Round 1

Reviewer 1 Report

The subject of the manuscript is interesting, and a number of analyses have been performed by the authors to support their hypothesis. Nevertheless, data analysis should be improved and various assumptions that are not supported by the results should be thoroughly revised.

Below some detailed comments:

 

Abstract

The term “bacterial fertilizer” should be reformulated since evidence of plant growth promotion by Bacillus amyloliquefaciens QSB-6 is not provided by this study.

Bacterial name missing “verify the effects of QSB-6”

 

Introduction

This study is based on the assumption that “Fusarium was one of the main soil-borne pathogenic fungi that cause ARD” (line 44), therefore most if the study is oriented towards this fungus. Nevertheless, a number of studies reports that the role of Fusarium towards plants is not so strict, but rather may also be associated with good soil health in permanent tree crops, for example:

Bacon, C.W., Yates, I.E., 2006. Endophytic Root Colonization by Fusarium Species: Histology, Plant Interactions, and Toxicity BT  - Microbial Root Endophytes, in: Schulz, B.J.E., Boyle, C.J.C., Sieber, T.N. (Eds.), . Springer Berlin Heidelberg, Berlin, Heidelberg, pp. 133–152. doi:10.1007/3-540-33526-9_8

Therefore this concept should be reformulated in the manuscript.

 

Results

Three orchards have been tested in this study, but data on the effect of the Bacillus QSB-6 strain treatment on the growth of apple plants have been analysed separately per each site (results in paragraph 3.1), in this way it is difficult to draw conclusions on the effect of strain QSB-6.

In Data analysis paragraph it is reported that “Comparisons were made using the least significant difference (LSD) test” (line 231), while the note in Table 2 reports that “Numbers followed by the same letter were not significantly different based on Duncan Multiple Range Test”. It is not clear which statistical test was performed to compare plant growth parameters. Furthermore, in Table 2, T1 and T2 treatment groups share the same letter in almost all conditions, meaning that the differences between the groups are not statistically significant, how can the authors say that the bacterial fertilizer treatment (T2) significantly promoted plant growth if it did not differ from control (T1)??

 

Authors state that “strain QSB-6 can reproduce normally after inoculating the soil” (line 362) but no specific viability test was performed.

 

Diversity indices described in paragraph 3.6 (lines 387-391) would benefit from bibliographic references.

Data analysis of diversity indices should be revised, since in line 392 it is reported that “The diversity of soil fungi communities was significantly increased” , while in the abstract “QSB-6 can significantly reduce the diversity of soil fungi” line 23. Simpson’s index does not differ between T1 and T2 in fungi. Same for Shannon index of bacterial communities.

 

Discussion

The first part of the discussion (lines 417-433) should be moved to the introduction.

It is stated that “This study found that the addition of strain QSB-6 can significantly reduced the content of the main phenolic acids in the soil” lines 482-483, but the significance of the results reported in figure 3 is not reported.

 

Conclusion

Which “pot study”? reference missing.

Strain XNRB-3? Line 519

 

English language should be revised.

Author Response

Response to Reviewer 1 Comments

 

 

Point 1: The term “bacterial fertilizer” should be reformulated since evidence of plant growth promotion by Bacillus amyloliquefaciens QSB-6 is not provided by this study.

Bacterial name missing “verify the effects of QSB-6”

Response 1: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 2: This study is based on the assumption that “Fusarium was one of the main soil-borne pathogenic fungi that cause ARD” (line 44), therefore most if the study is oriented towards this fungus. Nevertheless, a number of studies reports that the role of Fusarium towards plants is not so strict, but rather may also be associated with good soil health in permanent tree crops, for example:

 

Bacon, C.W., Yates, I.E., 2006. Endophytic Root Colonization by Fusarium Species: Histology, Plant Interactions, and Toxicity BT  - Microbial Root Endophytes, in: Schulz, B.J.E., Boyle, C.J.C., Sieber, T.N. (Eds.), . Springer Berlin Heidelberg, Berlin, Heidelberg, pp. 133–152. doi:10.1007/3-540-33526-9_8

 

Therefore this concept should be reformulated in the manuscript.

 

Response 2:  Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 3: Three orchards have been tested in this study, but data on the effect of the Bacillus QSB-6 strain treatment on the growth of apple plants have been analysed separately per each site (results in paragraph 3.1), in this way it is difficult to draw conclusions on the effect of strain QSB-6.

 

Response 3: Thank you for your constructive comments. In this experiment, three orchards with different textures (loam, sandy loam and clay loam) were selected to evaluate the biocontrol effect of strain QSB-6, and it was found that strain QSB-6 had the most significant effect on the growth promotion of replanted apple young trees under the condition of loam soil (TY), followed by sandy loam (QX) and clay loam (LZ). This was consistent with the results of previous studies (Sheng, 2020). The occurrence of Apple replant disease was the most serious under clay loam conditions, followed by sandy loam and loam. However, strain QSB-6 also exerts a good biocontrol effect under the clay loam condition. The overall growth of the replanted apple saplings treated with T2 was better than that with T1(Figure 1). From this, it can be concluded that the QSB-6 bacterial fertilizer treatment can promote the growth of replanted young apple trees. Thank you very much!

