Symbiotic Performance and Characterization of Pigeonpea (Cajanus cajan L. Millsp.) Rhizobia Occurring in South African Soils

Round 1

Reviewer 1 Report

 

The paper by Bopape describes the isolation and partial characterization of rhizobium strains isolated from soils to Pigeonpea.

The bacteria isolated from nodules were identified as putative rhizobia by 16S sequencing. This revealed that non rhizobial bacteria are found in nodules, in agreement with recent literature.

This paper gives interesting results on the pigeonpea associated rhizobia in South Africa as Rhizobium and Bradyrhizobium where mostly found in nodules following a trapping experiment.  

These were tested on two cultivars, one indigenous and one improved cultivar. Interestingly the authors found that the indigenous cultivar responded better to the rhizobia trapped from soil.

To test the interaction between the isolated rhizobia and the pigeonpea plants the authors performed a symbiotic assay in Leonard jars and measured, shoot dry weight, root dry weight, nodule number and the nodule dry weight. The corresponding fresh weights are also given. The dry weights are interesting proxy to measure the interaction between legume plants and rhizobia when /N content or Acetylene reduction assays are not available (see below).

The paper needs some improvement in the presentation and in the research part.

Minor points:

Line 104, replace lab. By laboratory

Line 108 : landraces that were originally…

Line 106-109 and Table 1: in the text the authors talk about 2 improved germplasm and 3 landraces while in the Table (and the rest of the paper) there are only two germplasm used. Please clarify.

Also decide if you talk about landraces or indigenous germplasm in the rest of the paper.

Line 115: which medium? Please add reference/composition or indicate if this is water agar.

Line 207: incubated with one of the presumptive rhizobia.

Line 250: in the current study are likely new to science.

Line 315: replace Table 1 by Table 3

Line 325: replace Table 2 by Table 4

Line 437: Replace Table 4 by Table 3

Line 455-456: please rephrase this sentence.

Figure 2 and 3: Please remove the line number inside the figure. This is not readable tis way.  

 

There are a number of questions concerning the work and principally the nodulation test.

I did not understand if the nodulation test were done one time or three time. The authors indicate in te Mat Met section that 3 seeds were planted by Leonard Jar and in the Table (3 and 4) legends that the values are means of three replicates. Does it mean 9 plants or the replicates correspond to the three plants in each Leonard Jar. If this is three plants, I think it is not enough for conclusions. Especially that we have no information on the variation inside the three replicates.

As indicated above the dry weight is a good proxy for the symbiotic efficiency. But the symbiosis can change the shoot to root ratio and the good value to estimate the gain in growth provided by the symbiosis is the plant (shoot plus root) dry weight. As indicated above ARA and carbon/nitrogen ratios are also good indicators for the symbiotic efficiency but are not always available for the laboratories.

In addition, peoples also measure nodule numbers and nodule dry weight. Nodule number is not always a good proxy for the symbiotic efficiency.

No nodule formed on the root indicates a non-symbiotic interaction. Interestingly some of the strains tested here (for ex 14a1p5, 13b1p4 and 13bp3 are nod+ (low nodule number) on the indigenous genotype and nod- on the improved genotype and inversely for other strains. This is not indicated and discussed in the manuscript. This reflects the presence of low nodulation efficient strains in the soil.

In contrast, a high nodule number may reflect higher symbiotic efficiency but can also be misleading as efficient infection can be also link to inefficient nitrogen fixation and in this case, plants will contain a high nodule number but nitrogen fixation for the plant will be poor. Efficient nitrogen fixation should be reflected by a high plant dry weight. For example, strain 8a2p3 will form 10 nodules per plant (indigenous) for a plant dry weight around 1 gr and strain 33ap4 will form 21 nodules per plant for a plant dry weight around 0.4 g. This difference in efficiency is also reflected by the nodule dry weight per nodule. If the plant produces numerous small nodules, they are probably inefficient. Big nodules are generally efficient nitrogen fixing nodules.

