (application 5); 9 May-petal fall (application 6); 20 May-1st cover (application 7); 29 May-2nd cover (application 8).\n**All values are disease incidence and the means and standard errors of at least 10 sets of cluster leaves or 10 fruit collections across four replicate trees. Values within columns followed by the same letter are not significantly different (P < 0.05) according to LSMEANS procedure in SAS 9.4 with an adjustment for Tukey’s HSD to control for family-wise error.\nin SAS 9.4 with an adjustment for Tukey’s HSD to control for family-wise error.\n5. Protection assays against E. amylovora were performed by spraying plants with RAA just before and after inoculation, whether in greenhouse or in field trials. This procedure suggests that RAA is not stable, however, authors suggest in the discussion section that the high lipophilicity of RAA likely contributes to better persistence on flower and leaf surface. You should provide data regarding the stability of the RAA.\nResponse to the reviewer:\nWe appreciate the reviewer’s interest in RAA’s stability. While our current study did not include specific stability assays, we have revised the discussion to state, 'Additionally, the higher lipophilicity of RAA, likely resulting in superior penetration, cellular uptake, and adherence to plant surfaces, promises effective control of fire blight at lower doses.\nMinor comments\nPage 2: please, provide the https links for\nFAO and WFP 2021\nIndustry outlook 2023, US apple\nFAOSTAT\nResponse to the reviewer:\nLinks added (see page 2).\nPage 3: You wrote “RAA effectively reduced the incidence of fire blight in some trials”\nThis sentence is surprising since RAA is as effective as streptomycin in all the presented trials (Table 2 and 3). Does it mean that some trials were not presented in the results?\nResponse to the reviewer:\nThank you for noting this wording. We have revised the sentence to ‘Our greenhouse and field experiments showed that RAA effectively reduced the incidence of fire blight, in several trials to a level comparable to streptomycin and kasugamycin.' (page 3)\nPage 4:\nMLSA analysis should be based on Pseudomonas core genome since the analysis was performed with sequenced strains.\nANI value is not sufficient to assign a strain to a species, you should also indicate the coverage.\nResponse to the reviewer:\nFor the purpose of species identification, we intentionally used MLSA because there are Pseudomonas-specific guidelines regarding the marker genes to be included (DOI: 10.1007/978-3-319-31198-2_1; 10.1016/j.meegid.2017.10.026). As such, the use of these specific genes, rather than the core genome, is preferred. In addition to the molecular phylogeny, the genes analyzed all met the sequence similarity thresholds suggested for Pseudomonas species identification. Moreover, in the MLSA phylogeny presented, 0617-T307 and all other Pseudomonas soli strains form a monophyletic clade with 100% bootstrap support. Changing the phylogenetic analysis to using the core genome cannot produce a higher level of support.\nTo address the concern that these Pseudomonas-specific guidelines of MLSA included only a limited number of loci, the ANI analysis was based on whole-genome comparison, which is similar to the core genome analysis suggested by the review comment. When compared to the type strain of Pseudomonas soli (LMG 27941; NCBI genome accession GCF_900110655.1), strain 0617-T307 shares 88.3% coverage and 95.2% ANI. When compared to another characterized strain of Pseudomonas soli (LBp-160603; NCBI genome accession GCF_003205305.1), strain 0617-T307 shares 96.6% coverage and 99.5% ANI. These similarity values are above the general thresholds of 70% coverage and 95% ANI suggested for bacteria (DOI: 10.1002/mlf2.12088). In contrast, 0617-T307 and the most closely related sister species Pseudomonas entomophila shares 78.4% coverage and 89.1% ANI. Based on these findings, we are confident with the identification of strain 0617-T307 as a member of Pseudomonas soli.\nTo address this review comment, the coverage value was added to the main text.\n[main text revision]\nPage 4:\nChange: “0617-T307 shared 95.3% nucleotide identity (ANI) with the P. soli type strain. This value is above the 95% ANI guideline suggested for delineating prokaryotic species”\nTo: “0617-T307 shared 88.3% coverage and 95.3% nucleotide identity (ANI) with the P. soli type strain. These values are above the guidelines of 70% coverage and 95% ANI suggested for delineating prokaryotic species”\nAlso change ref #33 to (DOI: 10.1002/mlf2.12088)\nPage 6 and Table 2:\nIt is surprising that RAA effects in greenhouse tests are not dose dependent as in field trials. Could you comment.\nResponse to the reviewer:\nThe crabapple cultivar Malus 'Snowdrift' is highly susceptible to Erwinia amylovora, and its delicate flower petals present specific challenges in greenhouse settings. However, Malus 'Snowdrift' is especially useful for these assays as it can be induced to flower multiple times a year, unlike apple, which blooms only once annually. This extended flowering capacity significantly facilitates greenhouse testing. Given that E. amylovora infects primarily through flowers, flower assays are the most reliable approach for evaluating efficacy.\nIn our greenhouse trials, higher doses of RAA occasionally led to minor phytotoxic effects on these fragile petals, likely contributing to the absence of a clear dose-dependent response. This contrasts with the more resilient and dose-responsive apple and pear flowers observed in field trials.\nPage 11:\nPlease provide references or composition for LB medium and potato dextrose agar\nResponse to the reviewer:\nThe composition has been added to the main text.