Effects of Chemotherapy on the Elimination of Various Viruses and Viroids from Grapevine

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The MS entitled “Effects of chemotherapy on the elimination of various viruses and viroids from grapevine” presents the results from experiments directed to finding of an appropriate approach for the sanitation of grapevine genotypes infected in mixed infections in various combinations with several viral and viroid pathogens. The antiviral activity of three chemotherapeutics, individually or in combinations, has been examined.

The conceptualization of the MS is well-augmented and the methodology is elaborated accurately and appropriately to the aim of the study.

The results are presented in details.

The establishment of new grapevine plantations with healthy planting material is a basic precondition for sustainable and qualitative yield. Since grapevine is a host of numerous viruses and viroids, very often, under field conditions it is difficult a healthy grapevine to be find because of that the determination of the virus status of the mother plants is the first step in the production of planting material. The sanitation of cultivars and clones from viral agents, aiming to obtain healthy nuclear stocks are essential work in the production of virus-free plants. In that respect the current MS gives valuable results concerning the sanitation of grapevine from viruses and viroids.

I think this MS is acceptable for publication after the precising of some points.

Specific comments

The virus/viroid names should be written in binominal nomenclature according to the recent rules of ICTV.

M&M

L103 – L105 It would be good if you notice that the genotypes included in the research are wine cultivars/clones suitable for production of qualitative wines.

L128 - L130 Please, define more precisely if each microplant was analyzed individually or the tested samples were composed from a certain number of microplants.

Results

Please, carefully check and consider Table 4. For example, you presented 5.5 % virus free plants from Kadarka P131 A1 pre-rooted and the same time 0.00 % elimination efficiency of ribavirin in HSVd and GYSVd-1. Could you explain that and other similar cases? Probably, in the term “virus-free” you do not included the viroids. Maybe you have to specify that you mean the percent of plantlets free only from viruses.

L255-256 “In the case of ribavirin, besides different concentrations…”, you used ribavirin only in concentration 25 mg/l.

L271 Is this success “…and GPGV (from 75 to 62.5%)…”? Please explain.

Indeed, the sanitation of the infected plant hosts is a very difficult accomplishment, especially in cases of co-infection with numerous viruses and viroids and nevertheless of the above comments, I think this research deserve to be accepted for publication.

Author Response

The authors would like to thank the reviewer for the helpful comments.

Specific comments of reviewer 1

1. The virus/viroid names should be written in binominal nomenclature according to the recent rules of ICTV.

We corrected the virus/viroid names in the manuscript based on the ICTV nomenclature.

M&M

2. L103 – L105 It would be good if you notice that the genotypes included in the research are wine cultivars/clones suitable for production of qualitative wines.

We supplemented the text:

For the experiments, based on preliminary virus test information (data not shown), five mother plants from four wine grape cultivars (Vitis vinifera L.)—Furmint P51 A1 and Furmint P51 ÜH2; Kadarka P131 A1; Kékfrankos Kt. 1/2 A1; and Sárfehér A1—were selected in 2022 and used to establish an in vitro culture. These cultivars are well known in Hungarian viticulture and provide excellent quality wines.

3. L128 - L130 Please, define more precisely if each microplant was analyzed individually or the tested samples were composed from a certain number of microplants.

We supplemented the text:

Each in vitro plant produced directly from the mother plants were tested with RT-PCR. Before each experiment, the presence of detected viruses and viroids was verified by RT-PCR in the used in vitro plants, individually.

Results

4. Please, carefully check and consider Table 4. For example, you presented 5.5 % virus free plants from Kadarka P131 A1 pre-rooted and the same time 0.00 % elimination efficiency of ribavirin in HSVd and GYSVd-1. Could you explain that and other similar cases? Probably, in the term “virus-free” you do not included the viroids. Maybe you have to specify that you mean the percent of plantlets free only from viruses.

We added the following supplement to Tables 4, 5, and 6: virus-free %: percentage of plantlets free from viruses, excluding viroids.

5. L255-256 “In the case of ribavirin, besides different concentrations…”, you used ribavirin only in concentration 25 mg/l.

We modified the sentence:

In the case of ribavirin, we also tested the effect of pre-rooting on virus elimination, which we found to be variable.

