Bioprospecting Fluorescent Pseudomonas from the Brazilian Amazon for the Biocontrol of Signal Grass Foliar Blight

Round 1

Reviewer 1 Report

In this paper, the authors assembled a collection of 24 Pseudomonas strains isolated from Amazonian suppressive soils. They studied the ability of these strains to inhibit the plant pathogenic fungus Rhizoctonia solani and to control signal grass foliar blight. Three strains (Amana, Poto and Yara) were extremely effective at inhibiting R. solani mycelial growth and sclerotia germination. In addition, these three strains provided disease suppression activity during in planta experiment. Then, the authors used 16s/rpoB sequencing to identify the strains and characterized several activity (protease, chitinase, siderophore etc.) using simple biotests.

In general, I like the manuscript and I find it quite well written. My biggest problem is that the phylogenetic analyses were not done properly. These data should be analyzed in a correct way to bring light into the phylogenetic distribution of these strains in the Pseudomonas genus.

Here are my comments :  

L50 : Colon rot ? You probably mean stolon rot (such an unfortunate mistake !)

L62 : I suggest toning down a bit the “biological control as the best alternative’’. Biocontrol is interesting, but it’s efficacy and reliability in the field still remain somewhat problematic. I suggest using the words “attractive alternative” or ‘’one of the best’’.

L67-68  : Are these soils naturally suppressive to signal grass foliar blight in particular, or to a lot of soilborne diseases ? Is the suppressiveness triggered by agricultural practice (e.g. monoculture or something else) or is it a long-lasting suppressiveness like in the Châteaurenard soils? Considering that these soils are “undisturbed” I don’t believe signal grass was ever planted here. How do you know it is suppressive ? For a soil to be suppressive, the pathogen should be present as well as sensible host. I think you should better introduce these soils (are there publication already available on these soils?). In addition, I find that choosing to focus on Fluorescent Pseudomonas is not that evident. Did you focus on these microorganisms because of their role in other suppressive soils (take-all of wheat an others) or because you had reason to believe they are involved in your case as well?

L70-71 : Replace “Pseudomonas fluorescens and P. putida” by “Members of the P. fluorescens and P. putida groups” (or something like this). There are not a lot of bona fide Pseudomonas fluorescens with biocontrol ability. Most of these strains belong to other species and have been reclassified. I suggest reading this publication : The use of Pseudomonas spp. as bacterial biocontrol agents to control plant disease (https://biblio.ugent.be/publication/8719375/file/8719376.pdf). It presents the different taxonomic groups/subgroups of Pseudomonas with biocontrol abilities.

L76 : Replace by “fungal pathogen-inhibiting enzymes”                        

L83 : There are quite a lot of studies using Pseudomonas strains to control Rhizoctonia disease, I was able to find over a hundred of them, for example :

Pseudomonas sp. CMR12a (and other biocontrol Pseudomonas spp.) against damping-off disease on Chinese cabbage (caused by Rhizoctonia solani AG 2-1) :

https://sfamjournals.onlinelibrary.wiley.com/doi/full/10.1111/1758-2229.12310?casa_token=lj_Crh-ahAsAAAAA%3AEXF58H5lSuJtcBBMcdfH8b_GzTmserJ-JUFxhNpy5eRO-zBTxbaym9UmwccZ1BeB4abOydepv1Rwww

Pseudomonas sp. CMR12a (and other biocontrol Pseudomonas spp.) against two AGs of R. solani

https://apsjournals.apsnet.org/doi/abs/10.1094/PHYTO-11-10-0315

 Various Pseudomonas strains against R. solani :

https://www.sciencedirect.com/science/article/pii/S1049964412000771?casa_token=Zb_D7Oinu3AAAAAA:GoUhOudiP9nu8x2lPQPvoTfObgKnkECEhA9gAgZus0R4P0cFbwneEQyw8ub0V9IoVEM0cUiY

You should modify these sentences to center it on your pathosystem (signal grass and Rhizoctonia). There are not a lot of paper on that.

