Exploring Microbial Diversity in Forest Litter-Based Fermented Bioproducts and Their Effects on Tomato (Solanum lycopersicum L.) Growth in Senegal

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LINE(S)

CONCERNS/ISSUES

Abstract

General

The abstract is a standalone section and should explicitly state the problem the authors are addressing. However, this abstract falls short of meeting this important expectation.

Line 20

Obey the rules of structuring sentences by avoiding to begin with acronyms.

Lines 25-28 Results showed that regardless of the litter 25 geographical collection site, the dominant bacterial taxa in the BMs belonged to the phyla 26 Firmicutes (27.75–97.06%) and Proteobacteria (2.93–72.24%), predominantly represented by 27 Bacilli (14.41–89.82%), α-proteobacteria (2.83–72.09%), and Clostridia (0.024–13.34%)

The sentence is too long. Consider breaking it up to improve readability and eliminate ambiguity.

Lines 31-32:Variations in taxa abundance were influenced by the litter’s origin and the carbon ingredients used in BM preparation

The authors should specify how variations in litter origin and carbon components influenced taxa abundance, avoiding generalization statements.

Introduction

Lines 47-48:In Senegal, family farming supplies local and national markets with fresh, perishable agricultural products

Follow the basic rules of sentence structure.

Lines 48-55: However, the high added value of these crops, combined with their high sensitivity to pests leads producers to use unsustainable farming methods. These methods heavily rely on imported chemical inputs (fertilizers, pesticides), exposing the environment and consumers to multiple pollutants

This seems to resemble empirical assertions that have been articulated after an investigation. Did the authors themselves conduct the investigation? If it was not, please provide the appropriate citations.

Lines 55-57: However, some, including the use of mineral fertilizers and agrochemical products, have high costs and harmful effects on the environment.

The word however has been overused in this paragraph

Lines 61-64: Effective Microorganisms (EM) technology, first described by Teruo Higa in  is a robust and versatile approach already tested in Latin America and Southeast Asia (https://www.emrojapan.com/what/), but relatively unknown in sub-Saharan Africa

This sentence is quite lengthy. Secondly, is this technology unfamiliar in sub-Saharan Africa, or is it simply not being used effectively?

Line 73: ….found that EM at concentrations of 1% and 2% improved seed germination and seedling vigor in Albizia saman and Acacia auriculiformis

The plant Albizia saman should be italicized according to the rules of botanical nomenclature.

Lines 83-84: Tomato is a second horticultural crop in Senegal and play an important socio-economic role in the Senegalese economy.

tomato ranks second after which horticultural crop? There's no need to mention the second if the authors don't specify the first.

Iines 86-87: The production has remained relatively stable over the past decade, 86 reaching 151,000 metric tons in 2022, with an average yield of 1.8 metric tons per hectare

the authors should provide the current production figures based on the most recent FAOSTAT data.

Lines: 89-91: Therefore, for this study, we produced forest-fermented litter products which we de-89 scribed as Local Beneficial Microorganisms (BMs) by fermenting in anaerobic conditions 90 the local litter collected in different agroecological regions of Senegal.

Remove personalization in this sentence

Lines 94-95: Then, the study aims first to characterize the microbial community biodiversity of 94 the BMs……

Delete the word ‘then’ from the sentence and secondly, what was the aim of this study? To characterize or to identify the BMs?

Materials and Methods

General

This section should be written in a reported manner

Lines 100-103: Local Fermented Forest Litters that we will call Local Beneficial Microorganisms 100 (BMs) used in this study are mixed cultures of different components with forest litters 101 collected in four different agroecological regions of Senegal following a climatic gradient 102 (Error! Reference source not found.)

The text should be redrafted in a reported manner, with precise documentation of how sampling was conducted along the climatic gradient, including the number of samples collected and whether the samples were variable or equal.

Lines 107- Figure 1

Figure 1 is not clear and not cited. The legend needs revision to fully describe the figure in a legible font type and size. Additionally, the sampling locations are shown but lack sufficient contrast.

Lines 122-124: Thus, a second fermentation phase is undertaken to create an activated liquid product by mixing 2.1 kg of the fermented product with 42 L of water, 1.05 kg of yogurt, and 123 2.1 kg of sugarcane molasses. The containers are then hermetically sealed and left to ferment anaerobically at room temperature for 7 to 14 days until the pH drops to 4 or lower. Once ready, the liquid BMs product is coarsely filtered through a sieve to remove plant 126 debris and stored at room temperature. Thus, a total of 15 local liquid-fermented products, prepared with litters of different origins and various carbon sources, were obtained .

Write in a reported manner. Additionally, I am concerned about the liquid fermented products. Were they analyzed to determine their nutrient levels before use? How did you ensure that the carbon sources were free of other nutrients that could impact your study, and how did you address this issue?

