Effects of Multi-Species Microbial Inoculants on Early Wheat Growth and Litterbag Microbial Activity
, Thomas F. Döring 1, Giorgio Masoero 2, Enrico Ercole 3 and Annamaria Bevivino 4
Round 1
Reviewer 1 Report
In short: In my opinion the authors should rewrite anew the methodology. They should provide only brief data about the multiplication of the microorganisms: medium, aeration (if any), time and temperature of incubation, agitation speed (with the use of magnetic stirrer horizontal/orbital shaker or other equipment), how they separate biomass from the broth, the moisture content of the zeolite carrier and the final concentration of the microbial inoculum in the carrier. The methodology regarding the preparation of Trichoderma inoculum is a good description, apart of inoculum density. Due to the very high density of the bacteria/fungi obtained in CM0001 broth, the authors should check the method of microbial density counting. In the results section, the authors should unify the scheme to present the data. This will be helpful to compare the data. This will of course be connected with rewriting the results section.Comments for author File: 
Comments.pdf
Author Response
Reviewer 1
Answer: We thank the reviewer for the valuable comments.
In short: In my opinion the authors should rewrite a new the methodology.
Answer A: We rewrote the microbial methodology (section 2.1) giving more details on selection, cultivation, and preparation of the microbial consortia for seed inoculation. See lines 91-172.
They should provide only brief data about the multiplication of the microorganisms: medium, aeration (if any), time and temperature of incubation, agitation speed (with the use of magnetic stirrer horizontal/orbital shaker or other equipment)
Answer B: We have provided the required data giving more information on the multiplication of microbial strains. See lines 111-126.
how they separate biomass from the broth, the moisture content of the zeolite carrier and the final concentration of the microbial inoculum in the carrier.
Answer C: We have specified the requested information along the text (see lines 126-135 and lines 154-159) and in Table 1.
The methodology regarding the preparation of Trichoderma inoculum is a good description, apart of inoculum density. Due to the very high density of the bacteria/fungi obtained in CM0001 broth, the authors should check the method of microbial density counting.
Answer D: We have revised and specified the information regarding the inoculum density. See lines 146-153.
In the results section, the authors should unify the scheme to present the data. This will be helpful to compare the data. This will of course be connected with rewriting the results section.
Answer E: Following the standard protocol for the evaluation of factorial field trials in agronomy, results of both trials were first evaluated in one joint data set. Depending on the outcome of the ANOVA, results were either shown over the main factors only (no interaction), or separated according to the significant interaction indicated by ANOVA. Hence, interactions between two or more experimental factors (microbial inoculation, substrate, and trial) are only shown when significant. In case of missing interactions only the main effects are presented. For the statistical reasons we prefer to stick to the method of data presentation in the results section. To clarify this issue, we have given a short amendment in the material and methods section. Please see section “statistical evaluation 2.5” (See lines 276-279).
Below, I put my comments:
- Line 97 – ‘a proven interspecific compatibility’ in the mentioned article there is antibiois test on one medium – so the authors should use therm a lack on antibiosis between strains.
Answer: changed to ‘lack on antibiosis test’. See lines 97-98.
- Lines 100 – 101 – Table 1 The authors obtain a gigantic cells density after a 24 or 72h of incubation. The inoculum with a cell density of 1010 or more, is likely to have a form of soft gel. It is impossible to obtain that concentration of biomass in only simple multiplication process. For example, according to the data from Bergey's Manual of Bacteriology SE vol 2B pp: 400– 402, the typical size of A. chroococcum and A. vinelandii cells varies from 1.6-2.5 x 3-5. So, the volume of single-cell varies from 6 to 24 cubic micrometers. The 1.1 x 1011 cfu x ml-1 (so single and small conglomerate of cells) should vary between 66% to 269 % of the total volume of broth. Also, this is impossible to obtain that amount of biomass from broth consisting 8 g (per liter) of peptones/meat extracts (typical density of bacterial cells is 1,1 cm3 x g-1 and typical composition of the bacterial cel[l] is 70% of water and 30% of other material like proteins etc).