6. L271 Is this success “…and GPGV (from 75 to 62.5%)…”? Please explain.

We corrected the sentence:

"led to a slight change in the effectiveness of elimination for GFkV (from 50 to 62.5%) and GPGV (from 75 to 62.5%),"

Reviewer 2 Report

Comments and Suggestions for Authors

Overall assessment

The manuscript addresses virus elimination in grapevine using three chemotherapeutic agents (2-thiouracil, ribavirin, and zidovudine) in in vitro cultures, combined with small RNA HTS and RT-PCR. The topic is relevant and potentially useful for grapevine sanitation, but the manuscript requires substantial revision in terms of (i) rationale for treatment design, (ii) clarity and logic of the Results, and (iii) depth of the Discussion.

Major comments

• Rationale for antiviral concentrations and combinations

The basis for the chosen concentrations and combinations of ribavirin, zidovudine, and 2-thiouracil is not explained. Please clarify whether doses were chosen from the literature or from preliminary trials, and explain why a range of ribavirin concentrations was not evaluated.

• Structure and interpretation of the Results

The Results section is difficult to follow and often reads as a description of experiments rather than a guided presentation of findings. It should be reorganized and made more interpretative.

• Discussion not sufficiently developed

The Discussion is too brief and mostly descriptive. It should more clearly emphasize the main contribution of the study (e.g. relative performance of ribavirin vs. 2-thiouracil and zidovudine, including phytotoxicity), discuss which viruses are more difficult to eliminate and whether this depends on cultivars, and indicate briefly how these viruses/viroids impact growth and yield to underline the practical relevance.

Abstract and keywords

The abstract is overly detailed, listing all viruses/viroids with multiple percentage ranges. These details should be condensed and grouped; the abstract should state the main patterns.

“Elimination” and “efficacy” must be briefly defined (e.g. Elimination means virus undetectable by RT-PCR, or?).

The abstract would benefit from a clearer structure: short background → aim → methods → key grouped findings → concise conclusion/novelty.

The strong “first report” claim should be checked against the literature and, if necessary, slightly moderated.

Keywords should include the plant material (e.g. Vitis vinifera) and better reflect the scope of chemotherapeutic virus elimination in vitro.

Introduction

Verify that 2-thiouracil is correctly described (e.g. as a guanosine analogue); if not, correct this.

Improve the logical flow of lines 77–82 and avoid overgeneral statements in lines 92–100; explicitly state that chemotherapeutic efficacy is virus- (and possibly cultivar-) dependent.

Give the full name of “small RNA HTS” at first mention.

Materials and Methods

For the small RNA HTS workflow, add key details for reproducibility: CLC Genomic Workbench version, NCBI database version/date, E-value cutoff for viral hits, and whether default assembly/mapping parameters were used.

Rephrase lines 132–134 so it is clear that CTAB was used for RNA extraction, not plant propagation.

Consider moving the description of plant material/in vitro culture (current Section 2.3) to section 2.1, and specifying the criteria for selecting the five mother plants. The subtitle could indicate the link to virus elimination experiments.

In Section 2.4, clarify how chemotherapeutic agents were applied (tissue type, duration, presence in the medium), provide supplier and catalog information, and explicitly define what is meant each time by “the same MS medium” in line 181.

Results

Revise Table 4 according to standard scientific formatting and remove visible Excel artifacts.

In Sections 3.1–3.3, add interpretation of the data (patterns of infection, treatment effects on survival and elimination), not just procedural description. Reconsider how shoot survival and virus elimination are presented—either as separate subsections or with a title that reflects both; improved tables or figures could help highlight key trends.

Avoid subjective wording such as “Unfortunately” in Section 3.4, and clarify whether “varieties” refers to cultivars (e.g. line 189).

Discussion

Expand the Discussion to interpret the findings more fully.

For example:

Emphasize the relative performance of ribavirin versus 2-thiouracil and zidovudine, including phytotoxicity versus efficacy trade-offs.

Discuss which viruses were particularly difficult to eliminate (and possible reasons) and whether this varied with cultivar.

Briefly connect virus/viroid presence to potential effects on growth and yield.

When mentioning “other chemotherapeutic agents” in line 295, specify which ones or provide examples.

Author Response

The authors would like to thank the reviewer for the detailed and helpful comments.

