RejuAgro A: A novel antimicrobial for fire blight control of pome fruits and beyond

doi:10.21203/rs.3.rs-5050621/v1

Download PDF

Article

RejuAgro A: A novel antimicrobial for fire blight control of pome fruits and beyond

https://doi.org/10.21203/rs.3.rs-5050621/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

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), a novel antimicrobial produced by Pseudomonas soli 0617-T307, showing potent activity against E. amylovora, including streptomycin-resistant strains. RAA demonstrated efficacy comparable to streptomycin in greenhouse and field trials, effectively reducing fire blight incidence. Furthermore, RAA displayed broad-spectrum activity against diverse plant bacterial and fungal pathogens. The RAA biosynthesis gene cluster in P. soli was identified, revealing key genes essential for its production. RAA presents a promising alternative to traditional antibiotics, potentially enhancing sustainable apple and pear production and addressing antibiotic resistance concerns.

Biological sciences/Biotechnology

Biological sciences/Microbiology/Applied microbiology

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 hectares (FAO and WFP 2021). In the United States, apples are the second most consumed fruit after bananas, with an estimated production of 5 million tons in the 2023-24 crop year (Industry Outlook 2023, USApple). Global pear (Pyrus communis, P. pyrifolia) production in 2022 was 26.5 million tons, with China growing over half of the world’s supply (FAOSTAT). The United States ranked second with 0.58 million tons.

Fire blight is a disease that severely hampers apple and pear production and is prevalent in all growing regions of the United States as well as in Europe, Central Asia, the Middle East, New Zealand, South Korea, and China ^1–6. 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 production ⁷. The pathogen E. amylovora grows epiphytically on flowers before infecting the flower base ^8–10. 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) ^{11, 12}. Streptomycin sulfate and oxytetracycline hydrochloride are the primary antibiotics deployed in the battle against fire blight. However, the significant importance of antibiotics in human medicine adds complexity to their application in plant agriculture. Concerns include increased risks of developing and spreading antibiotic resistance among bacteria, antibiotic residues in produce, and environmental issues like soil and water pollution, which may prompt future regulatory actions ^12–15. 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 2014 ¹⁶. 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 russeting ¹⁷. Streptomycin-resistant E. amylovora strains are present in almost all major pome fruit producing regions in the U.S., including California, Michigan, Washington, and New York ^{14, 18–22}, and strains of E. amylovora with resistance to oxytetracycline were recently isolated in California ²³. This widespread resistance raises concerns about the potential transfer of antibiotic resistance genes to human pathogens and the natural microbial community. Furthermore, the extensive utilization of the same antibiotics across plant, animal, and human health sectors diminishes effectiveness and risks the long-term viability of these treatments, underscoring the need for alternative management approaches in agriculture. Given these challenges, the discovery of new and effective antimicrobial agents is urgent and critical for ensuring sustainable pome fruit production ^{16, 24–30}, along with that of other crops that depend extensively on streptomycin and oxytetracycline for disease management ^{14, 31}.

In this research, we identified a novel antimicrobial compound, RejuAgro A (RAA), produced by the bacterium Pseudomonas soli 0617-T307, isolated from soil in Wisconsin, U.S. RAA demonstrated strong antimicrobial activities against all tested E. amylovora strains, including those resistant to streptomycin. Our greenhouse and field experiments showed that RAA effectively reduced the incidence of fire blight, in some trials to a level comparable to streptomycin. The chemical structure of RAA and the gene cluster encoding RAA biosynthesis were characterized. We also determined that RAA is effective against a wide range of phytopathogenic bacteria and fungi, suggesting its potential application in the management of various plant diseases.

Identification of Pseudomonas soli 0617-T307

To discover novel antimicrobial compounds effective against E. amylovora, we isolated over 40,000 bacteria from a wide range of soil samples. These samples were collected from diverse natural settings throughout Wisconsin, encompassing forests, lake shores, and marshlands. A culture extract from each isolate was obtained using ethyl acetate and was tested for antibiosis activity against 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 (MLSA) utilizing the 16S rRNA, gyrB, rpoB, and rpoD genes, 0617-T307 was classified as a member of Pseudomonas soli ³². 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 (Extended Data Fig. 1a). Finally, whole genome sequencing (Extended Data Fig. 1b) revealed that 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 ³³, 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 a novel 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 (data not shown). 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). Since this compound has not been previously described, we have designated it as novel and named it RejuAgro A (RAA).

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

To assess the antimicrobial potency of RAA, the minimum inhibitory concentration (MIC) of HPLC-purified RAA was determined for several E. amylovora strains. The MIC for the less virulent strain Ea1189 and the highly virulent strain Ea110 ^{34, 35} 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) ¹⁹ 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.

