The Effect of Mycorrhiza Fungi and Various Mineral Fertilizer Levels on the Growth, Yield, and Nutritional Value of Sweet Pepper (Capsicum annuum L.)
, Michał Tartanus 1, Robert Rosa 1,*, Anna Zaniewicz-Bajkowska 1, Henryk Dębski 2, Alena Andrejiová 3 and Andrii Dydiv 4
Round 1
Reviewer 1 Report
The effects of top dressing doses and the inoculation of root system with mycorrhizal fungi on the growth and yield of sweet pepper and the content of nutrients in fruits were stuided in this paper, which is useful to clear the interaction between top dressing doses with AMF inoculation. Some informations are not clear, which should be revised in follows.
1. keywords are not appropriate, plant growth parameters can be concised to plant growth; quality fruits can be changed into nutrient quality;
2. line 120 can be changed as, each treatment has 4 replicates plots, so the number of plots was 36.;
3. in 2.1, it is better to increase plant growth season inculding when the seedlings breeding and when they were transplanted;
4. in results part, it is better to compare the AMF infection rate of roots in AMF inoculation MS and MP and WM.
Author Response
The authors would like to thank the Reviewer for the comments and the evaluation of the manuscript, which made it possible to improve it and adapt it to the high requirements of the publishing house. Below we include our explanations and responses to your comments.
Ad. 1. As suggested, the Keywords were corrected.
Ad. 2. It was corrected according to Reviewer's note.
Ad. 3. Thank you for your remark. We will include it in our future experiments.
Ad. 4. The authors thank you very much for paying attention to this matter. During subsequent experiments with AMF or other symbiotic organisms we will certainly remember about it. In the present experiment, the degree of AMF infestation of the root system was not investigated. The experiment was purely agronomic. Its aim was to determine the optimal way of using mycorrhizal fungi with the lowest possible mineral fertilizer doses in order to obtain the highest yield without reducing its quality.
Reviewer 2 Report
The manuscript authored by Franczuk et al. investigated the impacts of arbuscular Mycorrhizal Fungi (AMF) and various mineral fertilizer levels on the growth, yield and nutritional value of sweet pepper (Capsicum annuum L.). The results showed that AMF inoculation of the root zone resulted in high sweet pepper yields of good quality. Meanwhile, the top-dressing dose of 50% applied during seedling production to plots with AMF resulted in a significant increase in the content of potassium, calcium and magnesium.
Some suggestions:
I suggest the authors should add some figures that may beneficial for readers to get the main point.
Note the spaces in the manuscript.
N-NO3 should be “N-NO3”.
It is better to provide some AMF colonization figures.
Author Response
The authors would like to thank the Reviewer for the time devoted to reading and analyzing the manuscript and for invaluable comments, which have been used to improve the quality of the manuscript.
During the experiment, no observations were made regarding the development of mycorrhiza. The research was agronomic and its aim was to investigate the effect of commercially available AMF vaccine on possible reduction of mineral fertilizer use without a significant decrease in pepper fruit yield and its quality. Reviewer's suggestion is very valuable and in the future will contribute to expanding the scope of our research, including research on other symbiotic microorganisms. There are few available studies on mycorrhization in pepper cultivation, which is why in the article we included literature on the use of mycorrhizal vaccines in the cultivation of other plant species.
Other comments of the reviewer's were taken into account while rewriting the manuscript.
- The Materials and Methods section has been expanded,
- The Discussion has been expanded,
- Editorial errors have been fixed.
Reviewer 3 Report
This manuscript presents a study on the influence of commercial AM fungal inoculum and different doses of fertilizer application on the growth, yield, and fruit quality of sweet bell pepper over three growing seasons. The results of the study indicate that inoculation of the commercial AM fungal inoculum increased the plant growth, nutrient uptake, and yield of bell pepper. Moreover, AM fungal inoculation reduced the fertilizer application for bell pepper production by 75% in addition to maintaining the fruit quality. Though the results are interesting, there are certain considerations mentioned below that need attention.
1. The first and foremost concern of the study is the nature of the experiment performed. It is not clear if the greenhouse experiments performed during the three growing seasons were conducted in pots or plots as both these terms appear throughout the text. The plot in horticulture means the small area of ground covered by the crop and pots means the containers in which plants are grown. In the present study, the plants were grown in plots as per line 121 and pots as per Lines 138–147. Therefore, please clarify if this is a pot culture study or a study performed in greenhouse plots.
2. The content and propagule number in the commercial inoculum Mykoflor needs clarification. The number of AM fungal taxa in the inoculum is specified as six and the number of infective propagules is 10000 per gram of inoculum as per the manufactures specification. However, other studies in the literature using this commercial inoculum have reported different AM fungal taxa and infective propagule numbers (https://doi.org/10.3390/agronomy11112355; https://doi.org/10.1007/s00572-019-00905-2; https://doi.org/10.1139/CJPS2012-239). Please clarify if the inoculum content was checked for the label specifications.
