Effects of Pre-Emergence Application of Organic Acids on Seedling Establishment of Weeds and Crops in Controlled Environments

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript “Effect of pre-emergent organic herbicides on the germination of crop and weed competitor species under controlled environments" presents some original and timely findings into the pre-emergent herbicidal effects of organic acids viz., citric and lactic acid, on selected weed and crop species under controlled conditions. The study has potential relevance for sustainable weed management and organic farming systems, aligning with EU pesticide reduction goals. However, there are significant issues that need to be addressed before the manuscript is suitable for publication.

Major Comments for Revision:

1. The manuscript claims novelty (e.g., “for the first time”), but does not clearly articulate the central hypothesis or what gap in knowledge this study addresses. It is suggested kindly clearly articulate it in the introduction.
2. The manuscript uses inconsistent and sometimes confusing terminology (e.g., interchangeably calling both “crops” and “weeds”). It is suggested that you clearly define in the materials and methods which species are considered crops versus weeds and adhere to that terminology throughout.
3. The manuscript lacks clarity on key aspects of the experimental design.

How many pots/petri dishes per treatment per species?

How were environmental conditions controlled and monitored?

Was there any blocking/randomization?

I suggest that you please indicate a clear-cut schematic or table of the experimental layout. Also clarify if each concentration of each acid was replicated separately for each species.

4. Statistics: The calculation of IC₅₀ values is presented, but the method for IC₅₀ estimation is not described (e.g., which model/software used). Confidence intervals for IC₅₀ values are missing.
5. The potential pH-mediated effects of acid treatments on soil are discussed hypothetically in the Discussion, but not empirically measured. It is suggested that either include soil pH measurements after acid application or explicitly state as a limitation. Alternatively, suggest in the Conclusion that future work should monitor pH shifts and their impact on nutrient availability.
6. The study was conducted under controlled conditions with growth chambers and pot experiments, but the conclusions imply direct relevance to field conditions. Please avoid overgeneralization. Rephrase sentences like “citric acid is a cost-effective alternative” to emphasize that field trials are still needed.
7. Figures 1–6 lack clarity and professional formatting:

Axis labels are too small or missing units.

Legends are overly long and repetitive.

Please consolidate some figures to improve readability.

Use color-coded or symbol-coded bar charts for clearer comparison.

Consider showing dose-response curves (concentration vs inhibition %) for key species.

 

8. The Discussion reiterates many results and lacks a concise structure. Please restructure Discussion with subheadings like: “Efficacy on Weeds,” “Selectivity to Crops,” “Mechanisms and pH Hypothesis,” “Implications for Organic Farming.” Avoid repeating earlier results; focus on mechanistic interpretation and broader relevance.
9. There are numerous grammatical, syntactical, and typographical errors.

“Noteworthy, citric acid…” → “Notably, citric acid…”

“are warranting attention” → “warrant attention”

A thorough English language and style editing is essential, preferably by a native speaker or professional editing service.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Major Comments for Revision:

1. The manuscript claims novelty (e.g., “for the first time”), but does not clearly articulate the central hypothesis or what gap in knowledge this study addresses. It is suggested kindly clearly articulate it in the introduction.

This is true. It also needs to appear in the Abstract:

Below I have included the Abstract:

“Within the framework of organic acid alternatives to chemical herbicides, pre-emergence weed control research is scare. Citric acid (CA) and lactic acid (LA), considered significantly less effective than pelargonic acid (PA) and acetic acid (AA) from post-emergence (foliar spraying) studies, have largely been disregarded. This in vitro study was aimed at comparing the effects of 5-20% AA, AA + essential oils, PA, CA and LA, respectively, on radicle emergence inhibition (direct spraying of seeds) and shoot emergence inhibition (application to peat) on both weeds (perennial ryegrass, green foxtail, common vetch and chicory) and crops (soft wheat, alfalfa and millet). All tested compounds demonstrated concentration-dependent and species-specific inhibitory effects on shoot emergence inhibition, with CA and LA (IC₅₀ range: 3.4-19.3%) showing a comparable efficacy to PA and AA (IC₅₀ range: 3.1-35.9%). Results also showed that CA and to a lesser extent LA, were less inhibitory to wheat (CA IC₅₀=62.5%; LA IC₅₀=35.9%) and alfalfa (CA IC₅₀=57.8%; LA IC₅₀=44.1%) shoot emergence. CA and LA show potential promise for pre-emergence weed control in field-testing, either on a false seedbed in pre-crop sowing or concurrently with soft wheat and alfalfa sowing. Investigating organic compound herbicidal effects on crops of interest warrants attention.”

.”

 

The Introduction has been modified in order to better articulate the gaps in knowledge and the objectives. Please read this part of the Introduction below.

“…Of particular interest to the present investigation are organic acid-based organic com-pounds, including acetic acid (AA), pelargonic acid (PA), citric acid (CA) and lactic acid (LA). In the EU, only PA has been registered as a plant protection product with recognized herbicidal use in agricultural crops (Reg. (EC) No 1107/2009) [15]. Although AA (vinegar) is not officially registered in the EU as a herbicide, it is used for non-selective herbicidal activity and can be found in many advertised commercial “weed killer” formulations. CA and LA are not officially recognized or registered as herbicides under EU regulations.

Post-emergence herbicides are administered in the control of germinated weed plants to disrupt cell membranes of vegetative tissues, whereas pre-emergence herbicides are administered to target the early stages of weed growth, preventing the development of the embryonic radicle, the shoot, or both, thereby inhibiting seedling establishment [3,13]. Research on post-emergence foliar applications indicated that CA was less effective than AA and PA [16, 25-26], and as a consequence CA has received less consideration due to this lower efficacy. Furthermore, there is a lack of research specifically examining the pre-emergence herbicidal activity of CA and LA on weed seeds. Our interest in examining the potential pre-emergence herbicidal effectiveness is be-cause seed banks are reservoirs of both newly shed weed seeds and persistent older seeds that contribute to the persistence of invasive plant infestations [7]. Given the increasing interest in sustainable herbicide research within the context of integrated weed management strategies, the use of organic compounds could be administered to a false (stale) seedbed for pre-emergence control of seedling establishment before sowing of crop plants, as was reported for post-mergence control [3,17,21,24,27]. Alternatively, organic compounds could be administered to reduce weed seeding establishment concurrently with crop sowing. The latter strategy would necessitate examining the impact of these organic compounds on crop plant seeds, for which information on many crop species is still lacking.

The present study was, therefore, aimed at investigating the in vitro effects from the administration of different concentrations (5, 10 and 20%) of AA, AA + eugenol and geraniol (AA+EG), CA, LA and PA in inhibiting radicle and shoot emergence of four common weeds (perennial ryegrass, common vetch, green foxtail and common chicory) and three well-recognized crops (soft wheat, millet and alfalfa [lucerne]). The objective was to evaluate whether the organic acid compounds possess potential for future field trials, and more specifically, whether they could potentially be used in a false (stale) seedbed technique to firstly control weed shoot emergence prior to sowing the crop plants or whether there is the possibility of using them concurrently with crop sowing.

 

The manuscript uses inconsistent and sometimes confusing terminology (e.g., interchangeably calling both “crops” and “weeds”). It is suggested that you clearly define in the materials and methods which species are considered crops versus weeds and adhere to that terminology throughout.

The confusing terminology has been removed throughout the manuscript from the Introduction to the Discussion. The plant species are clearly distinguishable as 3 crop species and 4 weed species, respectively.

 

2. The manuscript lacks clarity on key aspects of the experimental design.

How many pots/petri dishes per treatment per species?

How were environmental conditions controlled and monitored?

Was there any blocking/randomization?

I suggest that you please indicate a clear-cut schematic or table of the experimental layout. Also clarify if each concentration of each acid was replicated separately for each species.

We have opted to describe the experimental design without a Table.

 In the Methods section, the following was written:

“2.4. Growth chamber parameters and experimental design

To investigate the pre-emergent herbicidal effects of the organic compounds on seedling establishment, two different experimental trials were adopted. The first experimental trial was conducted in Petri dishes to assess either the presence or absence of radicle emergence on agar after directly spraying the seeds with the organic com-pounds. In the second trial, the organic compounds were sprayed on moistened peat in pots and not directly on the seeds. The presence or absence of shoot emergence (seedling establishment) was then assessed. Both experimental trials were conducted in a growth chamber during the period from March to September 2023.

2.4.1 Growth chamber parameters

Experiments were performed in a growth chamber at the Department of Agricultural and Food Sciences (DISTAL-University of Bologna). Above the bench surface were three fluorescent tube lights (FH21830, 21 W, warm white 830, 85 cm, 116 w; OSRAM SpA, Milan, Italy), installed at a height of 60 cm from positioning of the Petri dishes and pots. From a spectrometric analysis (Miniature Fibre Optic, USB2000+UV-VIS, Ocean Optics, Milan, Italy), light quality was characterized by two major spectral profile peaks in green and red and a minor peak in blue. Mean irradiance or photon flux density (PPFD) was 71.65 µmol s-1 m-2 (portable luxmeter, Delta OHM, Padova, Italy). The lighting was set to a 16/8 h (day/night) photoperiod. The in-ternal temperature was maintained at 23°C, and the relative humidity was 70-75%.

2.3.2 Experimental design for the assessment of radicle emergence in Petri dishes

For the assessment of the presence or absence of a radicle, tests were performed in 90-mm diameter glass petri dishes with 15 ml of 1.5% (w/v) agar. The agar in the Petri dishes was sterilized for 30 min inside a VWR® PCR Workstation (containing 2 internal UV tubes [254 nm, 25 W each] and 1 UV tube in a UV Air Recirculator [254 nm, 8 W] offering decontamination action by UV inactivation of airborne and surface-bound contaminants (Avantor, Milan, Italy). Seeds of all species tested were rinsed in distilled water containing 5% sodium hypochlorite for 3-5 min, followed by distilled water, prior to being placed on the agar. Each Petri dish contained 20 seeds. Treatments were carefully applied to each Petri dish at an application rate of 1000 L/ha using a portable 2 L manual pressure sprayer (LeroyMerlin.it Ref 84438959). The untreated CTRLs were sprayed with water only. After 7 days, seeds with radicles were counted following treatment/water exposure.