Table 1

Antimicrobial efficacy of RejuAgro A against various *E. amylovora* strains
Erwinia amylovora strains^a	MIC (µg/mL)
	RejuAgro A	Streptomycin
Erwinia amylovora Ea1189	5	20
E. amylovora Ea110	5	5
E. amylovora CA11	10	> 100
E. amylovora DM1	10	> 100
E. amylovora Ea88	10	> 2000
^a Relevant characteristics and references for the E. amylovora strains are as follows: Ea1189: virulent strain used in laboratory studies, isolated in Germany; Ea110: virulent strain used for the field trials in Michigan; CA11 and DM1: streptomycin-resistant strains containing Tn5393 with the transposon on the acquired plasmid pEa34 that can grow at 100 µg/mL streptomycin; Ea88: a spontaneous streptomycin-resistant strain with a mutation in the chromosomal rpsL gene that can grow at 2000 µg/mL streptomycin.

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. axonopodis pv. citri and the bacterial spot pathogen of tomato X. campestris pv. vesicatoria XV-16 were 5 and 2.5 µg/ml, respectively (Extended Data Table 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 to 40 µg/ml, which are higher than those for streptomycin (Extended Data Table 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 (Extended Data Table 1). Together, these results suggest that RAA is a potent, broad-spectrum antimicrobial effective against a panel of bacterial and fungal plant pathogens, offering a promising control option for many different plant diseases.

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

To determine whether RAA could effectively suppress fire blight on apple and pear, a series of experiments were conducted in greenhouse or field settings. In greenhouse assays, different concentrations of RAA were applied to open flowers of crabapple trees (Malus ‘Snowdrift’), followed by inoculation with E. amylovora. Water control flowers inoculated with E. amylovora developed fire blight at an incidence of 60.3%, whereas the incidence on flowers treated with 5 or 10 µg/ml of RAA was 37.8% or 49%, respectively. These values were comparable to that of streptomycin, where 47.4% of flowers were diseased (Table 2).

Table 2

Incidence of fire blight in crabapple trees (Snowdrift) treated with RAA and streptomycin at various concentrations in the greenhouse.
Name of treatment	Concentration (ppm)	Infection rate (%)
CK (Water)	-	60.3 ± 3.1* a
RAA5	5	37.8 ± 0.4 c
RAA10	10	49.0 ± 0.8 b
Streptomycin	100	47.4 ± 1.1 b
The infection rate (%) was evaluated 5 days after inoculating with Erwinia amylovora from water control (CK), RAA at 5 ppm (RAA5), RAA at 10 ppm (RAA10), and streptomycin at 100 ppm.
*Values within columns followed by the same letter are not significantly different (P ≥ 0.05).

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 µg/ml 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. 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 to 33%; Table 3). For example, in four of the five trials (2022 CT, 2023 CT, and 2023 NY, 2023 CA), the efficacy of RAA at 20 ppm was similar to 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 a promising alternative to antibiotics in fire blight management.

Table 3

Evaluation of RAA for fire blight control under field settings
Treatment and concentration	2022 MI-apple	2022 CT-apple	2023 CT-apple	2023 NY-apple	2023 CA-pear	2024 CA-pear
Treatment and concentration	Blossom blight (% incidence)
Untreated or water control	85.0 a*	82.4 a	64.4 a	69.5 a	4.1 a	35.2 a
Streptomycin (100 µg/ml)	-	32.2 c	15.1 b	18.0 b	-
Kasugamycin (100 µg/ml)	9.0 d	-	-	-	0.3 b	7.5 c
RAA (5 µg/ml)	56.5 b	76.9 ab	-	-	-	-
RAA (10 µg/ml)	52.0 bc	35.4 bc	35.6 b	12.5 b	-	-
RAA (20 µg/ml)	29.5 c	33.0 c	28.4 b	7.0 b	0.3 b	-
RAA (30 µg/ml)	-	-	18.8 b	-	-	12.4 b
*Values within columns followed by the same letter are not significantly different (P ≥ 0.05).

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 AntiSMASH ³⁶. This analysis led to the identification of ten putative secondary metabolite biosynthetic gene clusters (BGCs 1–10) (Extended Data Table 2). Among them, BGCs 3–8, and 10 are predicted functions in synthesizing previously characterized antimicrobials, while the functions of BGCs 1, 2, and 9 have not been characterized.

