Trick of the Trade: Unveiling the Importance of Feedstock Chemistry in Trichoderma-Organic Amendments-Based Bio-Stimulants
Round 1
Reviewer 1 Report
This ms. assessed the effect of the effect of Trichoderma harzianum in combination with biochar or organic feedstocks on growth of four plants. I have the following concerns: 1. Five feedstocks were adopted. However, the information about the four except AC is very poor. For example, pelleted fish meal, how or where did you got it. 2. information about T. harzianum strain T22 is needed. 3. More assay about the biostimulatory effect is needed. I suggested to add bioassay of pot experiments. Now, only in filter paper disks. 4. the title and conclusions should be more close to present results.
Author Response
This ms. assessed the effect of the effect of Trichoderma harzianum in combination with biochar or organic feedstocks on growth of four plants. I have the following concerns: 1. Five feedstocks were adopted. However, the information about the four except AC is very poor. For example, pelleted fish meal, how or where did you got it. 2. information about T. harzianum strain T22 is needed. 3. More assay about the biostimulatory effect is needed. I suggested to add bioassay of pot experiments. Now, only in filter paper disks. 4. the title and conclusions should be more close to present results.
ANSWER: We would like to thank the reviewer for his valuable comments and for his precise concerns. In our study, we deliberately chose to conduct experiments using plant growth on filter paper disks to focus on isolating the pure effect of mixing organic amendments with Trichoderma on the plant. The decision to use filter paper disks was made to minimize potential confounding factors that could arise from using soil pots. By using filter paper disks, we aimed to create a controlled environment where we could observe the direct impact of the organic amendments and Trichoderma on the plant's growth without the influence of soil chemistry or the resident microbial community. Soil pots could introduce additional variables, making it challenging to ascertain whether observed effects were solely due to the amendments and Trichoderma or were influenced by other soil-related factors. However, a further step of evaluation will be carried out with pot experiment but after a first screening based on filter paper bioassay. This concept is reported now in the Conclusions as follows: “Furthermore, future studies will be needed to evaluate the biostimulant efficacy through pot tests and subsequent field trials”.
Regarding the detailed description on the four used organic amendments except AC, we agree with the reviewer, and for this reason, the manuscript was adjusted as follows: “Five feedstocks with different chemical properties were selected: (1) pelleted fish meal, hereafter referred to as fish. This is a nitrogen rich feedstock (N=6.09%) produced from fish by-products and largely used to feed farm animals; (2) Medicago sativa straw (referred to as Medicago); (3) Zea mays straw (referred to as Maize); (4) biochar prepared from sawdust of the deciduous tree Fagus sylvatica and pyrolyzed at 550°C (referred to as Biochar); (5) acti-vated carbon (obtained from Sigma-Aldrich Co., referred to as AC )”. About Trichoderma, the text was integrated as follows: “The feedstocks used alone (i.e., biochar, AC, fish, Medicago, and Maize) and the six mix-tures (AC + fish, AC + Medicago, AC + Maize, biochar + fish, biochar + Medicago, biochar + Maize) were then used without and with inoculum of a commercial T. harzianum strain T22 obtained from the SAMAGRI Company (https://www.samagri.it/)”. Finally, the title was changed in: “Trick of the trade: Unveiling the importance of feedstock chemistry in Trichoderma-organic amendments based bio-stimulants”.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Rev.Feedstock Trichoderma Biochar
Review of Manuscript ID: horticulturae-2528549
Title: Trick of the trade: Unveiling the importance of feedstock chemistry in Trichoderma-Biochar based bio-stimulants.
By: Giuseppina Iacomino, Giuliano Bonanomi, Riccardo Motti1, Mohamed Idbella
Generally, Soil organic amendments (various plant residues, Biochar) which provide crop plants with important nutrients are well known to induce of soil microbial populations activity (among them various antagonistic microorganisms) which are involved in plant protection against plant pathogens (microbial control). Trichoderma. is known to be one of the antagonistic fungi. In order to find out the positive and negative factors of the various amendments, Trichoderma, the Authors investigated separate and combinations of each amendment also with Trichoderma.
