Characterizations of Newly Isolated Erwinia amylovora Loessnervirus-like Bacteriophages from Hungary

Comments and Suggestions

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Answer

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Referee 1

Burst size should be specified

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Burst size is included

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I do not understand this sentence. Apparently, UV-tolerance should be evaluated under controlled conditions. What is novel?

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Sentense is corrected.

Expanation about UV test is also expended in manuscript.

 Answer:

Irradiation dose depends on many factors such as wavelength, lamp power according to manufacture, distance from the lamp, irradiation time, and the lamp’s age because the lamp power will change. In many experiments, not all factors are considered, and some aspects are missed, which makes results not comparable because of missing parameters; they will not be repeated. In some examinations, it is given only wavelength and Time, no lamp power, no distance (Park J, 2018. Luo et al., 2023), or is given only lamp power and Time and no distance and no wavelength (Jo SJ et al., 2023) is the wavelength, Time, lamp power according to manufacture, distance (Born Y et al., 2015, Fu J, 2023). However, because of the aging of the lamp, power is still decreasing. Irradiation dose may vary, even if you use the same lamp, and after some extended period, power will be less. So, we offer to measure lamp intensity (Watt/cm2) at a chosen distance, at a given moment, and the variable time of exposure, and calculate the exact exposed irradiation doses (J/ cm2) and its effect on phage viability.

Park J, Lee GM, Kim D, Park DH, Oh CS. Characterization of the Lytic Bacteriophage phiEaP-8 Effective against Both Erwinia amylovora and Erwinia pyrifoliae Causing Severe Diseases in Apple and Pear. Plant Pathol J. 2018 Oct;34(5):445-450. doi: 10.5423/PPJ.NT.06.2018.0100. Epub 2018 Oct 1. PMID: 30369854; PMCID: PMC6200048.

Luo J, Xie L, Yang M, Liu M, Li Q, Wang P, Fan J, Jin J, Luo C. Synergistic Antibacterial Effect of Phage pB3074 in Combination with Antibiotics Targeting Cell Wall against Multidrug-Resistant Acinetobacter baumannii In Vitro and Ex Vivo. Microbiol Spectr. 2023 Aug 17;11(4):e0034123. doi: 10.1128/spectrum.00341-23.

Jo SJ, Kim SG, Park J, Lee YM, Giri SS, Lee SB, Jung WJ, Hwang MH, Park JH, Roh E, Park SC. Optimizing the formulation of Erwinia bacteriophages for improved UV stability and adsorption on apple leaves. Heliyon. 2023 Nov 8;9(11):e22034. doi: 10.1016/j.heliyon.2023.e22034

Born Y, Bosshard L, Duffy B, Loessner MJ, Fieseler L. Protection of Erwinia amylovora bacteriophage Y2 from UV-induced damage by natural compounds. Bacteriophage. 2015 Jul 24;5(4):e1074330. doi: 10.1080/21597081.2015.1074330.

Fu J, Li Y, Zhao L, Wu C and He Z.Characterization of vB_ValM_PVA8, a broad-host-range bacteriophage infecting Vibrio alginolyticus and Vibrio parahaemolyticus. Front. Microbiol. 2023; 14:1105924. doi: 10.3389/fmicb.2023.1105924.

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 UV explanation:

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Why it was important to check PCR amplification with already published primers? You isolated new phages and it is quite unlikely that they will match previous primers. Table 2 show only negative results and thus is unnecessary.

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About PCR corected in manuscript, table is deleted.

Answer: During isolation process, when we started picking up isolated plaques, we have no idea they were knew phages or not, so to continue with probably new ones we tested it with PCR. Because if there were positive results, we will not choose such phages for farther characterizations. as well as to check purity of the phages and avoid cross-contamination.

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Isolation of phages Ea PF 7 and Ea PF 9 should be described here. What was the source, how they were isolated?

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Information is added in manuscript

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Figure 2 Panels A and B are of poor quality, better resolution is required

Figures changed

Figure 3 and 4. What is shown in panel C? Should be specified in figure legend

Corrected in manuscript

 Figure 6 is of poor quality. Since the genomes are 98% identical, it is enough to show one of them.

Corrected, Only figure of  Ea PF7 is presented now

More detail genome analysis should be presented. For example, what was “functional proteins”, did you find genes relevant to lysogeny etc.