To pinpoint which BGCs are involved in RAA biosynthesis, mutations were generated in BGC 1, BGC 2, BGC 7, BGC 8, and BGC 9 (Extended Data Fig. 2a). Results indicated that the mutation of BGC 9 abolished RAA production. However, the mutation in BGC 1, 7, and 8 did not affect RAA production, although mutation in BGC 2 showed a slightly reduced production of RAA (Extended Data Fig. 2b). Since BGC 9 comprises six genes, we designated them ras1-6 (Fig. 3a), which stands for RAA biosynthesis genes. 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 Extended Data Fig. 4), suggesting that these genes are essential for RAA biosynthesis. Additionally, the cell-free culture supernatant extract from the ∆ras1 strain lacked inhibitory activity against E. amylovora strain Ea110 (Fig. 1a). Moreover, co-culturing Ea110 with ∆ras1 resulted in no inhibition of E. amylovora growth (Fig. 1b). Single deletion mutations of ras2 or ras5 resulted in partial reductions in RAA production, yet double mutation of ras2 and ras5 led to the complete abolishment of RAA synthesis (Fig. 3b). This observation suggests that Ras2 and Ras5 contribute to the biosynthesis of RAA with overlapping functions. It should be noted that the more substantial reduction in RAA production in ∆ras5, as opposed to ∆ras2, indicates the dominant role of Ras5 in RAA biosynthesis. Finally, the altered phenotypes in RAA biosynthesis observed in ras mutants could be restored through the complementation of selected genes (Fig. 3c), which confirms their roles in RAA biosynthesis. The predicted functions of the genes present in BGC 9 are listed in Extended Data Table 3.

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 and 2c). This compound, with a molecular formula of C₁₂H₈N₂O₆S and a molecular weight of 276.2, 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 (Extended Data Fig. 3).

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 mutation of various ras genes. Our results showed that RAB was not detected in the ∆ras2∆ras5 double mutant but was detected in wild type, ∆ras2, and ∆ras5 strains (Fig. 4a, b), suggesting that Ras2 and Ras5 are both involved in RAB biosynthesis and their functions are likely redundant. Adding RAB to the culture medium did not affect RAA production in the WT and ∆ras2 strains but partially restored the reduced RAA production in ∆ras5 and in ∆ras2∆ras5 (Fig. 4c-f). This confirms that Ras5 has a more dominant role in RAA biosynthesis than Ras2. RAB production was not detected in ∆ras1, ∆ras3, ∆ras4, and ∆ras6 (Fig. 4a and Extended Data Fig. 5), and supplementation of RAB did not restore RAA production in these strains (data not shown). This suggests that Ras1, Ras3, Ras4, and Ras6 function downstream of RAB in the RAA biosynthesis pathway. Collectively, these results revealed the biosynthesis pathway: ras2 and ras5 overlap in their function in synthesizing the intermediate RAB, while ras1, ras3, ras4, and ras6 are responsible for the subsequent conversion of RAB to RAA (Fig. 4g).

The discovery of RAA offers new potential perspectives and solutions for plant disease management and could significantly impact agricultural activities. The current widespread use of the same antibiotics to treat human, animal, and plant diseases could shrink the pool of effective treatment options, emphasizing the need for new management tools. To reduce resistance development to antibiotics that are used in human medicine, it is imperative that the plant agricultural sector gradually reduces its reliance on commonly used antibiotics such as oxytetracycline and streptomycin for the management of bacterial diseases. This strategic shift is essential for preserving the efficacy of these critical drugs for future generations, ensuring their continued effectiveness in human medicine. In light of this, RAA emerges as a novel approach to managing plant diseases. It exhibits prominent antimicrobial activity against bacterial, Oomycota, and fungal phytopathogens (Table 1 and Extended Data Table 1), and thus, has broad-spectrum efficacy. This new compound not only has the potential to provide a sustainable and effective solution for managing plant diseases but can also have a crucial role in preserving the effectiveness of antibiotics for human health applications.

In this research, RAA demonstrated efficacy in managing fire blight in field trials, with the notable advantage of requiring a lower dosage compared to streptomycin. The molecular weight of RAA is 185.2 Da, which is lower than that of streptomycin with a molecular weight of 581.6 Da. The smaller molecular size potentially enhances the ability of RAA to penetrate plant tissues more effectively, offering an advantage in its distribution within the plant system for improved disease control. Physicochemical properties of compounds such as the pKa and the lipid-water partition coefficient (log Kow, a marker of polarity or membrane permeability) significantly influence their translocation through the cuticle when applied to flowers ^{37 38}. RAA with a pKa of 7.76 and a log Kow of 1.5 likely has superior translocation ability compared to streptomycin (pKa of 10 and log Kow of -7.5). The higher lipophilicity and partial non-ionized state of RAA suggest better penetration through the waxy plant cuticle and more efficient movement across cell membranes. Additionally, the higher lipophilicity of RAA likely contributes to better retention and persistence on flower and leaf surfaces. These properties, resulting in superior penetration, cellular uptake, and adherence to plant surfaces, promise effective control of fire blight at lower doses.

