Antimicrobial Multiresistant Phenotypes of Genetically Diverse Pseudomonas spp. Isolates Associated with Tomato Plants in Chilean Orchards

Round 1

Reviewer 1 Report

The study Córdova et al. describes the isolation copper and antibiotic-resistant Pseudomonas bacteria from tomato growing areas localized in different regions of central Chile. The manuscript (MS) is well written, in general, and the experimental methodology described is coherent and robust. Therefore, the study merits publication in the Horticulturae journal. However, there are a number of aspects that need to be addressed before my endorsement of this MS. They are as follows: 

1. The authors describe the identification of Pseudomonad bacteria in agricultural scenarios having increased tolerance to copper toxicity and a relatively limited scope of antibiotic-resistance. How do these bacteria compare to the wide-spread development antibiotic resistance in several other bacteria, including multi-drug resistant human bacterial pathogens? Is the problem equally serious? Is it similar, worse/better than other tomato producing regions of the world? Please, elaborate.

2. It is unclear why isolates obtained from sources other than tomato plants and seedlings (e.g., lettuce, cucumber, soil and irrigation water) were included in the study (see Table 1). The authors should also notice that no further mention of these isolates is made in the MS.

3. How variable is the bacterial population present in tomato plants/orchards in a given season/year? Does the population density, diversity and/or antibiotic/copper resistance vary with the season? Is it subject to selective pressure? Please elaborate.

4. The use of the word “pesticide” in the Abstract is misleading. The latter considering the definition of pesticide as “… any substance or mixture of substances intended for preventing, destroying, repelling, or mitigating any pest”, where a pest is many times construed as a damaging insect or unwanted species of plants or animals. Perhaps this word should be changed to “bactericide” for clarity sense.

5. How easily do endophytic bacteria acquire antibiotic/copper resistance compared to bacteria living in other plant contexts? How likely is their role in conferring these properties to the surrounding population? Please consider that the virulence of several phytopathogens is significantly modified if they conform part of the endophytic microorganism population.

6. The authors fail to clearly indicate the possible sources of antibiotic/metal resistance (i.e., soil amendment with manure; reuse of wastewater for irrigation; other bacterial pathogens, such as Erwinia amylovora or Xanthomonas campestris; refer, for example, to Scaccia et al. 2021. Trends in Plant Science, 26: 1213-1226) detected in the Pseudomonas isolates described in the MS.

7. Lines 123-125 in Materials and Methods: The two sampling dates are separated by ca. two years. Why was this relatively large time-gap produced? Please explain.

8. Lines 136 in Materials and Methods: is “cc” a better way to indicate cubic centimeters than “cm^3”?

9. Line 137 in Materials and Methods: Please change H2O to H₂O.

10. Line 139 in Materials and Methods: The authors restrict the identification of metal and antibiotic resistant bacteria by using the highly selective King’s B medium. Can antibiotic resistance /metal toxicity tolerance be present in non-culturable bacteria? Could these bacteria contribute to the dissemination of these properties to other bacteria/microorganisms? Please elaborate.

11. Line 141 in Materials and Methods: Please change “hours” to “h”.

12. Line 142 in Materials and Methods: The use of a word different from “shine” (e.g., “luminosity”, “luster”, “brightness”, etc.) here and in other sections of the MS is suggested.

13. Line 145 in Materials and Methods: Something is wrong here. What does “growth and maintenance” supposed to mean or indicate?

14. Line 174 in Materials and Methods: Please use the journal´s referencing format; i.e [49], instead of "Sarkar and Guttman, 2004".

15. Line 186 in Materials and Methods: Please change “found” to “confirmed” or similar word.

16. Lines 212 and 213 in Materials and Methods: The meaning of MIC is unclear. Please redefine. How can the OD₆₀₀ be less than that observed at "0 h"? The authors should be aware that “…(MICs) are defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation”.

17. Line 214 in Materials and Methods: It is unclear why this parameter was used to determine MIC to copper. Please notice that this particular concentration was not included in the Cu toxicity assay.

18. Lines 397-399 in Results: Please rephrase this sentence to clarify its meaning.

19. Figure 1: Is there any particular reason why the sampling was done in the regions shown in the map?

20. Table 1: Was the development of pathological symptoms associated with the presence of known phytopathogenic Pseudomonas bacteria? Please define signs of illness/pathological symptoms with greater detail. Perhaps, by using a predetermined scale. The term "recently dead" is unclear; please modify it.

21. Table 2: Did the identification procedure employed permit the assignation of a particular pathovar to the P. syringae bacteria included in this group?

22. Lines 493-494 in Discussion: Regarding the possibility that some previously beneficial/non-pathogenic Pseudomonas species may turn virulent, what arguments can the authors present to support their suggestion that the pathological symptoms observed could have been caused by these bacteria? What conditions are responsible for this shift? Could the effect of other bacterial/fungal pathogens be ruled-out? Please elaborate.

23. Lines 523-528 in Discussion: On the basis of the arguments employed here, it is strongly suggested that the authors complement this study with genomic/ transcriptomic/ proteomic /metabolomic/ physiologic assays designed to identify the possible mechanisms that underlie the metal and antibiotic resistant phenotypes observed here. The authors should be aware that most of the discussion concerned with the nature of the metal and antibiotic-resistance observed in the Pseudomonas isolates is of a highly speculative nature.

24. Lines 538-554 in Discussion: This, and other paragraphs included in this section, should be streamlined to facilitate the comprehension of the complex and diverse information that is discussed. The authors should also consider transferring a sizeable portion of this information to the "Introduction".

25. Line 586 in Discussion and others: Please define the term "environmental bacteria".

26. Lines 631-633 in Discussion: In the context of this paragraph, please describe with greater detail what short- and long-term alternatives to these control practices could be implemented in tomato-producing areas to reduce the generation of Cu/metal and antibiotic-resistant bacteria.

27. Lines 666-667 in Conclusion: What is the experimental support of the suggestion that multi-resistant Pseudomonas present in tomato producing areas and other commercial agricultural scenarios could act as vectors of resistance genes to human pathogens?

28. In the References section: Please revise the reference included to correct deviations from the journal´s referencing format. Refer, for instance to lines 739 and several others, where scientific names need to be italicized; to line 754 and others, where the name of the journal should be spelled in full, and line 769 and others, where a comma should be added between the journal´s title and the year.

Author Response

Dear reviewer 1, we greatly appreciate your comments. They have contributed to improve our manuscript. Below we will answer each of the observations

1. The authors describe the identification of Pseudomonad bacteria in agricultural scenarios having increased tolerance to copper toxicity and a relatively limited scope of antibiotic-resistance. How do these bacteria compare to the wide-spread development antibiotic resistance in several other bacteria, including multi-drug resistant human bacterial pathogens? Is the problem equally serious? Is it similar, worse/better than other tomato producing regions of the world? Please, elaborate.

Pseudomonads in general have a reputation for being highly resistant to antimicrobial compounds, and Pseudomonas associated with plants (like P. syringae) are no exception. Antimicrobials such as copper and streptomycin have been used for decades to control bacterial infections of crop plants (Lamichhane et al., 2018; Sundin and Wang, 2018). The selection of bacterial strains resistant to bactericidal compounds seems to be the main cause of failure in control of fytopathogens with conventional treatments. Once the resistance genes are acquired, the frequency of resistant strains increases progressively due mainly to horizontal gene transfer between bacteria (Behlau et al, 2012; Xu et al, 2013). For example, strains of bacterial phytopathogens as Erwinia amylovora, Erwinia carotovora, Xanthomonas campestris, P. syringae pv. lachrymans, P. syringae pv. papulans, P. syringae pv. syringae, and P. syringae pv. actinidae resistant to streptomycin have been isolated in North and South America (Xu et al., 2013; Sundin and Wang, 2018).

There are several studies demonstrating resistance to agrochemicals of diferent phytopagens and confirming that this is a global threat to agricultural production (Lamichane et al., 2018; Sundin and Wang, 2018).

Comments were added in the introduction section as follows:

“Although resistance to antibiotics is a serious problem throughout the world, there are a limited number of studies on this topic in bacteria associated with agricultural crops in Chile (Esterio et al., 2007; Altimira et al., 2012), being this study, the first associated with tomato crops in agricultural environments of our country.”