\nPage 12:\nAlthough you have a complete genome it seems from your explanation that you blasted on NCBI with contigs. Why? not clear to us.\nConcerning species identification, you should provide a MLSA tree based on core genome and not only the ANI but also the coverage. A 100% ANI with a 5% coverage does not classify you strain within the P. soli species.\nResponse to the reviewer:\nThank you for your question. While we did achieve a complete circular genome assembly, the BLAST analysis against the NCBI database was part of our validation and comparative genomics process. This step wasn't actually using multiple contigs. We were comparing our fully assembled circular genome against reference sequences to identify the closest taxonomic matches and to verify the assembly's accuracy. The polished assembly was compared against curated NCBI references to establish evolutionary relationships and confirm the genomic structure.\nPlease refer to the earlier response. We followed the Pseudomonas-specific guidelines to infer the MLSA tree and utilized fastANI analysis to compare the whole genomes. The coverage values are provided.\nPage 14 and 15:\nWe imagine that the treatments applied to the field trials are the same as the one performed during the greenhouse assays but it is just a guess. Could you clarify this point.\nResponse to the reviewer:\nYes. They are.\nThe strains used for trials assays in Michigan, New York and Connecticut are different and in California the setting involves natural infections. We are concerned that the known strains may not have the same virulence. Could you perform a comparative plant test with all the strains used.\nResponse to the reviewer:\nWe appreciate the reviewer’s comment. To thoroughly evaluate RAA's efficacy, independent field trials were conducted over three years across diverse locations, including Connecticut, Michigan, New York, and California. These trials utilized locally adapted apple and pear cultivars, regional farm practices, and various E. amylovora strains across distinct climate zones in the Western and Eastern U.S. While regional variations in E. amylovora strains may result in differences in virulence, this multi-location approach was designed to provide a realistic assessment of RAA’s performance under varied environmental and agricultural conditions. This enables researchers to observe RAA’s effectiveness in managing fire blight across different local contexts.\nPage 18\nCould you explain on which basis/which medium P. soli in counter selected when you co-cultivate E. amylovora and P.soli and then quantify E. amylovora. May be there is no counter selection and it is just on a visual basis? If so, provide a picture of the two types of colonies in supplemental data.\nResponse to the reviewer:\nThe E. amylovora utilized in this investigation harbors a pML123 plasmid conferring kanamycin resistance. Co-cultures of E. amylovora and P. soli were subjected to selection on LB medium supplemented with kanamycin (50 µg/mL). Under these selective conditions, P. soli exhibited no growth. Colonies that developed on the selective medium were exclusively composed of the E. amylovora cells and were enumerated to determine colony-forming units (CFU). Changes have been made in the \"Methods\" section (page 21) with appropriate citation of reference material.\nL507 : please correct OD5900 to OD590\nResponse to the reviewer:\nCorrected.\nPage 27: Extended Table 1\npv. pruni\npv. juglandis\nP. parmentieri and P. atrosepticum are not producing soft rot on multiple crops. These two species have been isolated only from potato.\nSubspecies do not longer exist within the Pectobacterium genus. Change to P. brasilisense and P. carotovorum\nResponse to the reviewer:\nChanged to X. campestris pv. pruni.\nChanged to X. arboricola pv. juglandis 219.\nP. parmentieri and P. atrosepticum were changed to “Produces soft rot, primarily in potatoes”.\nChange to P. brasilisense and P. carotovorum as advised.\nTable page 28\nGive a number to this table\nWhy streptomycin was not tested on Phytophtora and Venturia?\nResponse to the reviewer:\nStreptomycin is primarily effective against Gram-negative bacterial pathogens, with some activity against certain Gram-positive bacteria. However, it does not show efficacy against fungi, oomycetes, or other non-bacterial pathogens.\nPage 29: extended data Table3\nThe P. soli species has been described as producing a secondary metabolites called Xantholysin (see doi: 10.1371/journal.pone.0062946 and 10.1016/j.syapm.2014.07.003 ) but this compound is not indicated on the table, could you clarified.\nResponse to the reviewer:\nThe absence of Xantholysin in Extended Data Table 3 is likely due to differences in the antiSMASH version used during the initial analysis. The table lists biosynthetic gene clusters (BGCs) predicted by antiSMASH v6.0.1. To improve resolution, we reanalyzed the P. soli T307 genome with antiSMASH v7.1.0, which identified five additional putative BGCs. Among these, BGC region 6 shows 25% similarity to the known compound MA026 (PubChem CID: 10285742).\nCrucially, phylogenetic comparisons (10.1371/journal.pone.0062946) reveal that MA026 and Xantholysin share 90% BGC similarity, and their structures differ by only two consecutive amino acids. Additionally, Xantholysin-like lipopeptides have been isolated from Pseudomonas sp. RtIB026, supporting the possibility that BGC region 6 in P. soli T307 encodes a structurally related variant.\nFinally, the BGC for RAA synthesis (initially identified in antiSMASH v6.0.1) corresponds to BGC 13 in the updated v7.1.0 analysis.\nPage 29: extended data Table 3\nThe blast results are clear enough to indicate these functions on the main text and on Fig3. From these functions, you could probably speculate the possible biosynthetic pathway(s).