The diminished effectiveness and agricultural dependence on conventional antibiotics have heightened concerns about environmental antibiotic resistance, posing risks to environmental and human health ^{12, 15}. Currently, available alternatives include copper products, hydrogen peroxide, peroxyacetic acid, sulfur, and essential oils, as well as biological control agents ^{39, 40}. However, these options are limited due to variable efficacy and the risk of phytotoxicity ^{2, 41–43}. The exclusion of streptomycin and other antibiotics from organic cultivation in the U.S. highlights the need for effective alternative treatments, steering organic producers toward biological control strategies. Biological control agents such as microorganisms antagonistic to E. amylovora can protect against infection ^{27, 30, 44–55}, but the performance of biocontrol agents is inconsistent across U.S. regions with generally better efficacy in the West compared to the East. For instance, BlightBan A506 significantly reduced fire blight by 40 to 80% in the Pacific Northwest over a six-year period, whereas in the Eastern U.S., it only resulted in a 9.1% reduction ⁴¹. Bloomtime Biological E325 also displayed variable control efficacy across different states. These inconsistencies emphasize the challenges of biocontrol agents relative to traditional antibiotics ³⁹.

Over three consecutive years, field studies were conducted in Connecticut, Michigan, New York, and California, covering both the Western and Eastern U.S. These studies included a range of local apple and pear cultivars to provide a comprehensive assessment of RAA performance under diverse conditions. The incidence of fire blight in the Western U.S., exemplified by California, is highly variable depending on environmental conditions, especially temperature, in a particular season, and rattail bloom contributes to frequent disease outbreaks, making fire blight a serious annual disease problem. The Eastern U.S., with a humid environment, has conditions more consistently favorable for the spread of fire blight. The effectiveness of RAA in controlling fire blight across these different climates and pome fruit cultivars highlights its adaptability for good efficacy. In the 2023 New York field trial, RAA with an infection rate of 7% demonstrated superior effectiveness in suppressing fire blight, compared to 18% for streptomycin, despite being used at a significantly lower concentration (20 ppm versus 100 ppm). This finding is particularly relevant considering that strains of streptomycin-resistant E. amylovora were consistently identified in apple-producing regions across New York ²⁰, underscoring the potential of RAA as a more effective alternative in areas where streptomycin is failing.

We have identified the BGC of RAA by constructing insertion mutants according to the antiSMASH prediction. In P. soli 0617-T307, BGC 9 was found to be essential for RAA synthesis, as deleting the ras1, ras3, ras4, or ras6 genes within the BGC completely abolished RAA production. Despite BGC9 being categorized as a type III polyketide synthase (T3PKS) gene cluster, the specific functions of its constituent genes remain largely unexplored, complicating the current efforts to delineate the biosynthetic pathway of RAA. Nonetheless, the identification of another compound, RAB, provides clues about how RAA is being synthesized. RAB is composed of two linked RAA molecules but misses the thiomethyl-group (-SCH₃) of RAA. RAB is an essential intermediate for RAA synthesis because RAA production can be rescued when RAB is supplied in ∆ras2∆ras5. This suggests that the larger RAB molecule is first synthesized followed by modifications by other enzymes in the RAA biosynthesis pathway.

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

Microbial strains, plasmids, primers, and media

Microbial strains and plasmids used in this study are listed in Table 1, and Extended Data Tables 1 and 4. 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 at 28°C. X. axonopodis 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. 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), while P. infestans was grown on RYE medium (dry rye berries, 60 g/L and sucrose, 20 g/L) at room temperature (22 ºC). Oligonucleotide primers used for cloning are listed in Supplementary Tables 1 and 2.

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 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 prokaryote 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 is made using MUMmer4 ⁵⁶. Each contig × NCBI reference MUM comparison was filtered requiring an exact match length of at least 2 kb. The dotplot was generated with MUMmer4 by computing maximal exact matching, match clustering, and alignment extension between the contig and the single best-match NCBI sequence. The assembly features of the polished assembly were depicted by CIRCOS ⁵⁷. Annotation of coding regions (genes on forward and reverse strands) including ORFs was determined by PROKKA ⁵⁸. 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) ⁵⁹. A low error-rate assembly was generated after three error-type corrections. The high-quality 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 NCBI RefSeq database. The marker genes were parsed from the genome sequences and analyzed according to the guidelines established for Pseudomonas ³². The procedure for molecular phylogenetic analysis was based on that described previously ⁶⁰. Briefly, the multiple sequence alignment was performed using MUSCLE v3.8.31 (10.1093/nar/gkh340) and the maximum likelihood phylogeny was inferred using PhyML v3.3.20180621 (10.1093/sysbio/syq010). The genome-wide average nucleotide identity was calculated using FastANI v1.1 ³³.

Construction of deletion and complementation strains

Deletion mutants Δras1 to Δras6 were generated using a double cross-over gene knock-out method as previously described ⁶¹. 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 Table 1), respectively. The two fragments were fused by overlapping PCR and cloned into a suicidal 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 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 does 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) are listed in Supplementary Tables 1 and 2.

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. 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 Table 1). The amplified sequence was cloned into the suicide vector pEX18-GmR with the restriction sites XbaI and EcoRI. Subsequent steps of homologous recombination in deletion mutant strain were done as described in the knock-out method. The complementation of mutants was confirmed by PCR and DNA sequencing.