Recent studies have focused in the posible role of environmental bacteria as sources of antimicrobial resistance genes (ARGs) for human pathogens with worrying results. In this sense, the problem of resistant environmental bacteria appears to be as serious as the resistance in human pathogens. For example, in the work of Forsberg et al (2012), multidrug-resistant soil bacteria containing resistance cassettes against five classes of antibiotics (β-lactams, aminoglycosides, amphenicols, sulfonamides, and tetracyclines) that have perfect nucleotide identity to genes from diverse human pathogens are described. This identity encompasses noncoding regions as well as multiple mobilization sequences, offering not only evidence of lateral exchange but also a mechanism by which antibiotic resistance disseminates. In this context, the environment and soil are important past, current, and potentially future reservoirs of clinically relevant antimicrobial resistant bacteria and thus must be considered in the evaluation of risk factors contributing to the global spread of antimicrobial resistance (Thanner et al., 2016).

Comments were added in the introduction section as follows:

“Several studies support the idea that environmental bacteria, including those associated to edible plants, play a role as vector of ARGs, mainly by its movilization troguh the food chain (Thanner et al., 2016). An example of this is the presence of antimicrobial resistant bacteria in fresh fruits and vegetables (including tomato) for human consumption (Ruimy et al., 2010; Allydice-Francis et al., 2012; Sun et al., 2021). Members of genera such as Enterobacter, Acinetobacter, Pseudomonas, Staphylococcus, Burkholderia, Serratia, Stenotrophomonas, and Bacillus, often associated with crops, may harbor antibiotic-resistance genes (Scaccia et al., 2021).”

Thus, there is strong evidence to suggest that crops are a potential vehicle for resistant bacteria to come into contact with the human microbiota. Some authors even suggest that these resistant bacteria could be a source of genetic material for lateral gene transfer subsequent to ingestion, giving rise to antibiotic resistant human pathogens (Taylor and Reeder, 2020). So, we can say that the problema of enviromental or crop associated resistant bacteria is closely related to the problem of antibiotic resistant human pathogens.

Concerning to the question of the situation in other tomato producer regions:

It is difficult to compare with the rest of the world in terms of tomato producing regions, since there are few specific studies for bacteria associated with this crop. In the discussion section of the manscript, the results are compared with those found in other similar studies of Pseudomonas with resistant phenotypes.

Comments were added in the introduction section as follows:

“Althoug we can mention the study of Hwang et al (2005) focused in Pseudomonas syringae isolated from different crops including tomato, betwwen years 1935-1998 and from different countries including USA, UK, Japan, Canadá, Switzerland, Zimbabwe, Ethipia, Yugoslavia, Greece. Overall, the results show, that from a total of 95 strains, 75%, 58%, 38%, 16%, 8% and 1% showed resistant phenotypes against copper, ampicillin, chloramphenicol, rifampicin, streptomycin and kanamycin-tetracycline, respectively. This demosntrated that the problema of antimicrobial resistance is present worldwide even in plant associated bacteria and since at leats 20 years ago. Recent studies, have confirmed that this is a problema common for different bacterial species.”

For example. Corzo-Ariyama et al., (2019) analyzed more than 300 E. coli strains isolated from the jalapeño pepper, tomato, and cantaloupe farm environments, in Northeast Mexico. They found that antibiotic resistance to tetracycline (23.2%) and ampicillin (19.9%) was high, and 3.5% of the strains presented resistance to more than 5 antibiotics, revealing a health risk for consumers.

Comments were added in the introduction section as follows:

“Also, the study of Sun et al., (2021) in fresh tomatoes ready for human consumption in the Chinese market, found a total of 191 ARGs and 10 mobile genetic elements (MGEs) were detected on fresh tomato surfaces. Their results indicated that fifteen bacterial families might be the potential hosts of ARGs. These results are a call to pay more attention to ecological environment impacts of ARGs and ARB on the surfaces of vegetable or fruit.”

Finally, all togheter the mentioned studies support the idea that crops, such as lettuce, carrot, radish, cucumber and tomato, among others, can host antibiotic-resistant bacteria, being a worldwide problem, as these resistant bacteria are potential vectors of transmission to humans.

Comments were added in the introduction section as follows:

“Finally, in relation with the seriousness of the antimicrobial resistance problem in agricultural bacteria compared with human pathogens, we share the opinion of Scaccia et al. (2021) that, under the One-Health concept (humans, animals, and environment), the environment contamination with antibiotic-resistant bacteria cannot be dissociated from its potential transmission to humans. So, we consider that, the presence of environmental bacteria resistant to antibiotic should be considered a risk as serious as antibiotic resistance in human pathogens.”

2. It is unclear why isolates obtained from sources other than tomato plants and seedlings (e.g., lettuce, cucumber, soil and irrigation water) were included in the study (see Table 1). The authors should also notice that no further mention of these isolates is made in the MS.

Although the study focused on Pseudomonas associated with tomato plants, it also wanted to include and explore those bacteria associated with the agricultural environment where these plants were found. For this reason, samples of plant that were grown in the same greenhouse (crop environment) in the surroundings of the tomato plants, and that were handled by the same agricultural staff, so they could be susceptible to being colonized by the same microorganisms, were also taken.

It has been observed in other studies that different bacteria, including Pseudomonas, are capable of surviving in the environment outside their host (Monteil, 2013; Ruinelli, 2019; Corzo-Ariyama, 2019), so it is possible to isolate them from samples such as soil, water or plant debris. In this case, soil, water and other vegetables located in the same orchard as the tomato plants were considered.

This information was included in the materials and methods section as follows:

“Several studies have shown the ability of Pseudomonads to survive in the environment outside their host plants, even in non-agricultural habitats (Monteil, 2013; Ruinelli, 2019). For these reasons, samples of irrigation wáter, soil and other vegetables grown in the same agricultural environment were included for their analysis (Table1)”

3. How variable is the bacterial population present in tomato plants/orchards in a given season/year? Does the population density, diversity and/or antibiotic/copper resistance vary with the season? Is it subject to selective pressure? Please elaborate.

In the present study, samples of the same orchard in different seasons or years were no taken, so reliable conclussions related to the bacterial diversity or antimicrobial resistance associated to the tomato plants can’t be drawn from the data obtained.From the reviewed literature, no long-term population studies of the microbiota associated with tomato are reported, so it is difficult to compare between one season and another. In general, there are studies on changes in the bacterial community associated with tomato due to the effect of some external factor (such as a fertilizer or a pathogen) (Jiang et al., 2019; Zheng et al., 2019), however, there has been no deepening of how the bacterial community associated with tomato changes in different seasons or how it is antimicrobial resistance would be affected depending on the season.

Regarding the selective pressure, it could be considered constant during the cultivation period in Chile. In the sampled orchards, the applications of agrochemicals are generally carried out periodically, however, the number of products applied, the products themselves, the number of applications, and dose may vary depending on the preferences of each farmer. In this sense we consider important to mention that there is increasing information about occurrence of co-selection of metal and antibiotic resistance determinants. In this sense, it is possible that even in the absence of selective antibiotic pressure, the genetic determinants are maintained by the cells, due to, for example, the presence of cupric bactericides or metal contamination in the soil (Altimira et al., 2012; Seiler and Berendonk, 2012; Mazhar et al., 2021)

This information was included in the Discussion section as follows

“This study was the first associated with tomato crops to evaluate the phenotypic and tolerance to antimicrobial compounds in agricultural environments in Chile.The samples of the same orchard in different seasons or years were no taken, so reliable conclussions related to the bacterial diversity or antimicrobial resistance associated to the tomato plants can’t be drawn from the data obtained. However, there has been no deepening of how the bacterial community associated with tomato changes in different seasons or how it is antimicrobial resistance would be affected depending on the season.”

“In the sampled orchards, the applications of agrochemicals are generally carried out periodically, however, the number of products applied, the products themselves, the number of applications, and dose vary depending on the preferences of each farmer. In this sense, it is possible that even in the absence of selective antibiotic pressure, the genetic determinants are maintained by the cells, due to, for example, the presence of cupric bactericides or metal contamination in the soil (occurrence of co-selection of metal and antibiotic resistance determinants) (Altimira et al., 2012; Seiler and Berendonk, 2012; Mazhar et al., 2021)

4. The use of the word “pesticide” in the Abstract is misleading. The latter considering the definition of pesticide as “… any substance or mixture of substances intended for preventing, destroying, repelling, or mitigating any pest”, where a pest is many times construed as a damaging insect or unwanted species of plants or animals. Perhaps this word should be changed to “bactericide” for clarity sense.