The amendments were mixed with water and shake incubated for 2 or 100 days.
The incubated amendments mixtures were separated to the solid part and liquid.
Biochar and other organic feedstocks were characterized by 13C-CPMAS NMR spectroscopy, to characterize the chemical properties of the organic matter, To analyze the relationship between the effect of T. harzianum and the chemistry of the organic feedstocks, a comprehensive correlation analysis was performed between TII and the organic carbon, total nitrogen, C/N ratio, pH, EC, and 13C CPMAS NMR fractions of the studied material.
The liquid parts of the various treatments were used to evaluate the stimulatory or inhibitory effects compared to water control on root growth of several crop plants.
The authors present very detailed, accurate, and statistically analyzed data of many detailed combinations (summarized in several tables and Figs). A very interesting gradual change of effects of the various treatment combination from a high increase of root growth (showing stimulatory effects) to a high inhibitory effect (showing inhibitory effects). But the data are not sufficient to draw reasoning conclusions.
Comments
However, the manuscript does not include an important part of discussion, mainly as the authors indicate that additional research and information are required to understand what the important data differences of treatments of the present research mean. What are the main factors causing the differences among the treatments are involved?
An additional question regarding the relevance of the incubation conditions of the organic matters (with or without) Trichoderma. The main problem is that the conditions of the water suspension of the amendments in the flasks are considerably different from that in the natural environment in soil. The microbial composition in any soil is considerably more complicated than that in the amendment suspension in the flask. It is expected that soil microbial composition in natural soil environment would undoubtedly degrade the organic amendments in a different way and may decompose the stimulatory and or the inhibitory factors produced in the flask environments. On the other hand, may lead to more favorable conditions for the crop plants. So, the actual correct data from the flask conditions may not represent the natural condition of crop plant growth conditions. The authors may compare similar experiments in natural soil conditions.
For this reason, the data of the present manuscript may be a part of a wider research.
[Why only Trichoderma? Bernard Glick, initiated important research around 1980 (and continues to publish innovative results in collaboration with many researchers until today) on PGPR (Plant Growth Promoting Rhizobacteria). Single isolates or consortia of isolates have been developed for plant growth promotion and plant protection against pathogens as well as extended to other fields. (many studies on the mode of action of these isolates and used by growers in the fields and also in hydroponics). It may be suggested for the authors to try working also with PGPR]
Therefore, I cannot recommend to publish the manuscript in its present form.
Comments for author File: Comments.docx
Minor editing of English language required
Author Response
Generally, Soil organic amendments (various plant residues, Biochar) which provide crop plants with important nutrients are well known to induce of soil microbial populations activity (among them various antagonistic microorganisms) which are involved in plant protection against plant pathogens (microbial control). Trichoderma. is known to be one of the antagonistic fungi. In order to find out the positive and negative factors of the various amendments, Trichoderma, the Authors investigated separate and combinations of each amendment also with Trichoderma.
The amendments were mixed with water and shake incubated for 2 or 100 days.
The incubated amendments mixtures were separated to the solid part and liquid.
Biochar and other organic feedstocks were characterized by 13C-CPMAS NMR spectroscopy, to characterize the chemical properties of the organic matter, To analyze the relationship between the effect of T. harzianum and the chemistry of the organic feedstocks, a comprehensive correlation analysis was performed between TII and the organic carbon, total nitrogen, C/N ratio, pH, EC, and 13C CPMAS NMR fractions of the studied material.
The liquid parts of the various treatments were used to evaluate the stimulatory or inhibitory effects compared to water control on root growth of several crop plants.
The authors present very detailed, accurate, and statistically analyzed data of many detailed combinations (summarized in several tables and Figs). A very interesting gradual change of effects of the various treatment combination from a high increase of root growth (showing stimulatory effects) to a high inhibitory effect (showing inhibitory effects). But the data are not sufficient to draw reasoning conclusions.