\nResponse to the reviewer:\nThe functional characterization of these genes and the proposed biosynthetic pathway for RAA production have been incorporated into the main text and revised Figure 4g, which implies the sequential enzymatic transformations and intermediate compounds involved in this process. Therefore, the original Extended Data Table 3 was removed.\nPage 38 Fig2\nOn the panel Fig2c, could you add the test with the purified RejuAgroB\nResponse to the reviewer:\nWe appreciate the reviewer’s suggestion. We incorporated one panel representing the result of the purified RAB against Ea110a.\nPage 40 Fig3\nGive details about the genes of the cluster\nResponse to the reviewer:\nWe appreciate the reviewer’s request for additional details regarding the genes within the biosynthetic cluster. The relevant information has been incorporated into the result of “Identification of the RAA biosynthesis gene cluster in P. soli” on Page 7 and the legend of Fig. 3a.\nPage 44 Extended Data Fig1\nChange the MLSA tree for a core genes tree\nRemove the part B that is not informative but indicate in the main text the size of the P. soli genome.\nAdd a table where you compare the ANI and the coverage of all the P. soli strains with that of the outgroup P. entomophila.\nResponse to the reviewer:\nPlease refer to the previous comment.\nThe ANI table has been added.\nExtended Data Table 1 | Average nucleotide identity (ANI)\nStrain name ID 1 2 3 4 5 6 7\nP. entomophila L48 1 - 89.1 88.9 89.1 89.0 88.9 88.9\n0617-T307 2 78.4 - 95.2 99.5 95.3 95.2 95.3\nP. soli SJ10 3 75.5 84.3 - 95.2 99.2 98.5 98.6\nP. soli LBp-160603 4 78.3 96.6 83.7 - 95.3 95.2 95.2\nP. soli 57B-090714 5 79.0 89.0 89.4 88.9 - 98.8 98.8\nP. soli BCW_7429 6 76.1 84.9 88.9 85.8 90.5 - 99.5\nP. soli LMG 27941 7 78.4 88.3 88.5 88.6 93.3 94.8 -\nGenome-wide average nucleotide identity (ANI) analysis. Coverage values corresponding to alignment fractions between two genome assemblies are listed below diagonal. ANI values are listed below diagonal. The novel strain 0617-T307 reported in this study shares 88.3% coverage and 95.3% ANI with the type strain of Pseudomonas soli, strain LMG 27941, and higher than 95% ANI with all other representative strains of P. soli. In comparison, 0617-T307 shares 78.4% coverage and 89.1% ANI with the type strain of Pseudomonas entomophila, strain L48. 1, P. entomophila L48; 2, 0617-T307; 3, P. soli SJ10; 4, P. soli LBp-160603; 5, P. soli 57B-090714; 6, P. soli BCW_7429; and 7, P. soli LMG 27941.\nPage 46 Extended data Fig2\nThe BGC9 cluster is not the same as in Fig3. Please clarify.\nResponse to the reviewer:\nPlease see our earlier clarification on Comment 3.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse to the reviewer:\nThank you for informing us about the co-reviewing process. We appreciate the Nature Communications initiative to involve and recognize Early Career Researchers in peer review, and we are grateful for the thoughtful and constructive feedback provided."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In the previous review, this reviewer pointed out the lack of data providing insight into RAA biosynthesis and the absence of data regarding the molecular target of RAA. In response to these comments, the authors conducted new experiments and have obtained results that can be considered a certain level of progress. In addition, some figures that had previously been referenced as “data not shown” are now provided in this submission.\nThe biosynthesis of RAA and the identification of its molecular target will likely be addressed in the authors’ future studies. In particular, in vitro experiments will be essential to fully elucidate the biosynthetic pathway of RAA.\nHowever, the following discussion presented by the authors regarding RAA biosynthesis is completely incorrect.\n------\nNotably, the complete abolishment of RAA production observed in Δras1, Δras3, Δras4, and Δras6 mutants suggests a high degree of interdependence among these enzymatic components. This \"all-or-none\" phenotype provides evidence that these enzymes might function as a tightly integrated enzymatic complex, wherein the disruption of any individual component results in the collapse of the entire biosynthetic pathway. This observation aligns with established models of secondary metabolite biosynthesis, where sequential enzymatic steps often exhibit strict substrate channeling and functional coupling.\n-------\nSpecifically, “the claim that the observed all-or-none phenotype serves as evidence for the formation of a biosynthetic enzyme complex” is entirely flawed and should be removed from the Discussion. This is because, in most cases, disruption of individual biosynthetic enzyme genes leads to a complete loss of the final product, regardless of whether the enzymes form a complex. Supporting this, it is well established that individual enzymes can often catalyze their respective biosynthetic reactions in vitro, even in the absence of complex formation.\nIn this context, the results presented in Fig. 4 warrant further discussion. When RAB was added to cultures of the ras2 or ras5 deletion mutants, RAA production was restored. This observation is consistent with the biosynthetic pathway proposed by the authors (Fig. 4g). In contrast, the addition of RAB to cultures of the ras1 or ras3 deletion mutants did not restore RAA production. These contrasting outcomes should be carefully considered and discussed in greater depth, as they may provide important clues regarding the functional roles of the respective genes in the proposed pathway.\nLine 232 and Fig. 3 legend.\nhydroxymethylglutaryl-CoA enzyme --> hydroxymethylglutaryl-CoA synthase\nFig. 4 legend.