Taxonomy-guided selection of Paraburkholderia busanensis sp. nov.: a versatile biocontrol agent with mycophagy against Colletotrichum scovillei causing pepper anthracnose

ABSTRACT This study introduces a novel approach based on the taxonomy-guided selection of bacterial biocontrol agents from a known beneficial taxonomic group. Following 16S rRNA screening, we focused on the genus Paraburkholderia, which harbors strains with large genomes and versatile benefits to plants. A strain designated P39 was selected, identified, and characterized for its biocontrol activity against Colletotrichum scovillei. Strain P39 exhibited antagonism against C. scovillei by producing compounds, including volatiles, with antifungal activity, both in vitro and on pepper fruits. Genomic, physiological, and biochemical analyses revealed that the selected strain represents a novel species, named Paraburkholderia busanensis. Genomic analyses provided insights into the fitness and biocontrol activities of the selected strains. Moreover, P39 displays mycophagy, consuming fungal mycelia and transforming them into bacterial biomass, particularly in nutrient-poor media supplemented with fungal mycelia. The genome harbored chitin and N-acetylglucosamine utilization genes, suggesting a proposed pathway for the utilization of fungal cells as a nutrient source. Microscopic observations further supported the ability of this strain to rupture and damage fungal hyphae, depriving them of their cellular constituents. This study successfully demonstrated the implementation of a taxonomy-guided approach for the selection of bacterial strains for biocontrol. These findings contribute to our understanding of biocontrol strategies, bacteria-fungi interactions, and the identification of Paraburkholderia busanensis sp. nov. as a potential candidate for the biocontrol of pepper anthracnose. Additionally, this strain serves as a valuable resource for antifungal compounds and volatiles, and for the study of bacteria-fungal interactions and mycophagy. IMPORTANCE Traditional control methods for postharvest diseases rely on fungicides, which cause human health and environmental concerns. This study introduces a taxonomy-guided strategy for selecting biocontrol agents. By focusing on Paraburkholderia group, which harbors diverse plant-beneficial strains, the inadvertent selection of harmful strains was circumvented, thereby obviating the need for laborious in vitro screening assays. A highly promising candidate, strain P39, has been identified, exhibiting remarkable biocontrol activity against Colletotrichum scovillei. Through comprehensive genomic, physiological, and biochemical analyses, P39 was characterized as a novel species within the Paraburkholderia genus and designated Paraburkholderia busanensis. Moreover, these findings deepen our understanding of bacterial-fungal interactions, as they elucidate a potential pathway for the utilization of fungal chitin, thereby enhancing our understanding of bacterial mycophagy. P. busanensis is a promising source of antifungal volatiles and putative novel secondary metabolites.

endochitinase activity decreases, compared to treatment where no mycelium was added.

In line 415, what means fragments ?
In line 440, more and more recent bibliographic citations can be added.
In lines 441-443, specify a bit about species and mechanisms.
Reviewer #3 (Comments for the Author): I commend the authors of this research article for executing a study with such depth, utilizing a number of different techniques for novel biocontrol agent selection and characterization.The characterization of the biocontrol agent is thorough and unveils several important metrics for understanding antagonistic activity against the target pathogen and disease.I especially appreciated the development of a potential mechanism of action for chitin utilization.I couldn't find any major issues with the methods or conclusions, but do have some minor comments and suggested changes, below.
Lines 83-84: "Among the examples of registered products based on biocontrol agents" This is not a complete sentence.
Line 150: "dipping in a fresh pepper bacterial suspension" Suggest changing to "dipping a fresh pepper in a bacterial suspension".
Line 362: "To conduct taxonomy-guided screening" Suggest changing to "To conduct the taxonomy-guided screening" Lines 576-577: "After analyzing the 16S rRNA gene sequence, it was not possible to identify P39 in a specific species."Suggest changing to "as a specific species" Lines 644-646: pH and NaCl increments are mentioned, but temperature increments are not.Table 1: "Acession" should be "Accession" Table 4: "Root nodule", "root nodule", inconsistent capitalization of "root"."+ W" not used in table but mentioned in caption.Lines 890-891: "+ W, weakly positive; + W, weak positive" mentioned twice in caption.Lines 890-891: "Positive... negative" different capitalization used in caption.
Line 934: "and incubated in on sealed square dish" Suggest changing to "and incubated in a sealed square dish" Line 970: "P.busanensis" should be italic.
Line 981: "circular map of the P. busanensis P39 genome." the word "the" should not be italic.Lines 1022-1023: "showing intact mycelia and fungal undepleted cellular contents without cell wall damage" Suggest changing to "showing intact mycelia with undepleted cellular contents and without cell wall damage" Line 1025: "rupture in the mycelial" Suggest changing to "rupture in the mycelia" Line 1027: "The red arrows indicating points of rupturing fungal cell wall" Suggest changing to "The red arrows indicate points of fungal cell wall rupture"