L106 : King’s Medium B Agar (or something like this to clearly state the presence of agar).

L117/L161/L179 :  Canada (and not Canadian)

L119 : Were the sclerotia grown with light or in the dark ? Please precise.

L153-155 : Please state how many hours of light.

L192 : add a space after 0.1

L195  : You should add a space before °C (please check it thoroughly in the text)

L253 : Do you really want the first sentence of the Results to be : ‘’There was no difference between replicates of the experiment” ??? You should reintroduce the fact that you isolated bacteria from a suppressive soil etc. (a few sentences).

L253-268 : I would like to see the results for each Pseudomonas strains somewhere (It could be in supplementary).

L309-L321 : My biggest issue with this paper is the phylogenetic analysis, which is not done properly. With rpoB and 16s rDNA gene sequences, you can nearly identify your strains at the species level and gather a lot of information about the 24 Pseudomonas strains. This was not done and you hardly present any information in your phylogenetic tree. In addition, you present some of your strains as ‘’new species of Pseudomonas’’ but you don’t provide any information to back this claim. For a phylogenetic tree to provide information regarding the phylogenetic placement of your strains, you need to include a lot of type strains.

The genus Pseudomonas is divided in several phylogenetic groups, including the P. fluorescens, P. putida and P. aeruginosa groups. The P. fluorescens group is subdivided in ~10 subgroups (e.g. the P. protegens, the P. chlororaphis and the P. corrugata subgroups). You can find details about the phylogeny of the genus Pseudomonas in the following publications (oldest to newest) : Mulet 2010 (https://sfamjournals.onlinelibrary.wiley.com/doi/full/10.1111/j.1462-2920.2010.02181.x?casa_token=SXQBRs_R8j4AAAAA%3A9P0W5Bg31e0vMC7cfKh1AYW3HCxbHLkYF0wCWw9Z4fBIlL2apfUtzOxa0qDGgEXEXULkQPD_yXKbIA ); Garrido-Sanz 2016 (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150183 ); Hesse 2018 (https://sfamjournals.onlinelibrary.wiley.com/doi/abs/10.1111/1462-2920.14130 ) and Girard 2021 (https://www.mdpi.com/2076-2607/9/8/1766 )

In your cases, you want for each strain to i) identify the closest type strain and ii) found in which group/subgroup your strain belong. I suggest you do the following :

I/- For each strain, blast the rpoB sequence on NCBI blast. You need to do the BLAST search only on type material sequences (there is an option in the interface to do so). Depending on the closest type strain, you can identify the group/subgroup to which your strain belong.

II/- Include the closest type strains in your tree for each strain. The best is to provide an additional tree in supplementary with a lot of type strains (e. g. 50 for each major Pseudomonas group, 100 in your cases) to visualize how your strains are distributed in these phylogenetic groups.

Additionally, you don’t want to use UPGMA for phylogenetic trees like this, use instead  neighbour-joining (NL) or Maximum-likelihood (ML). NL is way faster and provide most of the time similar results. You should not collapse the branches (when building the tree with Geneious, write ‘’0” in the Support Threshold %).

Also, you should add the group/subgroup on your tree instead of these arbitrary clade.

L320-321 : P. chlororaphis belong to the P. fluorescens group.

L362-L364  : Reference ? Any soil can be described as “rich in unknown bacterial species”

L364-366 : As mentioned previously, I would like to see more information regarding the phylogenetic placement of these two strains. From which species in the P. putida group are they the closest ?

L452-453 : It would have been nice to have access to these data for the review of this manuscript.

Author Response

Firstly, we would like to thank reviewer #1 for the excellent contribution on reviewing our manuscript, specially concerning the hints for implementing a more thorough phylogenetic analyses. 

I will approach item by item from the reviwers comments

L50: Collar rot is the correct term. Sorry for the unfortunate mistake.