An interesting result of the research here is that some inoculated plants (see inoculation by strain 26bp3) grow less than the non-inoculated control despite forming few nodules indicating a parasitic behavior of this strain. This result is an argument for the inoculation with efficient nitrogen fixing strains.

I suggest that the authors include the fresh weight values in the supplementary data, because they are normally not used for assessing nitrogen fixation.

For each genotype they could produce two figures that could be easier to visualize for the reader. One Figure would give the plant dry weight, with each bar of the graph split in two parts with root and shoot dry weight represented by different color. This way the ratio between root and shoot is directly visualized.

The second Figure will show the nodule number next to the single nodule dry weight. This representation will also reflect the symbiotic efficiency (infection efficiency and nitrogen fixation efficiency).

Please also indicate in these figures, together with the strain number if it is a Rhizobium (Rh) or a Bradyrhizobium (Br). This can help the interpretation of the results for the readers.

I believe the figures (graphs) are easier to interpret for the reader.

I do not understand why the figures 2 and 3 are not presented in the first paragraph of the results. Please introduce them in this part of the manuscript.

In summary I think that this work is very interesting but if the work has been done with only three plants per combination it is not enough for publication. If the experiment was repeated three times (9 plants) it is ok but the organization and presentation of the manuscript should be improved.

 

Author Response

Dear Review

We sincerely thank you for the positive and constructive comments that have helped to improve the quality of our manuscript. We agree with all the comments and we have incorporated them in the manuscript and list of responses document herewith attached.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript entitled "Symbiotic performance and characterization of pigeonpea (Cajanus cajan L. Millsp.) rhizobia occurring in South African soils" screened for nodulation of pigeonpea using indigenous rhizobia in soils and evaluated their performance on locally sourced and imported genotypes of the legume. The topic is relevant, and the subject is very interesting. The authors did a lot of work, and the methodology used is adequate for the objectives of the study. The results are of interest and support the conclusions. That being said, the manuscript has the potential to be accepted. However, there is still some minor issues need to be addressed before the paper could be accepted as follows:

 

Comments

Line 114-115: “The treated seeds” delete treated.

Line 120: What does it mean by green house?

Line 126: Why Congo red?? Please clarify.

Line 222: Add a reference https://doi.org/10.1007/s42729-021-00727-2

Tables 1&2: I suggest using three digits after the decimal point and aligning the numbers. For Duncan letters, do not use superscript.

Tables 1&2: The titles are excessively long. Try to concise them.

The authors should cite Tables and Figs in the discussion section.

Line 496: A brief conclusion should be added by the authors in a separate section.

Kind Regards.

Author Response

Dear Review

We sincerely thank you for the positive and constructive comments that have helped to improve the quality of our manuscript. We agree with all the comments and we have incorporated them in the manuscript and list of responses document herewith attached.

Author Response File: Author Response.docx

Reviewer 3 Report

Reviewer’s comments

The aim of this paper is searching out symbionts of grain legume Pigeonpea (Cajanus cajan (L.) Millsp. This is a very important and interesting topic for understanding proper productivity of pigeonpea in South Africa. Nevertheless, there are some comments regarding the submitted manuscript:

 

Major comments:

Point 2.1: Please provide in the form of a table accurate geographic data such as the latitudes and longitudes of the places from which the soil samples were taken. Please add information what South African provinces there were? Please add information how the soil samples were transported and stored in the lab and how long? There is only information that samples were kept in a cooler box, until transportation to the lab. What happened during transportation and later?

Point 2.2 Reconstruction of phylogeny based on the 16S gene is currently insufficient to publish the results in a reputable journal as Agronomy. For some strains, the authors also used only part of recA gene encoding the housekeeping recombinase A enzyme. These studies are insufficient. The results should be complemented by studies of other important housekeeping genes as previously published (e.g. in Sajnaga, Symbiosis 80, 245–255 (2020). doi: 10.1007/s13199-020-00668-x).