 

Sheng, Y.F.; Wang, H.Y.; Wang, M.; Li, H.; Xiang, L.; Pan, F.B.; Mao, Z.Q. Effects of soil texture on the growth of young apple trees and soil microbial community structure under replanted conditions. Horticultural Plant Journal 2020, 6(3), 123-131.

Point 4: In Data analysis paragraph it is reported that “Comparisons were made using the least significant difference (LSD) test” (line 231), while the note in Table 2 reports that “Numbers followed by the same letter were not significantly different based on Duncan Multiple Range Test”. It is not clear which statistical test was performed to compare plant growth parameters. Furthermore, in Table 2, T1 and T2 treatment groups share the same letter in almost all conditions, meaning that the differences between the groups are not statistically significant, how can the authors say that the bacterial fertilizer treatment (T2) significantly promoted plant growth if it did not differ from control (T1)??

Response 4: Thank you for your constructive comments. According to your suggestion, the data has been analyzed. In Table 2, the plant height and ground diameter of the T1 and T2 treatments of the LZ orchard were also different, but they were not significant, and there were significant differences in the number of branches and the average branch length. Figure 1 showed that the overall growth of the replanted apple saplings treated with T2 was better than that with T1. The insignificant difference between LZ orchard treatments may be related to the high severity of continuous cropping under clay loam conditions (Sheng, 2020) and the slower growth and reproduction of strain QSB-6. It can also be seen from the results in Table 3 that the number of bacteria in the LZ orchard treated with T2 was significantly lower than that in the QX and TY orchards. There were significant differences between the T1 and T2 treatments of QX and TY orchards. Thank you very much!

 

Point 5: Authors state that “strain QSB-6 can reproduce normally after inoculating the soil” (line 362) but no specific viability test was performed.

 

Response 5: Thank you for your constructive comments. In this experiment, microbial culture counts were carried out on the rhizosphere soil of different treatments using the dilution method of plate counting. It was found that the number of bacteria in the rhizosphere soil of the inoculated strain QSB-6 was significantly higher than that of other treatments, and the results of the previous potting treatments were consistent (Duan, 2021), and most of the bacterial colonies cultured on the plate were consistent with the morphology of strain QSB-6. A single colony with the same morphology was randomly picked and sequenced, and the similarity with the strain QSB-6 was found to be 100% (results not shown). so it was concluded that strain QSB-6 can reproduce normally after inoculating the soil, and the addition of the carrier also provided nutrients for strain QSB-6 to grow normally in the soil. Thank you very much!

Duan, Y.N.; Chen, R.; Zhang, R.; Jiang, W.T.; Chen, X.S.; Yin, C.M.; Mao, Z.Q. Isolation, identification, and antibacterial mechanisms of Bacillus amyloliquefaciens QSB-6 and its effect on plant roots. Frontiers in microbiology 2021, 12.

 

Point 6: Diversity indices described in paragraph 3.6 (lines 387-391) would benefit from bibliographic references.

 

Response 6: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 7: Data analysis of diversity indices should be revised, since in line 392 it is reported that “The diversity of soil fungi communities was significantly increased” , while in the abstract “QSB-6 can significantly reduce the diversity of soil fungi” line 23. Simpson’s index does not differ between T1 and T2 in fungi. Same for Shannon index of bacterial communities.

 

Response 7: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Simpson, Shannon, and Brillouin Index are all used to measure the diversity of microorganisms. There are limitations to using a single index to measure diversity.  Shannon index is closely related to richness and more sensitive to sparse species, while Simpson index is closely related to evenness and more sensitive to enriched species (Magurran, 1998; Xu, 2011).  No difference in Simpson index between T1 and T2 treatments can only indicate that the addition of organic carrier and bacterial fertilizer resulted in the similarity of the enriched species (the relative important value of newly added species is higher than 0.72) in the rhizosphere soil, while the Shannon and Brillouin Indexes of the T2 treatment were significantly lower than those of the T1 treatment. No difference in Shannon index between T1 and T2 treatments can only indicate that the addition of organic carrier and bacterial fertilizer results in the similarity of sparse species (the relative important value of newly added species is less than 0.72) in rhizosphere soil, while the Simpson and Brillouin Indexes of T2 treatment were significantly higher than those of T1 treatment. Thank you very much!

 

Magurran, A. E. (1988). Ecological diversity and its measurement. Princeton university press.

 

Xu, Q., Zhang, F., Xu, Z. Q., Jia, Y. L., & You, J. M. (2011). Some characteristics of Simpson index and the Shannon-Wiener index and their dilution effect. Pratacultural Science, 4, 527-531.

 

Point 8: The first part of the discussion (lines 417-433) should be moved to the introduction.

 

Response 8: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 9: It is stated that “This study found that the addition of strain QSB-6 can significantly reduced the content of the main phenolic acids in the soil” lines 482-483, but the significance of the results reported in figure 3 is not reported.