\nRAB stand --> RAB standard", "reviewer_3": "This revised version of the manuscript addresses many questions. Notably, the presentation of the cluster and the biosynthesis pathway of RAA has improved significantly. It clearly establishes that RAA is a new molecule that expands the range of antimicrobial molecules that can be used against microbes. This raises the question of whether RAA should be used for agricultural purposes or reserved for human health, given that some bacterial pathogens are already resistant to many clinically available antibiotics. In this sense, the fact that streptomycin-resistant E. amylovora strains are still sensitive to RAA is very promising. Clearly, RAA could help to combat clinically resistant human pathogens. While I understand that the author's primary aim was to provide a solution for farmers, the potential utilisation of RAA for human health should also be discussed in the 'Discussion' section. On this topic, I requested that the sensitivity of human pathogens (Salmonella, E. coli, Yersinia, Mycobacterium, etc.) to RAA be tested. I regret that this was not performed, but I still think it should be. These are simple experiments that will open up new perspectives for RAA.\nRegarding the sensitivity of fungal pathogens to RAA, the additional data presented are inconclusive, as RAA was always used in combination with other compounds, and all treatments were effective. A properly designed field trial should be performed to reach this conclusion. I do not think you could state in the discussion that\n“Additionally, our 2023 and 2024 field assays in New York demonstrated that RAA effectively controlled apple scab caused by the fungal pathogen V. inaequalis. Due to the resistance of V. inaequalis to conventional fungicides, managing apple scab typically requires a combination of treatments. RAA’s consistent efficacy over both years highlights its potential role in integrated fungal disease management (Supplementary Tables 4 and 5).”\nFinally, I think the section on resistance development to RAA in the discussion should be improved. The fact that glutathione (GSH) protects against RAA indicates that resistance could develop by inactivating the thiol reactive group of RAA, and this should be mentioned.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "Request from Dr. Chuanfu An, Senior Editor Nature Communications, examine Reviewer #2 suggestions and do the following:\nHave the authors appropriately addressed the reviewer concerns/questions/edits?\ni) Reviewer #2 Pg. 8 “In culture based assays, the authors….Please state that these assays were completed.”\nCompleted The authors have revised the manuscript to comply with the reviewers request. Indicating that the experiments were repeated in three independent experiments. In addition, data from field trials on apple scab from 2023/2024, was incorporated to support the fact that RAA has fungicidal activity (Supplementary Tables 4 and 5).\nii) Reviewer Pg. 8-9 “The efficacy of RAA in planta appears to be statistically similar to streptomycin….?\nCompleted The authors complied with the reviewers request and made the adjustments on the manuscript pg. 17 in regards to Regulaid and citric acid. Supplementary Table 3 shows the 2024 RAA evaluation on 19 year old apple trees that compares streptomycin to RAA with and without citric acid. This goes towards addressing the reviewers concerns.\nIdentified Problem In Supplementary Table 3, 2024 Cornell trial on Idareds, how did the “untreated” treatment obtain 77.6% incidence of blossom blight. Is this actually a positive water control? Were the trees artificially inoculated with E. amylovora and the “untreated” is actually a treatment. What was the concentration of the pathogen sprayed onto all the treatments?\niii) Pg. 8 Reviewer #2 “ ….In the Source Data spreadsheet Table 3_NY2023, the RAA treatments are labelled RAA10B and 20B. What does ‘B’ mean?\nNot addressed by authors. Not sure what this refers to.\niv) Pg. 9 Reviewer #2 “….data for the two other replicate experiments conducted on crab apple trees in the greenhouse were not presented…..Did the repeat experiments yield similar results?\nCompleted Data for the two additional replicates on crab apple were included.\nv) Reviewer #2 “As is, RAA reduced the incidence of fire blight only 18-37% and streptomycin provided only 22% disease relative to the check …Are values correct and complete…..”.\nAuthors replied that the values are correct and reflect the difficulty of achieving higher efficacy consistent efficacy results in the greenhouse.\nIn the expert opinion of this reviewer based on 22 years experience with fire blight efficacy trials under in vitro, in vivo, and in planta conditions. Fire blight trials under greenhouse conditions are difficult due to the environment and simply never provide relevant efficacy data for fire blight. The golden standard for fire blight efficacy trials is always the field trial. In the experiment, streptomycin provided 22% control, it is a perfect example why greenhouses are such poor environments for fire blight efficacy trials. I would highly recommend to the authors to delete the greenhouse trials since they have plenty of excellent field efficacy data.\nvi) Pg. 10 Reviewer #2 “the authors frequently refer to RAA as an antimicrobial, but they do not define the difference.”\nPartially answered by the authors. The authors provide to the reviewer their definition of an “antimicrobial”; and this is inserted on page 6 (L148-L150) of the manuscript.\nHowever, the reviewer requested the authors to define “antibiotic” and state the difference between microbial and antibiotic in the manuscript.