Finctinal proteins are more specified in manuscript.

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Discussion essentially repeats the Results. I would recommend here to compare the new phages with other known to infect Erwinia amylovora.

Changed style of discussion

Referee 3

However,

for Erwinia species there have already numerous phages been characterized attacking these

pathogens, so that the novelty of this paper is limited.

Based on the DNA blasting our phages are related only with Korean phage Fifi 044, Fifi 451 (Park J. et al.), and SNAUB 27( Kim SG et al.) Besides the main aim of the study to have well-characterized phages for further usage as an anti-bacterial biocontrol agent, by publishing this work, we aim to formally recognize that the same phages have also been isolated in Hungary, contributing to the broader knowledge of their geographical distribution and relevance.

Park J, Kim B, Song S, Lee YW, Roh E. Isolation of Nine Bacteriophages Shown Effective against Erwinia  amylovora in Korea. Plant Pathol J. 2022 Jun;38(3):248-253. doi: 10.5423/PPJ.NT.11.2021.0172. Epub 2022 Jun 1. PMID: 35678058; PMCID: PMC9343912.

Kim SG, Lee SB, Jo SJ, Cho K, Park JK, Kwon J, Giri SS, Kim SW, Kang JW, Jung WJ, Lee YM, Roh E, Park SC. Phage Cocktail in Combination with Kasugamycin as a Potential Treatment for Fire Blight Caused by Erwinia amylovora. Antibiotics (Basel). 2022 Nov 6;11(11):1566. doi: 10.3390/antibiotics11111566. PMID: 36358221; PMCID: PMC9686651.

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However, some experimental approaches have to be redesigned to draw the conclusions the

authors made.

We repetied some stability test with Double agar overlay as well.

Table 2:

This table only shows negative data and is therefore not really helpful. Please add positive

controls for the various phage tested. Why does the title of the table say Ea PF8?

Table is deleted

Table 3:

What is the reference strain for the EOP data? Plating on Ea4/82?

Refference stain was Ea 4/82, added in manuscript

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Figure 1:

The TEM picture does not give enough information to draw the conclusion that EaPF7 has a

contractile tail. The authors have to show more than only 1 phage particle and specifically

compare individuals with contracted vs. non-contracted tails. What is the size of the scale

bar?

Because of bad quality we do not include TEM photo of phages in contravted position . Now we added it.

Fig. 2

This figure (a and b) shows growth curves of the infected bacteria together with an

uninfected control. Basically, phage infection only leads to a growth delay and probably the

cultures reach the same OD after 2 more hours.

The authors do not give any explanation

why the growth delay is rather similar no matter whether an MOI of 1 or 10^-7 is used. This

is rather strange as it is not understandable that at these low MOIs (only one phage particle

per 1-10 million of bacteria) the culture growth is inhibited substantially (answered) .

Peng et al. investigated the effects of different MOIs (Multiplicity of Infection) on bacterial growth and observed that even at a very low MOI of 0.001, bacterial growth was completely inhibited during 6 hours.

 Building on these findings, we decided to explore even lower, more extreme MOIs to identify the threshold at which phages are no longer effective in suppressing bacterial growth. This approach aims to pinpoint the  optimal MOI for the phages under investigation. short latent period combined with a large burst size likely explains the exceptional results observed at extremely low MOIs. These characteristics enable efficient phage propagation even when the initial phage concentration is minimal, ensuring rapid infection cycles and high phage yields relative to the available host population.

Peng Q, Ma Z, Han Q, Xiang F, Wang L, Zhang Y, Zhao Y, Li J, Xian Y, Yuan Y. Characterization of bacteriophage vB_KleM_KB2 possessing high control ability to pathogenic Klebsiella pneumoniae. Sci Rep. 2023 Jun 17;13(1):9815. doi: 10.1038/s41598-023-37065-5. PMID: 37330608; PMCID: PMC10276810.

The authors have

to use fresh cultures rather than diluted overnight cultures. A lysis curve has to be shown.

Our overnight cultures were prepared by inoculating a single colony of E. amylovora from LB agar plates into 5 mL of LB broth, followed by incubation for 15 hours. This ensured that the bacteria were viable and active, as confirmed by their robust growth in subsequent turbidity and phage assays. Bacterial culture approximately 10⁸ CFU/ml in this case was used.