 

Response 9: Thank you for your constructive comments. According to your suggestions, the relevant data of Figure 3 has been added to the supplementary materials. Thank you very much!

 

Point 10: Which “pot study”? reference missing.

 

Response 10: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 11:Strain XNRB-3? Line 519

 

Response 11: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 12:English language should be revised.

 

Response 12: Thank you for your constructive comments. According to your suggestions, We have revised our manuscript by EditorBar Language Editing, Beijing for English writing. Thank you very much!

 

EditorBar Language Editing No. 35, Tsinghua East Road, Beijing, China 100083 Email: runse@editorbar.com Phone: +86-10-5620-8614

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Manuscript Information

 

ID	LE202112080129
Editing date	2021-12-14
Title	Effects of Bacillus amyloliquefaciens QSB-6 on the growth of replanted apple trees and soil microbial environment
Corresponding author	Zhiquan Mao
Language writing before editing	□ Very poor      □ Poor      □ Fair      ■ Good      □ Very good      □ Excellent
Recommendation after language editing	■ Submitting to target journal directly □ Submitting to target journal after minor revision □ Re-editing required after major revision □ Not suitable for publication
Overview comments	This manuscript has been thoroughly edited for English word choice, grammar, and sentence structure. Please see a small number of comments to the authors in the manuscript margins. Very best wishes for continued research success!

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Senior Editor

The Pennsylvania State University

Horticulture

 

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Editor in Chief

Editorbar Language Editing, Beijing, China

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We are truly grateful to you critical comments and thoughtful suggestions on our manuscript, comprehensive the comments of reviewer # 2 and 3, we have improved and revised some of the content in the manuscript, and checked the full text. Please review again during your busy schedule. Thank you very much! The specific changes are as follows:

Response to Reviewer 2 Comments

 

Point 1:  The entire manuscript is poorly adapted to the TEMPLATE requirements of the Horticulture journal, please pay special attention to the References section, but also to the formatting of Tables and chapter titles and sub-headings. 


 

Response 1: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 2: Keywords should be in alphabetical order and should not repeat words appearing in the title of the manuscript.

 

Response 2: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 3: Tables 2, 3 and 4 are difficult to read, please add lines between LZ, QX and TY treatments (Tables 2 and 3) and between Fungi and Bacterial treatments (Table 4).

 

Response 3: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 4: In Tables 3 and 4 and Figures 2-5, abbreviations should be explained or referred to Table 2.

 

Response 4: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

 

 

 

 

 

 

 

Response to Reviewer 3 Comments

 

Point 1: The introduction is clear except for one key matter that weaves its way throughout this manuscript: What is the contribution of pathogenic fungi to ARD. In the Abstract (line 9) the authors ascribe ARD to soil fungi – “mainly caused by soil-borne fungal diseases”. In the Introduction (from line 33) the factors contributing to ARD are described as much more diverse. In line 75 the authors hypothesize that introduced microbes will suppress disease-causing fungi, thereby alleviating ARD. While tree growth parameters are significantly higher in the QSB-6 treatments, the fungal count data reported does not support a primary role for these fungo in ARD. To be considered for publication, these interrelated factors will need to be clarified.

 

Response 1: Thank you for your constructive comments. ARD has been attributed to a variety of biotic and abiotic causal factors, but current studies suggest that biotic factors such as fungi play a leading role in disease development. This has been widely demonstrated in other studies via soil pasteurization and the application of biocides (Garbeva et al., 2004). The previous research of our laboratory found that the relative abundance of F. oxysporum and F. solani was the largest difference after methyl bromide fumigation and replanted treatments in clay loam (Sheng, 2020). Quantitative analysis based on qPCR confirmed that the wide distribution of F. oxysporum in replanted soil around Bohai Gulf, and the isolated F. moniliforme (F. verticillioides), F. oxysporum, and F. proliferatum isolated from replanted apple soil around Bohai Gulf were highly pathogenic to seedlings of Malus hupehensis, which demonstrated that Fusarium was one of the main soil -borne pathogenic fungi that cause ARD in China (Liu, 2013; Yin, 2017; Wang, 2018; Duan, 2021). The discussion also mentioned that long-term continuous cropping leads to the transformation of the soil microbial community structure from “bacterial type” to “fungal type”, which destroys the soil microbial community structure, leading to an increase in harmful pathogens. The results of this experiment found that the inoculation strain QSB-6 can significantly reduce the number of soil fungi, increase the ratio of soil bacteria/fungi, and transform the soil to a "bacterial type" (Qin, 2017). The community structure of bacteria and fungi was also significantly different from that of continuous cropping soil. Based on the results of qPCR data, the abundance of soil Fusarium (F. moniliforme, F. solani, F. oxysporum, and F. proliferatum ) inoculated with the strain QSB-6 was also significantly reduced, and the content of phenolic acids in the soil was also significantly reduced. Previous studies have found that phlorizin can promote the growth of F. moniliforme (Yin, 2017). Based on the above results, it can be concluded that the inoculation strain QSB-6 can inhibit the reproduction of harmful Fusarium in the soil, thereby reducing ARD. Thank you very much!