Response: We would like to extend our sincere gratitude to the reviewer for taking the time to review our research and provide valuable feedback on the validity of our data analysis approach. Moreover, we greatly appreciate your concerns, and we would like to assure you that we have treated them with the utmost seriousness and robustness.
However, the manuscript does not include an important part of discussion, mainly as the authors indicate that additional research and information are required to understand what the important data differences of treatments of the present research mean. What are the main factors causing the differences among the treatments are involved?
Response: We fully concur with the reviewer's insightful comment, emphasizing the necessity for additional clarification in the discussion section to elucidate the underlying factors contributing to the observed differences among treatments. To address this crucial point, we have incorporated the following sentence into the discussion section: “This indicates that the level of decomposition of organic amendments might be a key factor in determining their compatibility with Trichoderma inoculation.” Lines 344-346
And: “Indeed, to fully understand the factors causing differences among the treatments and to optimize the agricultural use of Trichoderma, further research is essential. Future studies should explore the population density of T. harzianum in different organic matrices during the decomposition process to identify the most suitable combinations of organic substrates. Additionally, investigating the microbial composition of the nutrient-rich coating formed during biochar co-composting could shed light on the mechanisms behind the growth-promoting effect of co-composted biochar.” Lines 376-382.
And: “Overall, our study highlights the complexity of the interaction between Trichoderma, organic amendments, and plants. The varying responses observed in different combinations underscore the need for careful consideration when applying Trichoderma in agricultural practices. By gaining a deeper understanding of the factors influencing these interactions, we can develop more effective and reliable strategies for utilizing Trichoderma as a bio-stimulant to enhance crop growth and agricultural productivity.” Lines 387-393.
An additional question regarding the relevance of the incubation conditions of the organic matters (with or without) Trichoderma. The main problem is that the conditions of the water suspension of the amendments in the flasks are considerably different from that in the natural environment in soil. The microbial composition in any soil is considerably more complicated than that in the amendment suspension in the flask. It is expected that soil microbial composition in natural soil environment would undoubtedly degrade the organic amendments in a different way and may decompose the stimulatory and or the inhibitory factors produced in the flask environments. On the other hand, may lead to more favorable conditions for the crop plants. So, the actual correct data from the flask conditions may not represent the natural condition of crop plant growth conditions. The authors may compare similar experiments in natural soil conditions.
For this reason, the data of the present manuscript may be a part of a wider research.
Response: We appreciate the valuable insights provided by the reviewer regarding the relevance of incubation conditions for organic matters in the presence or absence of Trichoderma. We acknowledge that the natural soil environment is significantly more complex and dynamic than the controlled flask conditions, encompassing a diverse microbial community that could influence the degradation of organic amendments and affect crop plant growth differently. By conducting experiments in controlled flask conditions, we intended to isolate and evaluate the direct impact of Trichoderma and its interactions with organic amendments on crop plant growth. The controlled environment allowed us to examine the pure effect of Trichoderma without the potential confounding factors present in the natural soil, thus providing valuable insights into its potential as a growth-promoting agent in agricultural practices. Nevertheless, we understand the reviewer's suggestion for future studies to include experiments conducted under natural soil conditions. These additional investigations would complement our findings and provide a more comprehensive understanding of how Trichoderma interacts with organic amendments and influences plant growth in more realistic agricultural settings.
[Why only Trichoderma? Bernard Glick, initiated important research around 1980 (and continues to publish innovative results in collaboration with many researchers until today) on PGPR (Plant Growth Promoting Rhizobacteria). Single isolates or consortia of isolates have been developed for plant growth promotion and plant protection against pathogens as well as extended to other fields. (many studies on the mode of action of these isolates and used by growers in the fields and also in hydroponics). It may be suggested for the authors to try working also with PGPR]
Response: We acknowledge the significant contributions of Bernard Glick and the extensive body of research on PGPR, demonstrating their potential for plant growth promotion and plant protection against pathogens. PGPR have indeed shown promising results and have been widely utilized by growers in various agricultural settings, including both field and hydroponic systems. While our current study focused on investigating the effects of Trichoderma in combination with organic amendments on plant growth, we recognize the potential benefits of incorporating PGPR in our future research endeavours. The utilization of PGPR alongside Trichoderma could provide a complementary approach to enhance plant growth, strengthen plant defences against pathogens, and further improve overall crop productivity. Additionally, studying the interactions between PGPR and Trichoderma in the context of our research objectives could yield valuable insights into potential synergistic effects and lead to innovative and sustainable agricultural practices. Therefore, based on the reviewer's valuable suggestion, we will consider conducting further research involving PGPR in our future studies. Exploring the mode of action and effectiveness of PGPR, either as single isolates or consortia, in combination with Trichoderma and organic amendments would undoubtedly contribute to a more comprehensive understanding of plant-microbe interactions and foster advancements in modern agricultural practices.