The suggestion was accepted, and we replace the word "pesticide" with "bactericide" in the text.

5. How easily do endophytic bacteria acquire antibiotic/copper resistance compared to bacteria living in other plant contexts? How likely is their role in conferring these properties to the surrounding population? Please consider that the virulence of several phytopathogens is significantly modified if they conform part of the endophytic microorganism population.

In general, and based on the revised literature, the possibility of endophytic bacteria to acquire genetic determinants of resistance, relies mainly on the probability of these bacteria to enter in contact with genetic material harbouring these deterinants or with other bacteria which coul act as a donor of genetic material. The occurrence of horizontal gene transfer (HGT) as a major evolutionary event has been described in various studies. However, reports about HGT events in endophytes are limited. Some recent studies confirmed the possibility of HGT between endophytic bacteria and also between epiphytic and endophytic bacteria.

A recent study performed by Karmakar et al. (2019) showed that endophytic bacteria isolated from green leafy vegetables (GLV's) were found to be resistant to a wide range of antibiotics (AB) and heavy metals (HM). Bacterial identity revealed the association of both plant beneficial and human pathogenic bacteria as an endophyte with GLV's. Also, the authors mentioned that vertical and horizontal gene transfer between introduced and native bacteria is the crucial factor in enhancing their fitness under abiotic stress conditions.

Tiwari et al. (2020) reviewed and discuss several studies that confirmes events of HGT between endophytic bacteria. For example; a study of, 116 arsenite-resistant bacteria isolated from P. vittata roots, showed that the exchange of arsenite transporter genes, possibly occurred through HGT between endophytic bacteria (Gu et al., 2018). A similar study reported that the gene transfer between endophytic bacteria residing inside T. durum and Z. mays, induced toluene degradation and promoted plant growth (Wang et al., 2010). Also, the work of Nongkhlaw et al. (2015) showed molecular evidence for HGT among the endophytes and epiphytes associated with Ethnomedicinal Plants.

So, despite there are a few works speaking of HGT in endopytic bacteria, the growing knowlenge about this topic suggest that it is posible that endophytic bacteria acquire genetic resistance determinants from other endophytes or even from epiphytic bacteria associated with plants.

This topic was included in the Discussion section as follow:

“Since horizontal gene transfer (HGT) is one of the mechanisms that contributes the most to the spread of resistance determinants, recent studies demonstrating HGT events between endophytic and epiphytic bacteria associated with plants are of importance, considering that endophytic bacteria associated with edible plants could easily enter in contact with human associated bacteria trough the food-chain (Nongkhlaw et al., 2015; Karmakar et al.,2019)”.

6. The authors fail to clearly indicate the possible sources of antibiotic/metal resistance (i.e., soil amendment with manure; reuse of wastewater for irrigation; other bacterial pathogens, such as Erwinia amylovoraor Xanthomonas campestris; refer, for example, to Scaccia et al. 2021. Trends

Antibiotic and copper resistance developed by phytopathogenic bacteria is mainly due to the excessive use of these bactericides to control the disease caused by Pseudomonas (Lamichhane et al., 2018; Sundin and Wang, 2018). In general, were the genetic basis for resistance has been examined, antibiotic resistance in plant pathogens has most often evolved through the acquisition of a resistance determinant via horizontal gene transfer. For example, the strAB streptomycin-resistance genes occur in Erwinia amylovora, Pseudomonas syringae, and Xanthomonas campestris, and these genes have presumably been acquired from nonpathogenic epiphytic bacteria colocated on plant hosts under antibiotic selection. The location of essentially the same genetic element in different genera of plant pathogens isolated from distinct crop hosts and from different continents is confirmatory evidence of the role of horizontal gene transfer (HGT) in the dissemination of antibiotic resistance in these pathosystems.  Several studies demonstrate that plant disease control agents such as streptomycin also affect the native phyllosphere and soil microflora and further indicated that ARGs can be selected in epiphytic bacteria in antibiotic-sprayed plant habitats and could provide a route of acquisition by plant pathogens (Sundin and Wang, 2018).

Also, it must be considered the effect of environment contamination with antimicrobials. Different studies argued that bacteria exposed to human-derived biological contamination may susceptible to acquire resistance genetic determinants under selective pressure (Scaccia et al., 2021). In this sense, irrigation water is generally regarded as one of the important bacterial contamination sources in vegetable growth during the preharvest phase. Also, the use of manure in soil and other soil contamination, as well as the presence of antimicrobials in any component of the agricultural environment, could lead to the selection of resistant bacterial strains and must be considered in the evaluation of risk factors contributing to the global spread of antimicrobial resistance (Thanner et al., 2016).

Taking into account the comment of the reviewer, we include the following information in the introduction section:

“Althoug, the excessive use of agrochemicals has been pointed out by different authors as the main cause of resistance development, other possible sources of antibiotic/metal resistance need to be considered. Several studies demonstrate that plant disease control agents such as antibiotics or copper, also affect the native phyllosphere and soil microflora and further indicated that antibiotic resistance genes can be selected in epiphytic bacteria in antibiotic-sprayed plant habitats and could provide a route of acquisition by plant pathogens (Sundin and Wang, 2018). Also, it must be considered the effect of environment contamination with antimicrobials, for example, as a consequence of the use of manure in soil or reuse of waste water for irrigation. Different studies argued that bacteria exposed to human or animal-derived biological contamination may susceptible to acquire resistance genetic determinants under selective pressure (Scaccia et al., 2021) and must be considered in the evaluation of risk factors contributing to the global spread of antimicrobial resistance (Thanner et al., 2016)”.

7. Lines 123-125 in Materials and Methods: The two sampling dates are separated by ca. two years. Why was this relatively large time-gap produced? Please explain.

The time gap is produced by the interest in collaborating with institutions and laboratories whose researchers isolated and characterized Pseudomonas spp. from different agricultural areas of Chile, in different times. It seemed important for us to expand the number of bacterial isolates, to incorporate this information in the work.

8. Lines 136 in Materials and Methods: is “cc” a better way to indicate cubic centimeters than “cm^3”?

The suggestion was accepted and “cc” was changed for “cm³”.

9. Line 137 in Materials and Methods: Please change H2O to H₂

The suggestion was accepted, and the word changed.

10. Line 139 in Materials and Methods: The authors restrict the identification of metal and antibiotic resistant bacteria by using the highly selective King’s B medium. Can antibiotic resistance /metal toxicity tolerance be present in non-culturable bacteria? Could these bacteria contribute to the dissemination of these properties to other bacteria/microorganisms? Please elaborate.

Although no reports of non-culturable bacteria associated with plants in which antimicrobial resistance genes were described were found in the reviewed literature, this possibility cannot be completely ruled out.

However, the study focuses its interest on the genus Pseudomonas, among other reasons, because it is one of the most abundant associated with tomato and because it has well-known phytopathogenic representatives that affect tomato production, such as those belonging to the P. syringae complex. Non-cultivable bacteria limit their study in terms of determining their resistant phenotype precisely because they are not cultivable, although resistance genes can be identified through metagenomics (Altimira et al., 2012; Fleischmann et al.,2021), it would be more complex to verify a resistant phenotype.

In the case of bacteria in the viable but non culturable (VBNC) state, it has been reported cases of human pathogens, like E. coli and P. aeruginosa, which present antimicrobial resistance and are able to retain virulence (Fu et al., 2020). But information about plant associated non-culturable bacteria is very limited concerning to the antimicrobial resistance.

Generally, for the control of non-cultivable phytopathogenic bacteria such as Candidatus Phytoplasma spp. and Candidatus Liberibacter spp., due to their obligate phloem habitat, antibiotics and copper are not widely used. The most frequent use of antibiotics has been made in citrus plants, injecting them into the trunks to control ´Ca. Liberibacter spp.´. However, this practice has shown low efficiency and is very expensive. Definitely, the control of both kind of pathogens is based almost exclusively on prevention of the infection (Li et al., 2021).