 

Garbeva, P. V., Van Veen, J. A., and Van Elsas, J. D. (2004). Microbial diversity in soil: selection of microbial populations by plant and soil type and implications for disease suppressiveness. Annu. Rev. Phytopathol. 42, 243-270.

Sheng, Y.F.; Wang, H.Y.; Wang, M.; Li, H.; Xiang, L.; Pan, F.B.; Mao, Z.Q. Effects of soil texture on the growth of young apple trees and soil microbial community structure under replanted conditions. Horticultural Plant Journal 2020, 6(3), 123-131.

 

Wang, G.S.; Yin, C.M.; Pan, F.B.; Wang, X.B.; Xiang, L.; Wang, Y.F.; Mao, Z.Q. Analysis of the fungal community in apple replanted soil around Bohai Gulf. Horticultural Plant Journal 2018, 4(5), 175-181.

 

Yin, C.M.; Xiang, L.; Wang, G.S.; Wang, Y.F.; Shen, X.; Chen, X.S.; Mao, Z.Q. Phloridzin promotes the growth of Fusarium moniliforme (Fusarium verticillioides). Scientia Horticulturae 2017, 214, 187-194.

 

Duan, Y.N.; Chen, R.; Zhang, R.; Jiang, W.T.; Chen, X.S.; Yin, C.M.; Mao, Z.Q. Isolation, identification, and antibacterial mechanisms of Bacillus amyloliquefaciens QSB-6 and its effect on plant roots. Frontiers in microbiology 2021, 12.

 

Qin, S., Yeboah, S., Cao, L., Zhang, J., Shi, S., & Liu, Y. (2017). Breaking continuous potato cropping with legumes improves soil microbial communities, enzyme activities and tuber yield. PloS one, 12(5), e0175934.

 

Liu, Z. The Isolation and Identification of Fungi Pathogen from Apple Replant Disease in Bohai Bay Region and the Screening of the Antagonistic Trichoderma Strains. Master's Thesis. Shandong Agricultural University, China. 2013.

 

Point 2: The fumigation treatment clearly led to substantial growth increase of the trees, but the fungal counts and qPCR of specific fungo show that fumigation did not kill a large proportion of the fungi. This raises the question what the contribution of fungi are to suppression of apple tree growth – linking in with the point of the previous paragraph.

 

Response 2: Thank you for your constructive comments. Although chemical fumigation treatment can effectively kill most of the fungi in the soil, after seven months, the soil itself will recruit some fungi to form a new microbial community, and the number of fungi in the soil will slowly increase. Compared with the replanted soil, the number of fungi in LZ, QX, and TY orchards after fumigation treatment was reduced by 40.38%, 68.44% and 49.08%, respectively. The numbers of F. solani and F. oxysporum both reduced by about 50%, which was consistent with the results of Sheng (2020). Compared with the replanted soil, the number of fungi in sandy loam, loam and clay loam fumigated by methyl bromide was reduced by 43.50%, 38.94% and 63.91%, respectively. The number of F. solani and F. oxysporum also reduced significantly. In addition, the fungal composition of the soil after the fumigation treatment is obviously different from that of the replanted soil. In the fumigation treatment, the abundance of of Gp4, Ohtaekwangia, Opitutus, Mortierella and Synchytrium are higher, and the abundance of Fusarium is significantly reduced, which can effectively controlled cucumber Fusarium wilt disease Li , 2016). It shows that the fumigation treatment can improved the soil fungus community, reduced the abundance of the ARD-related pathogen Fusarium in the soil, and promote the growth of replanted young apple trees.Thank you very much!

 

Sheng, Y.F.; Wang, H.Y.; Wang, M.; Li, H.; Xiang, L.; Pan, F.B.; Mao, Z.Q. Effects of soil texture on the growth of young apple trees and soil microbial community structure under replanted conditions. Horticultural Plant Journal 2020, 6(3), 123-131.

 

Li, R., Shen, Z., Sun, L., Zhang, R., Fu, L., Deng, X., & Shen, Q. (2016). Novel soil fumigation method for suppressing cucumber Fusarium wilt disease associated with soil microflora alterations. Applied Soil Ecology, 101, 28-36.

Point 3: The nature of the QSB-6 supplementation must be clarified. Was the cow dung and straw sterile or not when the culture was added? As this mixture was incubated warm for a day, non-sterile cow dung – straw microbiota could well have responded to the nutrients remaining in the bacterial culture, as well as to the culture, so that the mixture applied to soil before tree planting was either QSB-6 alone, or a diverse mixture of microorganisms.

 

Response 3: Thank you for your constructive comments. In Materials and Method 2.1, it has been mentioned that cow dung and wheat straw are fully sterilized when adding the inoculum. The bacterial density finally reached 5.0×10⁹ CFUg^-1_. The carrier can support the survival of strain QSB-6 while inhibiting the growth of target pathogens, thereby improving the performance of strain QSB-6 for plant disease control (Malusá, 2012; Wei, 2015). The raw materials (cow dung and wheat straw) in the formula are cheap and easy to obtain, and the fermentation level is high, which provides a good foundation for its large-scale industrial production (Smith, 1992; Vijayaraghavan, 2016). Thank you very much!