The experimental trial was composed of 7 plant species (3 crops and 4 weeds), comprising 5 different organic compound (AA, AA + GE, CA, LA and PA) treatments at 4 different concentrations (0, 5, 10, 20%) with 3 replicates (for each species, treatment and concentration), representing a total of 420 Petri dishes. As such, the experiments were performed separately for each treatment at a time. The positioning of the Petri dishes for species, concentrations and replicates (84 samples) were according to a completely randomized design.

2.4.3. Experimental design for the assessment of shoot emergence in pots

For the assessment of the percentage shoot emergence, seeds were sown in pots (6 cm diameter) with a commercial peat substrate that was wet with distilled water. Prior to sowing, seeds were incubated for 5 min in distilled water containing 5% sodium hypochlorite for 3-5 min, followed by rinsing in distilled water. Each pot contained 20 seeds. Treatments were sprayed at day 1 at an application rate of 1000 L/ha using a portable 2 L manual pressure sprayer. The untreated CTRLs were sprayed with water only. Water was sprayed on all pots to wet the peat after 5 days to ensure germination. After 10 days, the numbers of apical tips that had emerged from the peat after expo-sure to the treatments/water were recorded [32].

As with the Petri dish trial, the peat trial was performed on the 7 plant species, comprising 5 different herbicide treatments at 4 different concentrations with 3 replicates.  The experiments were performed separately for each treatment at a time. Similar, to the Petri dish trials, the positioning of the pots for species, concentrations and replicates (84 samples) were performed according to a completely randomized design.”

 

4. Statistics: The calculation of IC₅₀ values is presented, but the method for IC₅₀ estimation is not described (e.g., which model/software used). Confidence intervals for IC₅₀ values are missing.

The calculation is now presented in the Methods section as follows:

“The concentration required for 50% radicle and shoot emergence inhibition (IC₅₀), respectively, was estimated for each treatment applied to each species. Using the individual percentage radicle and shoot emergence values for each different treatment (5, 10 and 20% compared to the untreated baseline 0%), the absolute IC₅₀ values (expressed in %) were estimated using the regression equation of the concentration-response curve as reported previously [33].”

 

5. The potential pH-mediated effects of acid treatments on soil are discussed hypothetically in the Discussion, but not empirically measured. It is suggested that either include soil pH measurements after acid application or explicitly state as a limitation. Alternatively, suggest in the Conclusion that future work should monitor pH shifts and their impact on nutrient availability.

We have cited this as a limitation to the study. We present the potential pH-mediated effects of acid treatments on peat hypothetically, but indicate that the mechanisms were not measured in the present study and warrant future research attention

 See an excerpt of the Discussion

“The mechanisms of action of the tested organic compounds on radicle and shoot emergence were not considered, and represent a limitation of the present study. The following aspects warrant further attention. Of interest, increased inhibition of wheat shoot emergence (40%) with 20% LA contents were not evident for CA………continue… An explanation as to why CA and LA inhibited the radicle emergence of grass weeds but not soft wheat also remains to be investigated. Seed coat characteristics (permeability) can affect how herbicides impact plants, with some stimulating germination and others inhibiting germination [37].”

 

6. The study was conducted under controlled conditions with growth chambers and pot experiments, but the conclusions imply direct relevance to field conditions. Please avoid overgeneralization. Rephrase sentences like “citric acid is a cost-effective alternative” to emphasize that field trials are still needed.

The Reviewer has suggested we divide the Discussion into subdivisions which speak of effects of the organic compounds on weeds and effects of the organic compound on crops. Then we were advised to relate the possible implications of the in vitro results to future field studies. We have attempted to do this in the Discussion, which has been rewritten. Previous work focused on the post- emergence effects of mostly acetic acid and pelargonic acid in either in vitro studies or field trials. We have compared the pre-emergence effects from these preliminary in vitro studies to the post-emergence effects for the same compounds. However, then we speak about the possible implications to future field trials.

We were careful to use terms such as a “potentially”, “may” and “can” fully aware that field trials are needed.

See an excerpt of the Discussion

“The present results indicated potential for field testing. In vitro, AA, CA, LA and PA showed approximately 90% radicle emergence inhibition and 60-90% shoot emergence inhibition of the weed species at 1000 L/ha. Field administration of AA (20%) for the control of weeds at an application rate of 935 L/ha was shown to be effective in controlling weeds prior to crop sowing [17], as was 10% AA three days before sowing soft wheat (at an application rate of 1600 L/ha) with no negative effects on the wheat [24]. Post-emergence herbicide control of PA applied to a false seedbed in field studies was also reported [21,27]. It is feasible that pre-emergent weed control with AA, PA, LA and CA on a stale seedbed may represent a potentially cost-effective alternative to post-emergent weed control, and warrants field investigations. Notably, CA did not inhibit seedling establishment of either soft wheat or alfalfa, showing potential for the suppression of weed establishment at the time of crop sowing. LA can also potentially be applied for weed control at the time of sowing of alfalfa. Since PA is more costly [21] and also inhibitory to alfalfa, CA and LA warrant testing in the field to investigate their potential as more cost-effective pre-emergence alternatives to PA. Although LA was shown to be more phytotoxic to wheat shoot development than PA and CA, if potentially used at concentrations that ensure weed control without negative impacts to wheat shoot development, it may similarly show potential in field testing. The preliminary studies indicated that none of the weed control compounds warrant consideration during millet sowing. Given that weed control compounds impacted differently on the seedling establishment of the three crops tested, more preliminary studies are a requisite to investigate impacts on crop species of interest prior to implementing field studies.”

 

7. Figures 1–6 lack clarity and professional formatting:

Axis labels are too small or missing units.

Legends are overly long and repetitive.

Please consolidate some figures to improve readability.

Use color-coded or symbol-coded bar charts for clearer comparison.

Consider showing dose-response curves (concentration vs inhibition %) for key species.

We thank you very much for this suggestion. The graphs have all been restructured to follow the suggestions to improve both clarity and professional formatting.

The Reviewer is invited to the read the Results section, which has been completely rewritten.

The presentation of the graphs has been adjusted to reflect concentration vs inhibition for each treatment for the 3 crop species together, followed by the weed species together.

The percentage radicle emergence (Figure 1) and the percentage shoot emergence (Figure 4) of the untreated CTRL treatments for all 7 plant species have been provided. The percentage radicle emergence inhibition for the crops (Figure 2) and the weeds (Figures 3) was provided for each treatment between 5-20% (evaluated as the percentage radicle inhibition relative to the corresponding untreated CTRLs set to 0% inhibition). The percentage shoot emergence inhibition for the crops (Figure 5) and the weeds (Figures 6) was provided for each treatment between 5-20% (evaluated as the percentage shoot inhibition relative to the corresponding untreated CTRLs set to 0% inhibition).

 

 The Discussion reiterates many results and lacks a concise structure. Please restructure Discussion with subheadings like: “Efficacy on Weeds,” “Selectivity to Crops,” “Mechanisms and pH Hypothesis,” “Implications for Organic Farming.” Avoid repeating earlier results; focus on mechanistic interpretation and broader relevance.

This is true. We have completely rewritten the Discussion. Although the Discussion was not divided into above-mentioned subheadings, we followed the “subheadings” suggested in developing the Discussion.  The Reviewer is kindly requested to review the complete Discussion in the manuscript.

 

8. There are numerous grammatical, syntactical, and typographical errors.

“Noteworthy, citric acid…” → “Notably, citric acid…”

“are warranting attention” → “warrant attention”

A thorough English language and style editing is essential, preferably by a native speaker or professional editing service.

Comments on the Quality of English Language: The English could be improved to more clearly express the research.

The article was thoroughly checked by the English mother tongue author.  

 

Reviewer 2 Report

Comments and Suggestions for Authors

1. agronomy-3734943-peer-review-v1 titled “Effect of pre-emergent organic herbicides on the germination of 2 crop and weed competitor species under controlled environ-3 ments

 

By: Mattia Alpi, Anne Whittaker, Elettra Frasinetti, Enrico Toschi, Giovanni Dinelli and Ilaria Marotti

 

Dear Editor,

I have read the above referenced ms and found the following:

Title:

1. Short running title is absent.
2. The main title is vague but can be modefied to be more precise, constructive and indicative.

Abstract:

Certain terms used may be changed for better expressed e.g.

Pre-emergent change into pre-emergence

Oganic herbicides change into organic chemicals or organic chemicals of potential use as herbicides since the chemicals used are not registered as herbicides although a large number of articles was reported on their herbicidal activities.

weed competitor species change to weeds since weeds compete with crop plants for growth factors and their competition is well known and documented but its severity depends on weed species and density.

“respectively” was used in different improper places where is not desired

The difference is unclear between: Effect on the germination (application by direct spraying) and shoot emergence (application to soil). What is the difference? Both applied either on peatmoss or agar medium.

No clear cut difference between germination and shoot emergence. How authors differentiate between both while germinated seeds were only counted?

Authors should clearly differentiate between weed elimination (eradication) and weed control. In the present work weed control is more correct and not elimination.

Keywords: why only wheat and ryegrass were indicated and not other crops and weeds tested?

Introduction:

Bioherbicide usually more referred to or predominantly made from living microbial organisms (bacteria, viruses, and fungi) or their secondary metabolite by-products (phytotoxins)

Plant part-derived by-products (allelochemicals such as essential oils) are chemicals released and mediated through environment and referred to as phytotoxins.

Weed Elimination Vs. weed control

Organic acid herbicides. Are these acids registered and used as herbicides or still under testing? If herbicides please add references.

Pelargonic acid and essential oils are also considered as organic herbicides, which are broadly classified as herbicides made from plant- or mineral-based chemicals, and also comprise organic acid-based ingredients (acetic acid, citric acid and caprylic acid). Please add reference/s.