\nRegulators and researchers definition for antibiotic is “ a chemical substances produced or derived from microorganisms that kill or inhibit the growth and replication of bacteria”. The explanation is needed on page 6, to avoid the confusion, RAA is an antimicrobial not an antibiotic. By the way, I noticed that Reviewer #3 was also confused, since they asked in their reviewers’ comments “why didn’t you test streptomycin against fungi”.\nvii) Pg. 10 Reviewer #2 states “ The driver of this research is to provide material for fire blight management. Many of the hazards described for the use of two clinical antibiotics streptomycin and non-clinical kasugamycin ….are not supported in literature.\nNot completed/inadequate answer This is an excellent and critical point raised by reviewer and the answer provided by the authors requires editing. Majority of the manuscript references in this section, McManus et. al. (2002), Sundin et. al. (2016), Sundin and Wang (2018) and Sundin and Bender (1996) are out of date when it comes to 2025 concepts on the use of antibiotics in agriculture and more specifically usage in apple orchards.\nRecommendation, I would highly recommend that the authors cite a number of current references in scientific literature that deal with antibiotics in fire blight management. Particularly, Zeng…Rezzonico, Sundin and Peil (2024) J. Plant Pathol. 106: 811-822. This article provides an excellent reviews on fire blight best management practices and the direction being taken. Antibiotics are still encouraged in high humidity/high disease pressure areas; nonetheless, the world is moving away from agricultural use of antibiotics (One Health) and this needs too be addressed in this manuscript with 1-2 sentences.\nviii) Pg.11 Reviewer #2 “…do authors have information on the potential for spontaneous mutation resulting in tolerance..”\nCompleted Response provided by authors is acceptable.\nix) Pg. 11 Reviewer #2 “…Is there evidence that other eukaryotes, such as humans, fish….product”\nCompleted Response provided by authors is acceptable.\nx) Pg. 12 Reviewer #2 “Have the authors tested the sensitivity of these A. pullulans strains to RAA” “This would be important to growers that may want to integrate RAA with other methods for fire blight management.”\nNot completed The authors did not provide a credible response. Lines L306-L309 were inserted the manuscript discussion, stating simply that Blossom Protect has some efficacy in managing fire blight.\nThe authors tested the effect of RAA on A. pullulans strains but the inhibitory effect of RAA on these strains was not inserted into the manuscript.\nxi) Pg. 12 The authors statement to the reviewer “Therefore, growers aiming to integrate both RAA and A. pullulans into fire blight management programs should apply these treatments at separate times”.\nComment to authors The RAA manuscript focuses on the use of RAA for the control of blossom blight; therefore, it is used at the same time as Blossom Protect. The authors should state clearly in L309 that RAA is not compatible with Blossom Protect for fire blight management at bloom time. Did they test any other commercially available biological in the USA? Did they tested RAA against Bacillus subtilis (Serenade Opti) and Pantoea agglomerans E325 (Bloomtime)? Is this chemical compatible with biologicals registered for fire blight management …should be stated in a sentence.\nIs the manuscript suitable for publication?\nManuscript is suitable for publication, once the authors reply/address reviewer #2 questions: iii, vi, v, vii and x.\nMy comment xi is certainly optional."}, "author_response": "Response to the reviewer:\nWe appreciate the reviewer's detailed and constructive comments. This is the part we have been working on and have obtained some clues since the second submission. We have revised Fig. 4g and the corresponding description to address the reviewer's concerns.\nResults section (revised):\nThe 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 (PaaI family thioesterase) is predicted to perform the final hydrolytic step to release RAA or its immediate precursor (Fig. 4g).\nDiscussion section (revised):\nNotably, 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 in downstream of 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. Further studies are required to enhance our understanding of the detailed mechanisms underlying RAB and RAA biosynthesis.\nLegend section (revised):\ng, Schematic representation of the proposed RAA biosynthesis pathway. The proposed sequential enzymatic cascade involves six key enzymes functioning in a coordinated metabolic sequence. Initial substrate modification is proposed to occur via Ras2 (enoyl-CoA hydratase) and Ras5 (acyl-CoA dehydrogenase), generating RAB that serves as a primary metabolic intermediate. Structural modification of RAB is proposed to be mediated by the catalytic action of Ras1 (hydroxymethylglutaryl-CoA synthase), and Ras3 (asparagine synthase)-mediated amidation, introducing essential amino functionality. Subsequent redox modifications by Ras4 (NAD(P)/FAD-dependent oxidoreductase) are considered to facilitate electronic reconfiguration of the intermediate structure. Terminal processing by Ras6 (PaaI family thioesterase) is expected to catalyze thioester hydrolysis, liberating the mature RAA or closely related precursor molecule. Directional arrows indicate reaction progression and enzymatic transformation sequence. Additional auxiliary enzymes may contribute to RAA biosynthesis through supplementary biochemical modifications not depicted in this primary pathway.\n.\nLine 232 and Fig. 3 legend.\nhydroxymethylglutaryl-CoA enzyme --> hydroxymethylglutaryl-CoA synthase\nResponse to the reviewer:\nThank you for the reviewer’s comments. The changes have been made in the results and the legend.