Plant Broth- (Not Bovine-) Based Culture Media Provide the Most Compatible Vegan Nutrition for In Vitro Culturing and In Situ Probing of Plant Microbiota

Comments

Responses (line numbers refer to the file “diversity-944510 (1)-including English edits and revised marks.doc”*

To reduce the cultivation only to the R2A medium is extreme and in this form utterly wrong. There are a number of innovative methods, see e. g. Zervas, A., Zeng, Y., Madsen, A. M., & Hansen, L. H. (2019). Culturomics of Aerobic Photoheterotrophs in Wheat Phyllosphere Reveals Divergent Evolutionary Patterns of Photosynthetic Genes in Methylobacterium spp. Genome Biology and Evolution. 11(10):2895–2908. Therefore, the authors should be more cautious in the introduction not painting a B&W picture. These more sophisticated efforts have to be acknowledged and discussed. A convincing justification has to be given why only R2A medium was used for comparison. What about LB medium or a soil extract medium?

The article, as appeared in the title, is not primarily aiming at under estimating the great achievement of R2A as a very important culture medium with great achievements in the field of culturability. BUT underline the compatibility and feasibility of the use of the natural plant media in the form of plant broth to further explore the diversity of the plant microbiota.

This specific point was highlighted/acknowledged in the introduction Lines 152-154) and discussions (lines 1073-1078)

I am very surprised about the composition of the “R2A” medium used in this study because this is not the standard R2A medium used worldwide and sold by many companies. Why did the authors change the composition? Why did they add meat peptone?

R2A medium used is basically of Reasoner and Geldreich (Ref. 34) with a slight modification to further enrich the medium (https://assets.fishersci.com/TFS-Assets/LSG/manuals/IFU112543.pdf).

The text is adjusted and highlighted accordingly

(Material and Methods: lines 357-362)

Lines 21ff: what is here the connection to the human microbiome?

It is just to highlight progress of culturability of the plant microbiome compared to other environmental microbiomes. The sentence  (lines 25-27) was readjusted as follows:

“Despite recent advances of high-throughput methods, culturability is lagging behind other environmental microbiomes, notably human microbiome.”

Figure 2: this figure is not easy to read because the different amino acids cannot be distinguished. There are errors in the amino acid names: therionine, aspartic, glutamic should be threonine, aspartate and glutamate

Agree, the figure is very compact; however, and after improvement we request the understanding of the reviewer as it is pooling the nutritional profiles, macro- micro-molecules and major-minor-elements, of tested plants in a concise not wordy and lengthy form.

The figure is adjusted for better resolution and colours identification, and the names of amino acids are corrected as indicated.

Figure 4: only type strains should be used for comparison otherwise the chances of misidentifications are high. Please change.

Thanks for your recommendation. We reassigned the sequences including all respective type strains and corrected all related parts in the figures 5 and 6 and in the text as well

Figure 6 is in this resolution not readable and has to be improved

We removed the old Fig. 6 and the corresponding text

Lines 477ff: The statement “The widely used chemically synthetic culture media, on the other hand, contain defined chemicals of irrelevant composition of nutrients and other materials” is really a provocation because microbiologists spend more than a century optimizing the media for cultivation. Even if the authors only know R2A agar for comparison they have to be told that this medium is an excellent medium for cultivating bacteria from a broad range of habitats. But of course it is not perfect and this is why several thousand different media have been described and used for cultivation.

We do apologize for the misunderstanding.

We all acknowledge and appreciate the development of myriad formulas of artificial culture media and the unprecedented efforts of fellow researchers along the years to improve culturability of microorganisms in various environments. Over a century of successive efforts, now it exists a set of lab media that have been painstakingly and developed to date.

In our previous publications we used several formulas of such culture media. In this publication we particularly used R2A for comparisons, being reported as an excellent medium for cultivation of bacteria present in various environments. This is primarily to compare the performance of natural plant-only-based natural culture media to one of the best and well-known standard/artificial/designed culture media.

Accordingly, the related passage is re-edited to take into consideration the reviewer points of view (Discussion, lines 1073-1078):

“In contrast, the widely used standard culture media of designed and defined composition, on the other hand, contain prescribed substrates, possibly not very much related, required and/or demanded nutrients..”