On the other hand, for the control of Pseudomonas in crops in Chile, antibiotics are applied by spraying, with less possibility of coming into contact with bacteria present in the phloem, therefore it is highly probable that the transfer of resistance between phloem bacteria and Pseudomonas spp. (present only in the apoplast of plants) will not occur.

However, it must be taken into account that in the event that a non-culturable bacteria that contains genetic determinants of resistance to antimicrobials is associated with an edible plant, it is possible that it will come into contact with bacteria associated with humans through the food chain and serve as vectors of ARGs (Thanner et al., 2016; Karmakar et al.,2019; Scaccia et al., 2021).

11. Line 141 in Materials and Methods: Please change “hours” to “h”.

The suggestion was accepted.

12. Line 142 in Materials and Methods: The use of a word different from “shine” (e.g., “luminosity”, “luster”, “brightness”, etc.) here and in other sections of the MS is suggested.

The suggestion was accepted, and changed “shine” for “brightness”

13. Line 145 in Materials and Methods: Something is wrong here. What does “growth and maintenance” supposed to mean or indicate?

The suggestion was accepted, and was changed to the following paragraph

“The isolated strains were cultivated in solid KB medium or nutrient broth (NB) (meat extract 0.3%, peptone 0.5%) and incubated at 28 °C. For long-term maintenance, the isolated strains were cryopreserved and stored at -80ºC, in nutrient broth with glycerol in a 1:1 ratio”.

14. Line 174 in Materials and Methods: Please use the journal´s referencing format; i.e [49], instead of "Sarkar and Guttman, 2004".

The format mistake was corrected.

15. Line 186 in Materials and Methods: Please change “found” to “confirmed” or similar word.

The solicited change was made, and the word “found” was replaced by “confirmed”.

16. Lines 212 and 213 in Materials and Methods: The meaning of MIC is unclear. Please redefine. How can the OD₆₀₀be less than that observed at "0 h"? The authors should be aware that “(MICs) are defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation”.

In this case, we based our methodology in the work of Hwang et al. (2005) which works with P. syringae bacterial strains. According to what is described in the mentioned work, the definition of MIC was considered as the following “MIC is defined as the point where the OD of the bacterial culture at 48 h was the same or less than it was at 0 h” (Hwang et al., 2005). Since it is a methodology that used optical density to detect bacterial growth, a constant value will indicate no growth and a decreased value could indicate lisis or death. It is important to clatify that the initial OD₆₀₀ of the culture was fixed at 0.1.”

For more clarity, the follow information was added in the Materials and Method section:

“Each assay was started (0 h) with a fresh inoculum of the tested bacterial isolate fixing an initial OD₆₀₀ at 0.1”

And the definition of MIC was clarified as follow:

“MIC was defined as the point where the OD₆₀₀ of the bacterial culture at 24 h, 48 h and 72 h was equal to (indicating no growth) or less (indicating posible cell death or lysis) tan OD₆₀₀ at 0 h”.

17. Line 214 in Materials and Methods: It is unclear why this parameter was used to determine MIC to copper. Please notice that this particular concentration was not included in the Cu toxicity assay.

The cutoff MIC value to determine tolerance to copper was fixed according to the study of Nakajima et al. (2002), which established a MIC value of 0.75mM of CuSO₄ (equivalent to 48 µg/mL of Cu⁺²) for copper sensitive P. syringae. This agrees to the value established by Hwang et al., (2005) were P. syringae strains with MICs equal or less than 0.8 mM CuSO₄ (equivalent to 50 µg/mL Cu⁺²) were scored as copper sensitive. In other study, the cuttof MIC value was established at a concentration of 32 µg/mL of Cu⁺² (Gardan and Brault, 1993). These studies were referenced for a validated cuttof value. However, in practice, considering the concentrations used in our study, strains with MIC values ​​equal to or greater than 64 µg/mL of Cu⁺² were considered tolerant to copper, as it was a higher concentration than that used in other studies.

For more clarity, Materials and Methods section was modified as follow:

“A cuttof MIC value between 32-50 µg/mL of Cu⁺² has been considered for analyses in previous studies (Gardan and Brault, 1993; Nakajima et al., 2002; Hwang et al., 2005). Based on those criteria, in this study strains with MIC values ​​equal to or greater than 64 µg/mL of Cu⁺² was considered tolerant to copper”

18. Lines 397-399 in Results: Please rephrase this sentence to clarify its meaning.

The sentence was modified as follow:

“According to resistance phenotype, isolates were clustered into four groups (I-IV) (Figure 4). Cluster I contained twenty isolates, 75% resistant to rifampin and ampicillin, with 40% of isolates also being copper tolerant. Only two isolates (pJS5.5 and pAI.1) with resistance to a single antimicrobial (rif) were found in this group. Cluster II all the isolates were resistant to chloramphenicol and to ampicillin. Of the 34 isolates in this group, 52,9% were resistant to 4 antibiotics and 44,1% were resistant to 5 antimicrobials (Cu + antibiotics). Isolate JV.9r constituted cluster III, showing resistance to five antimicrobials (Cu, str, amp, rif and tet), being the unique isolate resistant to tetracycline. Finally, cluster IV was composed of 3 (33,3%) isolates sharing the resistance phenotype to streptomycin, rifampin, ampicillin and copper. Among them, seven (77,8%) resistance to three antimicrobials (Cu, str and rif). No isolates had resistance to chloramphenicol, gentamicin and tetracycline.”

19. Figure 1: Is there any particular reason why the sampling was done in the regions shown in the map?

Yes, because they are the most important areas for the production of tomato in Chile, equivalent to more than 10.400 ha of productive surface from a total of approximately 13.000 ha (odepa.gob.cl, “Boletín Hortalizas 2021”). It is important to mention that samples were obtained from the largest number of orchards that voluntarily wanted to collaborate with our scientific research.

For more clarity, Materials and Methods section was modified as follow:

“…bacterial isolates were recovered from vegetable tissue of tomato plants cultivated in greenhouses, open fields, and nurseries found throughout four regions of the Central Zone of Chile: Valparaíso, O´Higgins, Maule and Bío Bío (Figure 1). These regions were chosen, as they are the most important to produce tomato in Chile, equivalent to more than 75% of the national productive surface (odepa.gob.cl)”.

20. Table 1: Was the development of pathological symptoms associated with the presence of known phytopathogenic Pseudomonasbacteria? Please define signs of illness/pathological symptoms with greater detail. Perhaps, by using a predetermined scale. The term "recently dead" is unclear; please modify it.

In general, bacterial isolates related to phytopathogenic species were not always obtained when there were signs of disease. On the other hand, in the case of isolates close to P. syringae pv. syringae or P. viridiflava were found mostly in samples from symptomless plants. In this context, it should be noted that in the case of isolates identified as close to P. syringae pv. tomato, all of them indeed came from diseased plants. But a direct correlation cannot be made with all isolates.

It should be considered that external fators like temperature and humidity influences the development and severity of bacterial diseases in plants. In this sense, a phytopathogenic bacterium can remain a latent infection until the right conditions are met for the development of the disease.

For more clarity, the follow information was added to the description of Table 1:

“Signs of illnes: dark necrotic spots (with or without a clorotic halo) on leaves or stems, on plants that were still green.

Recently dead: In the final stage of the crop (prior to being discarded), corresponding to the state known as senescence.”

21. Table 2: Did the identification procedure employed permit the assignation of a particular pathovar to the  syringaebacteria included in this group?

Pathovars are bacterial isolates or a series of isolates with very similar characteristics, which differ from other isolates of the same species, on the basis of a specific pathogenicity in one or more host plants. This means that a pathovar is defined based on the result of pathogenicity tests. Then, there is no very well standardized procedure based on sequence analysis that allows unequivocally assigning a pathovar. In fact, some inconsistencies between the assignment of pathovar based on phenotypic characteristics (as ecology, physiology, and pathology) and the characterization based in molecular techniques (as DNA-DNA hybridization and phylogenetic analysis of housekeeping genes sequences), has generated controversy in the taxonomy of the genus Pseudomonas.