Therefore, I cannot recommend to publish the manuscript in its present form.
Response: We sincerely appreciate the time and effort the reviewer invested in evaluating our manuscript. We understand the reviewer's concerns and acknowledge the importance of their feedback in enhancing the quality of our work.
Author Response File: Author Response.docx
Reviewer 3 Report
Abbreviations must define at the first appearance: e.g.: AC in Abstract, or IBI in Introduction part.
In the “Introduction” part:
At rows 76-78.: I would omit the sentence which starts with “In general…”, because a lot of publication demonstrate the opposite of this statement.
At row 92.: lentil instead of lettuce
In the “Materials and methods” part:
From this part the characteristics of AC must move into Introduction part (rows 101-103), it belongs to that part, as characteristics of other feedstock.
The number of replications in case of different measurements must mention in this part!
It would be more perspicuous to demonstrate or summarise the different treatments in a table or in a figure.
Must clear precisely what kind of plant genera were used in the tests, especially in case of lentil/lettuce.
How was the bacterial/fungal contamination prevented throughout the experiment?
Why was the persistence of Tichoderma not tested in the experiment?
In the “Result” part:
Designation and order of tables and figures in the text is not consequent: the former mentioned tables are after the later mentioned figures, which are denoted as tables too in the text.
In Table 1. AC+EM means AC+Medicago, as in case of Biochar+Medicago, please change one of them, to be uniform.
In Figure 1. notification I would omit the definition of TII, which was described before in the text.
In the title of 1st figure the TII values were described in the presence and in the absence of Trichoderma. The latter is unreal, because in this case the effect of Trichoderma was represented.
3.1.
Were the effects of Trichoderma treatments on C-types investigated? It wold be informative to read, especially after the 100th day of the experiment.
Author Response
Abbreviations must define at the first appearance: e.g.: AC in Abstract, or IBI in Introduction part.
ANSWER: Done, we added abbreviation definition “Activated carbon” to abstract, line 18, and abbreviation definition “International Biochar Initiative” to introduction, line 55.
In the “Introduction” part:
At rows 76-78.: I would omit the sentence which starts with “In general…”, because a lot of publication demonstrate the opposite of this statement.
Done. Ok, the sentence was deleted.
At row 92.: lentil instead of lettuce
Done.
In the “Materials and methods” part:
From this part the characteristics of AC must move into Introduction part (rows 101-103), it belongs to that part, as characteristics of other feedstock.
ANSWER: After careful consideration, we have made the decision to remove this sentence from the manuscript. The reason for its removal is twofold: firstly, it appears repetitively in the introduction, and secondly, it is not relevant to the Material and Methods section. We believe that eliminating this sentence will improve the overall clarity and focus of the manuscript.
The number of replications in case of different measurements must mention in this part!
ANSWER: Ok, we have mentioned the number of replicates for our root growth experiment to be 5 replicates per each treatment. The manuscript was as follows: “Five replicates were used for each treatment, consisting of ten seeds in a Petri dish for each plant species. A total of 885 experimental units containing 8850 seeds were pre-pared (11 NPOA types and mixtures × 2 incubation times × 2 T. harzianum inoculum × 4 target species × 5 replicates plus the sterile water control)”. Lines 152-156.
For the chemical characterization, the following sentence was added to the manuscript: “For each chemical characterization, three replicates were conducted”. Lines 142-143.