The use of overnight cultures for Erwinia amylovora in similar assays, including turbidity measurements was done by Knecht et al, Savbri et al.,

Knecht LE, Born Y, Pelludat C, Pothier JF, Smits THM, Loessner MJ, Fieseler L. Spontaneous Resistance of Erwinia amylovora Against Bacteriophage Y2 Affects Infectivity of Multiple Phages. Front Microbiol. 2022 Aug 1;13:908346. doi: 10.3389/fmicb.2022.908346. PMID: 35979490; PMCID: PMC9376448.

Sabri M, El Handi K, Valentini F, De Stradis A, Achbani EH, Benkirane R, Resch G, Elbeaino T. Identification and Characterization of Erwinia Phage IT22: A New Bacteriophage-Based Biocontrol against Erwinia amylovora. Viruses. 2022 Nov 5;14(11):2455. doi: 10.3390/v14112455. PMID: 36366553; PMCID: PMC9698647.

The data are contradictory to the statement in fig. 2 c and d that the phage have extremely

short latent times(answered) .

The latent period data varies between 10, 20, and 30 minutes or more, depending on specific conditions.

10 minutes - Peng Q, Ma Z, Han Q, Xiang F, Wang L, Zhang Y, Zhao Y, Li J, Xian Y, Yuan Y. Characterization of bacteriophage vB_KleM_KB2 possessing high control ability to pathogenic Klebsiella pneumoniae. Sci Rep. 2023 Jun 17;13(1):9815. doi: 10.1038/s41598-023-37065-5. PMID: 37330608; PMCID: PMC10276810.

10 minutes - Jo SJ, Kim SG, Lee YM, Giri SS, Kang JW, Lee SB, Jung WJ, Hwang MH, Park J, Cheng C, Roh E, Park SC. Evaluation of the Antimicrobial Potential and Characterization of Novel T7-Like Erwinia Bacteriophages. Biology (Basel). 2023 Jan 23;12(2):180. doi: 10.3390/biology12020180. PMID: 36829459; PMCID: PMC9953017.

Less than 10 minutes - Tang M, Huang Z, Zhang X, Kong J, Zhou B, Han Y, Zhang Y, Chen L, Zhou T. Phage resistance formation and fitness costs of hypervirulent Klebsiella pneumoniae mediated by K2 capsule-specific phage and the corresponding mechanisms. Front Microbiol. 2023 Jul 19;14:1156292. doi: 10.3389/fmicb.2023.1156292. PMID: 37538841; PMCID: PMC10394836.

20 minutes - Fu J, Li Y, Zhao L, Wu C, He Z. Characterization of vB_ValM_PVA8, a broad-host-range bacteriophage infecting Vibrio alginolyticus and Vibrio parahaemolyticus. Front Microbiol. 2023 May 12;14:1105924. doi: 10.3389/fmicb.2023.1105924. PMID: 37250064; PMCID: PMC10213691.

35 minutes -  Eskenazi, A., Lood, C., Wubbolts, J. et al. Combination of pre-adapted bacteriophage therapy and antibiotics for treatment of fracture-related infection due to pandrug-resistant Klebsiella pneumoniae. Nat Commun 13, 302 (2022). https://doi.org/10.1038/s41467-021-27656-z

30 minutes - Zurabov F, Zhilenkov E. Characterization of four virulent Klebsiella pneumoniae bacteriophages, and evaluation of their potential use in complex phage preparation. Virol J. 2021 Jan 6;18(1):9. doi: 10.1186/s12985-020-01485-w. PMID: 33407669; PMCID: PMC7789013.

Fig 2 c and d are stated to show one step growth curves. This is not the case. No bacterial

counts are given and there are obviously two steps in the phage titer curves.

We consider that the 10-minute time gap is too short to reliably represent a two-step curve. The experiment was performed in triplicates and repeated three times using the double agar overlay method, sampling every 10 minutes. The data points that deviate from the expected trend in the graph are likely due to measurement inaccuracies. To address this, we have included error bars in the graphs for better representation of the variability.

One-step-

growth/infection curves cannot be achieved at an MOI of 0,01.