Pre-emergent herbicides target the early stages of weed growth, preventing the development of the radicle, the hypocotyl or coleoptile, or both, thereby inhibiting seedling establishment. Are these acting in this way or these chemicals absorbed by seeds in the soil and affect the readily growing embryo and thus prevent germination? I think they act on germinating seeds or prevent germination by killing the embryo.

            Objectives need to be clearly presented

 

Materials and Methods

Treatment preparation

“Acetic acid, pelargonic acid and lactic acid were prepared by adding distilled water to obtain final solutions at concentrations of 0, 5% (v/v), 10% (v/v) and 20% (v/v), respectively. Citric acid was prepared by adding distilled water to similarly obtain final concentrations of 0, 5% (w/v), 10% (w/v) and 20% (w/v), respectively”. These preperations are unclear or not well indicated. What “respectively” was used for?.

 

Experimental design

Data reported in this work were generated from two small experiments carriedout in a growth champer under controlled conditions. The first was in glass Petri-dishes and the second in small (6cm diameter) pots filled with peatmoss. Treatments were replicated 3 times. However, both experiments were not repeated.

The first experimental trial was conducted to assess germination and radicle growth. Seeds were placed on the surface of Agar medium. Treatments were applied by spraying directly onto the plate at corresponding volume of 1000 L/ha in order to wet all the seeds. How you insured that seeds were wetted? How  radical growth was determined? No measurments were shown.

The second experimental trial was conducted to assess the existence of a primary shoot upon germination. Seeds were sown in pots (6 cm diameter) with a commercial peat substrate. Treatments were sprayed at day 1 (T0) at 1000 L/ha directly onto the surface of the soil. Germinated seeds were counted, and efficacy of the treatments was evaluated as number of germinated seeds.

Methods were not detailed enough, I don’t know how seed germination was counted in pots filled with peatmoss?! How researchers were measuring existance of primary shoots while only germinated seeds were counted? However, no measurments were taken and thus no actual data were presented.

 

Results

Lines 153-165 are better placed in Material and Methods or Introduction section.

Line 161-163: The results were reported as percentage inhibition of either germination or shoot emergence for each of the individual plant species, respectively, for each of the organic herbicide concentrations (5, 10 and 20%).

How clculated? How authors differentiated between germination and shoot emergence while only germinated seeds were counted in both experiments as mentioned in methods? no maesurments were taken on shoots. What “respectively” referred to here?

In all Figs. The way that data were presented and bars were drawn is confusing and missleading.

Lines 231-233. Citric acid (5%) produced minimal inhibitory effects on soft wheat coleoptile and alfalfa hypocotyl emergence, with varying degrees of inhibition on the remaining species. How authors measured the inhibitory effects here??

Lines 235-237. minimal inhibitory effects on soft wheat coleoptile emergence, with increasing inhibition effects on all remaining species, particularly the weed competitors, perennial ryegrass, green foxtail and common vetch. How authors measured inhibitory effects on cloleoptile although no cloleoptile for vetch and cichorium weed species or crops other than wheat?

Lines 277-279. the I50 was similarly calculated using all the data (0-20%) of each herbicide to estimate the percentage required to inhibit the shoot emergence of 50% of the seedlings. How measured??

Lines 280-282. with no inhibitory effects on shoot emergence and significant inhibitory effects of the remaining species. How inhibitory effect was measured while no data were shown or no measurements were taken but only germination count??!!. How minimal or strong inhibitory effect was measured or judged???

Tables were not clear or easily understood but rather confusing.

 

Discussion

There is a large overlapping between results and discussion sections which needs reconsideration. A good part of the results was repeated in discussion section and there is a clear absence of authors opinion and explanation to the results obtained.

Results discussion is generally limited.

 

References

References in the list need to be stadarized and must follow a single system of writing. Some problems were highlightened in yellow and need to be checked and corrected.

 

 

In general, data presernted lacks ecological significance but rather generated from small two experiments conducted under artificial conditions and improper chemical application. The reported work neither innovative nor is new.

 

Thanks for giving the chance to evaluate this work and for trusting my opinion.

• Enclosed is the marked copy of the ms.

 

Comments for author File: Comments.pdf

Comments on the Quality of English Language

English can be much improved and be more understandable. Figs and tables are hard to follow or understand. The way they were calculated and presented is confusing.

Author Response

I have read the above referenced manuscript and found the following:

 

Title:

Short running title is absent.

A short running title is provided:

Pre-emergence herbicidal effects of organic acids on weeds and crops

 

The main title is vague but can be modified to be more precise, constructive and indicative.

The main title has been modified and now reads:

“Effects of Pre-Emergence Application of Organic Acids on Seedling Establishment of Weeds and Crops in Controlled Environments”

 

Abstract:

Certain terms used may be changed for better expressed e.g.

Pre-emergent change into pre-emergence

Pre-emergence, or alternatively the expression post-emergence, have now been used throughout the manuscript. Pre-emergent is no longer used.

 

Organic herbicides change into organic chemicals or organic chemicals of potential use as herbicides since the chemicals used are not registered as herbicides although a large number of articles was reported on their herbicidal activities.

We have included this aspect into the Introduction as follows:

Of particular interest to the present investigation are organic acid-based organic compounds, including acetic acid (AA), pelargonic acid (PA), citric acid (CA) and lactic acid (LA). In the EU, only PA has been registered as a plant protection product with recognized herbicidal use in agricultural crops (Reg. (EC) No 1107/2009) [15]. Although AA (vinegar) is not officially registered in the EU as a herbicide, it is used for non-selective herbicidal activity and can be found in many advertised commercial “weed killer” formulations. CA and LA are not officially recognized or registered as herbicides under EU regulations.

After this – we refer to the products tested as organic compounds, and not organic herbicides, throughout the manuscript. In many articles, pelargonic acid is referred to as a herbicide.

 

weed competitor species change to weeds since weeds compete with crop plants for growth factors and their competition is well known and documented but its severity depends on weed species and density.

This is true and we modified the manuscript to avoid the use of the term “weed competitor species”.

 

“respectively” was used in different improper places where is not desired

We noticed this from the manuscript version that you sent and this word has been eliminated from the improper places where it is not desired.

 

The difference is unclear between: Effect on the germination (application by direct spraying) and shoot emergence (application to soil). What is the difference? Both applied either on peatmoss or agar medium.

No clear-cut difference between germination and shoot emergence. How authors differentiate between both while germinated seeds were only counted?

We apologize for the confusion. We have no longer used the word “germination” but rather “radicle emergence inhibition” and “shoot emergence inhibition”.

In the Methods section we have written the following:

“2.4. Growth chamber parameters and experimental design

To investigate the pre-emergent herbicidal effects of the organic compounds on seedling establishment, two different experimental trials were adopted. The first experimental trial was conducted in Petri dishes to assess either the presence or absence of radicle emergence on agar after directly spraying the seeds with the organic com-pounds. In the second trial, the organic compounds were sprayed on moistened peat in pots and not directly on the seeds. The presence or absence of shoot emergence (seedling establishment) was then assessed. Both experimental trials were conducted in a growth chamber during the period from March to September 2023.”

 

Authors should clearly differentiate between weed elimination (eradication) and weed control. In the present work weed control is more correct and not elimination.

The term “weed control” has been used through the entire document. We no longer use weed elimination.

 

Keywords: why only wheat and ryegrass were indicated and not other crops and weeds tested?

This has been amended and the key words are as follows:

citric acid; lactic acid; pre-emergent weed control; shoot emergence; weeds; wheat; millet; alfalfa

 

Introduction:

Bioherbicide usually more referred to or predominantly made from living microbial organisms (bacteria, viruses, and fungi) or their secondary metabolite by-products (phytotoxins)

Yes, we defined bioherbicides as follows in the Introduction

“Focusing on chemical herbicide alternatives, bioherbicides are broadly defined as eco-friendly, biological herbicide control products, predominantly made from living microbial organisms (bacteria, viruses, and fungi) or phytotoxins derived from microbes, insects, or plant extracts [10].”

 

Plant part-derived by-products (allelochemicals such as essential oils) are chemicals released and mediated through environment and referred to as phytotoxins.

The confusion created was removed from the Introduction

 

Weed Elimination Vs. weed control

We have used the term weed control and not weed elimination throughout the manuscript.

 

Organic acid herbicides. Are these acids registered and used as herbicides or still under testing? If herbicides please add references.

Pelargonic acid and essential oils are also considered as organic herbicides, which are broadly classified as herbicides made from plant- or mineral-based chemicals, and also comprise organic acid-based ingredients (acetic acid, citric acid and caprylic acid). Please add reference/s.

In response to the comments above, the Introduction now reads

……“Instead, organic herbicides are defined as contact, non-selective (knock-down), broad-spectrum herbicides derived from plant- or mineral-based ingredients that act as natural eco-friendly alternatives to chemical herbicides [12]. Of particular interest to the present investigation are organic acid-based organic compounds, including ace-tic acid (AA), pelargonic acid (PA), citric acid (CA) and lactic acid (LA). In the EU, only PA has been registered as a plant protection product with recognized herbicidal use in agricultural crops (Reg. (EC) No 1107/2009) [15]. Although AA (vinegar) it is not officially registered in the EU as a herbicide, it is used for non-selective herbicidal activity and can be found in many advertised commercial “weed killer” formulations. CA and LA are not officially recognized or registered as herbicides under EU regulations.”

 

Pre-emergent herbicides target the early stages of weed growth, preventing the development of the radicle, the hypocotyl or coleoptile, or both, thereby inhibiting seedling establishment. Are these acting in this way or these chemicals absorbed by seeds in the soil and affect the readily growing embryo and thus prevent germination? I think they act on germinating seeds or prevent germination by killing the embry0

We did not investigate the mechanism of action of the pre-emergence organic compounds used. As such, we preferred to cite a more concise definition comparing post-emergence and pre-emergence herbicides. In the Introduction we stated the following:

“Post-emergence herbicides are administered in the control of germinated weed plants to disrupt the cell membranes of vegetative tissues, whereas pre-emergence herbicides are administered to target the early stages of weed growth, preventing the development of the embryonic radicle, the shoot, or both, thereby inhibiting seedling establishment [3,13].”