Greenhouse assays

Two-year-old cv. Snowdrift crabapple trees (Malus sp.) grafted onto cv. Dolgo rootstock and grown under greenhouse conditions (16-h light/8-h dark photoperiod at 28 ± 2°C and relative humidity of 60 ± 5%) were used in the experiments. At the 80% bloom stage, RAA solutions at 5 or 10 ppm with 0.12% (v/v) of surfactant (Regulaid; KALO, Overland Park, Kansas, U.S.) were sprayed directly onto the flowers in the evening. Flowers sprayed with water or 100 ppm of streptomycin (FireWall 50; AgroSource, Tequesta, FL) were used as negative or positive controls, respectively. A suspension of E. amylovora strain Ea110 (0.5 × 10⁵ CFU/ml) was prepared from an overnight culture in LB broth at 28°C and sprayed onto the flowers the next morning, and was followed by a second application of RAA, water, or streptomycin in the evening. The incidence of fire blight was determined 5 days after inoculation. Flower clusters were counted as infected if more than one flower showed symptoms. Three trees were used per treatment. Statistical significance was determined using the least significant difference (LSD) method and one-way analysis of variance (ANOVA) analysis for comparisons between treatments (P < 0.05). The experiments were repeated three times independently.

Field trials

Over three years, field trials were conducted in California, Connecticut, Michigan, and New York utilizing local spray application tools on regional apple or pear cultivars (Supplementary Data 1). In 2022, Michigan field trials were conducted on 5-year-old ‘Buckeye Gala’ apple trees on M.9 rootstock at the Northwest Michigan Horticultural Research Center near Traverse City. Treatments were applied using 11.34-liter backpack sprayers (Model 473-P, Solo; Newport News, VA) on 16 May (70 to 80% bloom) and 18 May (full bloom). Inoculation with E. amylovora was done at 80% bloom on 17 May by spraying the outer perimeter of each tree with a backpack sprayer using an aqueous suspension of E. amylovora strain Ea110 (1.0 × 10⁶ CFU/ml). Inoculation was conducted during the evening to ensure optimal conditions for bacterial survival. Blossom blight was assessed on 16 June. A total of 50 flower clusters from each of four replicate trees per treatment were evaluated for incidence of disease.

In 2023, a trial was conducted in New York at Cornell AgriTech in Geneva on 19-year-old ‘Idared’ apple trees on B.9 rootstock. Treatments were applied using a Solo 451 gas-powered mist blower (Solo Incorporated, Newport News, VA) calibrated to deliver 935.4 L ha^− 1 (1.9 L/tree) at 80% bloom (5 May) and full bloom/early petal fall (9 May). Trees were inoculated at 80 to 90% bloom (8 May) with E. amylovora strain Ea273 (1 × 10⁶ CFU/ml using a Solo 475-B backpack sprayer (Solo Incorporated). Disease was assessed on 2 June, and the incidence of fire blight was expressed as the number of blighted flowers out of five flowers in the cluster with 20 cluster assessments for six replicate trees per treatment for a total of 120 clusters per treatment.

In Connecticut, two trials were conducted at the Lockwood Farm of the Connecticut Agricultural Experiment Station in Hamden. In 2022, 40-year-old ‘Spartan’ apple trees were used. Treatments were sprayed using 18.92-liter backpack motorized sprayers (Solo 433, Newport News, VA) at 80–90% bloom on 6 May and at 100% bloom on 7 May, with approximately 1.9 liters per tree. Inoculation was conducted on 7 May by spraying E. amylovora strain MASHBO (1 × 10⁶ CFU/ml) before the second application. Fire blight incidence was determined on 24 May by calculating the percentage of infected flower clusters of the total flower clusters. 100–300 flower clusters were evaluated on each tree. In 2023, experiments were conducted on 33-year-old ‘Early Macoun’ apple trees. Treatments were sprayed on 20 April (80–90% bloom) and 21 April (100% bloom), and inoculation was done on 21 April before the second spray. Disease was evaluated on 18 May.100–300 flower clusters were evaluated on each tree.

In California, the efficacy of RAA against fire blight was evaluated on approximately 25-year-old ‘Bartlett’ pear trees in Live Oak. Treatments were applied using a backpack air-blast sprayer (SR 430; Stihl Inc., Virginia Beach, VA, U.S.) at 935.4 L ha^− 1 on 30 March (30% bloom) and 11 April (petal fall) 2023, and on 4 April (10% bloom) and 11 April (full bloom) 2024. Natural disease incidence was evaluated on 5 May 2023 or 18 April 2024, and the number of fire blight strikes on 100 flower clusters per replicate was counted.