Molecular techniques have allowed to reclassify strains and to clarify a more stable phylogenetic classification. However, in the work of Gomila et al. (2017) which included molecular analysis of 27 strains of P. syringae assigned to 15 different pathovars, the results showed that strains of a particular pathovar: (I) clustered together in one phylogenomic species, (II) clustered together with other species strains in one phylogenomic species or (III) affiliated to different phylogenomic species. The study highlights the need to reclassify the misclassified strains and to stablish a truly systematic taxonomy for P. syringae species that can be adopted by scientific community. Furthermore, is strongly complemented by the results presented by Morris et al. (2019) who propose that pathovar denominations do not correspond to the underlying biology of P. syringae and is misleading.

For these reasons and based on the results obtained and our experience, we could not assign a specific pathovar to these bacterial isolates.

22. Lines 493-494 in Discussion: Regarding the possibility that some previously beneficial/non-pathogenic Pseudomonasspecies may turn virulent, what arguments can the authors present to support their suggestion that the pathological symptoms observed could have been caused by these bacteria? What conditions are responsible for this shift? Could the effect of other bacterial/fungal pathogens be ruled-out?

Species like P. fluorescens and P. putida has been historically considered innocuous and also benefical for different crops. But, emergent studies, has demonstrated that some strains have the ability to develop opportunistic infections and can cause illness and death under certain conditions. The mechanism involved are still largely unknown, but cases of P. fluorescens and P. putida causing deasease has been confirmed (Dimartino et al., 2011; Mota et al., 2021). An example is the work of Dimartino et al. (2011) which demonstrate virulence traits in P. fluorescens biovar I and P. putida biovar A strains, which were able to induce leaf chlorosis, necrosis, or death of tomato plants under saline stress conditions. Other case is described in the study of Mota et al. (2021) were bacterial strains isolated from infected tissue samples of tomato plants with severe symptoms of pith necrosis and premature death (symptoms similar to those caused by Pseudomonas corrugata) in cultivation áreas of Paraná and Minas Gerais (Brazil), were characterized. The study revealed that the four isolated strains experimentally confirmed as pathogenic were identified by molecular characterization as Pseudomonas fluorescens and Pseudomonas putida.

Based on the revised literature, the authors pose as a possibility that Pseudomonas species generally known as saprophytes, could eventually developed an opportunistic deasease and be responsable of the illnes symptoms observed. But, based on the methodology used, the action of another pathogens like fungi or other bacteria, could not be rulet-out.

23. Lines 523-528 in Discussion: On the basis of the arguments employed here, it is strongly suggested that the authors complement this study with genomic/ transcriptomic/ proteomic /metabolomic/ physiologic assays designed to identify the possible mechanisms that underlie the metal and antibiotic resistant phenotypes observed here. The authors should be aware that most of the discussion concerned with the nature of the metal and antibiotic-resistance observed in the Pseudomonasisolates is of a highly speculative nature.

In Chile, information about bacteria associated with different agricultural crops is scarce, even the information of phytopatogen outbreaks is limited. The present study is, to the best of our knowledge, the first study in relation to microorganisms associated with tomato crops (specifically Pseudomonas) in Chile and their resistance phenotypes. In this context, this work constitutes a necessary initial and exploratory study that allows establishing a starting point and a warning for the need of future studies regarding bacterial resistance to antimicrobials in our country.

The main motivation of the study was to assess the susceptibility of tomato-associated Pseudomonas to the main agrochemicals (copper and streptomycin) used to control bacteriosis in these crops. Despite, resistance to these bactericides is reported in different parts of the world, there was no study on the matter in our country. The lack of information and the fact that the conventional treatments were not satisfactorily effective, led us to make a first approximation to assess the situation of antimicrobial resistance in Chilean crops. In this sense, the identification of the Pseudomonas associated to tomato crops and their resistance phenotypes constitute a valuable first step that allows to lay the foundations to continue the research in this matter in Chile.

It should be mentioned that most studies in the agricultural field are limited to analyzing bacterial resistance to agrochemicals such as copper and streptomycin. But, considering the global crisis of antimicrobial resistance, the idea of ​​analyzing the resistance of environmental bacteria to antimicrobials used in other fields such as veterinary and human medicine has gained strength. In this context, the information about environmental or phytopathogenic Pseudomonas showing resistant phenotyps to both antimicrobials used in agriculture and human/ veterinary medicine constitutes a wake-up call and an invitation to continue contributing to scientific research in our country.

Despite the genetic bases that support resistant phenotypes could not be addressed, the methodology to determine resistant phenotypes is used throughout the world for these purposes, so we consider that the information obtained satisfies the exploratory nature of this study. In this sense, we agree with what was outlined by the reviewer and in light of the results obtained, arise the need of future research in the field of genomic/transriptomic to delve into the resistance mechanisms and the genetic bases that support them.

Taking in to account the reviewer observation, the discussion section was edited to reduce the speculative nature of some parragraphs, mainly those related to the risk of resistance genes transfer between environmental bacteria and human pathogens and the possible role of environmental bacteria as resistance genes reservoir. Also, was clarifies that future studies are needed to support these ideas.

24. Lines 538-554 in Discussion: This, and other paragraphs included in this section, should be streamlined to facilitate the comprehension of the complex and diverse information that is discussed. The authors should also consider transferring a sizeable portion of this information to the "Introduction".

The observation of the reviewer was accepted, and the paragraph was simplified and moved to the introduction section.

25. Line 586 in Discussion and others: Please define the term "environmental bacteria".

The concept of environmental bacteria, for purposes of this work is defined as: Bacteria present in the environment. The environment in this case means the soil, water, air and sediments covering the planet and can also include the animals and plants that inhabit these areas.

For more clarity, besides the first time the concept of environmental bacteria was used in the manuscript we include the following information:

“environmental bacteria (undestood as bacteria present in the soil, water, air and sediments covering the planet, including animals and plants that inhabit these áreas)”

26. Lines 631-633 in Discussion: In the context of this paragraph, please describe with greater detail what short- and long-term alternatives to these control practices could be implemented in tomato-producing areas to reduce the generation of Cu/metal and antibiotic-resistant bacteria.

In the context of the mentioned paragraph, the follow information was added to discussion section:

“Faced with the imminent problem of antibiotic resistance, it is necessary to develop, implement or reinforce alternative control measures. In this context, reinforcement of preventive control of bacteriosis would allow to reduce the use of bactericides. In the nursery, it is critical the use of bacteria-free seeds and test seedlings before delivery to growers. At the producer level, it is opportune to consider carrying out crop’s rotation, cleaning of greenhouse structures, elimination of plant residues from previous production, use of resistant varieties, reduction of relative humidity inside the greenhouse and use of ozonated or ultrafiltered water for irrigation, as principal preventive management (Li et al., 2021; Mann et al., 2021). Also, the use of non-conventional control strategies could be implemented, like biocontrol or phagetherapy (Buttimer et al., 2017), to reduce the use of agrochemicals. Finally, it is considered of great importance to establish a program of antimicrobial resistance surveillance as a critical step within risk assessment schemes and for detecting new trends and emerging threats (Oniciuc et al., 2018)”.

27. Lines 666-667 in Conclusion: What is the experimental support of the suggestion that multi-resistant Pseudomonas present in tomato producing areas and other commercial agricultural scenarios could act as vectors of resistance genes to human pathogens?

Gene transfer between bacteria is a recurring phenomenon in different environments where microorganisms coexist and is considered one of the most likely mechanisms by which bacteria have acquired multiple resistance to antimicrobials (Sun et al., 2019). In the present work, the presence of phenotypes resistant to certain antibiotics for human use was determined through standardized methodologies. This suggests that these microorganisms must have genetic determinants that support this phenotype and that they could be transferable to human pathogens as well as to other microorganisms if they coexist in the same environment. This would not be surprising, considering studies reporting human pathogens (capable of surviving outside their host) that have acquired genes from the environment. The mentioned human pathogens include Vibrio cholera, Burkholderia cepacia, Legionnella pneumophila, Mycobacterium avium and Pseudomonas aeruginosa, have acquired some of their virulence factors as well as resistance to antibiotics in their environmental (non-human host) niches (Morris et al., 2007).

On the other hand, it has been widely described that bacteria from agricultural environments can come into contact with human pathogens through their mobilization through the food chain and transfer genetic determinants of resistance through horizontal gene transfer (Thanner et al., 2016; Karmakar et al.,2019; Scaccia et al., 2021).