 

Materials and Methods

Treatment preparation

“Acetic acid, pelargonic acid and lactic acid were prepared by adding distilled water to obtain final solutions at concentrations of 0, 5% (v/v), 10% (v/v) and 20% (v/v), respectively. Citric acid was prepared by adding distilled water to similarly obtain final concentrations of 0, 5% (w/v), 10% (w/v) and 20% (w/v), respectively”. These preparations are unclear or not well indicated. What “respectively” was used for?

This has been rewritten in the Methods section as follows:

“2.3. Preparation of treatment concentrations

Solutions of AA, PA, CA and LA were prepared individually by adding distilled water to obtain final concentrations of 5, 10 and 20% (v/v or w/v), as used previously [17,24]. The 0% concentration treatment contained only distilled water and represent-ed the untreated control (CTRL). The essential oils were added as adjuvants at a con-centration of 1.5% (v/v) eugenol + 1.5% (v/v) geraniol to the each of the different AA treatment concentrations (5, 10, 20%).”

 

Experimental design

Data reported in this work were generated from two small experiments carried out in a growth chamber under controlled conditions. The first was in glass Petri-dishes and the second in small (6cm diameter) pots filled with peatmoss. Treatments were replicated 3 times. However, both experiments were not repeated.

The first experimental trial was conducted to assess germination and radicle growth. Seeds were placed on the surface of Agar medium. Treatments were applied by spraying directly onto the plate at corresponding volume of 1000 L/ha in order to wet all the seeds. How you insured that seeds were wetted? How radical growth was determined? No measurements were shown.

The second experimental trial was conducted to assess the existence of a primary shoot upon germination. Seeds were sown in pots (6 cm diameter) with a commercial peat substrate. Treatments were sprayed at day 1 (T0) at 1000 L/ha directly onto the surface of the soil. Germinated seeds were counted, and efficacy of the treatments was evaluated as number of germinated seeds.

Methods were not detailed enough. I don’t know how seed germination was counted in pots filled with peatmoss?! How researchers were measuring existence of primary shoots while only germinated seeds were counted? However, no measurements were taken and thus no actual data were presented.

In the Methods section, the following was written:

“2.4. Growth chamber parameters and experimental design

To investigate the pre-emergent herbicidal effects of the organic compounds on seedling establishment, two different experimental trials were adopted. The first experimental trial was conducted in Petri dishes to assess either the presence or absence of radicle emergence on agar after directly spraying the seeds with the organic com-pounds. In the second trial, the organic compounds were sprayed on moistened peat in pots and not directly on the seeds. The presence or absence of shoot emergence (seed-ling establishment) was then assessed. Both experimental trials were conducted in a growth chamber during the period from March to September 2023.

2.4.1 Growth chamber parameters

Experiments were performed in a growth chamber at the Department of Agricultural and Food Sciences (DISTAL-University of Bologna). Above the bench surface were three fluorescent tube lights (FH21830, 21 W, warm white 830, 85 cm, 116 w; OSRAM SpA, Milan, Italy), installed at a height of 60 cm from positioning of the Petri dishes and pots. From a spectrometric analysis (Miniature Fibre Optic, USB2000+UV-VIS, Ocean Optics, Milan, Italy), light quality was characterized by two major spectral profile peaks in green and red and a minor peak in blue. Mean irradiance or photon flux density (PPFD) was 71.65 µmol s-1 m-2 (portable luxmeter, Delta OHM, Padova, Italy). The lighting was set to a 16/8 h (day/night) photoperiod. The in-ternal temperature was maintained at 23°C, and the relative humidity was 70-75%.

2.3.2 Experimental design for the assessment of radicle emergence in Petri dishes

For the assessment of the presence or absence of a radicle, tests were performed in 90-mm diameter glass petri dishes with 15 ml of 1.5% (w/v) agar. The agar in the Petri dishes was sterilized for 30 min inside a VWR® PCR Workstation (containing 2 internal UV tubes [254 nm, 25 W each] and 1 UV tube in a UV Air Recirculator [254 nm, 8 W] offering decontamination action by UV inactivation of airborne and surface-bound contaminants (Avantor, Milan, Italy). Seeds of all species tested were rinsed in distilled water containing 5% sodium hypochlorite for 3-5 min, followed by distilled water, prior to being placed on the agar. Each Petri dish contained 20 seeds. Treatments were carefully applied to each Petri dish at an application rate of 1000 L/ha using a portable 2 L manual pressure sprayer (LeroyMerlin.it Ref 84438959). The untreated CTRLs were sprayed with water only. After 7 days, seeds with radicles were counted following treatment/water exposure.

The experimental trial was composed of 7 plant species (3 crops and 4 weeds), comprising 5 different organic compound (AA, AA + GE; CA, LA and PA) treatments at 4 different concentrations (0, 5, 10, 20%) with 3 replicates (for each species, treatment and concentration), representing a total of 420 Petri dishes. As such, the experiments were performed separately for each treatment at a time. The positioning of the Petri dishes for species, concentrations and replicates (84 samples) were according to a completely randomized design.

2.4.3. Experimental design for the assessment of shoot emergence in pots

For the assessment of the percentage shoot emergence, seeds were sown in pots (6 cm diameter) with a commercial peat substrate that was wet with distilled water. Prior to sowing, seeds were incubated for 5 min in distilled water containing 5% sodium hypochlorite for 3-5 min, followed by rinsing in distilled water. Each pot contained 20 seeds. Treatments were sprayed at day 1 at an application rate of 1000 L/ha using a portable 2 L manual pressure sprayer. The untreated CTRLs were sprayed with water only. Water was sprayed on all pots to wet the peat after 5 days to ensure germination. After 10 days, the numbers of apical tips that had emerged from the peat after expo-sure to the treatments/water were recorded [32].

As with the Petri dish trial, the peat trial was performed on the 7 plant species, comprising 5 different herbicide treatments at 4 different concentrations with 3 replicates.  The experiments were performed separately for each treatment at a time. Similar, to the Petri dish trials, the positioning of the pots for species, concentrations and replicates (84 samples) were performed according to a completely randomized design.”

 

Results

The Results section has been rewritten in order to take into consideration all the reviewers’ comments. You are pleased requested to read the Results in the new uploaded version. Many of the comments made below have been addressed in the new version of the manuscript.

 

Lines 153-165 are better placed in Material and Methods or Introduction section.

This has been eliminated

Line 161-163: The results were reported as percentage inhibition of either germination or shoot emergence for each of the individual plant species, respectively, for each of the organic herbicide concentrations (5, 10 and 20%).

How calculated? How authors differentiated between germination and shoot emergence while only germinated seeds were counted in both experiments as mentioned in methods? no measurements were taken on shoots. What “respectively” referred to here?

Please refer to your last comment under Methods (or the article), where this is now explained.

The “respectively” has been removed.

 

In all Figs. The way that data were presented and bars were drawn is confusing and misleading.

The graphs have all been restructured to follow the suggestions to improve both clarity and professional formatting.

The Reviewer is invited to the read the Results section, which has been completely rewritten.

The presentation of the graphs has been adjusted to reflect concentration vs inhibition for each treatment for the 3 crop species together, followed by the weed species together.

The percentage radicle emergence (Figure 1) and the percentage shoot emergence (Figure 4) of the untreated CTRL treatments for all 7 plant species have been provided. The percentage radicle emergence inhibition for the crops (Figure 2) and the weeds (Figures 3) was provided for each treatment between 5-20% (evaluated as the percentage radicle inhibition relative to the corresponding untreated CTRLs set to 0% inhibition). The percentage shoot emergence inhibition for the crops (Figure 5) and the weeds (Figures 6) was provided for each treatment between 5-20% (evaluated as the percentage shoot inhibition relative to the corresponding untreated CTRLs set to 0% inhibition).

 

Lines 231-233. Citric acid (5%) produced minimal inhibitory effects on soft wheat coleoptile and alfalfa hypocotyl emergence, with varying degrees of inhibition on the remaining species. How authors measured the inhibitory effects here??

The results have been modified to eliminate this confusion.  

For example, in the results the following has been written:

“Thereafter, the percentage shoot emergence inhibition following the treatments (Fig-ures 5 and 6) was then calculated relative to the untreated baseline values of the respective CTRLs that were set to 0% inhibition.

CA, showed minimal percentage shoot emergence inhibition (5-20%) on soft wheat and alfalfa (Figures 5C, 5M), but was more phytotoxic to millet (Figure 5H).”…..continue

 

Lines 235-237. minimal inhibitory effects on soft wheat coleoptile emergence, with increasing inhibition effects on all remaining species, particularly the weed competitors, perennial ryegrass, green foxtail and common vetch. How authors measured inhibitory effects on cloleoptile although no cloleoptile for vetch and cichorium weed species or crops other than wheat?

The terminology used now is the percentage inhibition of shoot emergence. From the methods, the presence of the apical tip (shoot) that emerged from the peat for all plant species was reported for each pot. The efficacy of each treatment concentration was expressed as the percentage inhibition of shoot emergence.  

 

Lines 277-279. the I50 was similarly calculated using all the data (0-20%) of each herbicide to estimate the percentage required to inhibit the shoot emergence of 50% of the seedlings. How measured??

For each species and each treatment, the individual values on either radicle/shoot emergence were recorded for the 0,5,10,20% concentrations and the IC50 estimated by linear regression

 

Lines 280-282. with no inhibitory effects on shoot emergence and significant inhibitory effects of the remaining species. How inhibitory effect was measured while no data were shown or no measurements were taken but only germination count??!!. How minimal or strong inhibitory effect was measured or judged???