Lines 142: ……16S rRNA gene with the 515F/806R primers set and ITS gene with the ITS3F-142 ITS4R primers…….

Why did the authors sequence the 16S rRNA gene for bacterial identification and the ITS gene for fungal identification, considering that the two methods cannot resolve up to the species level?

Lines 147-148: We then produced the final OTU tables containing the number 147 of sequences per sample per OTU matching the designated taxonomic classification.

The authors need to remove personalization from the sentence.

Secondly, the sequence data should be deposited in public repositories for reference

Lines 152-153: The variety AMIRAL hybrid was used in the greenhouse test

The authors should state or indicate the conditions of the greenhouse experiment

Lines 160-162: The chlorophyll content of leaves was measured with a chlorophyll meter (SPAD-502 Plus), while plant height (cm) was measured from the ground to the tip of the stem with a tape measure.

The Authors need to state when Chlorophyll content was measured.

Lines 158-159: Each treatment was repeated 12 times including the un treated control which received 10 ml of water.

This statement is imprecise. Does it mean that each treatment was replicated 12 times, or does it indicate that each treatment was inoculated 12 times with BM?

Results

Figure 2b page 6

Figure 2b has not been cited in the text.

Figure 2

The legend does not explain how the relative abundance was normalized.

Lines 186-190: A diversity of fungi was observed more particularly in the composition of BMs made with ingredients from St-Louis which, in addition to the higher dominance of the genus 187 Acidea (77% to 82% in GL-G, SL-GM, and SL-R), contain the genera Gaeumannomyces (100% 188 for SL-M, 3% to 13% in SL-GM, SL-G and SL-R) and Knoxdavieasia (1 and 3% for SL-GM and SL-R)

The sentence is too long and lacks clarity.

In addition, the Bannoa genus is present in BMs from the Dakar region (DK-M, 190 DK-R, and DK-GM respectively 0.9%, 0.6%, and 4.2%) and BMs from the Saint Louis re-191 gion (SLR, SL-G, and SL-GM respectively 0.5%, 11.8%, and 6.2%). The commercial BM, BJ-192 CCS, included in our study was also mostly dominated by Firmicutes (97.06%) and Prote-193 obacteria (2.93 %) with 2 most dominant classes Bacilli (97.04%) and Alphaproteobacteria 194 (2.83%) while the fungi class was dominated by Leotiomycetes (99.99%). At the genus level, 195 BJ-CCS composition was dominated by Lactobacillus (96,9197%), Acetobacter (2,8377%), and 196 the fungus genus Acidea (99.99%).

The sentences is too long and lacks clarity.

Figure 3 line 203

The legend should be enhanced to indicate how the normalization of relative abundance was done

Line 217-218: ……..Principal Coordinates Analysis (PCoA), indicated that the samples 217 clustered according to their litter collection origin

the authors should provide the PCoA diagram so that readers can follow the manuscript, either within the text or as supplementary material.

Table 3 line 223

The table legend should be in a legible size and font. In addition the legend should wholly describe the contents

Figure 4: Growth of tomato seedlings treated with BMs products at 28 days after treatment. A: Untreated 238 control plants; F: Plants treated with NB-R; O: Plants treated with DK-G.

Improve the legend by making it more precise

Line 246-248: Interestingly, root volume increased by 59.21% for 246 DK-M, 84.32% for DK-G, 85.44% for DK-GM, and a whopping 97.35% for NB-R. Root sur-247 face area also saw significant increases:

Use a formal word instead of whopping

Figure 5 line 264: Effect of BMs on tomato plant shoot and root biomass: (a) fresh shoot biomass; (b) dry shoot biomass; (c); fresh root biomass; and (d) dry root biomass.

Enhance the legend by describing the analysis precisely

Discussion

Our results showed some variations in the structure of the BMs' microbial communities due to the litter's origin

How did your study vary with those of the other workers? State clearly and why:

General

The implications of this study have not been adequately addressed in this discussion

Conclusion

Lines 349-362

The conclusion merely summarizes the key findings but lacks a focus on future directions.

Response to Reviewer 1 Comments

Abstract

Point-by-point response to Comments and Suggestions for Authors

Comments 1:  The abstract is a standalone section and should explicitly state the problem the authors are addressing. However, this abstract falls short of meeting this important expectation.

Response 1: Thank you for your insightful comment. We acknowledge the importance of explicitly state the problem we are addressing; we review the abstract by adding the problem.

Lines 19-24: Reducing the use of chemical inputs (fertilizers, pesticides) in agriculture while maintaining crop productivity is the main challenge facing sub-Saharan African family farming systems. The use of effective microorganisms (EM) is among the various innovative approaches for minimizing chemical inputs and the environmental impact of agricultural production and protecting soil health while enhancing crop yields and improving food security.