\nResults section (revised):\nIdentification of the RAA biosynthesis gene cluster in P. soli\nThese six genes, designated as ras1-6, stand for RAA biosynthesis genes and encode hydroxymethylglutaryl-CoA synthase, enoyl-CoA hydratase, asparagine synthase, NAD(P)/FAD-dependent oxidoreductase, acyl-CoA dehydrogenase, and Paal family thioesterase, respectively (Fig. 3a).\nThe biosynthesis of RAA has two steps with RAB being an intermediate.\nRas1 (hydroxymethylglutaryl-CoA synthase) likely facilitates structural modifications to the RAB scaffold, while Ras3 (asparagine synthase) may mediate additional nitrogen incorporation or structural rearrangements.\nLegend section (revised):\nFig. 3| Identification of the biosynthesis gene cluster for RejuAgro A\na, Schematic of the RejuAgro A biosynthetic gene cluster (BGC 9) in P. soli 0617-T307. Yellow arrow bars, primary biosynthetic genes; a blue triangle indicates the putative promoter region of biosynthesis genes. The ras1-6 genes encode hydroxymethylglutaryl-CoA synthase, enoyl-CoA hydratase, asparagine synthase, NAD(P)/FAD-dependent oxidoreductase, acyl-CoA dehydrogenase, and Paal family thioesterase, respectively.\nFig. 4| RAB is a potential intermediate during the biosynthesis of RAA\ng, Schematic representation of the proposed RAA biosynthesis pathway. The proposed sequential enzymatic cascade involves six key enzymes functioning in a coordinated metabolic sequence. Initial substrate modification is proposed to occur via Ras2 (enoyl-CoA hydratase) and Ras5 (acyl-CoA dehydrogenase), generating RAB that serves as a primary metabolic intermediate. Structural modification of RAB is proposed to be mediated by the catalytic action of Ras1 (hydroxymethylglutaryl-CoA synthase), and Ras3 (asparagine synthase)-mediated amidation, introducing essential amino functionality. Subsequent redox modifications by Ras4 (NAD(P)/FAD-dependent oxidoreductase) are considered to facilitate electronic reconfiguration of the intermediate structure. Terminal processing by Ras6 (PaaI family thioesterase) is expected to catalyze thioester hydrolysis, liberating the mature RAA or closely related precursor molecule. Directional arrows indicate reaction progression and enzymatic transformation sequence. Additional auxiliary enzymes may contribute to RAA biosynthesis through supplementary biochemical modifications not depicted in this primary pathway.\n.\nFig. 4 legend.\nRAB stand --> RAB standard\nResponse to the reviewer:\nThank you for the reviewer’s comments. The change has been made in the legend.\nLegend section (revised):\nFig. 4| RAB is a potential intermediate during the biosynthesis of RAA\na, HPLC analysis (Method B, detected at 254 nm) of RAB stand, WT, ∆ras1, ∆ras2, ∆ras3, ∆ras4, ∆ras5, ∆ras6, and ∆ras2∆ras5. Stars indicate the peak of RAB. b, LCMS analysis of RAB standard, WT, ∆ras2, ∆ras5, and ∆ras2∆ras5 showing a peak at m/z 275 except ∆ras2∆ras5.\nReviewer #3 (Remarks to the Author):\nThis revised version of the manuscript addresses many questions. Notably, the presentation of the cluster and the biosynthesis pathway of RAA has improved significantly. It clearly establishes that RAA is a new molecule that expands the range of antimicrobial molecules that can be used against microbes. This raises the question of whether RAA should be used for agricultural purposes or reserved for human health, given that some bacterial pathogens are already resistant to many clinically available antibiotics. In this sense, the fact that streptomycin-resistant E. amylovora strains are still sensitive to RAA is very promising. Clearly, RAA could help to combat clinically resistant human pathogens. While I understand that the author's primary aim was to provide a solution for farmers, the potential utilisation of RAA for human health should also be discussed in the 'Discussion' section. On this topic, I requested that the sensitivity of human pathogens (Salmonella, E. coli, Yersinia, Mycobacterium, etc.) to RAA be tested. I regret that this was not performed, but I still think it should be. These are simple experiments that will open up new perspectives for RAA.\nRegarding the sensitivity of fungal pathogens to RAA, the additional data presented are inconclusive, as RAA was always used in combination with other compounds, and all treatments were effective. A properly designed field trial should be performed to reach this conclusion. I do not think you could state in the discussion that\n“Additionally, our 2023 and 2024 field assays in New York demonstrated that RAA effectively controlled apple scab caused by the fungal pathogen V. inaequalis. Due to the resistance of V. inaequalis to conventional fungicides, managing apple scab typically requires a combination of treatments. RAA’s consistent efficacy over both years highlights its potential role in integrated fungal disease management (Supplementary Tables 4 and 5).”\nFinally, I think the section on resistance development to RAA in the discussion should be improved. The fact that glutathione (GSH) protects against RAA indicates that resistance could develop by inactivating the thiol reactive group of RAA, and this should be mentioned.\nResponse to the reviewer:\nThank you for the reviewer’s insightful comments. We have addressed them as follows:\nDiscussion section (revised):\nRAA 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 novel, 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.\nDiscussion section (revised):\nAdditionally, 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 the results were promising, all treatments included multiple components. Further field trials using RAA alone are necessary to assess its standalone efficacy.\nDiscussion section (revised):\nAlthough 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.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse to the reviewer:\nWe thank the reviewer for their contribution to the peer-review process and for their time and effort in evaluating this manuscript.