Biological Control of Lettuce Drop (Sclerotinia minor Jagger) Using Antagonistic Bacillus Species
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe Manuscript ID: applmicrobiol-3176951 examines biological control of lettuce drop (Sclerotinia minor) using antagonistic Bacillus species. It addresses an important and interesting subject as it boost reliable and benign biocontrol methods which can contribute towards future soil health and biodiversity efforts. Redirecting research efforts toward exploring alternative treatment techniques that are effective and safe, particularly those that would be, most importantly, techno-economical viable options, is crucial for controlling plant pathogens like Sclerotinia minor especially in IPM schemes. Hence the antifungal activities of the four tested strains inhibited S. minor and the calculated inhibition effects were reported. The ability of those strains to produce certain compounds that distinguish these biocontrol agents and/or antagonize the pathogen was proved. So, the results demonstrate a promising potential for the suppression of the S. minor infection of Lactuca sativa and expand the knowledge on antimicrobial applications of Bacillus strains.
Generally, the subject is worth publication and the authors did a good job but further clarifications and insights should have improved this research. I’d suggest the following points:
1) It is better to detail in the introduction commercial fungicides (biological and chemical) used against the target pathogen (S. minor) along with their merits and demerits.
2) In the Materials and Methods (M&M) section, although the four bacterial species have accession numbers at The National Center for Biotechnology Information (NCBI), the authors should give a brief on how the bacterial species were isolated and REF(s) utilized for their identification.
3) Also, in M&M, before saying that “The pathogen was cultivated at 280C on yeast extract medium (10 g yeast extract, 20 g glucose and peptone, and 20 g agar) for 7 days.”, the authors should mention how the fungal pathogen was originally isolated from its source. Likewise, the REF they used for S. minor identification must be stated.
4) In the results, standards and methods used to measure various activities of the four Bacillus strains should be indicated in each related table (please, set self-explanatory tables).
5) Likewise, each figure should be self-explanatory; e.g. Figure 4. A) Siderophores production by B. subtilis, P. megaterum, B. safensis, and B. mojavensis strains, (do you mean respectively for numbers 1, 2, 3, and 4 ??) expressed as a halo zone on CAS agar plate. And so on for other figures.
6) The authors should mention other commercial biocontrol agents (BCAs) currently registered for Sclerotinia in lettuce such as BACSTAR™, CLARITY®, SERENADE®) that are all strains of Bacillus subtilis.
7) The authors should have discussed how to include their tested biocontrol agents in IPM. At least they should extensively mention the current disease management methods of lettuce drop. Measures of management would include avoid planting in fields with a history of Sclerotinia, for crop rotation - via cultivating non-host crops - to be effective against Sclerotinia, it must be coupled with a weed control program that minimizes the chances of a ‘green bridge’ establishing and allowing sclerotia to persist in fields (small grain crops like maize, wheat, barley, oats, sorghum are not susceptible to infection by Sclerotinia spp. Onions, maize and spinach are reported as discouraging Sclerotinia). Deep plowing to keep sclerotia from the soil surface and drip irrigation below the soil surface may alleviate the disease severity or avoid the infection. Finally, application of fungicides should be at early plant growth to best control S. minor.
8) Many typos and grammar mistakes were found in the MS and should be corrected, to name but a few:
i) …..and they can the degraded starch as result a clear zone…..
ii) Bacillus sp. involve in specific degradation processes became of proteins whose activities are required to fulfil a specific function (e.g. cell cycle. differentiation process. stress response)
iii) On the seventh DAI symptoms was (were) observed with
iv) Most REFs were repeated in the text by both themselves and their numbers; e.g. environmental factors (Foley et al. 2016) [5].
v) REF number was also repeated in the REF section; e.g. 1. [1] Hao,… 2. [2] Abawi,…
Therefore, I would suggest resubmitting after major revision.
Author Response
Dear reviewer,
Thank you for the questions asked, for the corrections made, for your time and effort. We send answers to the questions asked and the corrections we made. If the answers and corrections are sufficient, I hope the article will be published in the special edition.