I hope the above-mentioned adjustments have helped clear the confusion we created. The above sentences have been rewritten. What is intended is the percentage inhibition of shoot emergence. A minimal percentage inhibition of shoot emergence would be would mean that only 5-10% of the seeds would not produce shoots, as opposed to a 60-90% inhibition as noted for some treatments.

 

Tables were not clear or easily understood but rather confusing.

The Tables have been modified – please see the manuscript

 

 Discussion

There is a large overlapping between results and discussion sections which needs reconsideration. A good part of the results was repeated in discussion section and there is a clear absence of authors opinion and explanation to the results obtained.

Results discussion is generally limited.

The Discussion has been completely reconstructed. The reviewer is invited to consider the Discussion.

 

References

References in the list need to be standardized and must follow a single system of writing. Some problems were highlighted in yellow and need to be checked and corrected.

The references have been thoroughly checked and inconsistences corrected. According to the MDPI journal format, the abbreviated Journal title is provided in italics, followed by the year of publication in bold, followed by the journal volume in Italics, followed the page number. This has been done for each reference. I noticed that you highlighted Agronomy in yellow. The journal Agronomy of MDPI is not abbreviated and is written as Agronomy in full.

 

In general, data presented lacks ecological significance but rather generated from small two experiments conducted under artificial conditions and improper chemical application. The reported work neither innovative nor is new.

We advised to better articulate the gaps and knowledge and the objectives. Below is an excerpt of the Introduction which I hope will assist in understanding the significance of the present study.

As follows:

“…Of particular interest to the present investigation are organic acid-based organic com-pounds, including acetic acid (AA), pelargonic acid (PA), citric acid (CA) and lactic acid (LA). In the EU, only PA has been registered as a plant protection product with recognized herbicidal use in agricultural crops (Reg. (EC) No 1107/2009) [15]. Although AA (vinegar) it is not officially registered under EU regulations, it is used as non-selective herbicide and can be found in many advertised commercial “weed killer” formulations. CA and LA are not officially recognized or registered as herbicides under EU regulations.

Post-emergence herbicides are administered in the control of germinated weed plants to disrupt cell membranes of vegetative tissues, whereas pre-emergence herbicides are administered to target the early stages of weed growth, preventing the development of the embryonic radicle, the shoot, or both, thereby inhibiting seedling establishment [3,13]. The vast majority of studies have focused on testing the efficacy of the most well-studied post-emergence organic compounds, PA and AA (alone or in combinations with essential oils), on controlling germinated weed plants [6,12,14,16-24]. In research on post-emergence foliar applications, CA alone has been found to be significantly less effective compared to AA and PA [16,25-26] and as such CA has been considered to a lesser extent. To the best of our knowledge, the pre-emergence herbicidal efficacy of CA and LA alone on weed seeds has not been considered. Our interest in examining the potential pre-emergence herbicidal effectiveness is because seed banks are reservoirs of both newly shed weed seeds and persistent older seeds that contribute to the persistence of invasive plant infestations [7]. Given the increasing interest in sustainable herbicide research within the context of integrated weed management strategies, the use of pre-emergence organic compounds could be administered to a false (stale) seedbed to control weed seedling establishment in the period before the sowing of crop plants, as has been reported previously for post-emergence control [3,17,21,24,27]. Alternatively, organic compounds could be administered to reduce weed seeding establishment (pre-emergence) concurrently with crop sowing. The latter strategy would necessitate examining the impact of these organic compounds on crop plant seeds, for which information on many crop species is still lacking.

The present study was, therefore, aimed at investigating the in vitro effects from the pre-emergence administration of different concentrations (5, 10 and 20%) of AA, AA + eugenol and geraniol (AA+EG), CA, LA and PA in inhibiting radicle and shoot emergence of four common weeds (perennial ryegrass, common vetch, green foxtail and common chicory) and three well recognized crop species (soft wheat, millet and alfalfa [lucerne]). The objective was to evaluate whether the organic acid compounds possess potential for future field trials, and more specifically, whether they could potentially be used in a false (stale) seedbed technique to firstly control weed shoot emergence prior to sowing the crop plants or whether there is the possibility of using them concurrently with crop sowing.”

 

Though it is true, that the present studies were conducted in vitro, the results of this preliminary study highlight that when used as pre-emergent compounds, CA and LA have equivalent herbicidal efficacy against weeds. The implications show that these compounds warrant testing in field studies prior to the sowing the crops. Moreover, it was shown that application of CA incurred minimal inhibition of wheat and alfalfa shoot emergence. The implication here is that CA merits field testing concurrently with wheat and alfalfa sowing. Similarly, LA was effective at removing weed species with minimal inhibition to alfalfa shoot emergence and can potentially be tested in the field.  

 

 

Enclosed is the marked copy of the manuscript.

 peer-review-47874084.v1.pdf

We have taken the marked copy into consideration when correcting the manuscript.

 

Comments on the Quality of English Language

English can be much improved and be more understandable.

The English has been improved.

 

Figs and tables are hard to follow or understand. The way they were calculated and presented is confusing.

Yes, this is very true! As such, the Reviewer is invited to the read the Results section, which has been completely rewritten.

The presentation of the graphs has been modified to render the results clearer. The graphs have been designed to show the effects of all treatments for each species. Figure 2 and Figure 5, show the inhibition of radicle emergence and shoot emergence for the 3 crop species, respectively. Figures 3 and 6 show the inhibition of radicle emergence and shoot emergence for the 4 weed species, respectively. Figures 1 and 5 include the percentage radicle and shoot emergence, respectively, of the 7 plant species that were not treated with the organic compounds. The presentation of the results in this format were also requested by the other reviewers.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Editor and Authors,

I have a big concern about the terms organic and bioherbicides. In my opinion, this is not the same. Why do you see them as the same? Why do you classify pelargonic acid as a bioherbicide?

https://www.belchimcanada.com/Brochure/188/E/1.pdf

There is a thin line between these concepts. Maybe I'm seeing it wrong, so I'm interested in your opinion.

Line 42-44: Within the framework of the European Union (EU), in order to combat this problem, the Green Deal is aimed at reducing the use and risk of chemical pesticides by 50% by 2030 [5].

The EU withdraws the proposal to reduce the use of pesticides. Please see the link below:

https://www.britishagriculturebureau.co.uk/updates-and-information/eu-withdraws-proposal-to-reduce-the-use-of-pesticides/

Please rewrite above above-mentioned part in the introduction.

Line 84: Please change the term sprout to shoot.

Line 115: 0, 5% (v/v), Is it a mistake?

Line 131: Why did you use 1000 L/Ha? 200 to 300 l/ha of working fluid is generally used in arable crops. In orchards up to 500L/ha. Where is 1000 L/ha used?

Results: General, in an experiment with the effectiveness of the tested treatments on seed germination, a high percentage of weed seed inhibition was observed alongside a notable inhibition of crop germination. Although there was somewhat less inhibition in the growth of crop seedlings, significant phytotoxicity was still evident. Therefore, these treatments generally cannot be recommended for these crops based on these results. I would agree with your conclusion that more research is warranted to understand the mechanisms of action of these herbicides and their differential effects on weeds and crops. The current results give unclear answers, based on which no conclusions can be drawn. Therefore, my impression is that the work is unfinished.

Please, in the results, try to separate the effectiveness of the applied treatments on the weed species and the crops. Observe the impact on crops as a separate experiment, where you monitored the phytotoxicity on germination and growth of these seeds. Maybe that would help make the results clearer.

 

Author Response

I have a big concern about the terms organic and bioherbicides. In my opinion, this is not the same. Why do you see them as the same? Why do you classify pelargonic acid as a bioherbicide?

https://www.belchimcanada.com/Brochure/188/E/1.pdf

There is a thin line between these concepts. Maybe I'm seeing it wrong, so I'm interested in your opinion.

Thank you for raising that concern. We agree that the line is thin between the concepts. I was under the impression that pelargonic could be considered both a bioherbicide and an organic herbicide. Bio – because it is of natural origins. Organic - because it is of natural origins permitted for use in sustainable agriculture.

Regarding Beloukha®, which has pelargonic acid as the main ingredient, in the literature I read this – cited as follows: “Beloukha® is a bioherbicide registered in Europe since 2015, and the active ingredient is pelargonic acid (Torres-Pagán et al., 2024; paper title Herbicidal Potential of the Natural Compounds Carvacrol, Thymol, Eugenol, p-Cymene, Citral and Pelargonic Acid in Field Conditions: Indications for Better Performance).” Given that bioherbicide definitions are extended to include plant-based natural products extracts, and pelargonic acid is a naturally occurring fatty acid found in many plants, I was under the impression it could be considered both.

In the Introduction, we have adjusted my original thinking and modified the paragraph (Lines 57-71 of the original document) as follows:

“Focusing on chemical herbicide alternatives, bioherbicides are broadly defined as eco-friendly, biological herbicide control products, predominantly made from living microbial organisms (bacteria, viruses, and fungi) or phytotoxins derived from microbes, insects, or plant extracts [10]. Overall, bioherbicides have a shorter environmental persistence and are biodegradable with fewer risks to non-target organisms and ecosystems [13]. However, microbial bioherbicides are specifically shown to display a narrow control spectrum, pose a risk of genetic exchange, and are subject to climate-dependent environmental factors that affect the stability and spread of phytopathogens [11].  Instead, organic herbicides are defined as contact, non-selective (knock-down), broad-spectrum herbicides derived from plant- or mineral-based ingredients that act as natural eco-friendly alternatives to chemical herbicides [12].”

 

Line 42-44: Within the framework of the European Union (EU), in order to combat this problem, the Green Deal is aimed at reducing the use and risk of chemical pesticides by 50% by 2030 [5].

The EU withdraws the proposal to reduce the use of pesticides. Please see the link below:

https://www.britishagriculturebureau.co.uk/updates-and-information/eu-withdraws-proposal-to-reduce-the-use-of-pesticides/

Please rewrite above above-mentioned part in the introduction.