Disease incidence data for all field trials were subjected to ANOVA for a randomized block design using Generalized Linear Mixed Model (GLIMMIX) procedures of SAS (version 9.4; SAS Institute Inc., Cary, NC). All percentage data were subjected to arcsine square root transformation prior to analysis. Multiple comparisons for significant fixed effects (P < 0.05) were determined using the LSMEANS procedure in SAS with an adjustment for Tukey’s HSD to control for family-wise error.

Fermentation and compound extraction

P. soli 0617-T307 was grown in 500 ml of YME media at 28°C for 24 h. Subsequently, the seed culture was inoculated into a 20-liter fermenter (BioFlo IV, New Brunswick Scientific Co., NJ) containing 12 liters of YME media. 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 by 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-liter culture.

Compound separation and antimicrobial activity identification

The crude extract was dissolved in acetone and mixed with silica gel, which was loaded to a silica gel column (φ3.0 X 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 elute was monitored at UV 254 nm, fractions were collected by a time mode at 20 ml/tube, and 114 fractions/tubes (F1-113) 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 1:100 diluted with water (~ 10⁸ CFU/ml), spread onto LB agar plates, and 2 µl-droplets of each re-dissolved fraction were 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 elute was monitored at 254 nm using a DAD detector. For RAA, the gradient program 40 to 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

The structures of the two compounds were investigated by multiple analytical techniques, including high-resolution mass spectrometry (HR-MS), infrared spectroscopy (IR), ultraviolet spectroscopy (UV), 1D/2D nuclear magnetic resonance (NMR), and X-ray crystallography (Supplementary Data 2 and 3). RAA and RAB crystals were obtained through slow evaporation of their respective methanol solutions at room temperature. The X-ray diffraction analysis was performed at the Department of Chemistry, Marquette University, Milwaukee, WI, using an Oxford Diffraction SuperNova kappa-diffractometer equipped with dual microfocus Cu/Mo X-ray sources at 100K with Cu(Kα) radiation.

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 x 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 X 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 concentration.

LC-MS analysis

LC-MS analysis was performed using a Shimadzu LCMS-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 positive 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. Data acquisition and analysis were performed using LabSolutions software (Shimadzu, Japan).

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 at an optical density of OD₅₉₀₀ = 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 by colony forming unit assay.

The antimicrobial assay was conducted following the CLSI Antimicrobial Susceptibility Testing (AST) Standards, as outlined in CLSI document M07-A10 issued in January 2015. In brief, overnight bacterial cultures were diluted to an 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. 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 ºC). 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 3,500 rpm, and resuspended in sterile distilled water. Ten µl of this suspension was 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.

Acknowledgments

This work was supported by grants from the United States Department of Agriculture-National Institute of Food and Agriculture (NIFA) [grant no. 2020-70006-32999, 2023-51106-40960, 2023-70029-41268], the Discovery and Innovation Grant of the University of Wisconsin-Milwaukee, the Bradley Catalyst Grant, and the Bridge Grant from the University of Wisconsin-Milwaukee Research Foundation. We thank the Milwaukee Institute for Drug Discovery for their assistance with compound analysis, P. Engevold and J. Ali for their help with greenhouse and genetic complementation assays, and S. Kuchin for his support with statistical analysis.