The Manuscript ID: applmicrobiol-3176951 examines biological control of lettuce drop (Sclerotinia minor) using antagonistic Bacillus species. It addresses an important and interesting subject as it boost reliable and benign biocontrol methods which can contribute towards future soil health and biodiversity efforts. Redirecting research efforts toward exploring alternative treatment techniques that are effective and safe, particularly those that would be, most importantly, techno-economical viable options, is crucial for controlling plant pathogens like Sclerotinia minor especially in IPM schemes. Hence the antifungal activities of the four tested strains inhibited S. minor and the calculated inhibition effects were reported. The ability of those strains to produce certain compounds that distinguish these biocontrol agents and/or antagonize the pathogen was proved. So, the results demonstrate promising potential for the suppression of the S. minor infection of Lactuca sativa and expand the knowledge on antimicrobial applications of Bacillus strains.
Generally, the subject is worth publication and the authors did a good job but further clarifications and insights should have improved this research. I’d suggest the following points:
- It is better to detail in the introduction commercial fungicides (biological and chemical) used against the target pathogen ( minor)along with their merits and demerits.
[Answer]-Accepted New information about the advantages and disadvantages of biological control was added REF 9, 13, and 14
The development of sclerotia also depends on several factors like location of sclerotia along the soil profile, moisture, temperature, and microflora in the soil (Hao et al. 2003) [8]. In Bulgaria, the management of Sclerotinia fungi relies primarily on the strategic application of synthetic fungicides. Fungicide efficacy is affected by disease pressure, weather conditions, method of application, fungicide overuse and crop canopy. Resistant to Sclerotinia lettuce cultivars are currently not available. In an attempt to find alternative strategies for the management of the disease, four naturally occurring bacterial isolates were screened for antagonism to this pathogen. Biological control is a result of interaction among microorganisms and includes mycoparasitism, antibiosis, competition for site and nutrient and induced resistance. It is one of the most important non-chemical strategies evaluated for the management of several plant pathogens in several cropping systems like roughing of wilted plants, crop rotation with Brassicaceae plants and subsurface-drip irrigation (Subbarao, 1998) [9]. Against diseases, mycoparasites that attack both sclerotia and mycelia of Sclerotinia spp., have been evaluated both in laboratory and field conditions by utilization of Trichoderma spp. (Jones and Stewart, 1997) [9], Sporidesmium sclerotivorum (Del Rio et al. 2002) [10], and Coniothyrium minitans (Partridge et al. 2006). Trichoderma spp., are perhaps the most widely used mycoparasites and numerous commercial formulations exist (Elias et al. 2016) [11]. Biocontrol agents nowadays registered for Sclerotinia in lettuce are based on utilization of Bacillus subtilis as a biocontrl agent (BACSTAR™, CLARITY®, SERENADE®). One reason for biocontrol failure is the neutral to alkaline pH of the lettuce fields soils (Barrière, 2014) [13]. The other possible reason is that biocontrol for lettuce drop is also incompatible with chemical control because of the effects of fungicides on the biocontrol agent (Adam, 1999) [14]. Although, many studies have been conducted to determine the effects of chitinolytic fungi on the growth and development of several fungal pathogens. Larena and Melgarejo, 1996 [12] applied chitinolytic actinomycetes and bacteria for the control of lettuce drop disease with possible production of chitinase, β-1,3-glucanase.
- In the Materials and Methods (M&M) section, although the four bacterial species have accession numbers at The National Center for Biotechnology Information (NCBI), the authors should give a brief on how the bacterial species were isolated and REF(s) utilized for their identification.
[Answer]-added in M&M
The four bacteria were isolated from soil intercropping with camelina (Camelina sativa L.) and vetch (Vicia sativa L.) on the experimental field of Agricultural University Plovdiv. DNA isolation was performed using the HiPurA™ Bacterial Genomic DNA Purification Kit (Canvax, Spain) following the manufacturer's protocol with the addition of 5 μL lysozyme in the lysis buffer, flowed by 20 μL proteinase K). Concentrations of isolated DNA samples (50 - 70 ng/µl of DNA) were determined by Qubit fluorometer (Thermo Fisher Scientific). To achieve a correct species identification of isolates, was used sequencing the 16S rRNA gene with universal primers (27F and 1492R) in Microsynth (Balgach, Switzerland) and comparing the obtained sequences with the world databases (https://blast.ncbi.nlm.nih.gov/ and https://www.ezbiocloud.net/). The accession in the NCBI Genbank is under submission sub14450584/ 08.06.2024 as follows: B. subtilis JAN25-4 accession OR731944.1, B. megaterium JAN33-2 accession OR731941.1 (NCBI submission sub13929898/ 29.10.2023) and B. safensis CAM24K3 with accession PP797578.1 and B. mojavensis CAM23K1 accession PP797576.1.