L67-68: We rewrote this paragraph to reflect the exact origin of soil samples that were used in our study, which we postulated were naturally suppresive. These were not agricultural soils that have been found supressive as the case of the Châteaurenard.  In fact, signal grass has not been cropped in this undisturbed forest area. It is rather an invasive crop species in the Amazon ecosystem, so it is Rhizoctonia solani AG-1 IA an invasive plant pathogen. In consequence, thousands of hectares of signal grass pastures are now cropped in areas adjacent to the native forest. Our reasoning for sampling fluorescent Pseudomonas from these particular Amazon rainforest soils was that the bacteria with potential biocontrol abilities would be well adapted to that agroecosystem when delivered as biofungicides against the signal grass foliar blight disease.

Therefore, the new paragraph now reads: "Our study focuses on the biocontrol potential role of fluorescent bacteria from the genus Pseudomonas previously obtained from undisturbed and possibly naturally suppressive soils within areas of native rainforest from the Amazon. These fluorescent Pseudomonas with biocontrol abilities would be well adapted to that agroecosystem when delivered as biofungicides against the signal grass foliar blight disease on adjacent pastures areas." 

L70-71: Thanks for recommending the very helpful and updated reference on Pseudomonas as biocontrol agents. The paragraph has been rephrased and now reads: "Therefore, the lack of economically viable, ecologically sustainable and minimally effective strategies for disease management, makes biological control one of the best alternatives for controlling the foliar blight and sudden death on signal grass"

L76 : Adjusted to  “fungal pathogen-inhibiting enzymes”  as suggested.

L83 : The literature on biocontrol of Rhizoctonia diseases with strains of Pseudomonas is indeed very rich. Since we have  cited only a couple of examples limited to few AGs and crops,  we expanded these citations by incluing all the examples cited by the reviwer. We also centered the paragraph on the lack of information on the biocontrol of leaf blight of signal grass. Now this paragraph reads as follows: "

Several studies have already described the antagonistic potential of fluorescent Pseudomonas in the biocontrol of Rhizoctonia-like diseases such as the potato black scurf and root rot caused by R. solani AG-3 PT and AG-4 HGI  [19]; the maize foliar blight and banded leaf diseases [20], and the rice sheath blight [21], both caused by R. solani AG-1 IA; the damping-off disease on Chinese cabbage caused by R. solani AG 2-1 [47]; the root rot disease on snap beans) caused by R. solani AG 4-HGI [49],; and the wheat root rot caused by R. solani AG-8 and R. oryzae [50]."

"So far, there have been no studies focused on the biological control of the foliar blight disease caused by R. solani AG-1 IA on signal grass pastures in Brazil."

The minor changes suggested in the following lines were all implemented:

L106 : King’s Medium B Agar 

L117/L161/L179 :  Canada 

L119 : 12 h of light.

L153-155 : 12 h of light.

L192 : A space was added after 0.1.

L195  : A space was added before °C and in the remaining instances in the text.

L253 :  Following suggestions, we began the first phrase of results with the following paragraph now: "The first step to assess the potential of fluorescent Pseudomonas (from undisturbed and possibly naturally suppressive soils within areas of native rainforest from the Amazon) as biocontrol agents against the signal grass foliar blight disease was to select strains capable to inhibit mycelial growth and sclerotia germination of the pathogen R. solani AG-1 IA. "

L253-268 : The data requested by the reviewer in regard means comparison and the significant differences in mycelial growth and sclerotia germination inhibiton for each Pseudomonas strains is provided in a Supplementary material file (S2).

L309-L321 : By following all the steps proposed for a more proper phylogentic analysis, we were able to reveal a more clear phylogenetic placement of the Pseudomonas strains analyzed, either supporting the former observation that a number of these strains could possibly belong to new species not yet described or indicating the placement of other strains within species from groups or subgroups of the P. fluorescens lineage already described.