Concerning to the observation of the reviewer:  although there is information in the literature that supports the transmission of resistance genes between environmental bacteria and human pathogens, our results do not allow us to establish this as a conclusion, so the “conclusions section” was edited accordingly as follow:

“Numerous Pseudomonas spp. associated with tomato crops in Chile were found in this study. The isolated Pseudomonas spp. belonged to distinct species based on multilocus sequence analysis (MLSA) and showed diverse antimicrobial response patterns, with most of the isolates showing resistance to at least two of the tested antimicrobial agents. Antimicrobial resistance dissemination among bacterial populations is an increasing challenge worldwide. Antibiotic resistance in Pseudomonas isolates, mainly P. syringae, P. viridiflava and P. fluorescens species, recovered from vegetal tissue is of particular concern because these Pseudomonas spp. are considered phytopathogens or opportunistic pathogens. Based on our results, bacteria associated to crops should be at the center of antimicrobial resistance studies, and surveillance throughout the agricultural environment is needed to detect emerging multiresistant phenotypes”.

28. In the References section: Please revise the reference included to correct deviations from the journal´s referencing format. Refer, for instance to lines 739 and several others, where scientific names need to be italicized; to line 754 and others, where the name of the journal should be spelled in full, and line 769 and others, where a comma should be added between the journal´s title and the year.

Unfortunately, it was an error of the mendeley program. It was revised and corrected according to the format of the journal.

English edition certificate attached

 

References for reviewer

 

Scaccia N, Vaz-Moreira I, Manaia CM. The risk of transmitting antibiotic resistance through endophytic bacteria. Trends Plant Sci. 2021 Dec;26(12):1213-1226. doi: 10.1016/j.tplants.2021.09.001. Epub 2021 Sep 27. PMID: 34593300.

 

Forsberg KJ, Reyes A, Wang B, Selleck EM, Sommer MO, Dantas G. 2012. The shared antibiotic resistome of soil bacteria and human pathogens. Science 337:1107–1111. 10.1126/science.1220761. PubMed.

 

Morris, C. E., Kinkel, L. L., Xiao, K., Prior, P., & Sands, D. C. (2007). Surprising niche for the plant pathogen Pseudomonas syringae. Infection, Genetics and Evolution, 7(1), 84–92. https://doi.org/10.1016/j.meegid.2006.05.002.

 

Fleischmann, Susanne, Christian Robben, Thomas Alter, Peter Rossmanith, and Patrick Mester. 2021. "How to Evaluate Non-Growing Cells—Current Strategies for Determining Antimicrobial Resistance of VBNC Bacteria" Antibiotics 10, no. 2: 115. https://doi.org/10.3390/antibiotics10020115.

Ruimy R, Brisabois A, Bernede C, Skurnik D, Barnat S, Arlet G, Momcilovic S, Elbaz S, Moury F, Vibet MA, Courvalin P, Guillemot D, Andremont A. 2010. Organic and conventional fruits and vegetables contain equivalent counts of gram-negative bacteria expressing resistance to antibacterial agents. Environ Microbiol 12:608–615. 10.1111/j.1462-2920.2009.02100.x. PubMed.

 

Allydice-Francis, K.; Brown, P.D. Diversity of Antimicrobial Resistance and Virulence Determinants in Pseudomonas aeruginosa Associated with Fresh Vegetables. International Journal of Microbiology 2012, doi:10.1155/2012/426241.

 

Altimira, F.; Yá̃ñez, C.; Bravo, G.; González, M.; Rojas, L.A.; Seeger, M. Characterization of Copper-Resistant Bacteria and Bacterial Communities from Copper-Polluted Agricultural Soils of Central Chile. BMC Microbiology 2012, doi:10.1186/1471-2180-12-193.

 

Xu, Y.; Luo, Q. quan; Zhou, M. guo. Identification and Characterization of Integron-Mediated Antibiotic Re-sistance in the Phytopathogen Xanthomonas oryzae pv oryzae. PLoS One. 2013, 8(2), doi:10.1371/journal.pone.0055962.

 

Zheng, X.,  Liu, B., Zhu, Y.,  Wang, J., Zhang, H., and Wang, Z. Bacterial community diversity associated with the severity of bacterial wilt disease in tomato fields in southeast China. Canadian Journal of Microbiology. 65(7): 538-549. https://doi.org/10.1139/cjm-2018-063.

 

Jiang, S., Yu, Y., Gao, R., Wang, H., Zhang, J., Li, R., Long, X., Shen, Q., Chen, W., Cai, F., High-throughput absolute quantification sequencing reveals the effect of different fertilizer applications on bacterial community in a tomato cultivated coastal saline soil. Science of The Total Environment, Volume 687,2019,Pages 601-609,ISSN 0048-9697,https://doi.org/10.1016/j.scitotenv.2019.06.105.

 

Seiler, C. and Berendonk, T. U. (2012). Heavy metal driven co-selection of antibiotic resistance in soil and water bodies impacted by agriculture and aquaculture, Front Microbiol, 3:1–10.

 

Sohaib H. Mazhar, Xuanji Li, Azhar Rashid, JunMing Su, Junqiang Xu, Asker Daniel Brejnrod, Jian-Qiang Su, Yijian Wu, Yong-Guan Zhu, Shun Gui Zhou, Renwei Feng, Christopher Rensing. Co-selection of antibiotic resistance genes, and mobile genetic elements in the presence of heavy metals in poultry farm environments. Science of The Total Environment, Volume 755, Part 2, 2021,142702, ISSN 0048-9697, https://doi.org/10.1016/j.scitotenv.2020.142702.

 

Behlau, F., Canteros, B.I., Jones, J.B. et al. Copper resistance genes from different xanthomonads and citrus epiphytic bacteria confer resistance to Xanthomonas citri subsp. citri . Eur J Plant Pathol 133, 949–963 (2012). https://doi.org/10.1007/s10658-012-9966-8.

 

Sundin, G.W.; Wang, N. Antibiotic Resistance in Plant-Pathogenic Bacteria. Annual Review of Phytopathology. 2018, 56, 161–180. 10.1146/annurev-phyto-080417-045946.

 

Nakajima, M., Goto, M., & Hibi, T. (2002). Similarity between Copper Resistance Genes from Pseudomonas syringae pv. actinidiae and P. syringae pv. tomato. Journal of General Plant Pathology, 68(1), 68–74. https://doi.org/10.1007/pl00013056.

 

Gardan, L. "Brault. T., and Germain E., Copper resistance of Xanthomonas campestris pv. juglands in French Walnut orchards and its association with conjugative plasmids." Acta Hort (1993): 239-265.

 

Gu, Y.; Wang, Y.; Sun, Y.; Zhao, K.; Xiang, Q.; Yu, X.; Zhang, X.; Chen, Q. Genetic diversity and characterization of arsenic-resistant endophytic bacteria isolated from Pteris vittata, an arsenic hyperaccumulator. BMC Microbiol. 2018, 18, 42.

 

Wang, Y.; Li, H.; Zhao, W.; He, X.; Chen, J.; Geng, X.; Xiao, M. Induction of toluene degradation and growth promotion in corn and wheat by horizontal gene transfer within endophytic bacteria. Soil Biol. Biochem. 2010, 42, 1051–1057.

 

Nongkhlaw, 2015: Nongkhlaw, F.M.W., Joshi, S.R. Horizontal Gene Transfer of the Non-ribosomal Peptide Synthetase Gene Among Endophytic and Epiphytic Bacteria Associated with Ethnomedicinal Plants. Curr Microbiol 72, 1–11 (2016). https://doi.org/10.1007/s00284-015-0910-y.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and suggestions for authors

Manuscript ID: horticulturae-1793116

Title: Antimicrobial multiresistant phenotypes of genetically diverse Pseudomonas spp. isolates associated with tomato plants in Chilean orchards.

In this manuscript, authors assessed the diversity of Pseudomonas species associated with tomato plants from Chile and analyzed antimicrobial resistance among the isolated strains. In my opinion this work has merits to be published, but first it is necessary authors address some suggestions and comments about it:

1.       In the section “Instructions for Authors” of this journal, authors are informed that “The abstract should be a total of about 200 words maximum”, but the abstract of this manuscript has 312 words. It must be shortened to meet journal requirements.