Lines 491ff: If you have a look at the composition of R2A you will see that this is not “chemical synthetic”.  Some basal media could be called chemical synthetic because they are defined in their chemical composition

Agree, this is a difficult issue . However, throughout the manuscript “chemically-synthetic” is replaced by “Standard chemically-defined and artificial culture media”. let me hope that this term will be accepted by the reviewer

Comments

Responses

First the plant growth conditions, including substrate, origin of soil, and presence of nodules (on clover roots) are missing in the description. Are the plant grown in green-house or in field?. If roots of the legume plants included nodules it is normal to observed higher content of rhizobia in the endophyte population.

The sampled clover plants were grown in the Giza open fields at the experimental station of Faculty of Agriculture, Cairo university.

Yes nodules were present on the sampled and processed roots; which is confirmed by the isolation of more than 12 isolates of Rhizobium sp. throughout the study. Rhizobia were among the most common isolates,

The plant broth successfully recovered rhizobia. Among the sequenced isolates, Bacillus sp. and Rhizobium sp. were the most common on the tested plant-broth culture media (Discussion, lines 627-628).

The related text was adjusted accordingly (Materials and Methods, lines 133-135), and photo of root nodules is included in figure S3

For the writing the authors should avoid using words like excellent, exceptionally… like in paragraph 3.1. This part should be written in a neutral way giving only facts. I propose that an English native speaking person correct the whole text of the paper.

Agree, the paragraph is adjusted accordingly

English language of the manuscript was checked by an English language expert/service: bioXpress Editorial, Revision, Writing and Translation Services

In 3.3 the authors claim that the colonies are less slimy on the plant broth media. This is not really what is observed on Figure S3.

Agree, it is very relative

Slimy colonies were resolved on all tested culture media. However, their percentages were relatively higher on standard R2A, to the extent that colonies diffuse together in running slimy flocks (demonstrated in plates A of Fig. S3).

The text was adjusted and highlighted accordingly (Discussion, lines -1038588-589

The number of colonies identified from rhizosphere does not really change on the different media, but was significantly higher on plant broth (25ml) for the endophyllosphere. This higher recovery at the lowest broth concentration raises the question of testing even lower broth concentrations for this compartment.

Yes, further experiments, on cross-cultivation of endophytes on homologous and heterologous plant-based culture media, included concentrations of plant broth down to 5ml. Very interesting results are obtained and currently under preparation for publication.

The characterization (16S) of the isolated bacteria on the different media shows that the diversity was higher on plant broth media. This is the interesting point of the paper.  

Thanks

The quality of Figure 6 is very poor.

After improving the resolution of the figure, we prefer not to include it in the manuscript.  As the figure is solely based on presumptive taxonomic position of partial sequences of 16S rRNA sequences

I did not understand the meaning of the section 3.5 (Maldi-TOF MS). This section should be better written/explain.

The section of (Maldi-TOF MS) is edited and readjusted for better explanation, in results (lines 492-504) and discussion (641-655)

Line 478: irrelevant composition. This is the kind of sentence that should be removed from the text.

Thanks for your comment, text adjusted accordingly

(Discussions, lines 610-615)

R2A media (g L^-1)

Reasoner and Geldreich (1985)

Modified R2A  (g L^-1) ,         https://assets.fishersci.com/TFS-Assets/LSG/manuals/IFU112543.pdf

Comments

Casamino acids            0.5

Casein hydrolysate                 0.5

Roughly, both are similar, but there are processing differences- the real difference is filtering

Proteose peptone         0.5

Casein peptone                     0.25

Meat peptone                       0.25

All of these bacteriological pre-digested peptones  are of bovine source, not plant or yeast; being organic nitrogen source containing the short chained peptides & broad spectrum amino acids required for general microbial growth

Yeast extract                0.5

Yeast extract                        0.5

Dextrose,                         0.5

Dextrose                                0.5

Soluble starch             0.5

Soluble starch                     0.5

Dipotassium phosphate   0.3

Dipotassium phosphate      0.3

Magnesium sulfate     0.05

Magnesium sulfate               0.024

Sodium pyruvate         0.3

Sodium pyruvate                   0.3

Agar       15.0

Final pH 7 ± 0.2 @ 25 °C

Agar       15.0

Final pH 7 ± 0.2 @ 25 °C