 

Smith, R. S. (1992). Legume inoculant formulation and application. Canadian journal of microbiology, 38(6), 485-492.

 

Wei, Z., Huang, J., Yang, C., Xu, Y., Shen, Q., and Chen, W. (2015). Screening of suitable carriers for Bacillus amyloliquefaciens strain QL-18 to enhance the biocontrol of tomato bacterial wilt. Crop Protection, 75, 96-103.

 

Malusá, E., Sas-Paszt, L., and Ciesielska, J. (2012). Technologies for beneficial microorganisms inocula used as biofertilizers. The scientific world journal, 2012.

 

Vijayaraghavan, P., Arun, A., Al-Dhabi, N. A., Vincent, S. G. P., Arasu, M. V., & Choi, K. C. (2016). Novel Bacillus subtilis IND19 cell factory for the simultaneous production of carboxy methyl cellulase and protease using cow dung substrate in solid-substrate fermentation. Biotechnology for biofuels, 9(1), 1-13.

 

Point 4: What was the amount of this mixture added to the soil before planting? If it was a high proportion, the chemical properties of the cow dung themselves would have contributed to growth. I. this case the experiment was not about the effect of QSB-6 alone, but the combined effect of bacteria and nutrients. In that case, there should have been a control of cow dung – straw addition without QSB-6 added.

 

Response 4: Thank you for your constructive comments. In Materials and Method 2.3, it has been mentioned that the amount of application for each young tree is controlled at 1kg. This experiment set up a blank carrier treatment without adding strain QSB-6, namely T1, to further verify the effect of strain QSB-6 on replanting young apple trees and the soil microbial environment. Thank you very much!

 

Point 5: There are multiple cases of questionable numbers. For example, line 104 reports bacterial count of 5.0 × 10⁹ CFU·g^-1, whereas Table 3 reports counts of ~ 5 x 10^6 per gram. The actinomycete count is ten time as high as the bacterial count (Table 3), but actinomycetes are but a part of the bacteria overall. While different culture media were used, and we never get all the bacteria to form colonies on agar, this is still of concern. The Y axis labels of Fig 4 range from 10^5 to 10^11. I do not see how there can be 10^ fungal cells (or even target genes) per gram of soil unless it is pure fungi.

 

Response 5: Thank you for your constructive comments. The Chinese bio-organic fertilizer production standard stipulating that the functional microorganism content should be greater than 2.0 × 10⁷ CFU g⁻¹ dry formulation (Emmert and Handelsman, 1999). The bacterial count reported in line 104 is 5.0 × 10⁹ CFU·g^-1, which is the content of the strain QSB-6 in the bacterial fertilizer, which has reached the production standard. The counting units in Table 3 have been checked and modified.  The CFU per gram of dry soil was used as the measurement unit for the bacterial, fungal, and actinomycetes populations. The data in Figure 4 is based on the plasmid DNA concentration and the transformed copy concentration. For the specific formula, see Materials and Method 2.4.6. It is used to measure the difference in the relative abundance of Fusarium among different treatments. The value does not represent the number of Fusarium spores present in the soil. Specific primers and standard curves have been added in the supplementary materials.

 

Emmert, E. A., and Handelsman, J. (1999). Biocontrol of plant disease: a (Gram-) positive perspective. FEMS Microbiology letters, 171(1), 1-9.

 

Point 6: The manuscript is clear, but the manuscript is full of grammatical errors.

 

Response 6: Thank you for your constructive comments. According to your suggestions, We have revised our manuscript by EditorBar Language Editing, Beijing for English writing. Thank you very much!

 

Point 7: Line 105 – 106: Some of these values are very low – much lower than those reported for the soil. Are there typographical errors here in the units?

 

Response 7: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 8: Line 134 – as the sol for DNA extraction was reportedly kept at -80℃, these samples at 4℃ were more likely used for culturable counts and not microbial structure.

 

Response 8: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 9: Line 194: Are you certain this was from MO BIO? They were bought out by Qiagen about a decade ago, so unless the kits were that old, they were obtained from Qiagen.

 

Response 9: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 10: Line 194: Please specify what gene was amplified by PCR.

 

Response 10: Thank you for your constructive comments. Specific primers and standard curves have been added in the supplementary materials. Thank you very much!

 

Point 11: Line 198: Please specify what plasmid was used.

 

Response 11: Thank you for your constructive comments. Specific primers and standard curves have been added in the supplementary materials. Four Fusarium (F. moniliforme, F. solani, F. oxysporum, and F. proliferatum) DNAs were used as templates, and specific primers were used for PCR amplification. The purified PCR products were connected to pMD18-T vector (TaKaRa Biotech, China), transformed into E.coli competent cells DH5α (TaKaRa Biotech, China). After sequencing verification, the plasmid was extracted according to the method of the plasmid extraction kit (TaKaRa Biotech, China). Measure its concentration, and carry out a gradient dilution with sterile water. The standard curves were generated by plotting the cycle threshold (Cq) values obtained for each specific DNA concentration versus the log of the initial concentration of species DNA.Thank you very much!