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21-July-2023
Dear Editor, Subject: Submission of revised paper [Manuscript Number: Spectrum02426-23] Thank you for your email dated 22-Feb-2023 enclosing the reviewers' comments.We are sincerely grateful to both you and the reviewers for the insightful comments and suggestions for improvement.We have taken these recommendations seriously and have revised our manuscript accordingly.We are pleased to enclose the revised version of our manuscript titled "Taxonomyguided selection of Paraburkholderia busanensis sp.nov.: A versatile biocontrol agent with mycophagy against Colletotrichum scovillei causing pepper anthracnose".We are confident that the manuscript has significantly improved after the revisions.
In response to the comments, we have addressed each of the points raised, and have included the necessary changes in the accompanying revised manuscript.To ensure transparency, all changes were made using the track changes option in word processing.We appreciate the thorough review process and are grateful for the opportunity to improve our work based on the reviewers' feedback.

Reviewer Comments:
Reviewer 1: (Comments for the Author): The text in lines 122-125 sounds repetitive with lines 128-131.>> We thank the reviewer for dedicating time to thoroughly review our manuscript and for offering valuable suggestions.In response to the reviewer's feedback, we would like to clarify the methodology and address potential points of confusion.
Initially, we performed a preliminary screening dual-culture assay, evaluating a total of 33 Paraburkholderia-related strains.Through this screening, we identified strain P39 (as depicted in Figure 1B) as the most promising candidate.Subsequently, strain P39 was selected for further characterization, which included an additional dual-culture assay shown in Figure 2A and B.
To enhance clarity and eliminate potential ambiguity, we have explicitly stated that the results presented in Figure 1B correspond to the outcomes from the preliminary screening assay.By doing so, we aim to ensure that readers can discern the two separate stages of our investigation.
The sentence in L 123-124) was fixed for clarity.
Highlight some of the specific genetic characteristics for each chromosome.For example, size, %GC, CDS, cluster of biosynthetic genes, etc. >> As suggested, we have added supplementary table 2 containing the specific features and genetic characteristics for each chromosome.
In lines 222-225, this point deserves further analysis and discussion of why in the presence of mycelium of the fungus, endochitinase activity decreases, compared to treatment where no mycelium was added.
>> Thank you for your insightful comments.Concerning the observed slight decrease in the endochitinase activity, we propose that this reduction can be primarily attributed to the utilization of a portion of the enzyme activity during the metabolic processes involved in the degradation of the fungal mycelial cell wall, which contains chitin.Our mycophagy assay confirmed a decrease in the fungal mycelial weight in the presence of bacteria, and it is well-established that the utilization of chitin is a crucial aspect of this process.
In response to your recommendation, we have taken the opportunity to provide a more detailed explanation in our revised manuscript (see Line 225-227) to clarify this point.
>> The typo has been rectified to 100X and 1000X (L262) In line 415, what means fragments ?>> The typo has been rectified from "fragments" to "fragin", the name of the secondary metabolite (L415) In line 440, more and more recent bibliographic citations can be added.>> As per the reviewer's suggestion, we have incorporated more recent bibliographic citations that include additional similar observations (L 438 to 460).
In lines 441-443, specify a bit about species and mechanisms.>> Based on the reviewer's feedback, we have made improvements to the relevant section.We have included additional details about the specific species known to display mycophagy, and we have expanded upon the mechanisms and strategies employed by the selected species in the mycophagy activities (L 438 to 460).Moreover, we have incorporated relevant citations to support these additions.