We apologize as I was unaware of this amendment. We will remove the sentence completely.  The previous sentence has been replaced with the following sentence:

“Within the framework of the European Union (EU), the overall use of synthetic pesticides is a contentious issue, involving debates about environmental and human health risks, agricultural practices, and the balance between food security and sustainability [5].”

 

Line 84: Please change the term sprout to shoot.

This has been done

 

Line 115: 0, 5% (v/v), Is it a mistake?

Yes, it was a mistake.

In the Methods section it now reads:

2.3. Preparation of treatment concentrations

“Solutions of AA, PA, CA and LA were prepared individually by adding distilled water to obtain final concentrations of 5, 10 and 20% (v/v or w/v), as used previously [17,24]. The 0% concentration treatment contained only distilled water and represent-ed the untreated control (CTRL). The essential oils were added as adjuvants at a con-centration of 1.5% (v/v) eugenol + 1.5% (v/v) geraniol to the each of the different AA treatment concentrations (5, 10, 20%).”

 

Line 131: Why did you use 1000 L/Ha? 200 to 300 l/ha of working fluid is generally used in arable crops. In orchards up to 500L/ha. Where is 1000 L/ha used?

The working fluids of less than 400 L/ha are typically used in post-emergence studies as foliar sprays. If the organic compound is applied to remove weeds growing among crop plants, the crops plants can also be affected at application rates exceeding at an application rate exceeding 400 L/Ha. However, much higher application rates (5, 10, 20% acetic acid at either 187 L/ha or 935 L/ha) have been used to eliminate weeds in the period prior to the sowing of the crop plants (Webber et al., 2018 in the reference section, and cited as reference number 17). Higher application rates (10% acetic acid at 200, 400, 800, 1600 and 2400 L/ha) were used to remove weeds 3 days prior the sowing of a wheat crop with no effects on the wheat crop (Johnson et al.,2003 in the reference section and cited as number 24 of the reference list).

There is an excerpt from the Discussion

“The present results indicated potential for field testing. In vitro, AA, CA, LA and PA showed approximately 90% radicle emergence inhibition and 60-90% shoot emergence inhibition of the weed species at 1000 L/ha. Field administration of AA (20%) for the control of weeds at an application rate of 935 L/ha was shown to be effective in controlling weeds prior to crop sowing [17], as was 10% AA three days before sowing soft wheat (at an application rate of 1600 L/ha) with no negative effects on the wheat [24]. Post-emergence herbicide control of PA applied to a false seedbed in field studies was also reported [21,27]. It is feasible that pre-emergent weed control with AA, PA, LA and CA on a stale seedbed may represent a potentially cost-effective alternative to post-emergent weed control, and warrants field investigations.”

 

Results: General, in an experiment with the effectiveness of the tested treatments on seed germination, a high percentage of weed seed inhibition was observed alongside a notable inhibition of crop germination. Although there was somewhat less inhibition in the growth of crop seedlings, significant phytotoxicity was still evident. Therefore, these treatments generally cannot be recommended for these crops based on these results. I would agree with your conclusion that more research is warranted to understand the mechanisms of action of these herbicides and their differential effects on weeds and crops. The current results give unclear answers, based on which no conclusions can be drawn. Therefore, my impression is that the work is unfinished.

The work is complete. I will address each of the issues raised above. Firstly, we apologize for not having articulated the gaps in knowledge and the objectives effectively in the Introduction. This section has been rewritten along with the Results and Discussion. The reviewer is invited to read these sections. The Abstract provides the overall synopsis and reads

The Abstract:

“Within the framework of organic acid alternatives to chemical herbicides, pre-emergence weed control research is scare. Citric acid (CA) and lactic acid (LA), considered significantly less effective than pelargonic acid (PA) and acetic acid (AA) from post-emergence (foliar spraying) studies, have largely been disregarded. This in vitro study was aimed at comparing the effects of 5-20% AA, AA + essential oils, PA, CA and LA, respectively, on radicle emergence inhibition (direct spraying of seeds) and shoot emergence inhibition (application to peat) on both weeds (perennial ryegrass, green foxtail, common vetch and chicory) and crops (soft wheat, alfalfa and millet). All tested compounds demonstrated concentration-dependent and species-specific inhibitory effects on shoot emergence inhibition, with CA and LA (IC₅₀ range: 3.4-19.3%) showing a comparable efficacy to PA and AA (IC₅₀ range: 3.1-35.9%). Results also showed that CA and to a lesser extent LA, were less inhibitory to wheat (CA IC₅₀=62.5%; LA IC₅₀=35.9%) and alfalfa (CA IC₅₀=57.8%; LA IC₅₀=44.1%) shoot emergence. CA and LA show potential promise for pre-emergence weed control in field-testing, either on a false seedbed in pre-crop sowing or concurrently with soft wheat and alfalfa sowing. Investigating organic compound herbicidal effects on crops of interest warrants attention.”

Introduction now reads as follows:

“Post-emergence herbicides are administered in the control of germinated weed plants to disrupt cell membranes of vegetative tissues, whereas pre-emergence herbicides are administered to target the early stages of weed growth, preventing the development of the embryonic radicle, the shoot, or both, thereby inhibiting seedling establishment [3,13]. Research on post-emergence foliar applications indicates that CA is less effective than AA and PA [16, 25-26], and as a consequence CA has received less consideration due to this lower efficacy. Furthermore, there is a lack of research specifically examining the pre-emergence herbicidal activity of CA and LA on weed seeds. Our interest in examining the potential pre-emergence herbicidal effectiveness is be-cause seed banks are reservoirs of both newly shed weed seeds and persistent older seeds that contribute to the persistence of invasive plant infestations [7]. Given the in-creasing interest in sustainable herbicide research within the context of integrated weed management strategies, the use of organic compounds could be administered to a false (stale) seedbed for pre-emergence control of seedling establishment before sowing of crop plants, as was reported for post-mergence control [3,17,21,24,27]. Alternatively, organic compounds could be administered to reduce weed seeding establishment concurrently with crop sowing. The latter strategy would necessitate examining the impact of these organic compounds on crop plant seeds, for which information on many crop species is still lacking.

The present study was, therefore, aimed at investigating the in vitro effects from the administration of different concentrations (5, 10 and 20%) of AA, AA + eugenol and geraniol (AA+EG), CA, LA and PA in inhibiting radicle and shoot emergence of four common weeds (perennial ryegrass, common vetch, green foxtail and common chicory) and three well-recognized crops (soft wheat, millet and alfalfa [lucerne]). The objective was to evaluate whether the organic acid compounds possess potential for future field trials, and more specifically, whether they could potentially be used in a false (stale) seedbed technique to firstly control weed shoot emergence prior to sowing the crop plants or whether there is the possibility of using them concurrently with crop sowing. ”and the Introduction continues…….

 

It is true that phytotoxicity is evident on shoot emergence after exposure to the organic compounds. From the IC₅₀ results, only certain compounds could potentially be applied simultaneously with certain crop plants. In the modified Discussion, we discussed the effects of the compounds on the weeds, followed by the effects on the crops. The reviewer is invited to read the new version of the Discussion. Thereafter we discussed the potential application for field studies.

An excerpt of the Discussion is included below:

“The present results indicated potential for field testing. In vitro, AA, CA, LA and PA showed approximately 90% radicle emergence inhibition and 60-90% shoot emergence inhibition of the weed species at 1000 L/ha. Field administration of AA (20%) for the control of weeds at an application rate of 935 L/ha was shown to be effective in controlling weeds prior to crop sowing [17], as was 10% AA three days before sowing soft wheat (at an application rate of 1600 L/ha) with no negative effects on the wheat [24]. Post-emergence herbicide control of PA applied to a false seedbed in field studies was also reported [21,27]. It is feasible that pre-emergent weed control with AA, PA, LA and CA on a stale seedbed may represent a potentially cost-effective alternative to post-emergent weed control, and warrants field investigations. Notably, CA did not significantly inhibit seedling establishment of either soft wheat or alfalfa, showing potential for the suppression of weed establishment at the time of crop sowing. LA can also potentially be applied for weed control at the time of sowing of alfalfa. Since PA is more costly [21] and also inhibitory to alfalfa, CA and LA warrant testing in the field to investigate their potential as more cost-effective pre-emergence alternatives to PA. Although LA was shown to be more phytotoxic to wheat shoot development than PA and CA, if potentially used at concentrations that ensure weed control without negative impacts to wheat shoot development, it may similarly show potential in field testing. The preliminary studies indicated that none of the weed control compounds warrant consideration during millet sowing. Given that weed control compounds impacted differently on the seedling establishment of the three crops tested, more preliminary studies are a requisite to investigate impacts on crop species of interest prior to implementing field studies”

 

Please, in the results, try to separate the effectiveness of the applied treatments on the weed species and the crops. Observe the impact on crops as a separate experiment, where you monitored the phytotoxicity on germination and growth of these seeds. Maybe that would help make the results clearer.

The Reviewer is invited to the read the Results section, which has been completely rewritten.

The presentation of the graphs has been adjusted to reflect concentration vs inhibition for each treatment for the 3 crop species together, followed by the 4 weed species together.

The percentage radicle emergence (Figure 1) and the percentage shoot emergence (Figure 4) of the untreated CTRL treatments for all 7 plant species have been provided.  The percentage radicle emergence inhibition for the crops (Figure 2) and the weeds (Figures 3) was provided for each treatment concentration (evaluated as the percentage radicle inhibition relative to the corresponding untreated CTRLs set to 0% inhibition). The percentage shoot emergence inhibition for the crops (Figure 5) and the weeds (Figures 6) was provided for each treatment concentration (evaluated as the percentage radicle inhibition relative to the corresponding untreated CTRLs set to 0% inhibition).

Reviewer 4 Report

Comments and Suggestions for Authors

This manuscript presents the results of a series of petri dish and pot experiments on the germination and early growth of 7 plant species following spraying with several organic acid solutions at 3 concentrations, with the addition of eugenol and geraniol. I find that the methods are missing several significant details. Also, in my opinion, one series of each experiment is not enough to present robust results (e.g., only 60 seeds of the tested species in the Petri dish experiment, and their germination potential is not assessed, which is limiting specifically for weed species)

Lns. 57-60, pls give citations for the definition of bioherbicides

Ln. 60 – Why do you call essential oil a “by-product”? EOs are the main product obtained from different EO-bearing crops

Ln. 67 – cite a proper literature source for a definition of “organic herbicide”.