 

Point 12: Line 208: If you used 27f and 1492r then you amplified almost the entire 16S gene and not just one of its nine variable regions.

 

Response 12: Thank you for your constructive comments. The primers used in this study were used for the amplification of most eubacterial 16SrRNAs (Weisburg, 1991). Thank you very much!

 

Weisburg, W. G., Barns, S. M., Pelletier, D. A., & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of bacteriology, 173(2), 697-703.

 

Point 13: Line 227: As you report tRFLP data, why does it state here that the samples were sequenced? Do I assume that fragment lengths were determined using the same equipment used for Sanger sequencing?

 

Response 13: Thank you for your constructive comments. According to your suggestions, the method has been re-described. Thank you very much!

 

Point 14: Line 252: “became significant” – No, data are either significantly different or not. Please rephrase all your results in term of what treatments led to differences that were statistically different. You report ANOVA results, so this is a matter of revising the text.

 

Response 14: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 15: Figure 1: While the data in Table 2 report that trees in TY soil were larger, they look smaller in the figure. This may the result of how photographs were taken – from what distance or with what magnification. Unless some size marker can be added, I suggest removing this figure.

 

Response 15: Thank you for your constructive comments. The picture has been re-adjusted and modified. The picture was the result of artificial shooting with Canon PowerShotG16 camera. In order to ensure that the trees are displayed completely in the picture, the shooting distance is random, but each picture uses the same plant height measuring rule as a measuring tool. Can it be used as a size marker? If not, I can accept to delete this picture. Thank you very much!

 

Point 16: Section 3.2: Focus on the ratios of bacteria:fungi and actinomycetes:fungi as this is likely a critical part of why the trees grew better.

 

Response 16: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 17: Table 3: Actinomycetes cannot be more than bacteria overall. Please check yoru raw data again.

 

Response 17: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised, and corrected the counting unit. Thank you very much!

 

Point 18: Figure 3: Please explain in the figure legend the scale used for red to blue. Was this linear or logarithmic? Also, the names of the samples are abbreviated from your other way of naming samples, so please add a key.

 

Response 18: Thank you for your constructive comments. According to your suggestions, Figure 3 has been annotated in detail. Thank you very much!

 

Point 19:  Please check the unites for the four graphs – 10^5 to 10^11.

 

Response 19: Thank you for your constructive comments. According to your suggestions, I have recalculated the data, there is no error in the data unit, and the order of magnitude can refer to other articles in this laboratory (see below). Thank you very much!

 

Sheng, Y.F.; Wang, H.Y.; Wang, M.; Li, H.; Xiang, L.; Pan, F.B.; Mao, Z.Q. Effects of soil texture on the growth of young apple trees and soil microbial community structure under replanted conditions. Horticultural Plant Journal 2020, 6(3), 123-131.

 

Duan, Y.N.; Chen, R.; Zhang, R.; Jiang, W.T.; Chen, X.S.; Yin, C.M.; Mao, Z.Q. Isolation, identification, and antibacterial mechanisms of Bacillus amyloliquefaciens QSB-6 and its effect on plant roots. Frontiers in microbiology 2021, 12.

 

Point 20: Please define the approach taken to develop the cluster analyses in the figure legend.

 

Response 20: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 21: Line 448: “destroys” is a very strong word. It is more likely that there are shifts in the community composition, but a total destruction would require autoclaving, strong radiation or some other very aggressive treatment.

 

Response 21: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 22: Line 450: “flora” refers to plants. I do know that the word flora was used a lot in the past to refer to bacteria, and this is probably because in the early days of microbiology in Europe, non-medical microbiology was studied in botany departments. The preferred term in the more recent literature is “microbiota”.

 

Response 22: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 23: Line 458: The term “bacteriostatic” is not appropriate here. While these bacteria may have bacteriostatic properties as well, suppression of fungal growth would be fungistatic.

 

Response 23: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Editor, Dear Authors,

I am honored to be included as a reviewer of the manuscript entitled: "Effects of Bacillus amyloliquefaciens QSB-6 on the growth of replanted apple trees and soil microbial environment". I believe that the subject of the manuscript is interesting, and the research has been conducted reliably. The research methodology is presented in detail and the results are clearly discussed and summarized.

I have no serious substantive reservations. However, I believe that the manuscript should be technically improved.

Here are some of my comments:

1) The entire manuscript is poorly adapted to the TEMPLATE requirements of the Horticulture journal, please pay special attention to the References section, but also to the formatting of Tables and chapter titles and sub-headings.

2) Keywords should be in alphabetical order and should not repeat words appearing in the title of the manuscript.

3) Tables 2, 3 and 4 are difficult to read, please add lines between LZ, QX and TY treatments (Tables 2 and 3) and between Fungi and Bacterial treatments (Table 4).

