Survival Dynamics of Trichoderma longibrachiatum Tr58 in Conidia- and Chlamydospore-Amended Soils with Different Moisture Levels
Round 1
Reviewer 1 Report
The manuscript compared the survival dynamics of two types of Trichoderma longibrachiatum Tr58 spores, conidia and chlamydospores, under different soil water contents. However, major revision is needed before accepted.
1. The results were too little to support a full-size report article in Agriculture journal, only with germination and inhibition rate of Tr58 spores, and survival dynamics of Tr58 spores in soils with different water contents. More data or experiments must be supplemented for publication.
2. The authors claim to “confirmed that chlamydospores were more tolerant to soil fungistasis and greater application potential in disease control than conidia” in the conclusion. Yet only sterilized soil and non-sterilized natural soil were compared for their effects in Section 3.1. More factors of soil fungistasis such as pesticide residues in soil should be investigated. And more details of their potentials in disease control should also be included.
3. The Discussion should be re-organized to avoid the repetitive sentences “chlamydospores were higher than those of conidia in soil and showed a much higher resistance to soil fungistasis”. And more comparison of this study with other reports should be described other than just repeating “chlamydospores higher than conidia”.
4. There were too many references from 1950s to 1980s, is this meaning that the survival and proliferation of propagules of Trichoderma spp. fully investigated last century or need to be updated?
5. Some details of formats should be corrected including: Trichoderma should be italic, 106 should be 106, etc.
Author Response
Dear Editor and Reviewer,
Thank you both for your detailed work and comments. Accordingly, we have made the following revisions/corrections:
- The results were too little to support a full-size report article in Agriculture journal, only with germination and inhibition rate of Tr58 spores, and survival dynamics of Tr58 spores in soils with different water contents. More data or experiments must be supplemented for publication.
Response:
Trichoderma is widely used for the control of soil-borne disease. Soil moisture is an important environmental factor affecting the development of fungi and plants. However, the effect of moisture on the survival of Trichoderma especially on the survival of chlamydospores of Trichoderma in soil is not systematically studied. In this study, we reported the survival dynamics of T. longibrachiatum Tr58 in conidia- and chlamydospore-amended soils with different moisture levels. It includes three parts: (1) To determine the influence of soil moisture and sterilization on the germination of two propagules of Tr58. The results showed that soil moisture level was significantly correlated with the germination of Tr58 propagules regardless of the presence of other microbial populations in the soil. (2) to measure the Tr58 contents in soil with different moisture contents within 12 months after the application of conidia and chlamydospores. The result proved that the contents of Tr58 in chlamydospores-amended soils with different moisture content were all higher than those of conidia. (3) To measure the content of Tr58 in soil additionally 24 months after the application of Tr58 propagules. It was proved again that chlamydospore had stronger survival ability than conidia and which was also influenced by soil moisture content.
It is the first report on the effects of soil moisture on the survival of Trichoderma in conidia- and chlamydospore-amended soils, which provide theoretical basis and technical support for efficient utilization of Trichoderma preparations for the control of soil-borne disease. According to the reviewer's suggestion, the correlation analysis between soil moisture contents and spore germination rates was added to further support the conclusions of this paper (line 131-132,141-143, figure 1). Therefore, we consider this work new and can be published in Agriculture.
- The authors claim to “confirmed that chlamydospores were more tolerant to soil fungistasis and greater application potential in disease control than conidia” in the conclusion. Yet only sterilized soil and non-sterilized natural soil were compared for their effects in Section 3.1. More factors of soil fungistasis such as pesticide residues in soil should be investigated. And more details of their potentials in disease control should also be included.
Response: It is generally believed that the higher the content of Trichoderma in soil, the better the disease control effect can be achieved. Currently, Trichoderma commercial preparations are mainly made of conidia. Chlamydospores have higher survival and reproduction ability in soil than conidia, and therefore chlamydospores preparations have higher potential for disease control. The above content is added in Discussion section (line 256-261). We have studied and reported the effects of chemical pesticides on the germination and growth of chlamydospores of T. longibrachiatum Tr58 (see Introduction section, line 59-61).
- The Discussion should be re-organized to avoid the repetitive sentences “chlamydospores were higher than those of conidia in soil and showed a much higher resistance to soil fungistasis”. And more comparison of this study with other reports should be described other than just repeating “chlamydospores higher than conidia”.
Response: The discussion was re-organized to delete the duplicated content and compared the spore characteristics with those of Clonostachys rosea and Monilinia laxa reported (see Discussion section, line 287-289)
- There were too many references from 1950s to 1980s, is this meaning that the survival and proliferation of propagules of Trichoderma spp. fully investigated last century or need to be updated?