Comments 2:  Obey the rules of structuring sentences by avoiding to begin with acronyms.

Response 2: Thank you for your insightful comment.

Lines 26-27: Beneficial Microorganisms

Comments 3:  The sentence is too long. Consider breaking it up to improve readability and eliminate ambiguity

Response 3: Thank you for your suggestion. We have rephrased the sentence to:  

Line 32 Results showed that regardless of the litter geographical collection site, the dominant bacterial taxa in the BMs belonged to the phyla Firmicutes (27.75–97.06%) and Proteo-bacteria (2.93–72.24%). Within these groups, the most prevalent classes were Bacilli (14.41–89.82%), α-proteobacteria (2.83–72.09%), and Clostridia (0.024–13.34%). Key genera included Lactobacillus (13–65.83%), Acetobacter (8.91–72.09%), Sporolactobacillus (1.40–43.35%), and Clostridium (0.08–13.34%).

Comments 4:  The authors should specify how variations in litter origin and carbon components influenced taxa abundance, avoiding generalization statements.

Response 4: Thank you for your suggestion.

Lines 39-41 :“Although microbial diversity is relatively uniform across samples, the relative abundance of microbial taxa is influenced by the litter's origin. This is illustrated by the PCoA analysis, which clusters microbial communities based on their litter source.”

Introduction

Comment 5:  Follow the basic rules of sentence structure.

Response 5: Thank you for your suggestion. We have rephrased it to:

Lines 56-57: In Senegal, family farming plays a crucial role in supplying both local and national markets with fresh and perishable agricultural products.

Comment 6:  This seems to resemble empirical assertions that have been articulated after an investigation. Did the authors themselves conduct the investigation? If it was not, please provide the appropriate citations.

Response 6: Thank you for your valuable feedback. Citations are added in the text. Line 60

Comment 7:  The word however has been overused in this paragraph

Response 7: Thank you for your feedback on the abstract. We appreciate your remarks and replace it by “Nevertheless” line 64-65

Comment 8 :  This sentence is quite lengthy. Secondly, is this technology unfamiliar in sub-Saharan Africa, or is it simply not being used effectively?

Response 8: Thank you for your comment. The authors have revised the sentence. To respond to the question, the technology of EM is known but not for the largest farmers, so most are unfamiliar to it

Lines 69-75: “These bioproducts are formed by fungi and bacteria isolated from soil or litter and can coexist in a liquid fermentation medium. Effective Microorganisms (EM) technology, first described by Teruo Higa in [3,4], is a robust and versatile approach already tested in Latin America and Southeast Asia (https://www.emrojapan.com/what/). Although known by a few in sub-Saharan Africa, its use remains relatively limited. These bi-oproducts are based on the principle of simple fermentation by farmers using native microorganisms collected locally from forest litter areas.”

Comment 9:  The plant Albizia saman should be italicized according to the rules of botanical nomenclature

Response 9: Thank you for your comment;

Lines 83 “Albizia saman”

Comment 10: Tomato ranks second after which horticultural crop? There's no need to mention the second if the authors don't specify the first.

Response 10: Thank you for the comment. The authors specify the first horticultural crops in Senegal

Lines 92-94 :” Tomato is ranked as the second most important horticultural crop after onions in Sen-egal, playing a significant socio-economic role in the country's economy [19].”

Comment 11:  the authors should provide the current production figures based on the most recent FAOSTAT data.

Response 11: Lines 96-98: “The production has remained relatively stable over the past five years, reaching 152,950 metric tons in 2023, with an average yield of 1.7 metric tons per hectare [20].”

Comment 12:  Remove personalization in this sentence

Response 12: Thank you for pointing this out. Correction done on the manuscript lines 98-99

Comment 13:  Delete the word ‘then’ from the sentence and secondly, what was the aim of this study? To characterize or to identify the BMs?

Response 13: Thank you for your comment and question. “then” was removed from the sentence and sentence improved.

Lines 103-105:  “This study aims to perform microbial characterization of bioproduct BMs and to evaluate their ability to improve tomato production in Senegal”

Comment 14: The text should be redrafted in a reported manner, with precise documentation of how sampling was conducted along the climatic gradient, including the number of samples collected and whether

Response 14: Thank you for your comment. The text was updated Lines 107-109

Materials and Methods

Comments 15:  Most of the charts or graphs presented are difficult to interpret or do not fit the context of the data presented, making them difficult for readers to read and understand.

Response 15: Thank you for your feedback. However, we respectfully disagree with the comment regarding the interpretability and relevance of the charts and graphs. The visualizations used in our study are consistent with those found in similar research on this topic, and the format was chosen to effectively present complex data in a clear and concise manner. But we update the graphs title with more detail to facilitate understanding.