For our one-step growth curve experiments, we exclusively used a MOI of 0.01 to maintain consistency and ensure reproducibility. While it is common practice in the literature to conduct such experiments with MOIs ranging from 1.0 to 0.0001

 MOI = 1 - Peng Q, Ma Z, Han Q, Xiang F, Wang L, Zhang Y, Zhao Y, Li J, Xian Y, Yuan Y. Characterization of bacteriophage vB_KleM_KB2 possessing high control ability to pathogenic Klebsiella pneumoniae. Sci Rep. 2023 Jun 17;13(1):9815. doi: 10.1038/s41598-023-37065-5. PMID: 37330608; PMCID: PMC10276810.

MOI =0.1 - Fu J, Li Y, Zhao L, Wu C, He Z. Characterization of vB_ValM_PVA8, a broad-host-range bacteriophage infecting Vibrio alginolyticus and Vibrio parahaemolyticus. Front Microbiol. 2023 May 12;14:1105924. doi: 10.3389/fmicb.2023.1105924. PMID: 37250064; PMCID: PMC10213691.

 MOI = 0.01 -  Zurabov F, Zhilenkov E. Characterization of four virulent Klebsiella pneumoniae bacteriophages, and evaluation of their potential use in complex phage preparation. Virol J. 2021 Jan 6;18(1):9. doi: 10.1186/s12985-020-01485-w. PMID: 33407669; PMCID: PMC7789013.

 MOI= 0.01- Pertics BZ, Cox A, Nyúl A, Szamek N, Kovács T, Schneider G. Isolation and Characterization of a Novel Lytic Bacteriophage against the K2 Capsule-Expressing Hypervirulent Klebsiella pneumoniae Strain 52145, and Identification of Its Functional Depolymerase. Microorganisms. 2021 Mar 21;9(3):650. doi: 10.3390/microorganisms9030650. PMID: 33801047; PMCID: PMC8003838.

MOI-0.001 - Jo SJ, Kim SG, Lee YM, Giri SS, Kang JW, Lee SB, Jung WJ, Hwang MH, Park J, Cheng C, Roh E, Park SC. Evaluation of the Antimicrobial Potential and Characterization of Novel T7-Like Erwinia Bacteriophages. Biology (Basel). 2023 Jan 23;12(2):180. doi: 10.3390/biology12020180. PMID: 36829459; PMCID: PMC9953017.

 MOI=0.0001- Tang M, Huang Z, Zhang X, Kong J, Zhou B, Han Y, Zhang Y, Chen L, Zhou T. Phage resistance formation and fitness costs of hypervirulent Klebsiella pneumoniae mediated by K2 capsule-specific phage and the corresponding mechanisms. Front Microbiol. 2023 Jul 19;14:1156292. doi: 10.3389/fmicb.2023.1156292. PMID: 37538841; PMCID: PMC10394836.

Fig. 3

In the legend, Fig 3c is missing. How were the N0/Nt numbers calculated? It is not possible to

calculate reliable phage titers only from spot tests!

 (c) is added .

Calculating the logarithmic decrease of phage titer compared to the initial titer is a robust way to quantify and interpret the impact of environmental stress.

Logarithmic values transform significant differences in titer into manageable scales, making it easier to compare phage stability or resistance under different environmental conditions..

The primary concern is how much the titer decreases after stress exposure, not the absolute numbers. A focus on the logarithmic decrease ensures the emphasis remains on the effect of stress rather than variations in starting or ending titers.

For example:

• If 10¹⁰→10⁸, the decrease is 2 logs.
• If 10⁸→ 10⁶, the decrease is also 2 logs. Despite different initial and final values, the impact of the stress is the same.

Was repeated with Double agar-overlay.

Fig. 4

Same problems as for Fig. 3. In the legend, Fig 4c is missing. How were the N0/Nt numbers

calculated? It is not possible to calculate reliable phage titers only from spot tests!

c) is added .

Calculating the logarithmic decrease of phage titer compared to the initial titer is a robust way to quantify and interpret the impact of environmental stress.

Logarithmic values transform significant differences in titer into manageable scales, making it easier to compare phage stability or resistance under different environmental conditions..

The primary concern is how much the titer decreases after stress exposure, not the absolute numbers. A focus on the logarithmic decrease ensures the emphasis remains on the effect of stress rather than variations in starting or ending titers.

For example:

• If 10¹⁰→10⁸, the decrease is 2 logs.
• If 10⁸→ 10⁶, the decrease is also 2 logs. Despite different initial and final values, the impact of the stress is the same.

Was repeated with Double agar-overlay.