- Also, in M&M, before saying that “The pathogen was cultivated at 280C on yeast extract medium (10 g yeast extract, 20 g glucose and peptone, and 20 g agar) for 7 days.”, the authorsshould mention how the fungal pathogen was originally isolated from its source. Likewise, the REF they used for S. minor identification must be stated.
[Answer]-It was originally isolated from infected plants, cultivated in a greenhouse in Gradina village, Plovdiv, Bulgaria (42°07′58.8″ N, 25°12′00″ E). White, cottony fungal growth mycelia were observed on the basal plant surfaces and black structures called sclerotia were detected within the cottony growth (Fig. 1A). The pathogen was isolated and cultivated at 28°C on yeast extract medium (10 g yeast extract, 20 g glucose and peptone, and 20 g agar) for 7 days. Then, the conidia were collected by washing with cold sterile distilled water and used to prepare an inoculum at a concentration of 1x 104 spores/ml. S. minor infected plants were used in the plant pathogenicity tests according to Koch's postulates for the infection of the lettuce leaves is shown in Fig. 1 B. The 9 mm dicks were cut from the inoculated Petri discussed and used for re-infects the healthy lettuce leaves after surface sterilization (Fig. 1B). After infection, the fungal phytopathogen caused wilting and yellowing of lower leaves on 5–7 days of incubation and sclerotia were observed (Fig. 1 B). Figure 1C shows the healthy lettuce leaves.
- In the results, standards and methods used to measure various activities of the four Bacillus strains should be indicated in each related table (please, set self-explanatory tables).
[Answer]-Accepted and notes were added for each table
Note: a and b – indicate statistical references, * - indicates highly significant in p<0.05.
- Likewise, each figure should be self-explanatory; e.g. Figure 4. A) Siderophores production by subtilis, P. megaterum, B. safensis, and B. mojavensis strains, (do you mean respectively for numbers 1, 2, 3, and 4 ??) expressed as a halo zone on CAS agar plate. And so on for other figures.
[Answer]-Figure 4. A) Siderophores production by B. subtilis (1), P .megaterum (2), B. safensis (3) and B. mojavensis (4). Strains, expressed as a halo zone on CAS agar plate. B) S. minor filament growth, and С) changes in hyphal branching pattern after treatment with B. mojavensis siderophores on two layers CAS/PDA agar.
- The authors should mention other commercial biocontrol agents (BCAs) currently registered for Sclerotinia in lettuce such as BACSTAR™, CLARITY®, SERENADE®) that are all strains of Bacillus subtilis.
[Answer]-It is now mention in the Introduction
Biocontrol agents nowadays registered for Sclerotinia in lettuce are based on utilization of Bacillus subtilis as a biocontrl agent (BACSTAR™, CLARITY®, SERENADE®).
- The authors should have discussed how to include their tested biocontrol agents in IPM. At least they should extensively mention the current disease management methods of lettuce drop. Measures of management would include avoid planting in fields with a history of Sclerotinia, for crop rotation - via cultivating non-host crops - to be effective against Sclerotinia, it must be coupled with a weed control program that minimizes the chances of a ‘green bridge’ establishing and allowing sclerotia to persist in fields (small grain crops like maize, wheat, barley, oats, sorghum are not susceptible to infection by Sclerotinia Onions, maize and spinach are reported as discouraging Sclerotinia). Deep ploughing to keep sclerotia from the soil surface and drip irrigation below the soil surface may alleviate the disease severity or avoid infection. Finally, the application of fungicides should be at early plant growth to best control S. minor.
[Answer]-Accepted and the information was added to the conclusions
In the literature, the key management of Sclerotinia lettuce drop is taking in consideration records of disease incidence during several growing seasons. Cultural management practices include crop rotations with resistant nonhost plants, altering tillage practices, and using cover crops can help to reduce the risk of disease development (Peltier, 2012). Garza et al 2002 reported that crop rotation and no-tillage of soybeans are practically the most successful strategy of treatments for the prevention of sclerotinia stem rot in soybeans. The pathogen has more than 400 host plants and requires around 5 years of rotation of two non-host crops of Sclerotinia is necessary to decrease the infection level in sunflowers (Gulya, 1997). The research on the exploration of bacterial antagonists gains acceptance and application. The bacterial antagonists such as Trichoderma sp., Bacillus amyloliquefaciens BS6 and Pseudomonas sp. DF41 strains have well-studied mechanisms which play a role in the suppression of Sclerotinia infection both under field and greenhouse conditions (Peltier; Abdullah, 2008; Nandi, 2012). These strains produce extracellular metabolites like protease, hydrogen cyanide (HCN), and alginate as well as lipopeptides that suppress the development of Sclerotinia biocontrol (Berry, (2010).