3. The amount of AM fungal propagule in the inoculum is also confusing. Lines 134–135 of the methods state that one gram of the dry inoculum consisted of 10000 propagules. When 10000 propagules are mixed with 450 ml of water, each ml of the inoculum solution contains 22 propagules. If so, how could the addition of 3 ml of the inoculum solution result in 100 propagules (3 x 22 = 66) as mentioned in Line 131?
4. Another aspect that needs elucidation is whether the plants were grown on the same substrate during the three growing seasons or in new substrates during each growing season. This is important because if the plants were grown in the same substrate there could be the cumulative accumulation of AM fungal propagules that could have affected the nature of the treatments.
5. Some important parameters were not determined in the present study. For instance, fertilization is known to affect AM formation and function and unfortunately, the influence of fertilization on AM fungal symbiosis was not assessed in the present study.
6. Add one or two sentences of conclusion in the abstract based on the observations of the present study.
7. Revise the methods by providing additional details. Important information like the greenhouse conditions (light, temperature, humidity, etc.), source of bell pepper seeds, number of plants per pot, cultural practices (irrigation, etc.), and methods used to determine plant growth parameters are missing.
8. Parametric analysis like ANOVA requires normal distribution of data and it is not clear if the data were checked for homogeneity before statistical analysis. Further, mention the type of ANOVA (one-way, two-way, etc.) used and the reason for not including years as a factor in the study.
9. The discussion is mostly comparative in nature. In addition to comparing, discuss critically the reasons for the accordance or discordance of the results with other studies. Further, some of the arguments made are not acceptable. The increased plant growth and nutrient uptake in AM fungi-inoculated plants are attributed to increased root production. Generally, inoculation of AM fungi increases the efficiency of the root system; therefore, mycorrhizal plants invest less in root production than non-AM plants. However, AM pepper plants had similar or higher root mass compared to non-AM plants (Table 3) suggesting that AM fungi failed to increase the efficiency of the root system. This is also confirmed by the lack of a demonstrable correlation between root biomass and the majority of the nutrients in fruits.
Other comments:-
10. Lines 29, 34, and 36: Please check and correct them as pots.
11. Lines 38–39: Avoid using words that are already there in the title as keywords.
12. Lines 49–51: Please revise the definition of mycorrhiza. The term mycorrhiza denotes not only the association of AM fungi with plant roots but also several other fungi that associate with plant roots. Therefore, mycorrhiza is the association between certain soil fungi and plant roots.
13. Line 52: Not all land plants form AM symbiosis. A sizable number of plant species do not form this symbiosis (e.g., plants in the cabbage family).
14. Lines 50, 59–60: Use the abbreviations once introduced.
15. Lines 74–76: High dependence on AM fungi for what?
16. Lines 82–84: This is already there in the first paragraph of the introduction.
17. Line 104: Is there any specific reason for choosing this bell pepper cultivar? Previous studies have shown that the AM fungal benefits not only vary with plant species but also vary with the varieties/cultivars of the same species. Consider this aspect in the present study.
18. Lines 120–121: Is it plots or pots? See the main comments.
19. Line 122: The glomeromycotean fungi are now known as arbuscular mycorrhizal fungi and not vesicular-arbuscular mycorrhizal fungi.
20. Lines 125–126: Correct the names of the fungi in line with the current nomenclature.
21. Line 128: What are clean cultures? Did you mean pure cultures?
22. Line 129: Was it the roots or the whole plant that was dried? Again is it ‘ground to fine powder’?
23. Line 130: There are no details on the cited website.
24. Line 131–132: Indicate how the propagule number in the inoculum was determined.
25. Lines 135: This is unusually a very large number for one gram of the inoculum.
26. Line 145: Is there any specific reason for choosing this particular sweet pepper variety?
27. Lines 162–163: Were the roots checked for mycorrhization at the time of transplantation?
28. Line 166: Explain the abbreviation at its first mention. Do likewise for all related ones.
29. Table 3: Indicate if it is the wet weight or the dry weight.
30. Line 318: Mention the correlation analysis in the methods section.
31. Lines 324 and 329: Why notations are mentioned repeatedly? Remove them if not used.
32. Lines 458–460: Justify how this is applicable in the present study when the plants are grown in soilless substrates.
Author Response
We would like to thank Reviewer for accepting our manuscript for evaluation and for valuable comments that we have included in the manuscript to increase its value.