Response: Soil fungistasis was first reported in 1953, and many reports mainly focused on the mechanism of soil fungistasis from then on. The survival and proliferation of propagules of Trichoderma spp.in soil was not clear especially of chlamydospores. It was the first time to systematically study the effect of soil moisture on the survival of Trichoderma for comparing the characteristics of conidia and chlamydospore of Trichoderma in this report. The result provides guidance for the application of Trichoderma biocontrol preparations.
- Some details of formats should be corrected including: Trichoderma should be italic, 106 should be 106, etc.
Response: The Latin name of strains, case and superscript have been modified in revised manuscript.
Reviewer 2 Report
This manuscript presents a study examining the germination of conidia and chlamydospores Trichoderma longibrachiatum isolate Tr58 in soils of different moisture levels in two soil conditions (sterilized and unsterilized). The results of the study indicated that the germination of T. longibrachiatum propagules was lower in unsterilized soils compared to sterilized soils. Moreover, the presence of T. longibrachiatum was longer in the soil when inoculated as chlamydospores than as conidia. From the results, it was concluded that soil moisture of 10–20% was favorable for the long-term survival of T. longibrachiatum and chlamydospores were better propagule for application than conidia. Although these results are interesting there are certain concerns as mentioned below that need attention. As the comments were numerous I marked many of my comments directly on the annotated manuscript.
1. One of the major shortfalls of the study is that the experiment was conducted in one soil type. Previous studies have clearly shown that the influence of soil factors on fungal propagule growth and development can vary with soil types.
2. The term soil water should be replaced with soil moisture throughout the manuscript including the title.
3. Please do not repeat the words that are already there in the title as keywords.
4. Italicize the fungus name throughout the manuscript.
5. The methods need a thorough revision by providing additional details. Mention the type of statistical method used to assess the significance of the data in the methods. Moreover, use multiple comparison analyses like the ANOVA to assess the influence of the main factors and their interactions on the studied parameters.
6. Figures should be self-explanatory. However, it is not so in the current presentation. Mark the results of the statistical analysis in the figures.
7. Revise the discussion thoroughly by critically discussing the reasons for the observed results. Remove the general statements and the repetition of the results from the discussion. Some key factors were not measured in the present study. For instance, the factors that contribute to the fungistatic effect (e.g., soil microbial populations) of the soil were not assessed in the study. This renders the discussion on fungistatis highly speculative.
8. The reference list should be checked thoroughly as scientific names are not italicized and journal names are abbreviated for some and not for others.
9. The presentation has to be improved. I suggest the authors seek the help of a colleague with good proficiency in English while revising the manuscript.
Comments for author File: 
Comments.pdf
Author Response
Dear Editor and Reviewer,
Thank you for your patience and careful revision and constructive suggestions. Accordingly, we have made the following revisions/corrections:
Comments and Suggestions for Authors
This manuscript presents a study examining the germination of conidia and chlamydospores Trichoderma longibrachiatum isolate Tr58 in soils of different moisture levels in two soil conditions (sterilized and unsterilized). The results of the study indicated that the germination of T. longibrachiatum propagules was lower in unsterilized soils compared to sterilized soils. Moreover, the presence of T. longibrachiatum was longer in the soil when inoculated as chlamydospores than as conidia. From the results, it was concluded that soil moisture of 10–20% was favorable for the long-term survival of T. longibrachiatum and chlamydospores were better propagule for application than conidia. Although these results are interesting there are certain concerns as mentioned below that need attention. As the comments were numerous I marked many of my comments directly on the annotated manuscript.
- One of the major shortfalls of the study is that the experiment was conducted in one soil type. Previous studies have clearly shown that the influence of soil factors on fungal propagule growth and development can vary with soil types.
Response: Due to the long duration of this study and large number of samples needed for detection of Trichoderma content in soil, and this study focused on the effect of soil moisture on the survival of two types of Trichoderma spores. Therefore, only one soil type was used in this study.
- The term soil water should be replaced with soil moisture throughout the manuscript including the title.
Response: All “soil water” has been replaced with “soil moisture” in the revised manuscript.
- Please do not repeat the words that are already there in the title as keywords.
Response: Key words “fungal propagules” and “soil moisture level” was added, and “soil water content” was deleted.
- Italicize the fungus name throughout the manuscript.
Response: All fungus names have been changed to italic
- The methods need a thorough revision by providing additional details. Mention the type of statistical method used to assess the significance of the data in the methods. Moreover, use multiple comparison analyses like the ANOVA to assess the influence of the main factors and their interactions on the studied parameters.
Response: Microsoft Excel 2007 and DPS7.05 were used for statistical analysis. Pearson correlation analysis was added to assess the correlations between soil moisture levels and germination rates of propagules. The Pearson correlation coefficient were calculated. The results further confirmed that soil moisture content significantly affected the germination and growth of two type of Trichoderma propagules, and the survival ability of chlamydospore in soil was significantly higher than that of conidia. (line131-132,141-143, and figure 1).