\nReviewer #5 (Remarks to the Author):\nRequest from Dr. Chuanfu An, Senior Editor Nature Communications, examine Reviewer #2 suggestions and do the following:\nHave the authors appropriately addressed the reviewer concerns/questions/edits?\ni) Reviewer #2 Pg. 8 “In culture based assays, the authors….Please state that these assays were completed.”\nCompleted The authors have revised the manuscript to comply with the reviewers request. Indicating that the experiments were repeated in three independent experiments. In addition, data from field trials on apple scab from 2023/2024, was incorporated to support the fact that RAA has fungicidal activity (Supplementary Tables 4 and 5).\nii) Reviewer Pg. 8-9 “The efficacy of RAA in planta appears to be statistically similar to streptomycin….?\nCompleted The authors complied with the reviewers request and made the adjustments on the manuscript pg. 17 in regards to Regulaid and citric acid. Supplementary Table 3 shows the 2024 RAA evaluation on 19 year old apple trees that compares streptomycin to RAA with and without citric acid. This goes towards addressing the reviewers concerns.\nIdentified Problem In Supplementary Table 3, 2024 Cornell trial on Idareds, how did the “untreated” treatment obtain 77.6% incidence of blossom blight. Is this actually a positive water control? Were the trees artificially inoculated with E. amylovora and the “untreated” is actually a treatment. What was the concentration of the pathogen sprayed onto all the treatments?\niii) Pg. 8 Reviewer #2 “ ….In the Source Data spreadsheet Table 3_NY2023, the RAA treatments are labelled RAA10B and 20B. What does ‘B’ mean?\nNot addressed by authors. Not sure what this refers to.\nResponse to the reviewer:\nThank you for the reviewer’s comments. In response to the query, we have included the citric acid (B) in the Source Data spreadsheet, specifically in Table 2_NY2023.\nIn Supplementary Table 3 (2024 Cornell trial on ‘Idared’), the “untreated” treatment refers to the water control. This has been updated accordingly.\nIn Supplementary Table 3 (2024 Cornell trial on ‘Idared’), the following was added to the table legend:\nTrees were inoculated at 80 to 90% bloom with E. amylovora strain Ea 273 at a concentration of 1 × 106 CFU/ml.\niv) Pg. 9 Reviewer #2 “….data for the two other replicate experiments conducted on crab apple trees in the greenhouse were not presented…..Did the repeat experiments yield similar results?\nCompleted Data for the two additional replicates on crab apple were included.\nv) Reviewer #2 “As is, RAA reduced the incidence of fire blight only 18-37% and streptomycin provided only 22% disease relative to the check …Are values correct and complete…..”.\nAuthors replied that the values are correct and reflect the difficulty of achieving higher efficacy consistent efficacy results in the greenhouse.\nIn the expert opinion of this reviewer based on 22 years experience with fire blight efficacy trials under in vitro, in vivo, and in planta conditions. Fire blight trials under greenhouse conditions are difficult due to the environment and simply never provide relevant efficacy data for fire blight. The golden standard for fire blight efficacy trials is always the field trial. In the experiment, streptomycin provided 22% control, it is a perfect example why greenhouses are such poor environments for fire blight efficacy trials. I would highly recommend to the authors to delete the greenhouse trials since they have plenty of excellent field efficacy data.\nResponse to the reviewer:\nThank you for the reviewer’s comments. The greenhouse data have been removed to focus the analysis on field-based results, which more directly support the study’s conclusions.\nvi) Pg. 10 Reviewer #2 “the authors frequently refer to RAA as an antimicrobial, but they do not define the difference.”\nPartially answered by the authors. The authors provide to the reviewer their definition of an “antimicrobial”; and this is inserted on page 6 (L148-L150) of the manuscript.\nHowever, the reviewer requested the authors to define “antibiotic” and state the difference between microbial and antibiotic in the manuscript.\nRegulators and researchers definition for antibiotic is “ a chemical substances produced or derived from microorganisms that kill or inhibit the growth and replication of bacteria”. The explanation is needed on page 6, to avoid the confusion, RAA is an antimicrobial not an antibiotic. By the way, I noticed that Reviewer #3 was also confused, since they asked in their reviewers’ comments “why didn’t you test streptomycin against fungi”.\nResponse to the reviewer:\nWe appreciate the reviewer’s thoughtful comments. The issues raised have been carefully considered and addressed as below:\nUnlike 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 and promising option for managing a wide range of plant diseases.\nvii) Pg. 10 Reviewer #2 states “ The driver of this research is to provide material for fire blight management. Many of the hazards described for the use of two clinical antibiotics streptomycin and non-clinical kasugamycin ….are not supported in literature.\nNot completed/inadequate answer This is an excellent and critical point raised by reviewer and the answer provided by the authors requires editing. Majority of the manuscript references in this section, McManus et. al. (2002), Sundin et. al. (2016), Sundin and Wang (2018) and Sundin and Bender (1996) are out of date when it comes to 2025 concepts on the use of antibiotics in agriculture and more specifically usage in apple orchards.\nRecommendation, I would highly recommend that the authors cite a number of current references in scientific literature that deal with antibiotics in fire blight management. Particularly, Zeng…Rezzonico, Sundin and Peil (2024) J. Plant Pathol. 