Acanthamoeba castellanii alone is not a growth promoter for Hordeum vulgare

  1. Julia Sacharow
  2. Stefan Ratering
  3. Bellinda Schneider
  4. Alessandra Österreicher Cunha-Dupont
  5. Sylvia Schnell

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Abstract

Protists are important key players in the microbial loop and influence their environment by grazing, which leads to the return of nutrients into the soil and reduces pathogen pressure on plants. Specifically, protists on and around plant roots are important for plants’ development and growth. For this study, the fourth most important crop in the world, Hordeum vulgare , was selected. Seeds of H. vulgare were inoculated with Acanthamoeba castellanii alone or with additional soil bacteria at the beginning and during the experiment. The germination of the seeds and the growth of the plants in pouches were monitored over 3 weeks. No differences were found in leaf growth, root growth, root and leaf nitrogen content or ammonia content of the liquid from the pouches. In contrast, the relative increase in root and leaf dry weight showed a small difference compared to the controls. The results of this experiment demonstrated that seed inoculation with A. castellanii alone or with additional unidentified soil bacteria did not have a major effect on the growth and development of barley. Nevertheless, small changes in plant development were detected, indicating that A. castellanii should be considered for further investigation of co-inoculations with plant growth-promoting bacteria and additional nutrients.

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  1. Version published to 10.1099/acmi.0.000761.v3 on Access Microbiology
  2. Access Microbiology

    The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community.

  3. Access Microbiology

    Comments to Author

    The revised version can be accepted

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Very good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  4. Version published to 10.1099/acmi.0.000761.v2 on Access Microbiology
  5. Access Microbiology

    This is a study that would be of interest to the field and community. The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments. Please include more rigour criteria and resources in your methods section, as highlighted by the SciScore reports. Including RRIDs and negative statements to explain why things were not performed should increase the rigour and reproducibility of your work. You can find tips on how to improve your article here: https://sciscore.com/reports/Core-Report.php Please deposit the data underlying the work in the Society’s data repository Figshare account here: https://microbiology.figshare.com/submit. Please also cite this data in the Data Summary of the main manuscript and list it as a unique reference in the References section. When you resubmit your article, the Editorial staff will post this data publicly on Figshare and add the DOI to the Data Summary section where you have cited it. This data will be viewable on the Figshare website with a link to the preprint and vice versa, allowing for greater discovery of your work, and the unique DOI of the data means it can be cited independently. The language used is poor, which can cause ambiguity at times. Please carefully rewrite it. We offer a discounted translation service, Editage (https://www.editage.com/; see https://www.microbiologyresearch.org/prepare-an-article#13 for more information).

  6. Access Microbiology

    Comments to Author

    This study presents a good foundation to develop the study of the role of protists in plant health and growth. 1. Acanthamoeba culture described is not conventional. Have the authors optimised this type of culture method? Line 104. protists were viewed under the microscope. Not were microscoped. How was the Acanthamoeba assessed to be free of bacteria? The method is not referenced and it does not account for any live bacteria inside the amoeba. Typically, Acanthamoeba become axenic through plate isolations and cleared of bacteria through encystment and SDS washes. It would have been useful to comment on the bacteria content that Acanthamoeba are exposed to. 2. the results focus on plant health and this is pertinent to the objectives of the study. Table 4 has spelling mistake. It should be 'trophozoites' It would be useful to illustrate images of trophozoites and cysts. The presence of cysts suggests that Acanthamoeba might be stressed either through soil pH, nutrient availability. This is not explained in the manuscript. 3. The style and organisation of the paper are clear and communicate well the key findings 4. The analysis of the literature is appropriate