Fig. 5

It is not possible to calculate reliable phage titers only from spot tests! Apply plating assays

Calculating the logarithmic decrease of phage titer compared to the initial titer is a robust way to quantify and interpret the impact of environmental stress.

Logarithmic values transform significant differences in titer into manageable scales, making it easier to compare phage stability or resistance under different environmental conditions..

The primary concern is how much the titer decreases after stress exposure, not the absolute numbers. A focus on the logarithmic decrease ensures the emphasis remains on the effect of stress rather than variations in starting or ending titers.

For example:

• If 10¹⁰→10⁸, the decrease is 2 logs.
• If 10⁸→ 10⁶, the decrease is also 2 logs. Despite different initial and final values, the impact of the stress is the same.

Was repeated with Double agar-overlay.

Some spelling errors have to be corrected

• P3: Transmission Electron Microscopy

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Corected

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Quantitatively

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Corected

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• P4 : Nucleotide Blast

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Corrected

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Check the uniformity of the phage names: sometimes it is Ea PF7 and Ea PF9,

sometimes EA Pf7 and EA PF9 resp.

Checked and corrected

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Check the word phage titer instead of phage titter

Checked and corrected

Lines:101-103, 144,256,262

Referee 2

What is the number of strains

Line no. 14:

The number of strains is added

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Name the diseases caused by Ea.

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Done

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Both the phages are morphologically and genomically similar. These phages' lytic activity (host range) differs, including plaque formation. What is the 2% mismatch or difference in their genomes (98% similarity)?

Added more information in manucript

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What are the chemicals or products used to control? What type of resistance developed?

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Information is added in manuscript.

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It looks more general. Write about the advantages of using phages against plant diseases. 

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Information is added in manuscript

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Be consistent with writing Ea (italics or not).

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Corrected

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How many strains of Ea cause diseases in plants?

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To provide a precise count of pathogenic strains, it would require an exhaustive review of all identified E. amylovora strains. However, nearly all naturally occurring strains studied are considered pathogenic to some degree, given the bacterium's reputation as the causative agent of fire blight. There are a few reports of strains with reduced or no pathogenicity, typically due to mutations or loss of critical virulence genes.

Piqué N, Miñana-Galbis D, Merino S, Tomás JM. Virulence Factors of Erwinia amylovora: A Review. Int J Mol Sci. 2015 Jun 5;16(6):12836-54. doi: 10.3390/ijms160612836. PMID: 26057748; PMCID: PMC4490474.

Wang D, Korban SS, Pusey PL, Zhao Y. AmyR is a novel negative regulator of amylovoran production in Erwinia amylovora. PLoS One. 2012;7(9):e45038. doi: 10.1371/journal.pone.0045038. Epub 2012 Sep 18. PMID: 23028751; PMCID: PMC3445560.

Write the exact location. How many samples are collected?

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Done in manuscript

Answer:

Samples for bacteriophage isolation were collected in Hungary, Pecs, from the apple gardens, where fire blight symptoms were seen. 10 samples of apples and soil were collected.

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How were the phages prepared and what concentration

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Information is addeed in manuscript

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Why LB broth? It is usually done using a buffer

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Answer :

While buffer solutions are commonly used, there are numerous instances where LB broth or other media are utilized, as documented in various studies (Erol HB et al., 2022; Zurabov F et al., 2021; Jamal M et al., 2015; Capra ML et al., 2006). Notably, in some cases, the type of suspension medium is not explicitly specified for temperature-related phage tests (Peng Q et al., 2023; Kim S.G et al., 2022). Given this precedent, using media broth should not pose any issues. Furthermore, commercially available phage preparations, such as those from the Eliava Institute, are often suspended in broth (Villarroel J et al., 2017), reinforcing the suitability of broth media for this purpose.

Erol HB, Kaskatepe B, Ozturk S, Safi Oz Z. The comparison of lytic activity of isolated phage and commercial Intesti bacteriophage on ESBL producer E. coli and determination of Ec_P6 phage efficacy with in vivo Galleria mellonella larvae model. Microb Pathog. 2022 Jun;167:105563. doi: 10.1016/j.micpath.2022.105563. Epub 2022 May 2. PMID: 35513294.

Zurabov, F., Zhilenkov, E. Characterization of four virulent Klebsiella pneumoniae bacteriophages, and evaluation of their potential use in complex phage preparation. Virol J 18, 9 (2021). https://doi.org/10.1186/s12985-020-01485-w.