106: 811-822. This article provides an excellent reviews on fire blight best management practices and the direction being taken. Antibiotics are still encouraged in high humidity/high disease pressure areas; nonetheless, the world is moving away from agricultural use of antibiotics (One Health) and this needs too be addressed in this manuscript with 1-2 sentences.\nResponse to the reviewer:\nThank you for the reviewer’s valuable comments. The following changes have been made in response to your feedback:\nMcManus (2014) reported that streptomycin use for fire blight does not significantly affect bacterial community structure or increase resistance genes 24. However, the presence of streptomycin-resistant isolates in apple orchards raises concerns about the potential transfer of resistance genes to human pathogens 25. As highlighted in a recent review 26, 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.\n25. Ham H, Oh GR, Lee YH, Lee YH. Comparison of resistance acquisition and mechanisms in Erwinia amylovora against agrochemicals used for fire blight Control. Plant Pathol. J. 40, 525-536 (2024).\n26. Zeng, Q., Emeriewen, O., Rezzonico, F. 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).\nviii) Pg.11 Reviewer #2 “…do authors have information on the potential for spontaneous mutation resulting in tolerance..”\nCompleted Response provided by authors is acceptable.\nix) Pg. 11 Reviewer #2 “…Is there evidence that other eukaryotes, such as humans, fish….product”\nCompleted Response provided by authors is acceptable.\nx) Pg. 12 Reviewer #2 “Have the authors tested the sensitivity of these A. pullulans strains to RAA” “This would be important to growers that may want to integrate RAA with other methods for fire blight management.”\nNot completed The authors did not provide a credible response. Lines L306-L309 were inserted the manuscript discussion, stating simply that Blossom Protect has some efficacy in managing fire blight.\nThe authors tested the effect of RAA on A. pullulans strains but the inhibitory effect of RAA on these strains was not inserted into the manuscript.\nResponse to the reviewer:\nWe thank the reviewer for their valuable feedback. Revisions have been made accordingly, and the changes are addressed below:\nBlossom 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 54, 55. However, our compatibility tests revealed that RAA at 20 and 30 ppm inhibits A. pullulans, suggesting that these treatments should be applied separately if both are used in an integrated program.\nxi) Pg. 12 The authors statement to the reviewer “Therefore, growers aiming to integrate both RAA and A. pullulans into fire blight management programs should apply these treatments at separate times”.\nComment to authors The RAA manuscript focuses on the use of RAA for the control of blossom blight; therefore, it is used at the same time as Blossom Protect. The authors should state clearly in L309 that RAA is not compatible with Blossom Protect for fire blight management at bloom time. Did they test any other commercially available biological in the USA? Did they tested RAA against Bacillus subtilis (Serenade Opti) and Pantoea agglomerans E325 (Bloomtime)? Is this chemical compatible with biologicals registered for fire blight management …should be stated in a sentence.\nResponse to the reviewer:\nWe thank the reviewer for the valuable feedback. Revisions have been made accordingly in response to comment item x. As the combination assays of RAA with Serenade Opti (Bacillus subtilis) and Bloomtime (Pantoea agglomerans E325) were suggested as optional, they were not included in the current study. However, we plan to evaluate the compatibility of RAA with these commercially available biologicals in future experiments.\nIs the manuscript suitable for publication?\nManuscript is suitable for publication, once the authors reply/address reviewer #2 questions: iii, vi, v, vii and x.\nMy comment xi is certainly optional."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The issues I pointed out have been properly addressed.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "The authors have successfully complied to all the recommendations and suggestions made by this reviewer.\nThe only problem area that remains in this manuscript is the authors suggestion that Aureobasidium pullulans (Blossom Protect) can be used in an integrated program for the control of blossom blight. The manuscript suggestion that RAA and Blossom Protect be applied at different times is simply not possible. A. pullulans functions by covering the blossom hypanthium and preventing the ingress of the fire blight pathogen, in order to be effective the yeast cells need to be live and replicating during the entire bloom period. Based on the data presented in this manuscript RAA and biologicals may net be compatible for the control of dire blight pathogen on the blossom.\nThe author’s can simply say that “compatibility with biologicals for control of blossom blight needs to be studied further”."}, "author_response": "Response:\nWe thank the reviewer for the positive assessment and for confirming that the concerns have been adequately addressed.\nResponse:\nWe thank the reviewer for their contribution to the peer-review process and for their time and effort in evaluating this manuscript.\nResponse:\nWe appreciate the reviewer’s thoughtful and detailed comments. In response, we have revised the Discussion to clarify this point. The sentence has been modified to state:“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.”\nBlossom 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 54, 55. 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."}]

Decision letter