2.       the section “Instructions for Authors” of this journal, there is a Microsoft Word template, which was used by the authors, and there, authors are informed that: “Tables should be placed in the main text near to the first time they are cited. Figures should be placed in the main text near to the first time they are cited. A caption on a single line should be centered”. But in this manuscript all figures and tables are at the end of Results section, which makes it unfriendly for the reader of the article to be able to analyze what was written about a result with the data that is presented.

3.       In Material and Methods section, all the sources of chemicals and equipment need to be added or completed (add city and country).

4.       In Introduction section, line 49, the reference FAOSTAT, 2018 appears, but it is not in the reference list nor is it written as indicated in the instructions to authors with a number and in square brackets.

5.       Material and Methods section, in line 174, reference Sarkar and Guttman, 2004 is written, when what is indicated is to write the corresponding reference number in the text between square brackets, in this case it would be 44 according to the order of appearance, but the mentioned reference appears in the list of references with the number 47.

6.       Also through the materials and methods section, references are made to information in tables, referring to the protocol followed in the study, but this information is given as supplementary material that generally does not appear in the paper.

7.       In references section there are several corrections that need to be made to fit the format requested by the journal:

a.       In reference 25 all the names of the authors are in capital letters.

b.       several references (12, 17, 18, 21, 23, 32, 35, 36, 40, 46, 58, 72, 80, 81, 82, 86) do not have the year written in bold.

c.       some references (12, 23, 46, 72, 81) are incomplete and have the volume abbreviation written along with the volume number.

For all the above, I consider this manuscript should be Accept after minor revision (corrections to minor methodological errors and text editing)

Author Response

Dear reviewer 2, we greatly appreciate your comments. They have contributed to improve our manuscript. Below we will answer each of the observations

1. In the section “Instructions for Authors” of this journal, authors are informed that “The abstract should be a total of about 200 words maximum”, but the abstract of this manuscript has 312 words. It must be shortened to meet journal requirements.

The abstract was redrafted to comply with "Instructions for Authors".

The abstract has been opportunely modified.

“Tomatoes are susceptible to bacterial diseases, mainly related to some Pseudomonas syringae pathovars. Many Pseudomonas species are considered innocuous, but some have shown the ability to opportunistically infect tomato plants. Antimicrobial compounds have been used to control pathogenic organisms, which can lead to environmental selection of phenotypically resistant bacteria. We assessed the diversity of Pseudomonas species associated with tomato plants from Chilean orchards and analyzed antimicrobial resistance among the isolated strains. A total of 64 Pseudomonas isolates (P. syringae, P. viridiflava, P. fluorescens, P. koreensis, P. gessardii and P. azotoformans) were evaluated for their phenotypic resistance to seven antimicrobial compounds, including copper, streptomycin, and five other antibiotics typically not used in agriculture. The results showed that 95%, 86%, 70%, 53%, 45% and 1.6% of the isolates were resistant to rifampin, ampicillin, copper, chloramphenicol, streptomycin and tetracycline, respectively, with no isolates being resistant to gentamicin. 96.9% Pseudomonas isolates exhibited a multiresistant phenotype, at least two of the antimicrobials tested. The most frequent multiresistance phenotype was Cu-Str-Amp-Cm-Rif (23.4%). The presence of Pseudomonas strains tolerant to conventional bactericides, metals, and other anti-microbials makes these bacteria an emerging threat to the agriculture industry and to human health.”

2. the section “Instructions for Authors” of this journal, there is a Microsoft Word template, which was used by the authors, and there, authors are informed that: “Tables should be placed in the main text near to the first time they are cited. Figures should be placed in the main text near to the first time they are cited. A caption on a single line should be centered”. But in this manuscript all figures and tables are at the end of Results section, which makes it unfriendly for the reader of the article to be able to analyze what was written about a result with the data that is presented.

The format mistake was corrected.

3. In Material and Methods section, all the sources of chemicals and equipment need to be added or completed (add city and country).

In this study we do not adapt or use new methods. We utilized established methods and protocols that we're briefly describe and appropriately cited, as indicated in the instructions for authors. We hope we have met this requirement.

4. In Introduction section, line 49, the reference FAOSTAT, 2018 appears, but it is not in the reference list nor is it written as indicated in the instructions to authors with a number and in square brackets.

The format mistake was corrected.

5. Material and Methods section, in line 174, reference Sarkar and Guttman, 2004 is written, when what is indicated is to write the corresponding reference number in the text between square brackets, in this case it would be 44 according to the order of appearance, but the mentioned reference appears in the list of references with the number 47.

The format mistake was corrected.

6. Also through the materials and methods section, references are made to information in tables, referring to the protocol followed in the study, but this information is given as supplementary material that generally does not appear in the paper.

Supplementary tables with the mentioned information, were moved to Materials and Methods section.

7. In references section there are several corrections that need to be made to fit the format requested by the journal:

Unfortunately, it was an error of the mendeley program. It was revised and corrected according to the format of the journal.

For all the above, I consider this manuscript should be Accept after minor revision (corrections to minor methodological errors and text editing)

English edition certificate attached

Author Response File: Author Response.docx

Reviewer 3 Report

This article assessed the diversity of Pseudomonas species and pathovars associated with tomato plants from Chilean orchards and analyzed antimicrobial resistance among the isolated strains, considering potentially pathogenic strains as well as presumptively innocuous strains that may serve as environmental reservoirs of resistance genes to antimicrobial compounds. Some good results were obtained, but the format and language should be improved. Most importantly, although most of isolates were found, no solutions or suggestions on the problem were reported as for multi-resistance to antimicrobials in this article. So recommend the authors to add this content in the discussion how to avoid this problem in agriculture industry for human health.

In addition, the references were so many, which can be removed for not necessary reference.

The languages should be improved, for example,

P6 L259：Before 298, the comma should be added.

P6 L261: ‘[41]’ should be deleted in the results.

P6 L263: The word ‘see’ should be deleted.

P3 L272-L282, this paragraph is recommended to move to the “materials and methods”.  

The figures and tables should be inserted in the results.

Author Response

Dear reviewer 3, we greatly appreciate your comments. They have contributed to improve our manuscript. Below we will answer each of the observations

1. This article assessed the diversity of Pseudomonas species and pathovars associated with tomato plants from Chilean orchards and analyzed antimicrobial resistance among the isolated strains, considering potentially pathogenic strains as well as presumptively innocuous strains that may serve as environmental reservoirs of resistance genes to antimicrobial compounds. Some good results were obtained, but the format and language should be improved. Most importantly, although most of isolates were found, no solutions or suggestions on the problem were reported as for multi-resistance to antimicrobials in this article. So recommend the authors to add this content in the discussion how to avoid this problem in agriculture industry for human health.

To answer the question posed by the reviewer, the follow information was added to discussion section:

“Faced with the imminent problem of antibiotic resistance, it is necessary to develop, implement or reinforce alternative control measures. In this context, reinforcement of preventive control of bacteriosis would allow to reduce the use of bactericides. In the nursery, it is critical the use of bacteria-free seeds and test seedlings before delivery to growers. At the producer level, it is opportune to consider carrying out crop’s rotation, cleaning of greenhouse structures, elimination of plant residues from previous production, use of resistant varieties, reduction of relative humidity inside the greenhouse and use of ozonated or ultrafiltered water for irrigation, as principal preventive management (Li et al., 2021; Mann et al., 2021). Also, the use of non-conventional control strategies could be implemented, like biocontrol or phagetherapy (Buttimer et al., 2017), in order to reduce the use of agrochemicals. Finally, it is considered of great importance to establish a program of antimicrobial resistance surveillance as a critical step within risk assessment schemes and for detecting new trends and emerging threats (Oniciuc et al., 2018)”.

The languages should be improved, for example,

English edition certificate attached

P6 L259：Before 298, the comma should be added.

The suggestion was accepted.

P6 L261: ‘[41]’ should be deleted in the results.

The suggestion was accepted.

P6 L263: The word ‘see’ should be deleted.

The suggestion was accepted.

P3 L272-L282, this paragraph is recommended to move to the “materials and methods”.  

The suggestion was accepted.

 

The figures and tables should be inserted in the results.

Supplementary tables with the mentioned information, were moved to Materials and Methods section.