In response to the comments in the review we inform that:
Ad. 1. The experiment was conducted three times, i.e. in 2014, 2015 and 2016. This information is provided in line 157. An experimental unit (combination) in the system of experiments is called a plot. In our experiment, 12 plants were grown on each plot (3 m2), with one plant in each pot. Thus, 12 pots constituted one experimental plot. This information was added in line 170-171.
Ad. 2. Information concerning the number of AM fungal taxa in the inoculums specified as six and the number of infective propagules as 10000 per gram of inoculum was included in the manuscript on the basis of information received from the manufacturer together with the vaccine.
We did not check inoculum content in the specifications. Different types of mycorrhizal vaccines are used for different groups of plants. The articles recommended by Reviewer concern other plant species (strawberry plants, fruit tree nurseries). Mycorrhizal vaccines provided in the papers indicated by Reviewer have a different microbiological composition (they contain fewer species of mycorrhizal fungi). According to the certificate sent by Mykoflor and issued by the Institute of Soil Science and Plant Cultivation State Research Institute in Puławy, Poland, the product contains the following fungi: Glomus intraradices, G. mosseae, G. claroideum, G. Gigaspora sp. Entrophospora sp. The amount of live propagules in 1 g of dry vaccine is approx. 10,000 CFU.
Ad. 3. The inoculate solution was prepared using 1.5 g (not 1.0 gram) of dry inoculate in 450 ml of distilled water (lines 132-136), i.e. 15 000 propagules in 450 ml of distilled water. This means that in 1 ml there are approx. 30 propagules (exactly 33.333), and in 3 ml approx. 100 propagation units (line 183). This means that about 100 propagation units were used for each plant.
Ad. 4. In each growing season, pepper plants were grown on a new substrate. This information was added to section 2.1.
Ad. 5. The aim of the experiment was not to determine the degree of colonization of the root system by mycorrhizal fungi. Our specialty is not microbiological experiments but agrotechnical ones. The aim of our research was to determine the optimal way of using mycorrhizal fungi with the lowest possible mineral fertilizer doses in order to obtain the highest yield without reducing its quality.
Ad. 6. We agree with the remark that the summarizing sentence in the Abstract is missing. It has been added.
Ad. 7. The experiment was conducted in an unheated greenhouse. Thermal and light conditions were dependent on the weather prevailing in this area. The greenhouse was equipped with an automatic ventilation system (flaps in the roof and side walls), which allowed regulating the temperature inside during days with high sunlight. The temperature was maintained in the range of 21-28°C. This information was added to the manuscript. During the experiment, parameters such as humidity and light intensity were not recorded.
As suggested by Reviewer, information was added to sections 2.1 and 2.2 on source of bell pepper seeds, number of plants per pot, cultural practices (irrigation, etc.), and methods used to determine plant growth parameters.
Ad. 8. In the experiment, two-way ANOVA was used. The data were checked for uniformity. Before performing the ANOVA test, assumptions about the normality of distributions and uniformity of variance were also checked. The Kolmogorov-Smirnov test was used for normality of distributions, and the Levene test was used to check the assumption of uniformity of variance in subgroups. The tests showed that the assumptions of the ANOVA test were met at p≤0.05.
The manuscript does not present the effect of growing seasons because there were no significant differences in the values of the examined traits in individual years and the interaction of factors and the years of research. This was due to the similar conditions of growing pepper (unheated greenhouse) across the growing seasons.
Ad. 9. In the studies, mycorrhization during seedling production (at an early stage of pepper growth) resulted in a root system of a higher weight (by about 8%) compared to non-mycorrhized plants. The stimulating effect of mycorrhiza contributed to a greater range of the root system and its better penetration into the substrate. This may have contributed to better uptake of K, Mg, Fe and Zn confirmed by positive correlation factors. The relevant information is included in the Discussion.
Ad. 10-20. Corrected and supplemented in accordance with the reviewer's comments.
Ad. 20. The names of the fungi were corrected in line with the current nomenclature.
Ad. 21. Corrected according to the reviewer's comments.
Ad. 22. According to the information provided by Mykoflor, the roots of Plantago laceolata were dried and ground. It was corrected in the text.
Ad. 23. Corrected and supplemented in accordance with the reviewer's comments.
Ad. 24-25. According to the vaccine certificate provided by the manufacturer, in 1 g there was 10,000 propagules, so in 1.5g there was 15,000. Thus, 15,000 propagules were dissolved in 450 ml of H2O. In 1 ml of solution there were 33.3 propagules, therefore in 3 ml of solution there were about 100 propagules. This information was left unchanged in the text.
Ad. 26-31. Corrected and supplemented in accordance with the reviewer's comments.
Ad. 32. Rationale for writing the manuscript has been extended.
Round 2
Reviewer 3 Report
The authors have taken into consideration all the suggestions raised in my previous review and revised the manuscript accordingly. The manuscript can now be accepted for publication.