Major comments

• Rationale for antiviral concentrations and combinations

The basis for the chosen concentrations and combinations of ribavirin, zidovudine, and 2-thiouracil is not explained. Please clarify whether doses were chosen from the literature or from preliminary trials, and explain why a range of ribavirin concentrations was not evaluated.

We supplemented section 2.4:

In the case of ribavirin, we chose a concentration of 25 mg/l based on the literature, which was confirmed by our preliminary experiments. In the case of zidovudine, very different concentrations were used for different species, so we also tried to use a wider range (10-120 mg/l). Due to its phytotoxicity described in the literature, we tested 2-thiouracil in lower concentrations.

• Structure and interpretation of the Results

The Results section is difficult to follow and often reads as a description of experiments rather than a guided presentation of findings. It should be reorganized and made more interpretative.

We reorganized the Results section, and will provide details later in the detailed section.

• Discussion not sufficiently developed

The Discussion is too brief and mostly descriptive. It should more clearly emphasize the main contribution of the study (e.g. relative performance of ribavirin vs. 2-thiouracil and zidovudine, including phytotoxicity), discuss which viruses are more difficult to eliminate and whether this depends on cultivars, and indicate briefly how these viruses/viroids impact growth and yield to underline the practical relevance.

We reorganized the Discussion section, and will provide details later in the detailed section.

Abstract and keywords

1. The abstract is overly detailed, listing all viruses/viroids with multiple percentage ranges. These details should be condensed and grouped; the abstract should state the main patterns.

Abstract was corrected.

2. “Elimination” and “efficacy” must be briefly defined (e.g. Elimination means virus undetectable by RT-PCR, or?).

We supplemented the text:

Among the tested agents, ribavirin had the broadest elimination effect (the virus was not detectable by RT-PCR after 8 months) on most viruses.

It proved to be particularly effective (the virus was not detectable by RT-PCR in most of the tested plants.)

3. The abstract would benefit from a clearer structure: short background → aim → methods → key grouped findings → concise conclusion/novelty.

Abstract was corrected.

4. The strong “first report” claim should be checked against the literature and, if necessary, slightly moderated.

We checked the claim but couldn't find any publications that contradicted it.

5. Keywords should include the plant material (e.g. Vitis vinifera) and better reflect the scope of chemotherapeutic virus elimination in vitro.

We modified the keywords:

Keywords: Vitis vinifera; in vitro; antiviral agents; sRNA HTS; ribavirin; zidovudine; 2-thiouracil

Introduction

1. Verify that 2-thiouracil is correctly described (e.g. as a guanosine analogue); if not, correct this.

The authors thank you for pointing this out. We have corrected it:

Although 2-thiouracil, a nucleobase analogue of uracil in which the C-2 oxo group is replaced by a thioxo group, shows strong phytotoxic effects [11, 20],

2. Improve the logical flow of lines 77–82 and avoid overgeneral statements in lines 92–100; explicitly state that chemotherapeutic efficacy is virus- (and possibly cultivar-) dependent.

We have reconsidered and modified the above sections.

3. Give the full name of “small RNA HTS” at first mention.

We have corrected this in the abstract and also in the introduction:

Abstract: Viruses and viroids were identified in the mother plants of different grapevine cultivars by small RNA HTS (High-Throughput Sequencing), and RT-PCR verified the results.

Introduction: Their viromes were determined using small RNA HTS (High-Throughput Sequencing).

Materials and Methods

1. For the small RNA HTS workflow, add key details for reproducibility: CLC Genomic Workbench version, NCBI database version/date, E-value cutoff for viral hits, and whether default assembly/mapping parameters were used.

We supplemented the text:

For the bioinformatics analysis of the fastq files, we used CLC Genomic Workbench 20 /20.0.4 version (Qiagen). Adapter sequences were trimmed from the obtained reads. To assemble contigs from the nonredundant reads de novo, the assembly command (using default options: word size 20, bubble size 50, and simple contig sequences and min 35 nt length) in CLC was used. These contigs were aligned to reference genomes of grapevine viruses and viroids in the NCBI ) using the NCBI Plant hosted Viral Reference genomes (downloaded at 0512 2023) using the BLASTN algorithm. Viral hits were evaluated based on their E-value (hits with E E-value below 10-2) were considered as positives. Nonredundant and redundant reads were mapped to all of the viruses and viroids, which resulted in hits during the contig blast. Normalized read counts (read/1 million reads – RPM) for these viruses were determined together with the coverage of the genome by small RNA reads.