4) In Tables 3 and 4 and Figures 2-5, abbreviations should be explained or referred to Table 2.

I believe that the research presented in the manuscript is valuable and the manuscript has been prepared relatively carefully, so I believe it deserves to be published in the journal Horticulture after following the comments of the reviewer.

Author Response

Response to Reviewer 2 Comments

 

 

 

Point 1:  The entire manuscript is poorly adapted to the TEMPLATE requirements of the Horticulture journal, please pay special attention to the References section, but also to the formatting of Tables and chapter titles and sub-headings. 


 

Response 1: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 2: Keywords should be in alphabetical order and should not repeat words appearing in the title of the manuscript.

 

Response 2: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 3: Tables 2, 3 and 4 are difficult to read, please add lines between LZ, QX and TY treatments (Tables 2 and 3) and between Fungi and Bacterial treatments (Table 4).

 

Response 3: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 4: In Tables 3 and 4 and Figures 2-5, abbreviations should be explained or referred to Table 2.

 

Response 4: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

We are truly grateful to you critical comments and thoughtful suggestions on our manuscript, comprehensive the comments of reviewer # 1 and 3, we have improved and revised some of the content in the manuscript, and checked the full text. Please review again during your busy schedule. Thank you very much! The specific changes are as follows:

 

 

Response to Reviewer 1 Comments

 

Point 1: The term “bacterial fertilizer” should be reformulated since evidence of plant growth promotion by Bacillus amyloliquefaciens QSB-6 is not provided by this study.

Bacterial name missing “verify the effects of QSB-6”

Response 1: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 2: This study is based on the assumption that “Fusarium was one of the main soil-borne pathogenic fungi that cause ARD” (line 44), therefore most if the study is oriented towards this fungus. Nevertheless, a number of studies reports that the role of Fusarium towards plants is not so strict, but rather may also be associated with good soil health in permanent tree crops, for example:

 

Bacon, C.W., Yates, I.E., 2006. Endophytic Root Colonization by Fusarium Species: Histology, Plant Interactions, and Toxicity BT  - Microbial Root Endophytes, in: Schulz, B.J.E., Boyle, C.J.C., Sieber, T.N. (Eds.), . Springer Berlin Heidelberg, Berlin, Heidelberg, pp. 133–152. doi:10.1007/3-540-33526-9_8

 

Therefore this concept should be reformulated in the manuscript.

 

Response 2:  Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 3: Three orchards have been tested in this study, but data on the effect of the Bacillus QSB-6 strain treatment on the growth of apple plants have been analysed separately per each site (results in paragraph 3.1), in this way it is difficult to draw conclusions on the effect of strain QSB-6.

 

Response 3: Thank you for your constructive comments. In this experiment, three orchards with different textures (loam, sandy loam and clay loam) were selected to evaluate the biocontrol effect of strain QSB-6, and it was found that strain QSB-6 had the most significant effect on the growth promotion of replanted apple young trees under the condition of loam soil (TY), followed by sandy loam (QX) and clay loam (LZ). This was consistent with the results of previous studies (Sheng, 2020). The occurrence of Apple replant disease was the most serious under clay loam conditions, followed by sandy loam and loam. However, strain QSB-6 also exerts a good biocontrol effect under the clay loam condition. The overall growth of the replanted apple saplings treated with T2 was better than that with T1(Figure 1). From this, it can be concluded that the QSB-6 bacterial fertilizer treatment can promote the growth of replanted young apple trees. Thank you very much!

 

Sheng, Y.F.; Wang, H.Y.; Wang, M.; Li, H.; Xiang, L.; Pan, F.B.; Mao, Z.Q. Effects of soil texture on the growth of young apple trees and soil microbial community structure under replanted conditions. Horticultural Plant Journal 2020, 6(3), 123-131.

Point 4: In Data analysis paragraph it is reported that “Comparisons were made using the least significant difference (LSD) test” (line 231), while the note in Table 2 reports that “Numbers followed by the same letter were not significantly different based on Duncan Multiple Range Test”. It is not clear which statistical test was performed to compare plant growth parameters. Furthermore, in Table 2, T1 and T2 treatment groups share the same letter in almost all conditions, meaning that the differences between the groups are not statistically significant, how can the authors say that the bacterial fertilizer treatment (T2) significantly promoted plant growth if it did not differ from control (T1)??

Response 4: Thank you for your constructive comments. According to your suggestion, the data has been analyzed. In Table 2, the plant height and ground diameter of the T1 and T2 treatments of the LZ orchard were also different, but they were not significant, and there were significant differences in the number of branches and the average branch length. Figure 1 showed that the overall growth of the replanted apple saplings treated with T2 was better than that with T1. The insignificant difference between LZ orchard treatments may be related to the high severity of continuous cropping under clay loam conditions (Sheng, 2020) and the slower growth and reproduction of strain QSB-6. It can also be seen from the results in Table 3 that the number of bacteria in the LZ orchard treated with T2 was significantly lower than that in the QX and TY orchards. There were significant differences between the T1 and T2 treatments of QX and TY orchards. Thank you very much!