Reviewer 2:
(Comments for the Author): I commend the authors of this research article for executing a study with such depth, utilizing a number of different techniques for novel biocontrol agent selection and characterization.The characterization of the biocontrol agent is thorough and unveils several important metrics for understanding antagonistic activity against the target pathogen and disease.I especially appreciated the development of a potential mechanism of action for chitin utilization.I couldn't find any major issues with the methods or conclusions, but do have some minor comments and suggested changes, below.
>> We thank the reviewer for the positive and encouraging feedback on our research article.We appreciate your commendation of the depth of our study, the use of various techniques for biocontrol agent selection, and the thorough characterization provided.Your recognition of the potential mechanism of action for chitin utilization is truly motivating.We have carefully considered and addressed your valuable suggestions to enhance the manuscript further.
Lines 83-84: "Among the examples of registered products based on biocontrol agents" This is not a complete sentence.
>> Thank you for bringing this to our attention.We have addressed the issue by completing the sentence and including the example of registered products.Line 150: "dipping in a fresh pepper bacterial suspension" Suggest changing to "dipping a fresh pepper in a bacterial suspension".>> The sentence was corrected as recommended.
Line 362: "To conduct taxonomy-guided screening" Suggest changing to "To conduct the taxonomy-guided screening" >> The sentence was corrected as recommended.
Lines 576-577: "After analyzing the 16S rRNA gene sequence, it was not possible to identify P39 in a specific species."Suggest changing to "as a specific species" >> The sentence was corrected as recommended.
Lines 644-646: pH and NaCl increments are mentioned, but temperature increments are not.
>> Temperature increments were mentioned as recommended.
>> The pointed error was corrected.
>> The pointed typo was corrected.
Line 934: "and incubated in on sealed square dish" Suggest changing to "and incubated in a sealed square dish" >> The pointed typo was corrected.
>> The pointed typo was corrected.
Line 981: "circular map of the P. busanensis P39 genome." the word "the" should not be italic.Thank you for submitting your manuscript to Microbiology Spectrum.Before we can accept your manuscript for publication, there are few more changes recommended by the reviewers, those changes are accompanying this letter.When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only".Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me.Detailed instructions on submitting your revised paper are below.

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ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data.If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record.If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication of your article may be delayed; please contact the ASM production staff immediately with the expected release date.
The ASM Journals program strives for constant improvement in our submission and publication process.Please tell us how we can improve your experience by taking this quick Author Survey.Thank you for addressing the previous comments.Upon further inspection, I found several minor misspellings that should be addressed.
From the line numbers of the PDF: Line 24 "Paraburkholderi" misspelled Line 199 and 216 "busanesnsis" misspelled Line 374 "Paraburkhoderia" misspelled Lines 688, 967, 972 "imageJ" should be ImageJ Line 1030 "repressession" misspelled Reviewer #4 (Comments for the Author): I acknowledge the authors for their well-accomplished work regarding the identification and characterization of a new species of Paraburkholderia with potential as biocontrol agent.This is a comprehensive study contributing to the understanding of bacterial antagonism against fungal pathogens.I have some minor issues and suggestions.
Line 113, please include some info regarding site of bacterial pool isolation Line 131, "co-cultivation" is duplicated Line 141 and 400, I will argue that there is not a clear direct effect of volatiles over conidiation of C. scovillei (Fig. 3C), it seems that lesser conidia count only reflects fungal growth inhibition.Please refer Table 3 at the beginning of description of corresponding data (line 199-200) Line 413, I couldn´t find any reference in the manuscript or tables regarding the "various types of secretion system".It would be nice to indicate which kind of secretion systems are encoded by P. busanensis P39, particularly if there is a T3SS. Figure 7.I find this figure unnecessary.Data in Fig. 7A is better summarized in Table S2.In fact, data in Table S2 can be merged with Table 2.And for Fig. 7B, with only 1642 (22%) of CDSs categorized by RAST software, subsystem categorization is probably unreliable by not considering over 3000 CDS with functional assignments (according to Table 2).

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• Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file.• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process.Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript." Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me.If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
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Figure 1B and Fig 2B refer to the same thing although they do not fully agree.Need to be specified or corrected.

>>
The pointed typo was corrected.Lines 1022-1023: "showing intact mycelia and fungal undepleted cellular contents without cell wall damage" Suggest changing to "showing intact mycelia with undepleted cellular contents and without cell wall damage" >> The sentence was corrected as suggested.Line 1025: "rupture in the mycelial" Suggest changing to "rupture in the mycelia" >> The pointed typo was corrected.Line 1027: "The red arrows indicating points of rupturing fungal cell wall" Suggest changing to "The red arrows indicate points of fungal cell wall rupture" >> The sentence was corrected as suggested.-23R1 (Taxonomy-guided selection of Paraburkholderia busanensis sp.nov.: A versatile biocontrol agent with mycophagy against Colletotrichum scovillei causing pepper anthracnose) Dear Prof. Young-Su Seo: Comments for the Author):