Jamal M, Hussain T, Das CR, Andleeb S. Characterization of Siphoviridae phage Z and studying its efficacy against multidrug-resistant Klebsiella pneumoniae planktonic cells and biofilm. J Med Microbiol. 2015 Apr;64(Pt 4):454-462. doi: 10.1099/jmm.0.000040. Epub 2015 Feb 13. PMID: 25681321.

Capra ML, Quiberoni A, Reinheimer J. Phages of Lactobacillus casei/paracasei: response to environmental factors and interaction with collection and commercial strains. J Appl Microbiol. 2006 Feb;100(2):334-42. doi: 10.1111/j.1365-2672.2005.02767.x. PMID: 16430510.

Peng Q, Ma Z, Han Q, Xiang F, Wang L, Zhang Y, Zhao Y, Li J, Xian Y, Yuan Y. Characterization of bacteriophage vB_KleM_KB2 possessing high control ability to pathogenic Klebsiella pneumoniae. Sci Rep. 2023 Jun 17;13(1):9815. doi: 10.1038/s41598-023-37065-5.

Kim, S.G.; Lee, S.B.; Jo, S.J.; Cho, K.; Park, J.K.; Kwon, J.; Giri, S.S.; Kim, S.W.; Kang, J.W.; Jung, W.J.; et al. Phage cocktail in combination with kasugamycin as a potential treatment for fire blight caused by Erwinia amylovora. Antibiotics 2022, 11, 1566. https://doi.org/10.3390/antibiotics11111566

Villarroel J, Larsen MV, Kilstrup M, Nielsen M. Metagenomic Analysis of Therapeutic PYO Phage Cocktails from 1997 to 2014. Viruses. 2017 Nov 3;9(11):328. doi: 10.3390/v9110328. PMID: 29099783; PMCID: PMC5707535.

 What is the length of the raw reads and coverage?

. Line no. 138

Information is added in manuscript:

Raw reads

Coverage

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Results should start with the phage isolation process. How many samples were processed, how many phages were isolated, and how were these two phages chosen for this study? What was the host strain used for enrichment.

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Information is added in manuscript

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Table 2 is not necessary here. Add as supplemental. 

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Table 2 is removed

 Is it both the phages? Need more clarity in writing.

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Table 2 is removed

The plaque size looks the same in Figure 1 a,b.

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In case of Ea PF 9 plaques were more unform.

Formulation is changed

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 Is it a cocktail? If not, explain the activity of the phages individually

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Corrected

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and Lines 215-218

Table 3: It should be self-explanatory. What are the numbers inside the table?

Done and renamed as Table 2

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Figure 2 A, B: Definitely, there is an emergence of resistant mutants after 15 hours. Did the authors observe the same in overlay plates? Why not test the cocktail (mix two phages)?

After 24 hours of incubation, no mutant bacterial growth was observed with either the double-agar overlay method or the spot test method, making it challenging to conclude at this stage.

Usually, phage cocktails contain several phages. The next phase of the investigation will involve testing these two phages alongside existing phages, such as EaH1, EaH2, and L1, to explore the potential for cocktail formulation and assess any antagonistic or synergistic interactions.

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18. Figure C, D: The X-axis should be identical to compare.

Figure changed

 Figures 3 and 4: Why more than 60 min incubation is necessary and what difference is it making?

We conducted phage inoculations at various temperatures, extending the incubation period from 60 to 120 minutes. This adjustment was made to observe the temperature-dependent dynamics of phage behavior over a prolonged incubation period, potentially providing more insight into temperature effects on phage activity and host interactions. In the case of Ea PF9, the data revealed that after 90 minutes of incubation, the phage titer dropped to 0, indicating complete inactivation. This outcome aligns with the rapid loss of viability observed at higher temperatures, particularly at 55°C. .

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Genome analysis needs comparative analysis of these genomes. Only a 2% difference was noted between the phage genomes, which causes substantial differences in their lytic activity. So the question is on the receptor-binding domain.

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Done comparative analysis of these genomes

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21. The discussion fails to address the important findings in the results. 1) How do both these phages differ, especially the genome, 2) Why phage-resistant mutants, 3) How and why these two phages can be used in plant diseases ?

1)     Added in manuscript

2)     Added in manuscript

3)     Adde in manuscript in results and discusions and conclusion

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