Sun W et al (2023) Current Situation of Fire Blight in China. Phytopathology 113:2143–2151
van der Zwet T, Orolaza-Halbrendt N, Zeller W (2012) Fire blight: history, biology, and management. American Phytopathology Society, St. Paul, MN
Denning W (1794) On the decay of apple trees. New York Soc promotion agricultural arts manufacturers Trans 2:219–222
Soukainen M, Santala J, Tegel J (2015) First report of Erwinia amylovora, the causal agent of fire blight, on pear in Finland. Plant Dis 99:1033
Myung I-S et al (2016) Fire blight of apple, caused by Erwinia amylovora, a new disease in Korea. Plant Dis 100:1774–1774
Rhouma A, Helali F, Chettaoui M, Hajjouji M, Hajlaoui M (2014) First report of fire blight caused by Erwinia amylovora on pear in Tunisia. Plant Dis 98:158–158
Kharadi RR et al (2021) Genetic dissection of the Erwinia amylovora disease cycle. Annu Rev Phytopathol 59:191–212
Slack SM et al (2022) In-orchard population dynamics of Erwinia amylovora on apple flower stigmas. Phytopathology 112:1214–1225
Pusey P, Curry E (2004) Temperature and pomaceous flower age related to colonization by Erwinia amylovora and antagonists. Phytopathology 94:901–911
Malnoy M et al (2012) Fire blight: applied genomic insights of the pathogen and host. Annu Rev Phytopathol 50:475–494
McManus PS, Stockwell VO, Sundin GW, Jones AL (2002) Antibiotic use in plant agriculture. Annu Rev Phytopathol 40:443–465
Sundin GW, Castiblanco LF, Yuan X, Zeng Q, Yang CH (2016) Bacterial disease management: challenges, experience, innovation and future prospects: challenges in bacterial molecular plant pathology. Mol Plant Pathol 17:1506–1518
Rezzonico F et al (2024) Burning questions for fire blight research: I. Genomics and evolution of Erwinia amylovora and analyses of host-pathogen interactions. J Plant Pathol 1–14
Sundin GW, Wang N (2018) Antibiotic resistance in plant-pathogenic bacteria. Annu Rev Phytopathol 56:161–180
Sundin G, Bender C (1996) Dissemination of the strA-strB streptomycin‐resistance genes among commensal and pathogenic bacteria from humans, animals, and plants. Mol Ecol 5:133–143
Adaskaveg J, Förster H, Wade M (2011) Effectiveness of kasugamycin against Erwinia amylovora and its potential use for managing fire blight of pear. Plant Dis 95:448–454
Aldwinckle HS, Bhaskara Reddy MV, Norelli JL (2002) Evaluation of control of fire blight infection of apple blossoms and shoots with SAR inducers, biological agents, a growth regulator, copper compounds, and other materials. Acta Hortic 590:325–331
Norelli J, Holleran H, Johnson W, Robinson T, Aldwinckle H (2003) Resistance of Geneva and other apple rootstocks to Erwinia amylovora. Plant Dis 87:26–32
McGhee GC et al (2011) Genetic analysis of streptomycin-resistant (Sm^R) strains of Erwinia amylovora suggests that dissemination of two genotypes is responsible for the current distribution of Sm^R E. amylovora in Michigan. Phytopathology 101:182–191
Tancos K, Cox K (2016) Exploring diversity and origins of streptomycin-resistant Erwinia amylovora isolates in New York through CRISPR spacer arrays. Plant Dis 100:1307–1313
Chiou C, Jones A (1993) Nucleotide sequence analysis of a transposon (Tn5393) carrying streptomycin resistance genes in Erwinia amylovora and other gram-negative bacteria. J Bacteriol 175:732–740
Förster H, McGhee GC, Sundin GW, Adaskaveg JE (2015) Characterization of streptomycin resistance in isolates of Erwinia amylovora in California. Phytopathology 105:1302–1310
Sundin GW et al (2023) A novel IncX plasmid mediates high-level oxytetracycline and streptomycin resistance in Erwinia amylovora from commercial pear orchards in California. Phytopathology 12:2165–2173
Yuan X et al (2023) Erwinia amylovora type III secretion system inhibitors reduce fire blight infection under field conditions. Phytopathology 113:2197–2204
Gill J, Svircev A, Smith R, Castle A (2003) Bacteriophages of Erwinia amylovora. Appl Environ Microbiol 69:2133–2138
Lindow SE, McGourty G, Elkins R (1996) Interactions of antibiotics with Pseudomonas fluorescens strain A506 in the control of fire blight and frost injury to pear. Phytopathology 86:841–848
Pusey PL, Stockwell VO, Mazzola M (2009) Epiphytic bacteria and yeasts on apple blossoms and their potential as antagonists of Erwinia amylovora. Phytopathology 99:571–581
Pusey P, Stockwell V, Reardon C, Smits T, Duffy B (2011) Antibiosis activity of Pantoea agglomerans biocontrol strain E325 against Erwinia amylovora on apple flower stigmas. Phytopathology 101:1234–1241
Stockwell V, Johnson K, Sugar D, Loper J (2010) Control of fire blight by Pseudomonas fluorescens A506 and Pantoea vagans C9-1 applied as single strains and mixed inocula. Phytopathology 100:1330–1339
Stockwell V, Johnson K, Sugar D, Loper J (2011) Mechanistically compatible mixtures of bacterial antagonists improve biological control of fire blight of pear. Phytopathology 101:113–123
Vidaver AK (2002) Uses of antimicrobials in plant agriculture. Clin Infect Dis 34:S107–S110
García-Valdés E, Lalucat J, Pseudomonas (2016) Molecular and applied biology 1–23. Springer, Cham
Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S (2018) High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat commun 9:5114