Answer: We are sorry for the “error” in the former Table 1. Data on CFU mL-1 are not referred to microbial growth in rich medium but to values obtained after centrifugation and pellet resuspension for inoculum preparation. We have revised and clarified the methodology for the strain production and viable count and added a sentence on CFU mL-1 obtained after 24-72 h of microbial growth (see lines 122-135 and Table 1).
- This is very high amount of spores obtained without centrifu. How the authors did filter over the 10 ml of 2.2 x 1010 of cfu (spores and hyphae)? That concentration of spores (according to Watanabe (2002) Pictorial Atlas of Soil and Seed Fungi spores are globose to subglobose with the size of 2.1-3 µm) will be dense (it will be from 9 to over 30% of inoculum total volume) and stratify quickly?
Answer: We clarified and specified the method and description for the strain production and viable count. High spore concentrations were obtained only upon centrifugation. See lines 146-153.
- Table 1– Komagataella pastoris belongs to the yeast (there is no methodology how the authors multiply the yeast).
Answer: We have specified the information on yeast multiplication. See line 112.
- Table 1– What is the known physiological mode of action of PGP strains like TH01?
Answer: T. harzianum strain TH01 act as PGP for its efficacy to suppress disease and also to enhance plant growth (unpublished data).
- Table 1– Why the authors used biocontrol strains in experiments regarding plant promotion?
Answer: We also used biocontrol strains to extend the functionality of the microbial consortia. The well-designed SIMBA microbial consortia were planned to favor plant growth in the presence of biotic and abiotic stress. We hypothesized that they need to be more versatile than the individual microbial inoculants.
- Lines 129–120 – Could the authors describe how the strains could be stable in environment with 10% of moisture and stored in room temperature?
Answer: The stabilization procedure using micronized zeolite is the same used by the SME CCS-Aosta Srl., Quart (AO) -Italy, Franzione Olleyes, 11020, for the production of commercial microbial biofertilizer. We added data on CFU g-1 after stabilization in zeolite but, unfortunately, no data on long-term stability of strains composing MC_A and MC_B after stabilization in zeolite and storage at room temperature are available. The consortia, we used, were fresh, since they were produced shortly before field application, i.e. they were less than 10 days old.
- Lines:103–145 – there have to be information regarding of density of microbial inoculum added to the soil in the formulated form in zeolite!
Answer: The information on colony forming units (CFU) of microbial inoculants stabilized in zeolite were added. See Table 1.
- Table 2 – Sadly there is no data about nitrogen content and available (soluble) forms of elements.
Answer: We agree that it would have been interesting to include ammonium and nitrate contents of the soil. As a rough indicator we have now included the total nitrogen content (%) of the substrate before steaming, since mineral nitrogen contents have not been measured. Instead, we used the plant as indicator for soil mineral nitrogen content using the total nitrogen uptake of the shoot. The determination of the total nitrogen content was done according to the DIN EN 16168:2012 (Sludge, treated biowaste and soil – Determination of total nitrogen using dry combustion method). See Table 2.
- Line 152 – please remove ‘the by humid heat’ it is mentioned earlier that soil was steamed.
Answer: Done. See line 187.
- Line 167 – autoclaved – should be sterile
Answer: Done. See line 202.
- Lines 212-221 – Perhaps it will be easier if the authors briefly describe the methodology ie the phosphorus content was determined by XXXXX method with the use of YYYYY equipment.
Answer: We have reformulated and shortened the paragraph to clarify the methodology of determining wheat shoot P, K, and N content. see lines 248-262.
- Line 245 – please replace crop length to plants length.
Answer: Done. See lines 290-291.
- Table 3 – The table is very unclear for me. Some homogenous groups are missing (or the authors did not place them). In my opinion, there should be separate tables for each trial. Also, I am not able to compare the results from table 3 to the results from figure 1 and table 4 (different results presentation scheme).
Answer: As explained above, interactions between experimental factors were only shown in case of significance. In other cases only, main effects were shown. Please see answer E and lines 276-279.
- Tables 3, 4, 5 – all the tables should have the same scheme to compare the results!
Answer: Please see also the explanations given in answer E and lines 276-279.
- Line 348 – what is the ‘early crop growth'?
