Engineering plant microbiota can improve plant health and growth sustainably. Emergent approaches include rational Synthetic Communities (SynCom) design or soil amendments and specific agricultural practices to shift resident microbiota and to understand its impact (Moreira et al. 2023).
In this context, the impact of seed microbiota on the early stages of plant development is becoming an essential topic in the study of plant–microbiota interactions. Behind the well-studied seed-borne pathogens, the seed microbiota can host many other commensal and beneficial organisms that have been neglected in the past.
The study of Simonin et al. (2023) applies single isolates and synthetic communities (SynCom) on radish seeds to answer two key questions: what is the role of seed microbiota during the early stages of plant development? How can SynCom influence the seedling health and its microbiota? The study describes an elegant approach to cope with the variability of natural microbiota using SynCom following a gradient of complexity.
Overall, the study highlighted a contrasted impact of the bacterial strains when applied in isolation or SynCom. The composition and complexity of the SynCom had also an impact on plant seedlings. Importantly, contrasting evolution from seeds to seedlings was observed for 3 strains of Pseudomonas fluorescens within the SynComs, underlining the importance of intra-species level diversity and precluding any generalization of results at species level.
Moreira, Z. P. M., Chen, M. Y., Ortuno, D. L. Y., & Haney, C. H. (2023). Engineering plant microbiomes by integrating eco-evolutionary principles into current strategies. Current Opinion in Plant Biology, 71, 102316. https://doi.org/10.1016/j.pbi.2022.102316
Simonin, M., Préveaux, A., Marais, C., Garin, T., Arnault, G., Sarniguet, A., & Barret, M. (2023). Transmission of synthetic seed bacterial communities to radish seedlings: impact on microbiota assembly and plant phenotype. bioRxiv, 2023-02. ver. 3 peer-reviewed and recommended by Peer Community in Microbiology. https://doi.org/10.1101/2023.02.14.527860
DOI or URL of the preprint: https://doi.org/10.1101/2023.02.14.527860
Version of the preprint: 2
Thank you very much for the improved version that answered the comments made on the first version by reviewers.
There are still one minor comments on your response: could you indicate in the result section your analysis of index hopping which was lower than 0.01 % ? For the rest, all your responses are accepted (except discussion shortening by eliminating results - see comment of new reviewer).
The improved version of the paper has been reviewed by a third (and last) reviewer who highlighted its strenght and suggested some improvements that are relevant for the final version.
Could you please respond to the comments of this review in the same manner (point by point response highlighted in the text) ?
We thank you again for submitting this very interesting research work to PCI microbiology.
Looking forward your response and the new version of the article,
DOI or URL of the preprint: https://doi.org/10.1101/2023.02.14.527860
Version of the preprint: 1
We would like ot thank you for considering PCI Microbiology for your publication. It has been reviewed by two reviewers and myself. We all underlined the interest and quality of the work carried out but also identified some aspects that deserve to be improved.
Your publication is therefore accepted provided the requested adaptations are implemented in a second version of the manuscript.
You will find the comments of both reviewers and mine jointly with this message. Please prepare a point-by-point response to the comments raised with references in lines in the new version. In addition to the new clean version uploaded on the preprint server, thanks for providing a track-change version so it facilitates the analysis of your responses.
Review from recommender:
Overall, the study describes an elegant approach to cope with variability of natural microbiota using Syncom. The manuscript describes a well designed study with relevant interpretation.Proper controls have been used at different stages of the experiment but their interpretation could be stated/improved (see comment hereunder)
- Bacterial taxa were added and followed. There is no information on fungal or protists taxa. Do they have also a potential influence ? It might be interesting to add a sentence explaining their relative importance (or not) somewhere in the article (maybe in the introduction).
Line 31-32: “These results confirm that the plant core microbiome includes pathogenic and not only commensal or mutualistic taxa” -> It is a very interesting and strong statement. The pathogens can be detrimental in some conditions and at least in the conditions tested during the experiment. At evolutionary point of view, how can it be explained/interpreted as the presence of pathogens could negatively impact fitness and survival ?