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  7. Access Microbiology

    Comments to Author

    The authors in the present manuscript tested A. castellanii on barley seedling growth. However, they did not get good results that can prove this organism as an efficient barley growth enhancer. Though it is not always possible that A. castellanii be a good growth promoter for every plant, but the experiments adopted are not enough and well organized and performed that can prove this protist as a neutral for this plant. I have so many doubts about the work and I am against the publication of this manuscript in "Access Microbiology". Some of the points are listed here which are of serious concern. * The English level remains too low to be able to communicate the findings to the target audience. There are numbers of grammatical errors through-out the manuscript. I am not going to list them out - it is not the job of referees to ensure the readability of the manuscript. Each line needs a correction, that's why I strongly suggest the authors to contact some native English corrector to make the manuscript understandable and reader friendly. The whole manuscript should be re-written before moving for publication. * There should be the flow in each part of the manuscript. I think this is missed in the introduction part for describing protist as an important plant growth promoter. Authors have used old references for bioinoculants! There are tremendous investigations going on world -wide recently to find efficient strains of bacteria, fungi and even of protists. Azam et al., (1983) did not gave the concept of "microbial loop" for terrestrial ecosystem. They have coined this term for aquatic ecosystem. The concept of "microbial loop in soil" was given by Clarholm (1985) and it was break through investigation in soil protozoological studies. * Methodology missed detailed description, flow, and adoption of suitable techniques for protozoological studies. For example, The A. castellanii culture 1534/3, …………..using a counting-chamber… lots of grammatical mistakes. No flow in the collection, enrichment, and/or purification of protists… * Even hard to understand the procedure, the authors have followed. For example, "The protist concentration, the concentration of remaining bacteria in the purified protist culture…….." * Why Volvic water and not "distilled water". * How authors made culture of A. castellanii bacteria-free? The procedure they follow is not satisfactory to make the amoeba axenic. Amoeba carry always some accompanied bacterial strains and it is really hard to remove them with this simple procedure. * Concentration of bacteria counted in "counting chamber"? Is it possible? * Moreover, 630X? Really? Please cross check the objective of microscope and magnification. * L-116 Again how authors counted bacteria with counting chamber? * L-121 "The surface of the Hordeum vulgare "Odilla" seedlings….." seedlings? Or seeds? * L-133 The authors should gave some specific code to the treatments! * How many replications are there for each treatment? * Size of the pouch? * Which type of soil they filled in the pouch? Soil physico-chemical characteristics? * Why authors have used supernatant of A. castellanii, how they prepare this? Is there any specific purpose? * L-147 2 ml beaker? How much g of sample used for the analysis? From which part …Root or shoot or whole plant? * L-160-163 first sampling is after one day; then re-inoculation. For this, the authors have used 50 mL + 10 mL water + culture inoculation which is according to me is too much for small pouches. * After 7 days, again sampling and re-inoculation. Please make sure these much organisms have you inoculated to small pouches? And why re- and re-treatments? This is not recommended dose for bioinoculants. * There is no uniformity in the figures. The authors should follow only one representative method; either as fig 2 and 3 or as fig 4 (bar graphs). Where are error bars? Figure 2 is not readable. L- 184 There is difference between 7days and 14 days. Fig. 1 and even Fig 3 depict this. Then how authors stated that "there was no difference" in the results. * Tables are not in appropriate and publishable format. Even table caption and arrangements are not valid. Table 1- Treatments are not properly arranged. Caption is not suitable to table. Treatment code must be there to write the manuscript in a proper way. * There is no flow in discussion part. Not valuable arguments are given that justified the results of the present study. The references that are discussed, represents protist as an efficient plant growth enhancer. Not a single reference authors have discussed that supports their study. Even some of the references are not well understood by authors, such as, "Kreuzer et al., 2006". Repetition of sentence "This experiment showed that an inoculation of barley seeds………….. influence barley seedlings growth" ….. Language is very poor. For example, L- 364 what does authors mean by "difference in growth between best and worst growth treatments" A. Castellanii as a good "biological compound" …really could this organism be termed as compound?

    Please rate the manuscript for methodological rigour

    Poor

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  8. Version published to 10.1099/acmi.0.000761.v1 on Access Microbiology