2. Rephrase lines 132–134 so it is clear that CTAB was used for RNA extraction, not plant propagation.

We rephrased the sentence:

For PCR-based tests, total nucleic acids were isolated from leaves of the green-house-grown mother plants and in vitro propagated plants according to a simplified CTAB-based protocol [33].

3. Consider moving the description of plant material/in vitro culture (current Section 2.3) to section 2.1, and specifying the criteria for selecting the five mother plants. The subtitle could indicate the link to virus elimination experiments.

We moved Section 2.3 to 2.1, and subtitle was changed:

Establishment of in vitro cultures for virus elimination experiments

We specified the criteria for selecting the five mother plants:

The mother plants were selected to represent a wide range of viruses (nepoviruses, ampeloviruses, vitiviruses, maculaviruses, foveaviruses, trichoviruses), including those that are economically significant (GFLV, GLRaV-1, GVA, GFKV).

4. In Section 2.4, clarify how chemotherapeutic agents were applied (tissue type, duration, presence in the medium), provide supplier and catalog information, and explicitly define what is meant each time by “the same MS medium” in line 181.

Chemotherapy treatments were performed on the apical shoots (1-2 cm) of the micropropagated virus-infected plants using different concentrations and combinations of antiviral agents, as detailed in Table 2. Apical shoots of some clones (that showed symptoms of high phytotoxicity) were pre-rooted for two weeks on MS medium containing 0.8 mg/l IBA (isobutyric acid [12]) prior chemotherapy treatment. Thus, freshly cut shoot tips or pre-rooted shoot tips were placed on MS medium containing the antiviral agent.

After approximately 12 weeks of chemotherapy, the 2 mm shoot tips were removed from the treated plants, and were placed on MS media free for antiviral agent in petri dishes supplemented with 0.02 mg/l BA (benzyl-adenine) and 0.01 mg/l NAA (naphty-lacetic acid) for regeneration.

After three weeks, the growing shoots were transferred to MS medium free from growth regulators and containing only 1% sucrose and half the amount of macro salts. These shoots were maintained and propagated on the same medium. After 8 months, these plants were then tested using RT-PCR for the presence of the viruses and viroids that were detected at the beginning of the treatment. The ratio of infection after and before treatment allowed us to determine the efficacy of the chemotherapeutic agents.

We defined what is meant each time by “the same MS medium” in Section 2.1:

The shoots were then washed three times in sterile distilled water, and the shoot tips (about 0.5-1 cm in size) were prepared and placed on a solid MS medium consisting of half the amount of macro salts (0.826 g/l MS Macro Salt mixture, Duchefa-M0305), microsalts (1 g/l MS Micro-Salt Mixture, Duchefa-M0301), vitamins (MS Vitamin Mixture, Duchefa-M0409 according to the supplier’s instructions), 2% sucrose, solidified with 3 g/l gelrite (Duchefa-G1101) supplemented with 0.5 mg/l BA (benzyl-adenine) or 0.5 mg/l mT (meta-Topolin, [4]). We used the above-mentioned basic MS medium (hereinafter referred to as MS medium) with minor modifications in all experiments.

In section 2.4, we referred to section 2.1:

Apical shoots of some clones (that showed symptoms of high phytotoxicity) were pre-rooted for two weeks prior chemotherapy treatment on the MS medium (in Section 2.1) containing 0.8 mg/l IBA (isobutyric acid [12]) and 1% sucrose.

We provided supplier and catalog information of chemotherapeutic agents:

Chemotherapy treatments were performed on the apical shoots (1-2 cm) of the micropropagated virus-infected plants using different concentrations and combinations of antiviral agents (2-thiouracil – ACRO151841000; ribavirin – Duchefa R0182; zidovudine –  ACRO454980050), as detailed in Table 2.

Results

1. Revise Table 4 according to standard scientific formatting and remove visible Excel artifacts.

Tables 3, 4, 5, and 6 were revised.

2. In Sections 3.1–3.3, add interpretation of the data (patterns of infection, treatment effects on survival and elimination), not just procedural description. Reconsider how shoot survival and virus elimination are presented—either as separate subsections or with a title that reflects both; improved tables or figures could help highlight key trends.