It would be more perspicuous to demonstrate or summarise the different treatments in a table or in a figure.
ANSWER: Practically, the complete list of the treatments are reported in Table 1 and 2 as well as in figure 1. Therefore, reporting another Table/Figure with only the list of treatments would be redundant and cause an unnecessary extending of the text.
Must clear precisely what kind of plant genera were used in the tests, especially in case of lentil/lettuce.
ANSWER: We sincerely apologize for the confusion between lentils and lettuce. After careful review, we have rectified the error, and the correct term should be "lentils." We have made the necessary adjustments to the text to ensure accuracy and clarity. Thank you for bringing this to our attention.
How was the bacterial/fungal contamination prevented throughout the experiment?
ANSWER: Contamination by bacteria or fungi was diligently prevented throughout the experiment by strictly adhering to aseptic conditions and working under sterile conditions. As stated in the text, we utilized sterile filter paper and sterile distilled water for irrigation purposes. Furthermore, to minimize the risk of any additional contamination, the petri dishes were securely sealed with parafilm. These precautions were taken to maintain the integrity and reliability of our experimental results, ensuring that any observed effects were attributed solely to the variables under investigation.
Why was the persistence of Tichoderma not tested in the experiment?
ANSWER: We would like to clarify to the reviewer that our experiment focused on utilizing petri dishes under sterile conditions. This approach was deliberately chosen to isolate the study of Trichoderma without any external influences that could affect its persistence. By using petri dishes, we aimed to ensure a controlled environment where Trichoderma could thrive without interference.
However, we acknowledge the valid point made by the reviewer. If we were to conduct experiments in soil pot conditions, Trichoderma would inevitably encounter competition from other soil microbial communities for essential nutrients and space. In such cases, it would be crucial to assess Trichoderma's persistence over time under these more complex and challenging conditions. This point is currently highlighted in the Conclusions as follows: “The study of the population dynamics of Trichoderma interacting with the native mi-cro-biome will be a fundamental aspect for the development of functionalized organic amendments with significant bio-stimulation of plants”.
In the “Result” part:
Designation and order of tables and figures in the text is not consequent: the former mentioned tables are after the later mentioned figures, which are denoted as tables too in the text.
DONE. The figure and table order is now correct.
In Table 1. AC+EM means AC+Medicago, as in case of Biochar+Medicago, please change one of them, to be uniform.
DONE.
In Figure 1. notification I would omit the definition of TII, which was described before in the text.
ANSWER: We would like to clarify to the reviewer that the text in question is not intended as a definition but rather serves as an axis title essential for providing context and justification to the figure.
In the title of 1st figure the TII values were described in the presence and in the absence of Trichoderma. The latter is unreal, because in this case the effect of Trichoderma was represented.
ANSWER: We would like to apprise the reviewer that within the text, we have extensively detailed the methodology employed for calculating the TII, encompassing root growth both with and without Trichoderma incolum. therefore, we would consider it “real”. The caption of the figure clarify this point as follows: “…after 2 and 100 days of incubation in absence and presence of Trichoderma harzianum T22 inoculum. Plant responses is expressed with the Trichoderma Interaction Index: positive values indicating stimulation and negative value inhibition compared to the same organic feedstock but without the inoculum with T. harzianum”.
Were the effects of Trichoderma treatments on C-types investigated? It wold be informative to read, especially after the 100th day of the experiment.
ANSWER: We believe that it was the purpose of our experiment as well, checking in detail the results provided by the correlation heatmap (Figure 2) were organic feedstock chemistry is associated with the response to Trichoderma inoculum. Basically, we found a positive correlation between the TII (Trichoderma Interaction Index) of maize and the di-O-alkyl C type, between the TII of soybean and the aromatic C type, and between the carbonyl C types and the TII of lentils. Negative correlations were found between the TII of soybean and the alkyl C and methoxyl C types.