Cabrefiga J, Montesinos E (2005) Analysis of aggressiveness of Erwinia amylovora using disease-dose and time relationships. Phytopathology 95:1430–1437
Puławska J, Sobiczewski P (2012) Phenotypic and genetic diversity of Erwinia amylovora: the causal agent of fire blight. Trees 26:3–12
Blin K et al (2023) antiSMASH 7.0: new and improved predictions for detection, regulation, chemical structures and visualisation. Nucleic Acids Res 51:W46–W50
DeBoer G, Satchivi N, Retention (2014) Uptake, and Translocation of Agrochemicals in Plants 75–93. ACS
Kleier DA (1988) Phloem mobility of xenobiotics: I. Mathematical model unifying the weak acid and intermediate permeability theories. Plant Physiol 86:803–810
Johnson K, Stockwell V (1998) Management of fire blight: a case study in microbial ecology. Annu Rev Phytopathol 36:227–248
Johnson KB, Temple TN, Kc A, Elkins RB (2022) Refinement of nonantibiotic spray programs for fire blight control in organic pome fruit. Plant Dis 106:623–633
Sundin GW, Werner NA, Yoder KS, Aldwinckle HS (2009) Field evaluation of biological control of fire blight in the eastern United States. Plant Dis 93:386–394
Winkler A, Athoo T, Knoche M (2022) Russeting of fruits: Etiology and management. Horticulturae 8:231
Sugar D, Powers KA, Basile SR (2005) Mancozeb and Kaolin Applications Can Reduce Russet of 'Comice' Pear. HortTechnology 15, 272–275
Freimoser FM, Rueda-Mejia MP, Tilocca B, Migheli Q (2019) Biocontrol yeasts: mechanisms and applications. World J Microbiol Biotechnol 35:154
Gayder S, Parcey M, Castle AJ, Svircev AM (2019) Host range of bacteriophages against a world-wide collection of Erwinia amylovora determined using a quantitative PCR assay. Viruses 11:910
Gayder S, Parcey M, Nesbitt D, Castle AJ, Svircev AM (2020) Population dynamics between Erwinia amylovora, Pantoea agglomerans and bacteriophages: exploiting synergy and competition to improve phage cocktail efficacy. Microorganisms 8:1449
Gildemacher P et al (2004) Can phyllosphere yeasts explain the effect of scab fungicides on russeting of Elstar apples? Eur J plant Pathol 110:929–937
Heidenreich MM, Corral-Garcia M, Momol E, Burr T (1997) Russet of apple fruit caused by Aureobasidium pullulans and Rhodotorula glutinis. Plant Dise 81:337–342
Ippolito A, El Ghaouth A, Wilson CL, Wisniewski M (2000) Control of postharvest decay of apple fruit by Aureobasidium pullulans and induction of defense responses. Postharvest Biol Technol 19:265–272
Kunz S (2004) Ecofruit-11th international conference on cultivation technique and phytopathological problems in organic fruit-growing. 108–112 (Weinsberg/Germany
Leibinger W, Breuker B, Hahn M, Mendgen K (1997) Control of postharvest pathogens and colonization of the apple surface by antagonistic microorganisms in the field. Phytopathology 87:1103–1110
Slack SM, Outwater CA, Grieshop MJ, Sundin GW (2019) Evaluation of a contact sterilant as a niche-clearing method to enhance the colonization of apple flowers and efficacy of Aureobasidium pullulans in the biological control of fire blight. Biol Control 139:104073
Schnabel EL, Jones AL (2001) Isolation and characterization of five Erwinia amylovora bacteriophages and assessment of phage resistance in strains of Erwinia amylovora. Appl Environ Microbiol 67:59–64
Spadaro D, Droby S (2016) Development of biocontrol products for postharvest diseases of fruit: The importance of elucidating the mechanisms of action of yeast antagonists. Trends Food Sci Technol 47:39–49
Thompson DW, Casjens SR, Sharma R, Grose JH (2019) Genomic comparison of 60 completely sequenced bacteriophages that infect Erwinia and/or Pantoea bacteria. Virology 535:59–73
Marçais G et al (2018) MUMmer4: A fast and versatile genome alignment system. PLoS Com Biol 14:e1005944
Krzywinski M et al (2009) Circos: an information aesthetic for comparative genomics. Genome Res 19:1639–1645
Seemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069
Gil R, Silva FJ, Peretó J, Moya A (2004) Determination of the core of a minimal bacterial gene set. Microbiol Molecul Biol Rev 68:518–537
Chung WC, Chen LL, Lo WS, Lin CP, Kuo CH (2013) Comparative analysis of the peanut witches'-broom phytoplasma genome reveals horizontal transfer of potential mobile units and effectors. PLoS ONE 8(4):e62770
Huang W, Wilks A (2017) A rapid seamless method for gene knockout in Pseudomonas aeruginosa. BMC Microbiol 17:199

There is NO Competing Interest.

SupplementaryData.pdf
RejuAgro A: A novel antimicrobial for fire blight control of pome fruits and beyond Supplementary Data
ExtendedDataTables.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

RejuAgro A: A novel antimicrobial for fire blight control of pome fruits and beyond

Status:

Version 1

Abstract

Figures

Introduction

Results

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

Discussion

Methods

Microbial strains, plasmids, primers, and media

Genome sequencing, assembly, and annotation

Species identification

Construction of deletion and complementation strains

Greenhouse assays

Field trials

Fermentation and compound extraction

Compound separation and antimicrobial activity identification

RAA and RAB characterization

HPLC analytical methods

LC-MS analysis

Antimicrobial Assay

Declarations

Acknowledgments

References

Additional Declarations

Supplementary Files

Status:

Version 1