We modified the specified sections, re-edited the tables, and inserted a new section (3.5. The effect of pre-rooted shoots on phytotoxicity and chemotherapeutic efficacy).

3. Avoid subjective wording such as “Unfortunately” in Section 3.4, and clarify whether “varieties” refers to cultivars (e.g. line 189).

We deleted the incorrect word: Unfortunately

Line 189 was corrected:

Five mother plants representing four cultivated varieties were selected in such a way so that they contained viruses in combinations and that different virus families were represented.

Discussion

Expand the Discussion to interpret the findings more fully.

For example:

1. Emphasize the relative performance of ribavirin versus 2-thiouracil and zidovudine, including phytotoxicity versus efficacy trade-offs.

In conclusion, ribavirin is a widely applicable chemotherapeutic agent with mod-erate phytotoxicity at 25 mg/l, while the use of zidovudine and 2-thiouracil in combi-nation with ribavirin may increase the efficiency of elimination.

2. Discuss which viruses were particularly difficult to eliminate (and possible reasons) and whether this varied with cultivar.

Since GFLV is not a phloem-limited virus, removal of this nepovirus proved to be the most difficult of all the viruses tested. Ribavirin alone could not eliminate GFLV in our experiments, despite studies reporting its effectiveness [13, 45]. However, Dolatabadi et al. [46] reported similar results to ours, observing that GFLV could not be eliminated from the grapevine cultivar ‘Peykani’ using 25 mg/l ribavirin. These results suggest that the use of ribavirin on GFLV may be genotype-dependent, and a similar phenomenon may be the cause of failure in the Sárfehér and Furmint cultivars.

3. Briefly connect virus/viroid presence to potential effects on growth and yield.

We supplemented the discussion:

Virus-induced diseases cause significant economic damage. For example, grape-vine leafroll disease can cause losses of $2-23,000 per hectare even in cases of moderate infection [44]. It is therefore extremely important to produce propagating material that is free from viruses that cause significant economic damage. Among the available virus removal methods, chemotherapy is one of the most promising procedures.

4. When mentioning “other chemotherapeutic agents” in line 295, specify which ones or provide examples.

We corrected the sentence:

Ribavirin proved to be effective for elimination of GFkV, GPGV and GRSPaV, with results similar to those previously published for the application of ribavirin [5, 16-17].

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

 

This manuscript is giving information regarding usage of different chemotherapeutic agents for the elimination of pathological problems of grapevine.

 

Give information regarding mother plants.

 

On what kind of soil they were breed. Insert in the manuscript.

 

Did you use any fertilizer? If yes what kind.

 

Did you use any substances for the protection against pests?

 

How application of different chemotherapeutics agents influence on the sensory properties of grapes. Explain it in the manuscript.

 

How application of different chemotherapeutics agents influence on the content of biologically active compounds such as polyphenols in grapes. Explain it in the manuscript.

 

Is there any side effects of application chemotherapeutic agents on grapevine? Expalain it in the manuscript.

Author Response

The authors would like to thank for the helpful comments.

1. Give information regarding mother plants.

We added more information about mother plants:

For the experiments, based on preliminary virus test information (data not shown), five mother plants from four wine grape cultivars (Vitis vinifera L.)—Furmint P51 A1 and Furmint P51 ÜH2; Kadarka P131 A1; Kékfrankos Kt. 1/2 A1; and Sárfehér A1—were selected in 2022 and used to establish an in vitro culture. These cultivars are well known in Hungarian viticulture and provide excellent quality wines.

2. On what kind of soil they were breed. Insert in the manuscript.

We inserted the following sentence into the manuscript:

The mother plants were maintained under greenhouse conditions in a mixture of peat and perlite (2:1).

3. Did you use any fertilizer? If yes what kind.

Genezis NPK (11:11:18+16 S) was applied by irrigation.

4. Did you use any substances for the protection against pests?

Orange oil and potassium soap was used against pests.

5. How application of different chemotherapeutics agents influence on the sensory properties of grapes. Explain it in the manuscript.

We did not find any publications on how chemotherapy affects the sensory properties of grapes. This is probably because the technology is almost always applied to plants in vitro.

6. How application of different chemotherapeutics agents influence on the content of biologically active compounds such as polyphenols in grapes. Explain it in the manuscript.