Line 33-35: the impact of Syncom on seed and seedling microbiota is underlined. I interpret that the fitness is related to the plant but it can be stated more clearly -> “.. and cause strong plant fitness differences between native…”
Lines 77-78: the statement “Despite the crucial role of seeds for food production and maintenance of plant biodiversity, microbiome studies on the seed compartment are still a minority (Shade, Jacques and Barret 2017)”. The citation is 6 years-old, is it still the case in 2023 ? Were there many experiments in this timeframe ? Some examples are given further but it could be relevant to use past term if the 2017 reference is maintained or to state it is still the case today. Explaining new research in between would be interesting
Line 81 “Recent studies are starting to be published” -> recent studies have been published… + are there many other studies ? Only two are cited and it is not clear how they were selected for being highlighted
The introduction is clear but I missed some comments on the different compartments where bacteria can be present on seeds (surface or within tissue…). It is indirectly addressed when mentioning disinfection but it worth refining the information for the readers. Reading material and methods, the reader further understand the work is carried out on microbes present on the surface of the seed but what about the endophytes ? This could be added
Some information related to the radish seed microbiota can be added in the introduction
Bacterial taxa were added and followed. There is no information on fungal or protists taxa. Do they have also a potential influence ? It might be interesting to add a sentence explaining their relative importance (or not) somewhere in the article (maybe in the introduction).
Material and methods
Line 112-114: transferring the number of strains and their phyla, families and genera in part 1 of results
Line 138: how was this concentration selected ? Is it because it allowed to achieve concentration of strains or Syncoms comparable to concentration of native microbiota ?
Line 141: what was (were) the control ?
Line 143: “The bacterial cell density of the inocula and inoculated seeds (pool of 30 seeds) were assessed by plating on TSA 10% (CFU/mL or /seed)” -> at which timing were analysed the inoculated seeds ?
Line 153: the link goes to homepage of ISTA and not the protocol
Line 173: 35 cycles of PCR were carried out. It is more frequent to carry out 25 to 30 cycles to avoid representation bias at the end of exponential phase of the PCR. Can you comment on this specific point ?
Line 204: 14,116 reads
There is no information on the way the alien control (Lactococcus piscium) is used to monitor contamination among the samples. Could you explain how you took into account these results in the global analysis (for example to set up contamination threshold as recommended in the following guidelines on HTS use for plant pest detection : https://peercommunityjournal.org/articles/10.24072/pcjournal.181/) ?
-point 1 – comment 1 : The selection process is well described but you did not mention before in M&m that 523 strains were isolated and sanger sequenced for species identification (see comment for line 112-114)
- Point 1 – comment 2: Did you also took into account already known beneficial or pathogenic effect related to the selected species ?
- Point 2: a native microbiome (e.g. non disinfected seed) could have been useful as additional conditions. You pointed the high variability observed elsewhere (and discuss it) but it might have added interesting information to compare naive and Syncoms (as for the other analyses). It might be relevant to discuss this point (maybe first part of discussion) ?
- Figure 3A: I interpret the figure with the fact that seed and inoculum are very close to each other (superposition). It seems confirmed by fig 3D. Is it the case ?
- Line 300: “The control seeds were surface-disinfected but they still harbored a low bacterial diversity likely of endophytic bacteria, including ASVs of strains included in the SynComs because the strains selected have been isolated from the same radish genotype”. Does it also explain the much higher variability observed on fig 4A ?
Line 316: the genera names can be abbreviated (please check on the complete document)
Figure 4B: when looking the results of the control for the seedlings, it seems there are more than a median of 3 taxa. I understand therefore that “other taxa” corresponds to a single or two ASV ? If so, is it the same taxa between replicates or it varies ? It might worth to consider these “other taxa” for better overview of the results.
- Lines 450-456: discussion on P. fluorescens is interesting but It can be noted that there are pathogenic strains as well as beneficial strains from this species that are studied from a long time. Here, a more subtle distinction is raised.
- Lines 464-470: the detrimental effect of P. viridiflava might not be surprising as it is a known plant pathogen. Has it been selected on purpose to include a pathogenic strain ? If so, it could be mentioned in when explaining species selection
- Line 473-475: how were the core taxa defined ? It worth reminding the criteria for being considered as core taxa (prevalence threshold ?) to better contextualize this term (maybe adding the number of species considered as belonging to core taxa as a comparison: three bacterial strains among … species of the core taxa ? This comment is also related with previous comment on giving more details on core microbiota of seeds/seedlings.
- when mentioning Paenibacillus, “spp.” should be always added in the text (correction needed at least for line 488)