Ln. 69 – What is “organic acid herbicide”? Define and properly cite verifiable sources.

Lines 107-108: Please provide cultivars of crops.

Ln. 118-119 repeated acetic acid twice in one sentence. Also, what do you mean by the 1.5% concentration of essential oil compounds was “standard”

Ln. 129 – soaking for how long and in what?

Did you assess the germination percentage of weed seeds before the experiment?

Line 131, express the volume of spray also per Petri dish to give the scope of the treatment on a microscale. Do the same with the pot experiment.

Ln. 138 – seeds of which species? Be clear.

Ln. 138 How many seeds per pot? How did you assure all of them were germinable?

Ln. 143 – what was control, [precisely? Describe?

Ln. 143 – “the tegument was torn” – this expression is not fully biologically correct.

Ln. 184 acetic acid

The IC50 dose definition and calculation method are not explained in the Methods section. Table 1 caption gives I50 dose.

Table 1: Is it concerning the control? Additionally, what were the germination rates in the control treatments? This would be informative to show real values and provide an overview of the actual germination capability of the tested seed material for crops and weeds.

POT experiment – commercial peat substrate has a huge sorption capacity. How can you speculate on the effect of spraying in these conditions? It is not comparable to a Petri dish experiment, where agar is used. Additionally, the fact that the volume of spray in the pot is not given hinders the actual outcomes of the pot experiment.

Chapter 3.2 title should not be “in soil” but “ in peat”

Figure 4: How does the result look for the control treatment?

ln 277 – I50 or IC50?

Table 2 title – I50 or IC50?

Discussion is not fully relevant due to a lack of information in the methods and results chapters.

Author Response

This manuscript presents the results of a series of petri dish and pot experiments on the germination and early growth of 7 plant species following spraying with several organic acid solutions at 3 concentrations, with the addition of eugenol and geraniol. I find that the methods are missing several significant details. Also, in my opinion, one series of each experiment is not enough to present robust results (e.g., only 60 seeds of the tested species in the Petri dish experiment, and their germination potential is not assessed, which is limiting specifically for weed species).

 

Each of the seed species was tested both on agar and also in peat. For each concentration of each treatment, we tested 60 seeds. The Methods section has been rewritten in a more clear and concise manner. The missing details have now been included. We apologize for the lack of clarity. The reviewer is kindly requested to read this section in the uploaded version of the article.

In the Methods section, it is written:

“2.4. Growth chamber parameters and experimental design”

To investigate the pre-emergent herbicidal effects of the organic compounds on seedling establishment, two different experimental trials were adopted. The first experimental trial was conducted in Petri dishes to assess either the presence or absence of radicle emergence on agar after directly spraying the seeds with the organic compounds. In the second trial, the organic compounds were sprayed on moistened peat in pots and not directly on the seeds. The presence or absence of shoot emergence (seedling establishment) was then assessed. Both experimental trials were conducted in a growth chamber during the period from March to September 2023.”

 

Then the following headings were included

“2.3.2 Experimental design for the assessment of radicle emergence in Petri dishes”

“2.4.3. Experimental design for the assessment of shoot emergence in pots”

 

Essentially, for each species, each of the 5 treatments was performed at 4 treatment concentrations (0, 5,10 and 20%) with 3 replicates. This experimental design was performed to assess both radicle emergence inhibition (Petri dish) and shoot emergence inhibition (pot) trials. The total of 60 seeds was the number for each treatment concentration for each species for each type of experimental trial.

 

Lns. 57-60, pls give citations for the definition of bioherbicides

Bioherbicides have been defined in the Introduction as follows:

…” Focusing on chemical herbicide alternatives, bioherbicides are broadly defined as eco-friendly, biological herbicide control products, predominantly made from living microbial organisms (bacteria, viruses, and fungi) or phytotoxins derived from microbes, insects, or plant extracts [10].”

 

Ln. 60 – Why do you call essential oil a “by-product”? EOs are the main product obtained from different EO-bearing crops

This part of the sentence is incorrect and has been removed.

 

Ln. 67 – cite a proper literature source for a definition of “organic herbicide”.

Ln. 69 – What is “organic acid herbicide”? Define and properly cite verifiable sources.

“Instead, organic herbicides are defined as contact, non-selective (knock-down), broad-spectrum herbicides derived from plant- or mineral-based ingredients that act as natural eco-friendly alternatives to chemical herbicides [12].”

 

Below for the Methods

The reviewer is asked to check the Methods section which has been rewritten. I will respond to each comment below as well

 

Lines 107-108: Please provide cultivars of crops.

The cultivars of the crop were provided.

In the Methods as follows:

2.2. The selection of crops and weeds

“The three crop plants selected were T. aestivum L. var. Verna, P. miliaceum L. var. Blond and M. sativa L. var. Minerva.”

 

Ln. 118-119 repeated acetic acid twice in one sentence. Also, what do you mean by the 1.5% concentration of essential oil compounds was “standard”

The repetition of acetic acid has now been corrected. The term “standard” has been eliminated.

 In the Methods as follows:

“The essential oils were added as adjuvants at a concentration of 1.5% (v/v) eugenol + 1.5% (v/v) geraniol to the each of the different AA treatment concentrations (5, 10, 20%).”

 

Did you assess the germination percentage of weed seeds before the experiment?

The radicle emergence percentages for the experimental Petri dish experiment were assessed and presented in Figure 1 of the Results section. Then, the shoot emergence percentages for the experimental pot experiment were assessed and presented in Figure 4 of the Results section.

 

Collectively:

Ln. 129 – soaking for how long and in what?

Line 131, express the volume of spray also per Petri dish to give the scope of the treatment on a microscale.

In the Methods section

2.3.2 Experimental design for the assessment of radicle emergence in Petri dishes

“Seeds of all species tested were rinsed in distilled water containing 5% sodium hypochlorite for 5 min, followed by distilled water, prior to being placed on the agar. Each Petri dish contained 20 seeds. Treatments were carefully applied to each Petri dish at an application rate of 1000 L/ha using a portable 2 L manual pressure sprayer (LeroyMerlin.it Ref 84438959). The untreated CTRLs were sprayed with water only.”

We decided not to opt for providing this on a micro-scale because in published literature it is give in macro-scale.

 

Ln. 138 – seeds of which species? Be clear.

Ln. 138 How many seeds per pot? 20

Ln. 143 – what was control [precisely? Describe? Non-treated

In the Methods as follows:

Ln. 143 – “the tegument was torn” – this expression is not fully biologically correct.

In the Methods section it reads:

“2.3. Preparation of treatment concentrations

Solutions of AA, PA, CA and LA were prepared individually by adding distilled water to obtain final concentrations of 5, 10 and 20% (v/v or w/v), as used previously [17,24]. The 0% concentration treatment contained only distilled water and represent-ed the untreated control (CTRL). The essential oils were added as adjuvants at a con-centration of 1.5% (v/v) eugenol + 1.5% (v/v) geraniol to the each of the different AA treatment concentrations (5, 10, 20%).”

And then

“2.4.3. Experimental design for the assessment of shoot emergence in pots

For the assessment of the percentage shoot emergence, seeds were sown in pots (6 cm diameter) with a commercial peat substrate that was wet with distilled water. Prior to sowing, seeds were incubated for 5 min in distilled water containing 5% sodium hypochlorite for 3-5 min, followed by rinsing in distilled water. Each pot contained 20 seeds. Treatments were sprayed at day 1 at an application rate of 1000 L/ha using a portable 2 L manual pressure sprayer. The untreated CTRLs were sprayed with water only. Water was sprayed on all pots to wet the peat after 5 days to ensure germination. After 10 days, the numbers of apical tips that had emerged from the peat after expo-sure to the treatments/water were recorded [32].”

 

The IC50 dose definition and calculation method are not explained in the Methods section.

In the Methods section

“The concentration required for 50% radicle and shoot emergence inhibition (IC₅₀), respectively, was calculated for each treatment applied to each species. Using the individual percentage radicle and shoot emergence values for each different treatment (5, 10 and 20% compared to the untreated baseline 0%), the absolute IC₅₀ values (expressed in %) were estimated using the regression equation of the concentration-response curve as reported previously [33].”

 

 

Table 1 caption gives I50 dose.

IC₅₀ is defined as the inhibitory concentration at 50% (IC₅₀) and we intended to use this term. We apologize for the inconsistencies and confusion. This term is now used in a consistent manner throughout the manuscript.

 

Table 1: Is it concerning the control? Additionally, what were the germination rates in the control treatments? This would be informative to show real values and provide an overview of the actual germination capability of the tested seed material for crops and weeds.

In the Methods section

To calculate the IC50 percentages in Table 1, we wrote as follows in the Methods

“The concentration required for 50% radicle and shoot emergence inhibition (IC₅₀), respectively, was calculated for each treatment applied to each species. Using the individual percentage radicle and shoot emergence values for each different treatment (5, 10 and 20% compared to the untreated baseline 0%), the absolute IC₅₀ values (expressed in %) were estimated using the regression equation of the concentration-response curve as reported previously [33].”

Then in the Results Section

An excerpt

“In this trial, root emergence was estimated following the direct spaying of seeds with the tested organic compounds. Firstly, the radicle emergence percentage of the crop and weed seeds without organic compound treatments, was assessed. Radicle emergence after 7 days was evident in over 95% of the untreated CTRL crop and weed seeds, with the exception of common chicory with ca 80%. The percentage radicle emergence inhibition following the treatments was then calculated for each species (Figures 2 and 3). Radicle emergence inhibition at 5, 10 and 20% was calculated as a percentage, with the control group (CTRLs) set as the baseline at 0% inhibition.

It was easier to evaluate treatment-dependent inhibition in this way.