From a handful of Pseudomonas strains formely analyzed, we now included GenBank derived sequences of the 16S and rpoB genes from 77 type species spanning including the major groups P. fluorescens (subgroups P. chlororaphis, P. corrugata, P. fluorescens, P. fragi, P. gessardii, P. jessenii, P. mandelii and P. protegens), P. putida, P. asplenii, P. lutea and P. syringae besides P. aeruginosa used as outgroup. We also followed the reviewer´s recommendation concerning the reconstruction of the tree using NJ instead of UPGMA and we have not collapsed branches. This new phylogenetic tree, with resulting higher power for species resolution, replaces the former Figure 4.  

L320-321 : Phrase adjusted as suggested: "A fourth clade (clade IV) with bootstrap support contained the isolates Abati, Iracema, Juçara and Joaci, and could consist of a new subspecies comprising the P. chlororaphis subgroup within the P. fluorescens group."

L362-L364  : Reference included to support the assertion of high bacterial diversity in undisturbed Amazonian soils, as suggested: Cenciani et. al (2009) R. Bras. Ci. Solo, 33:907-916.

L364-366 :  Answering the reviewer question based on the new phylogeny reconstructed: " The phylogenetic analysis based on the joint analysis of the 16S [27] and rpoB [28,29] genes indicated a number of strains from yet undescribed fluorescent Pseudomonas species or subspecies, including the isolate Poti (independent but closely related to P. asplenii), which showed potential as biocontrol agent.  In addition, the two other Pseudomonas isolates with biocontrol potential, Amana (closely related to P. wayambapalatensis) and Yara (an independent clade closely related to the type species P. putida) fell into the Pseudomonas putida species complex.

L452-453 : The data requested is now provided as Supplementary material S1.

Thanks a lot for your precious contribution reviewing the first version of our manuscript.

Reviewer 2 Report

 In this manuscript, the authors reported the bioprospecting fluorescent Pseudomonas from the Brazilian Amazon for the biocontrol of signal grass foliar blight. The research is carried out accurately and the results were successfully obtained. However, there are several issues needed to be addressed.
 Q1: Line 109, check the writing throughout the manuscript and keep the writing in standard. e.g. change “DO 620” to “OD 620”.
 Q2: Line 141, why do the authors choose proportion of de 3:1? The authors should explain.
 Q3: In table 4, Amana, Poti and Yara produced siderophores in culture medium with low amount of iron. Kindly please mention whether experimental studies are carried out by adding the siderophores directly, and discuss what will happen in that case.
  Q4: The conclusion section was not sufficient to effectively summarize the content of the entire manuscript. This section needs to be more specific and detailed.

Author Response

Our answers to the reviewer # 2 requests:

 Q1: Line 109: “DO 620” was changed to “OD 620” all throughout the text.

 Q2: Line 141, in regard the choice of  a substrate with the proportion 3:1 substrate:vermiculite: Vermiculite is an inert material that, when added to the substrate, maintains moisture for a longer time. The proportion used was adjusted in previous experiments in order to maintain the substrate moisture for a longer period of time and was therefore adopted in all subsequent experiments.

 Q3: Yes, we adopted the standard procedure of checking siderophore production by the isolates Amana, Poti and Yara in culture medium with low amount of iron. The possibility of carrying out experiments by adding  siderophores directly to the culture medium would be plausible to test hypothesis on the significant biocontrol activity of an isolated and characterized  siderophore molecule obtained from our strains. That was not the case of our study, yet.

Q4: We expanded and detailed the conclusion section to effectively summarize the content of the entire manuscript, as suggested.

"Based on in vitro antagonism, three isolates (Amana, Poti and Yara) were selected for further in vivo assays. Multilocus phylogenetic analysis indicated that Amana and Yara grouped into the Pseudomonas putida group while Poti was grouped into the Pseudomonas asplenii group, and could well constitute a new Pseudomonas species. All three isolates produced siderophores and solubilized phosphate, while Amana and Poti showed protease and chitinase in vitro activity. Foliar application of P. putida Amana from Amazonian suppressive soils resulted in significant reduction of the foliar blight disease severity on signal grass."

Round 2

Reviewer 2 Report

• This paper can be accepted without any further changes.