Point 3 “They were grouped based on their different growth characteristics, particularly growth rate, colony shape and texture when cultured on YMA supplemented with Congo red. This approach allowed separation of the isolates into various groups” there is no information in point 2.1 what were the criteria for this division? how the colonies were to look like so that they could be divided in the right way. The matter of these criteria should be completed in the materials and methods. It is not clear how the authors divided the isolates into various groups. This should be clarified.

Line 247-248: Reconstruction of phylogeny based on the 16S gene is currently insufficient. These are very old publications for the current knowledge.

 

Minor comments

Line 16: Please remove “200”

Line 181: There are no spaces between sentences “…in this study.” and All sequences..”

Line 204: No information whether the medium was sterile? was the water that was watered sterile? Please briefly describe the growing conditions in the jars.

Lines 282-290: This information is unclear. Which bacteria influenced particular genotypes? What were the genotypes? It is difficult to understand what the authors mean. Please rearrange this paragraph to make it clearer.

Line 315: It should be Table 3

Line 325: It should be Table 4.

Line 344: Please correct from “…were also able form…” to: …were also able to form…

Lines 344-347: This information is unclear. It is difficult to understand what the authors mean.

Line 454: Please correct from “pigeonpeaa” to pigeonpea.

Lines 462-463: Members of the genus Burkholderia (β-proteobacteria) have recently been shown to be able to establish a nitrogen-fixing symbiosis. Burkholderia legume symbionts (also called β-rhizobia) are ancient in origin and are the main nitrogen-fixing symbionts of species belonging to the large genus Mimosa e.g. in Brazil. Please correct the sentence and find a newer reference than from 2008 (e.g. Beukes, Front Microbiol. 10, 1195 (2019). doi: 10.3389/fmicb.2019.01195, Jach, Biology, 11(5), 676 (2022); doi: 10.3390/biology11050676)

Line 465: “Another recent study” please remove the word “recent”. The 2016 research is not recent one.

Line 477. Please add dot after sp.

Line 482: Please correct from “nodulea” to nodule

 

There are no table numbers in the text. There is no information what description refers to which table.

 

Table 2: Please write “Isolate Number” or “Isolate No”

In conclusion, authors should plan future studies using consortia of these different bacteria (several strains together) to see if such a consortium will improve plant yield compared to using only a single strain. It should be added.

Author Response

Dear Review

We sincerely thank you for the positive and constructive comments that have helped to improve the quality of our manuscript. We agree with all the comments and we have incorporated them in the manuscript and list of responses document herewith attached.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

Major comments:

Point 2.1: Please provide in the form of a table accurate geographic data such as the latitudes and longitudes of the places from which the soil samples were taken. Please add information what South African provinces there were? Please add information how the soil samples were transported and stored in the lab and how long? There is only information that samples were kept in a cooler box, until transportation to the lab. What happened during transportation and later?

Point 2.2 Reconstruction of phylogeny based on the 16S gene is currently insufficient to publish the results in a reputable journal as Agronomy. For some strains, the authors also used only part of recA gene encoding the housekeeping recombinase A enzyme. These studies are insufficient. The results should be complemented by studies of other important housekeeping genes as previously published (e.g. in Sajnaga, Symbiosis 80, 245–255 (2020). doi: 10.1007/s13199-020-00668-x).

Point 3 “They were grouped based on their different growth characteristics, particularly growth rate, colony shape and texture when cultured on YMA supplemented with Congo red. This approach allowed separation of the isolates into various groups” there is no information in point 2.1 what were the criteria for this division? how the colonies were to look like so that they could be divided in the right way. The matter of these criteria should be completed in the materials and methods. It is not clear how the authors divided the isolates into various groups. This should be clarified.

Line 247-248: Reconstruction of phylogeny based on the 16S gene is currently insufficient. These are very old publications for the current knowledge.

 

Author Response

We sincerely thank you for the constructive suggestions that improved our manuscript significantly

Author Response File: Author Response.pdf