We did not find anything published on how chemotherapy affects biologically active compounds and polyphenols in grapes. This is most likely because this method is usually used in vitro on plants.

7. Is there any side effects of application chemotherapeutic agents on grapevine? Expalain it in the manuscript.

We added the following supplement to the introduction:

During in vitro virus elimination, plants are exposed to various stress factors, the se-verity of which depends on the in vitro conditions, the degree of wounding, the toxicity and concentration of the chemotherapeutic agent used, and the duration of exposure. This can result in low survival rates, stunted growth, chlorosis, morphological changes, etc. [11].

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Recommendation: The authors have adequately addressed the concerns raised in the first review. I therefore recommend acceptance after minor revision.

General assessment

The authors have revised the manuscript thoroughly and have addressed the main points raised in my previous review. Overall, the manuscript is now clearer, more logically structured, and scientifically sound.

Minor comments

Abstract

The abstract has been improved compared to the previous version. However, it is still detailed in terms of listing specific viruses and numerical elimination rates. I recommend further condensing these details and focusing more on general patterns (e.g. relative performance of ribavirin versus the other agents and the lack of efficacy against viroids).

The Results section is clearer than before, but in a few places it still reads as a description of tables. Please define, at first mention, what is meant by "virus elimination". Adding short interpretative statements highlighting key trends (virus-dependent and cultivar-dependent responses, as well as phytotoxicity versus efficacy) would further improve readability.

Tables and terminology

Please double-check table formatting and consistency in terminology (e.g. cultivar vs. clone, abbreviations, percentage notation). Minor layout refinements would improve clarity.

Author Response

The authors would like to take this opportunity to thank for the helpful comments and guidance.

Abstract

1. The abstract has been improved compared to the previous version. However, it is still detailed in terms of listing specific viruses and numerical elimination rates. I recommend further condensing these details and focusing more on general patterns (e.g. relative performance of ribavirin versus the other agents and the lack of efficacy against viroids).

We condensed the abstract in accordance with the recommendations.

2. The Results section is clearer than before, but in a few places it still reads as a description of tables. Please define, at first mention, what is meant by "virus elimination". Adding short interpretative statements highlighting key trends (virus-dependent and cultivar-dependent responses, as well as phytotoxicity versus efficacy) would further improve readability.

Virus elimination was defined in the tables 4,5,6 and in lines 264-265: … chemical was used for elimination (the virus was not detectable by RT-PCR after 8 months) alone

Line 219 was modified: Thus, we detected seven viruses…

Lines 237-246: we merged the paragraphs.

Lines 240-241: We added a short statement: „Since Kékfrankos was the only cultivar on which we observed phytotoxicity in the presence of zidovudine, this phenomenon is probably cultivar-dependent.”

Line 267: We added a short statement: „Another explanation could be the virus-dependent effect of 2-thiouracil.”

Lines 283-284: We added a short statement: „The results may therefore show the virus and cultivar dependence of the treatment.”

Lines 295-297: We added a short statement: „The results show that the combination of ribavirin and 2-thiouracil can reduce the virus dependence of ribavirin.”

Lines 306-309: We added a short statement: „The data show that pre-rooting alone increases the survival rate of shoot tips. This phenomenon can be explained by the fact that rooted plants are more resistant to the effects of chemotherapeutic treatments. However, the effects of chemotherapeutic compounds may also be reduced through the roots.”

Lines 312-315: We improved the readability: “The efficiency of GFkV and GRSPaV elimination from this clone also decreased, but the efficiency of GRSPaV elimination from the other four clones did not change. Our results show that the effect of pre-rooting on elimination efficiency is cultivar-dependent and generally reduces it.”

3. Tables and terminology

Please double-check table formatting and consistency in terminology (e.g. cultivar vs. clone, abbreviations, percentage notation). Minor layout refinements would improve clarity.

We checked the tables 4,5,6 and modified the headers and footers. We highlighted the positive results to improve clarity.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors, 

Insert in the revised version of your abstract answer on the question number 3. 

Author Response

The authors would like to take this opportunity to express their gratitude for the useful additions.

1. Insert in the revised version of your abstract answer on the question number 3. 

The following sentence was inserted into the first paragraph of section 2.2:

For nutrient supply, Genezis NPK (11:11:18+16 S, Bige Holding Kft.) was applied by irrigation.