- Figures should be self-explanatory. However, it is not so in the current presentation. Mark the results of the statistical analysis in the figures.
Response: The results of the statistical analysis were marked as an asterisk in figure 1 and figure 5.
- Revise the discussion thoroughly by critically discussing the reasons for the observed results. Remove the general statements and the repetition of the results from the discussion. Some key factors were not measured in the present study. For instance, the factors that contribute to the fungistatic effect (e.g., soil microbial populations) of the soil were not assessed in the study. This renders the discussion on fungistatis highly speculative.
Response:The discussion was re-organized to delete the duplicated content. The spore characteristics were compared with those of Clonostachys rosea and Monilinia laxa reported (see Discussion section, line 287-289).
Microbial population is an important that influence factor on the development of Trichoderma in soil. In this study, we first studied that sterilization of soil on the germination of two type of spores of Tr58, and the result showed that reducing soil microbial populations by sterilization was beneficial in relieving soil fungistasis (line 158-160; 255-257).
Based on the results of Tr58 contents detected in this study and the results of our field control experiments results (line 275-278, reference 21 and 22), It can be speculated that Trichoderma chlamydospore preparation has a stronger potential of disease control than conidia preparation.
- The reference list should be checked thoroughly as scientific names are not italicized and journal names are abbreviated for some and not for others.
Response: The references have been checked thoroughly and modified as required by the journal
- The presentation has to be improved. I suggest the authors seek the help of a colleague with good proficiency in English while revising the manuscript.
Response: The presentation has been improved with the help of a colleague with good proficiency in English. And we looking forward to seeking help of Language Editing of MDPI if necessary.
Round 2
Reviewer 2 Report
In the revised manuscript authors have considered the suggestions raised in my earlier review. One of my earlier comments was on the statistical treatment of the data.
My comment: Mention the type of statistical method used to assess the significance of the data in the methods. Moreover, use multiple comparison analyses like the ANOVA to assess the influence of the main factors and their interactions on the studied parameters.
The author's response was: Microsoft Excel 2007 and DPS7.05 were used for statistical analysis. Pearson correlation analysis was added to assess the correlations between soil moisture levels and germination rates of propagules. The Pearson correlation coefficient was calculated. The results further confirmed that soil moisture content significantly affected the germination and growth of two types of Trichoderma propagules, and the survival ability of chlamydospore in soil was significantly higher than that of conidia. (line131-132,141-143, and figure 1).
- My response: I still fail to find the type of statistical analysis used to assess the significance of variations among groups (statistical section of the methods). I suggest the use of multiple comparison analyses like the ANOVA involving factors like the propagule types and soil moisture content. It is not clear how to read the significant levels marked in Figure 1. Is it between propagule types? What about the significance of variation among soil moisture levels? Moreover, I don't see any statistical treatment of the survival rate of Trichoderma propagules with time. This experiment involves three factors propagule type, soil moisture levels, and time points. Therefore analyze and present the results of significance for differences among the propagule types, soil moisture, and time points and their interactions.
- Revise the results based on the results of the statistical analysis.
- Differentiate the sub-figures in figures 1 and 5.
Author Response
Response to Reviewer 2 Comments
Point 1: I still fail to find the type of statistical analysis used to assess the significance of variations among groups (statistical section of the methods). I suggest the use of multiple comparison analyses like the ANOVA involving factors like the propagule types and soil moisture content. It is not clear how to read the significant levels marked in Figure 1. Is it between propagule types? What about the significance of variation among soil moisture levels? Moreover, I don't see any statistical treatment of the survival rate of Trichoderma propagules with time. This experiment involves three factors propagule type, soil moisture levels, and time points. Therefore analyze and present the results of significance for differences among the propagule types, soil moisture, and time points and their interactions.
Response 1:
- Data in figure1 was analyzed with two-way ANOVA and was redrawn. Two factors are sterilization and moisture level of soils.
- In this study, we detected the content of Tr8 in soil within 12 consecutive months. The detection time was closely related to environmental temperature. As is known to all, temperature has an important impact on germination and growth of fungi spores. Considering that we focused on the influence of soil moisture on the survival of Tr58 in this paper, the time point was not taken as a factor in statistical analysis. However, we conducted a two-way ANOVA to analyze the spore types and soil moisture levels on the Tr58 content in soils at three timepoints of 2, 7 and 12 months. The related content and a new Figure 5 were added in 3.4.
- The data in Figure 6 (original Figure 5) was reanalyzed with one-way ANOVA and was redrawn.
Point 2: Revise the results based on the results of the statistical analysis.
Response 2: we have revised the results based on the results of the statistical analysis in 3.1 and 3.4.
Point 3: Differentiate the sub-figures in figures 1 and 5.
Response 3: Figures 1 and 6 (the original Figure 5) are redrawn and the difference significances are marked with lower case letters.
Author Response File: Author Response.pdf