Answer: ‘Early crop growth’ refers to the growth of wheat in the first weeks after germination. The time period on which the results presented were based included wheat growth for 63 days, i.e. until the transition of tillering to stem elongation. Despite being not a guarantee for higher yields, we think that an early established growth-advantage of inoculated plants is an important hint for beneficial microbial effects. This section was shifted to the results, based on the recommendation of another reviewer. Please see section 2.3 and 3.2 (e.g. lines 212-213; lines 304-306 and lines 314-316).
- Lines 496-498 – I recommend deleting reference no 71 in that sentence because the authors of reference no 71 inform that: „The effects on soil-test P and K and extractable Ca were small and variable, but the soils were already high in these elements.”
Answer: We agree that the reference does not adequately support the statement. We have deleted the reference in the respective sentences. See line 563.
Reviewer 2 Report
I have read this manuscript and in my opinion it requires a few of corrections.
Comments:
L347-354 This part of the text belongs to the Results section.
L372-380 This part of the text belongs to the Results section.
L389-397 This part of the text belongs to the Results section.
L421-427 This part of the text belongs to the Results section.
Author Response
Reviewer 2
I have read this manuscript and in my opinion it requires a few of corrections.
Answer: We thank the reviewer for the valuable comments.
Comments:
L347-354 This part of the text belongs to the Results section.
Answer: We have moved the respective parts to the results section as suggested. Please see lines 300-306.
L372-380 This part of the text belongs to the Results section.
Answer: We have moved the respective parts to the results section as suggested. Please see lines 381-388.
L389-397 This part of the text belongs to the Results section.
Answer: We have moved the respective parts to the results section as suggested. Please see lines 341-342 and lines 344-354.
L421-427 This part of the text belongs to the Results section.
Answer: We think that this section gives more an explanation than presenting results. It discusses the importance to include autoclaved MC and highlights also the relevance of the carrier material. For this reason, we prefer to the continuance of this section in the discussion.
Reviewer 3 Report
The revised manuscript studies the role of constructed microbial consortia on wheat yield. This topic is very interesting and each new studies on identification, description and application new or partially studies MC are very valuable for sustainable and improved agriculture.
I found the revised manuscript falling into the scope of Agronomy Journal. The work is written very carefully, precisely describing all information. The aim is clear, design perfect and all methodology (including statistics) is very good allowing for the repetition of this study. For that reason I found presented results genuine and convincing. Discussion is all well done and all makes me feel this is good piece of work well presented.
I have just tiny technical comments:
- in line 96 the word 'First' should be rather written in lowercase
- I noticed that text is bigger in lines 121-123
Author Response
Reviewer 3
The revised manuscript studies the role of constructed microbial consortia on wheat yield. This topic is very interesting and each new studies on identification, description and application new or partially studies MC are very valuable for sustainable and improved agriculture.
I found the revised manuscript falling into the scope of Agronomy Journal. The work is written very carefully, precisely describing all information. The aim is clear, design perfect and all methodology (including statistics) is very good allowing for the repetition of this study. For that reason, I found presented results genuine and convincing. Discussion is all well done and all makes me feel this is good piece of work well presented.
I have just tiny technical comments:
Answer: We thank the reviewer for the valuable comments.
in line 96 the word 'First' should be rather written in lowercase
Answer: Done. See line 96.
I noticed that text is bigger in lines 121-123
Answer: Done See lines 141-142.
Reviewer 4 Report
Dear Authors,
the manuscript is very interesting and depict very well and clearly (text, data and statistical analysis) the need of advanged biological and sustainable strategies to improve wheat growth. LBN evaluation is also very original and interesting. I really appreciate the manuscript in present form.
Best regards
Author Response
Reviewer 4
Dear Authors,
the manuscript is very interesting and depict very well and clearly (text, data and statistical analysis) the need of advanged biological and sustainable strategies to improve wheat growth. LBN evaluation is also very original and interesting. I really appreciate the manuscript in present form.
Best regards
Answer: We thank the reviewer for the valuable comment.
Round 2
Reviewer 1 Report
The authors studied the impact of microbial consortia on plant growth. However, for the next studies, I suggest checking the impact of single strains and the consortia. This will give some data about which strain ' works'. Sometimes the mixture of microorganisms will not work while the single strain will for example promote plant growth.