 

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

The referecences seemed to be so many, recommend to be reduced.

Author Response

Of our consideration.

We would like to thank the reviewer for his kind comments about our work and his valuable contribution to the improvement of it.

Next, we want to respond to your comments and suggestions:

Comment of the reviewer: “The references seemed to be so many, recommend to be reduced”.

In response to the reviewer's comment, we have reviewed the references cited throughout the work and decided to reduce some of them, mainly because they were considered redundant. Finally, 25 references were reduced from the original article, decreasing to 77.

Unfortunately, we had some problems with Mendeley, however we did a detailed review to correct any format mistake

Expecting to have solved what was requested by the reviewer, we remain attentive to your response regarding the evaluation of the updated version of the manuscript.

Best regards,

Dr. Gastón Higuera.

For reviewer's knowledge, the list of discarded references is detailed below:

Garrity, G.M.; Bell, J.A.; Lilburn, T. Order IX. Pseudomonadales. Bergey’s manual of systematic bacteriology, 2005, 2, 323.

Arnold, D.L.; Preston, G.M. Pseudomonas Syringae: Enterprising Epiphyte and Stealthy Parasite. Microbiology (United Kingdom) 2019, 165, doi:10.1099/mic.0.000715.

Tian, B.; Zhang, C.; Ye, Y.; Wen, J.; Wu, Y.; Wang, H.; Li, H.; Cai, S.; Cai, W.; Cheng, Z.; et al. Beneficial Traits of Bacterial Endophytes Belonging to the Core Communities of the Tomato Root Microbiome. Agriculture, Ecosystems and Environment 2017, 247, doi:10.1016/j.agee.2017.06.041.

Yu, Z.; Gunn, L.; Wall, P.; Fanning, S. Antimicrobial Resistance and Its Association with Tolerance to Heavy Metals in Agriculture Production. Food Microbiol 2017, 64, 23–32, doi:10.1016/J.FM.2016.12.009.

Griffin, K.; Campbell, P.; Gambley, C. Genetic Basis of Copper-Tolerance in Australian Pseudomonas syringae pv tomato. Australasian Plant Pathology 2019, doi:10.1007/s13313-019-00646-y.

Behlau, F.; Canteros, B.I.; Jones, J.B.; Graham, J.H. Copper Resistance Genes from Different Xanthomonads and Citrus Epiphytic Bacteria Confer Resistance to Xanthomonas citri Subsp. citri. European Journal of Plant Pathology 2012, 133, doi:10.1007/s10658-012-9966-8.

Cha, J.; Cooksey, D. Copper Resistance in Pseudomonas syringae Mediated by Periplasmic and Outer Membrane Proteins. Proc Natl Acad Sci U S A 1991, 88, 8915–8919, doi:10.1073/pnas.88.20.8915.

Goto, M.; Hikota, T.; Nakajima, M.; Takikawa, Y.; Tsuyumu, S. Occurrence and Properties of Copper-Resistance in Plant Pathogenic Bacteria. Japanese Journal of Phytopathology 1994, 60, doi:10.3186/jjphytopath.60.147.

Sundin, G.W.; Bender, C.L. Expression of the StrA-StrB Streptomycin Resistance Genes in Pseudomonas syringae and Xanthomonas campestris and Characterization of IS6100 in X. Campestris. Applied and Environmental Microbiology 1995, 61, doi:10.1128/aem.61.8.2891-2897.1995.

Jones, J.B.; Jackson, L.E.; Balogh, B.; Obradovic, a; Iriarte, F.B.; Momol, M.T. Bacteriophages for Plant Disease Control. Annu Rev Phytopathol 2007, 45, 245–262, doi:10.1146/annurev.phyto.45.062806.094411.

Epstein, F.H.; Jacoby, G.A.; Archer, G.L. New Mechanisms of Bacterial Resistance to Antimicrobial Agents. The New England journal of medicine, 1991. 324(9), 601–612. https://doi.org/10.1056/NEJM199102283240906

Pscheidt J.W.; Moore L.W. Diseases Caused by Pseudomonas syringae Available online: https://pnwhandbooks.org/plantdisease/pathogen-articles/pathogens-common-many-plants/bacteria-other-prokaryotes/diseases (accessed on 22 May 2022)

Vidaver, A.K. Uses of Antimicrobials in Plant Agriculture. Clinical Infectious Diseases 2002, 34, S107–S110, doi:10.1086/340247

Ruinelli, M.; Blom, J.; Smits, T.H.M.; Pothier, J.F. Comparative Genomics and Pathogenicity Potential of Members of the Pseudomonas syringae Species Complex on Prunus Spp. BMC Genomics 2019, 20, doi:10.1186/s12864-019-5555-y.

Morris, C.E.; Sands, D.C.; Vinatzer, B.A.; Glaux, C.; Guilbaud, C.; Buffière, A.; Yan, S.; Dominguez, H.; Thompson, B.M. The Life History of the Plant Pathogen Pseudomonas syringae Is Linked to the Water Cycle. ISME Journal 2008, 2, doi:10.1038/ismej.2007.113.

Anzai, Y.; Kudo, Y.; Oyaizu, H. The Phylogeny of the Genera Chryseomonas, Flavirmonas, and Pseudomonas Supports Synonymy of These Three Genera. International Journal of Systematic Bacteriology 1997, 47, 249–274, doi:10.1099/00207713-47-2-249.

Garrido-Sanz, D.; Meier-Kolthoff, J.P.; Göker, M.; Martín, M.; Rivilla, R.; Redondo-Nieto, M. Genomic and Genetic Diversity within the Pseudomonas fluoresces Complex. PLoS ONE 2016, 11, doi:10.1371/journal.pone.0150183.

Pernezny, K.; Kůdela, V.; Kokošková, B.; Hládká, I. Bacterial Diseases of Tomato in the Czech and Slovak Republics and Lack of Streptomycin Resistance among Copper-Tolerant Bacterial Strains. Crop Protection 1995, 14, doi:10.1016/0261-2194(94)00010-6.

Patil S, T.M. Antimicrobial Sensitivity Pattern of Pseudomonas fluorescens after Biofield Treatment. Journal of Infectious Diseases & Therapy 2015, 03, doi:10.4172/2332-0877.1000222.

Morris, C.E.; Kinkel, L.L.; Xiao, K.; Prior, P.; Sands, D.C. Surprising Niche for the Plant Pathogen Pseudomonas syringae. Infection, Genetics and Evolution 2007, 7, 84–92, doi:10.1016/j.meegid.2006.05.002.

Yan, S.; Liu, H.; Mohr, T.J.; Jenrette, J.; Chiodini, R.; Zaccardelli, M.; Setubal, J.C.; Vinatzer, B.A. Role of Recombination in the Evolution of the Model Plant Pathogen Pseudomonas syringae pv tomato DC3000, a Very Atypical Tomato Strain. Applied and Environmental Microbiology 2008, 74, 3171–3181, doi:10.1128/AEM.00180-08.

M’Barek Fatmi; Alan Collmer; Nicola Sante Iacobellis; John W. Mansfield; Jesus Murillo; Norman W. Schaad; Matthias Ullrich. Pseudomonas syringae Pathovars and Related Pathogens – Identification, Epidemiology and Genomics; 2008; ISBN: 978-1-4020-6901-7.

Luczkiewicz, A.; Kotlarska, E.; Artichowicz, W.; Tarasewicz, K.; Fudala-Ksiazek, S. Antimicrobial Resistance of Pseudomonas Spp. Isolated from Wastewater and Wastewater-Impacted Marine Coastal Zone. Environmental Science and Pollution Research 2015, 22, 19823–19834, doi:10.1007/s11356-015-5098-y.

Li, X.; Ruan, H.; Zhou, C.; Meng, X.; Chen, W. Controlling Citrus Huanglongbing: Green Sustainable Development Route Is the Future. Frontiers in Plant Science 2021, 12. 760481. https://doi.org/10.3389/fpls.2021.760481

Jensen, L.B.; Baloda, S.; Boye, M.; Aarestrup, F.M. Antimicrobial Resistance among Pseudomonas spp and the Bacillus Cereus Group Isolated from Danish Agricultural Soil. Environment International 2001, 26, doi:10.1016/S0160-4120(01)00045-9.