Author Response File: Author Response.docx
Reviewer 4 Report
The study has provided a very comprehensive insight into Trichoderma and organic feedstock based biostimulants and their efficacy in various mixtures, with the aim of further utilizing them for promoting the growth of various industrial crops. This study is well written, easy to read and the topic is very important in the context of green agriculture. The purpose of the study is clearly presented, well designed and described in detail. The discussion section should avoid repeating results, please discuss them in the context of the current literature.
Line 2: The title should be changed because it was not only the Trichoderma-biochar mixture that was tested as a biostimulant
Line 18: What does the abbreviation AC stands for?
Line 151: how seeds were prepared for the experiment? Were they sterilized?
Author Response
The study has provided a very comprehensive insight into Trichoderma and organic feedstock based biostimulants and their efficacy in various mixtures, with the aim of further utilizing them for promoting the growth of various industrial crops. This study is well written, easy to read and the topic is very important in the context of green agriculture. The purpose of the study is clearly presented, well designed and described in detail. The discussion section should avoid repeating results, please discuss them in the context of the current literature.
ANSWER: We would like to thank the reviewer for his comments. In order to improve our manuscript, we have tried to answer the following reviewer's concerns as below.
Line 2: The title should be changed because it was not only the Trichoderma-biochar mixture that was tested as a biostimulant
DONE. The title was changed as follows: “Trick of the trade: Unveiling the importance of feedstock chemistry in Trichoderma-organic amendments-based bio-stimulants”.
Line 18: What does the abbreviation AC stands for?
ANSWER: AC stands for “Activated carbon”, as indicated in Materials and Methods, line 100. Also, we have specified it in the abstract, line 18.
Line 151: how seeds were prepared for the experiment? Were they sterilized?
ANSWER: Sorry for forgetting this detail, the seeds surface has been sterilized. The text was accordingly amended as follows: “Five replicates were used for each treatment consisting of ten seeds, previously sterilized for 5 minutes in a 2% sodium hypochlorite solution, in a Petri dish for each plant species”.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
I suggest to accept this revised ms.
Author Response
We would like to express our heartfelt gratitude for your thoughtful review and acceptance of our manuscript." Your valuable insights and constructive feedback have significantly contributed to enhancing the quality aof our work. We truly appreciate your time, expertise, and dedication in evaluating our research.
Best regards
The corrsponding Ahuthor
Reviewer 2 Report
Despite the author's response I think that the data represented in the present reseach will be worthwhile when the additional research that I have indicated in my previous review testing in natural soil environment to show the differences will be sent to publication as a more balanced research.
Author Response
We deeply appreciate your meticulous review of our manuscript and your continued engagement in advancing the quality of our research. Your insights have been instrumental in refining our work and enhancing its scientific rigor. Your emphasis on the need to extend our investigations to natural soil conditions is well-taken. We concur with your viewpoint that the complex microbial composition and dynamic processes inherent to soil environments may yield nuanced outcomes different from those observed in controlled flask experiments. We are fully aligned with your recommendation to conduct experiments that mirror real-world agricultural settings, which will undoubtedly enrich the comprehensiveness and practical applicability of our findings. Recognizing the limitations of a single article to encompass the entirety of research pursuits, we want to assure you that our ongoing research initiatives are devoted to addressing the gaps you've identified. We are actively planning and executing studies that involve natural soil conditions, aiming to elucidate the intricate interplay between Trichoderma, organic amendments, and plant growth under more realistic scenarios. These forthcoming investigations will serve as essential components to complement and validate the controlled flask experiments detailed in our current manuscript. Your suggestion to explore the potential synergy between Plant Growth Promoting Rhizobacteria (PGPR) and Trichoderma is both astute and timely. We acknowledge the merits of such an approach, and we are eager to incorporate PGPR into our research agenda. By investigating the combined effects of these bio-stimulants within the context of organic amendments, we anticipate uncovering novel insights into fostering sustainable and effective agricultural practices. Your commitment to fostering balanced and impactful research is greatly appreciated, and your comments have ignited a robust drive within us to advance our studies in line with your recommendations. We extend our gratitude for your dedicated effort in reviewing our manuscript and for spurring us to undertake research that aligns with the highest scientific standards.