- Many typos and grammar mistakes were found in the MS and should be corrected, to name but a few:
[Answer]-Accepted and corrected.
- i)…..and they can the degraded starch as result a clear zone…..
- ii)Bacillus involve in specific degradation processes became of proteins whose activities are required to fulfil a specific function (e.g. cell cycle. differentiation process. stress response)
iii) On the seventh DAI symptoms was (were) observed with
- iv)Most REFs were repeated in the text by both themselves and their numbers; e.g. environmental factors (Foley et al. 2016) [5].
- v)REF number was also repeated in the REF section; e.g. [1]Hao,… 2. [2] Abawi,…
Therefore, I would suggest resubmitting after major revision.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript successfully highlights the selection of Bacillus strains based on their enzymatic activities and their potential roles as biocontrol agents. However, several areas require further clarification and investigation:
· The author is advised to thoroughly review the manuscript to correct spellings and grammatical errors.
· It is noted that "Sclerotinia spp." on line 48 should be italicized. Additionally, lines 92-93 contain scientific names that also require italicization. Throughout the manuscript, there are several cases where scientific names are not presented in italics.
· Figure 2B lacks error bars. It is suggested that the authors include error bars based on SE or SEM. Furthermore, statistical analyses should be added to compare data at different levels of significance (1% or 5%).
· The expression of cell-bound amylases is influenced by multiple factors but does not explore the specific regulatory mechanisms/pathways that affect amylase production across different Bacillus strains.
· The research primarily conducted under in vitro or pot conditions, does not address the long-term stability of the amylolytic activity of these strains in field conditions.
· The conclusion section of the manuscript is inadequately discussed and requires significant improvement. It would be beneficial to outline potential avenues for future research based on the findings. This could involve investigating the mechanisms of action of the identified strains, assessing their efficacy under field conditions, and exploring their interactions with various plant species.
Comments on the Quality of English LanguageThe author is advised to carefully review the manuscript to correct spelling and grammatical errors. Simple sentences should be used to improve the overall readability of the manuscript.
Author Response
Dear reviewer, Thank you for the questions asked, for the corrections made, for your time and effort. We send answers to the questions asked and the corrections we made. If the answers and corrections are sufficient, I hope the article will be published in the special edition.
The manuscript successfully highlights the selection of Bacillus strains based on their enzymatic activities and their potential roles as biocontrol agents. However, several areas require further clarification and investigation:
- The author is advised to thoroughly review the manuscript to correct spellings and grammatical errors.
[Answer] - Accepted Spelling and grammatical mistakes have been corrected.
- It is noted that "Sclerotinia spp." on line 48 should be italicized. Additionally, lines 92-93 contain scientific names that also require italicization. Throughout the manuscript, there are several cases where scientific names are not presented in italics.
[Answer] – Accepted all the latin names have been italicized/
- Figure 2B lacks error bars. It is suggested that the authors include error bars based on SE or SEM. Furthermore, statistical analyses should be added to compare data at different levels of significance (1% or 5%).
[Answer] Error bars have been added.
- The expression of cell-bound amylases is influenced by multiple factors but does not explore the specific regulatory mechanisms/pathways that affect amylase production across different Bacillus strains.
[Answer] Further analysis of the expression of genes involved in starch metabolism will be conducted and the influence of abiotic factors will be examined.
- The research primarily conducted under in vitroor pot conditions, does not address the long-term stability of the amylolytic activity of these strains in field conditions.
[Answer]-Primarily the research was conducted in vitro and pots experiments. We have applied those strains in the greenhouse in Gradina village but just one year. We need to repeat the experiments for a few years ahead to observe the sustainability of the results.
- The conclusion section of the manuscript is inadequately discussed and requires significant improvement. It would be beneficial to outline potential avenues for future research based on the findings. This could involve investigating the mechanisms of action of the identified strains, assessing their efficacy under field conditions, and exploring their interactions with various plant species.
[Answer]-Further experiments will include the application of those strains in the greenhouse with with Sclerotinia infestation. Phytopathogen suppression by the tested bacterial strains will be followed with microbiological and metagenomic studies.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsYes, I mean respectively for numbers 1, 2, 3, and 4 ?? but it is ok also in the new version.