The same procedure was followed for the shoot emergence pot trials

Then all the individual values were used in order to estimate the IC₅₀ % of the organic acid compounds necessary to inhibit 50% of either radicle emergence or shoot emergence

 

POT experiment – commercial peat substrate has a huge sorption capacity. How can you speculate on the effect of spraying in these conditions? It is not comparable to a Petri dish experiment, where agar is used. Additionally, the fact that the volume of spray in the pot is not given hinders the actual outcomes of the pot experiment.

In the pot experiments, the peat was made moist. Thereafter, a treatment application rate of 1000 L/ha was applied to each pot (see comment above). 

 

Chapter 3.2 title should not be “in soil” but “in peat”

The treatments are now referred to peat treatments throughout the manuscript

 

Figure 4: How does the result look for the control treatment?

The Results section has been rewritten. The presentation of the graphs has been adjusted to reflect concentration vs inhibition for each treatment for the 3 crop species together, and followed by the weed species together.

The percentage radicle emergence (Figure 1) and the percentage shoot emergence (Figure 4) of the untreated CTRL treatments for all 7 plant species have been provided.  The percentage radicle emergence inhibition for the crops (Figure 2) and the weeds (Figures 3) was provided for each treatment concentration (evaluated as the percentage radicle inhibition relative to the corresponding untreated CTRLs set to 0% inhibition). The percentage shoot emergence inhibition for the crops (Figure 5) and the weeds (Figures 6) was provided for each treatment concentration (evaluated as the percentage radicle inhibition relative to the corresponding untreated CTRLs set to 0% inhibition).

 

 

ln 277 – I50 or IC50?

Table 2 title – I50 or IC50?

IC₅₀ is defined as the inhibitory concentration at 50% (IC₅₀) and we intended to use this term. We apologize for the inconsistencies and confusion. This term is used throughout the manuscript.

 

Discussion is not fully relevant due to a lack of information in the methods and results chapters.

We hope that the Methods and Results are now much clearer, and can facilitate the understanding of the Discussion. The reviewer is invited to consider the Discussion.

 

 

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have revised the manuscript nicely. Congratulations to the authors for their efforts.

Author Response

The authors have revised the manuscript nicely. Congratulations to the authors for their efforts.

Thank you for the suggestions made

Reviewer 2 Report

Comments and Suggestions for Authors I have carefully read the revised copy of the above referenced ms. and checked all amendments, correction and edits done by authors. While I highly appreciate authors response and value the further work done on the ms. to have it better standing for publication but the main problem remains unsolved since further field at least a single experiment was suggested to verify the ecological importance of the results obtained under artificial conditions in a growth chamber. As I mentioned before, data reported were generated from two small experiments in Petri-dishes and in small pots filled with peatmoss. Results reported may be changed to the opposite in the field. Considering the limited experimental results for publication in a reputable journal is highly questionable since incomplete without being examined directly in the field and data obtained in a growth chamber were ecologically confirmed. Unless this part is completed, I think the ms. is unsuitable in its present form and sorry to decline recommending it for publication considering that herbicidal activity of the examined organic acids in the present work has been already reported by other workers and available in literature.  

Author Response

Please see attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

This form allows the manuscript to be published. I still do not agree with using organic acids in preemergence treatment. I believe that this amount of water and acid is irrational and economically unjustified. In my opinion, these researches have no future, but as research, they are acceptable.

Please see:

 Lines 126-137: This information is redundant for the M&M section.

Author Response

Dear Authors,

This form allows the manuscript to be published. I still do not agree with using organic acids in preemergence treatment. I believe that this amount of water and acid is irrational and economically unjustified. In my opinion, these researches have no future, but as research, they are acceptable.

We just wanted to highlight pre-emergence studies warrant attention. As to the amount of citric acid, we thank you for your input. Based on what you have said, we will keep in mind your comments for future research. We cannot exclude the fact that it won’t work, at the same time, small-field studies can be implemented to investigate further

 

Please see:

Lines 126-137: This information is redundant for the M&M section.

The information has been removed from the M&M section. We have included it in the Results as it provides the motivation for the choice of crops and weeds, which are then presented

The English could be improved to more clearly express the research.

I checked the manuscript to see where I can improve the English.

 

Reviewer 4 Report

Comments and Suggestions for Authors

Ln. 99. False and stale seedbeds are not synonyms. Check: https://www.physicalweeding.com/information/index.html

Since authors often refer to pot experiments as a form of stale seedbed, it is necessary to explain this term in the Introduction.

I also recommend removing the term “false seedbed” as it does not apply to your results.

Ln. 191-192 – How deep were seeds sown?

Ln. 462-463 – wheat at the growth stage in the experiment is rather using nutrients contained in its seeds, so it is less probable that CA affected this process.

Figure 1 and 4 – What is the seed number?

Titles of figures do not reflect whether the results come from a laboratory (agar) or pot (peat) experiment. Please correct that.

Author Response

Ln. 99. False and stale seedbeds are not synonyms. Check: https://www.physicalweeding.com/information/index.html.

I mistakenly interchanged the terminology. Thank you for bringing that to our attention. It is true that the stale seed bed is the one that receives treatment to remove weeds prior to crop sowing whilst the false seedbed uses tillage practices to remove the weeds.

 

Since authors often refer to pot experiments as a form of stale seedbed, it is necessary to explain this term in the Introduction.

The pot trials cannot exactly be compared to a stale seed bed. However, based on the pot trial results, different organic compounds may present potential for application in a stale seedbed for pre-emergence weed control. We have nonetheless included the definition of a stale seedbed in the Introduction as follows:

“….the use of organic compounds could be administered to a stale seedbed for pre-emergence control of seedling establishment before sowing the crop plants. A stale seedbed involves preparing the soil for the germination of the weeds, followed by an herbicidal method for weed control just before the sowing of the crop. The organic compounds with herbicidal activity are generally applied to the vegetative tissues for post-mergence control [3,17,21,24,27].”

 

I also recommend removing the term “false seedbed” as it does not apply to your results.

This is true. We have removed the term false seedbed from the manuscript

 

Ln. 191-192 – How deep were seeds sown?

All seeds were sown at depth of between 1-2 cm. This will be added to the methods.

There is a sentence that now reads:

“All seeds were sown at a depth of 1-2 cm in the peat.”

 

Ln. 462-463 – wheat at the growth stage in the experiment is rather using nutrients contained in its seeds, so it is less probable that CA affected this process.

The paragraph compared the effects of LA and CA on wheat at the same growth stage. Both organic compounds produced a minimal inhibition of radicle emergence in wheat after 7 days (they produced similar effects).  However, when CA or LA were sprayed on peat, CA did not inhibit wheat shoot emergence to the same extent as LA after 10 days. I then wrote this sentence to which I understand you are referring to:

“Alternatively, it is feasible that 10-20% LA in the peat trials lowered the pH to a greater extent than 10-20% CA, which may have affected nutrient uptake impacting on wheat shoot development. These aspects remain to be evaluated”

 You are saying that it is less probable. I will remove the sentence. I will leave the more probable reason. Nonetheless, I proposed that they be investigated.

 

Figure 1 and 4 – What is the seed number?

We were asked whether we tested the germinability of the seeds. The graphs show the germinability and Figures 1 and 4 show a total of 60 seeds for each species. For the set-up of the experiments, please refer to the Methods

The example of Figure is provided

Figure 1. The percentage germination of the untreated control (CTRL) seeds for three crop species and four weed species after a 7-day incubation in Petri dishes to test germinability (60 seeds for each species). Results were expressed as the mean percentage inhibition with standard deviations. The different letters (a to b) represent significant differences (p > 0.05, Tukey’s least significant difference test).

 

Titles of figures do not reflect whether the results come from a laboratory (agar) or pot (peat) experiment. Please correct that.

This has been included as follows:

Figure 2. The percentage radicle emergence inhibition of the crop seeds placed on agar in Petri dishes. Triticum aestivum L. (A-E), Panicum miliaceum L. (F-J) and Medicago sativa L. (K-O) were evaluated 7 days following treatments of acetic acid (A,F,K), acetic acid with eugenol + geraniol (B,G,L) citric acid (C,H,M), lactic acid (D,I,N) and pelargonic acid (E,J,O) at concentrations of 5, 10 and 20%, respectively (eugenol + geraniol at 1.5%).

Figure 3. The percentage radicle emergence inhibition of the weed seeds placed on agar in Petri dishes. Lolium perenne L. (A-E), Cichorium intybus L. (F-J), Setaria viridis L. (K-O) and Vicia sativa L. (A-E) were evaluated 7 days following treatments of acetic acid (A,F,K,P), acetic acid with eugenol + geraniol (B,G,L,Q) citric acid (C,H,M,R), lactic acid (D,I,N,S) and pelargonic acid (E,J,O,T) at concentrations of 5, 10 and 20%, respectively (eugenol + geraniol at 1.5%).

Figure 5. The percentage shoot emergence inhibition of the crop seeds sown in peat in pots. Triticum aestivum L. (A-E), Panicum miliaceum L. (F-J) and Medicago sativa L. (K-O) were evaluated 10 days following treatments of acetic acid (A,F,K), acetic acid with eugenol + geraniol and pelargonic acid (B,G,L) citric acid (C,H,M), lactic acid (D,I,N) and pelargonic acid (E,J,O) at concentrations of 5, 10 and 20%, respectively (eugenol + geraniol at 1.5%).

Figure 6. The percentage shoot emergence inhibition of the weed seeds shown in peat in pots. Lolium perenne L. (A-E), Cichorium intybus L. (F-J), Setaria viridis L. (K-O) and Vicia sativa L. (A-E) were evaluated 10 days following treatments of acetic acid (A,F,K,P), acetic acid with eugenol + geraniol (B,G,L,Q) citric acid (C,H,M,R), lactic acid (D,I,N,S) and pelargonic acid (E,J,O,T) at concentrations of 5, 10 and 20%, respectively (eugenol + geraniol at 1.5%).