 

Point 5: Authors state that “strain QSB-6 can reproduce normally after inoculating the soil” (line 362) but no specific viability test was performed.

 

Response 5: Thank you for your constructive comments. In this experiment, microbial culture counts were carried out on the rhizosphere soil of different treatments using the dilution method of plate counting. It was found that the number of bacteria in the rhizosphere soil of the inoculated strain QSB-6 was significantly higher than that of other treatments, and the results of the previous potting treatments were consistent (Duan, 2021), and most of the bacterial colonies cultured on the plate were consistent with the morphology of strain QSB-6. A single colony with the same morphology was randomly picked and sequenced, and the similarity with the strain QSB-6 was found to be 100% (results not shown). so it was concluded that strain QSB-6 can reproduce normally after inoculating the soil, and the addition of the carrier also provided nutrients for strain QSB-6 to grow normally in the soil. Thank you very much!

Duan, Y.N.; Chen, R.; Zhang, R.; Jiang, W.T.; Chen, X.S.; Yin, C.M.; Mao, Z.Q. Isolation, identification, and antibacterial mechanisms of Bacillus amyloliquefaciens QSB-6 and its effect on plant roots. Frontiers in microbiology 2021, 12.

 

Point 6: Diversity indices described in paragraph 3.6 (lines 387-391) would benefit from bibliographic references.

 

Response 6: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 7: Data analysis of diversity indices should be revised, since in line 392 it is reported that “The diversity of soil fungi communities was significantly increased” , while in the abstract “QSB-6 can significantly reduce the diversity of soil fungi” line 23. Simpson’s index does not differ between T1 and T2 in fungi. Same for Shannon index of bacterial communities.

 

Response 7: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Simpson, Shannon, and Brillouin Index are all used to measure the diversity of microorganisms. There are limitations to using a single index to measure diversity.  Shannon index is closely related to richness and more sensitive to sparse species, while Simpson index is closely related to evenness and more sensitive to enriched species (Magurran, 1998; Xu, 2011).  No difference in Simpson index between T1 and T2 treatments can only indicate that the addition of organic carrier and bacterial fertilizer resulted in the similarity of the enriched species (the relative important value of newly added species is higher than 0.72) in the rhizosphere soil, while the Shannon and Brillouin Indexes of the T2 treatment were significantly lower than those of the T1 treatment. No difference in Shannon index between T1 and T2 treatments can only indicate that the addition of organic carrier and bacterial fertilizer results in the similarity of sparse species (the relative important value of newly added species is less than 0.72) in rhizosphere soil, while the Simpson and Brillouin Indexes of T2 treatment were significantly higher than those of T1 treatment. Thank you very much!

 

Magurran, A. E. (1988). Ecological diversity and its measurement. Princeton university press.

 

Xu, Q., Zhang, F., Xu, Z. Q., Jia, Y. L., & You, J. M. (2011). Some characteristics of Simpson index and the Shannon-Wiener index and their dilution effect. Pratacultural Science, 4, 527-531.

 

Point 8: The first part of the discussion (lines 417-433) should be moved to the introduction.

 

Response 8: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 9: It is stated that “This study found that the addition of strain QSB-6 can significantly reduced the content of the main phenolic acids in the soil” lines 482-483, but the significance of the results reported in figure 3 is not reported.

 

Response 9: Thank you for your constructive comments. According to your suggestions, the relevant data of Figure 3 has been added to the supplementary materials. Thank you very much!

 

Point 10: Which “pot study”? reference missing.

 

Response 10: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 11:Strain XNRB-3? Line 519

 

Response 11: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 12:English language should be revised.

 

Response 12: Thank you for your constructive comments. According to your suggestions, We have revised our manuscript by EditorBar Language Editing, Beijing for English writing. Thank you very much!

 

EditorBar Language Editing No. 35, Tsinghua East Road, Beijing, China 100083 Email: runse@editorbar.com Phone: +86-10-5620-8614

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Manuscript Information

 

ID	LE202112080129
Editing date	2021-12-14
Title	Effects of Bacillus amyloliquefaciens QSB-6 on the growth of replanted apple trees and soil microbial environment
Corresponding author	Zhiquan Mao
Language writing before editing	□ Very poor      □ Poor      □ Fair      ■ Good      □ Very good      □ Excellent
Recommendation after language editing	■ Submitting to target journal directly □ Submitting to target journal after minor revision □ Re-editing required after major revision □ Not suitable for publication
Overview comments	This manuscript has been thoroughly edited for English word choice, grammar, and sentence structure. Please see a small number of comments to the authors in the manuscript margins. Very best wishes for continued research success!

Edited by

Christina E.W.

Senior Editor

The Pennsylvania State University

Horticulture

 

Certificate Issued by

Dr. Jason Qee

 

Editor in Chief

Editorbar Language Editing, Beijing, China

runse@editorbar.com   www.editorbar.com

Certificate link: www.editorbar.com/order/cert/LE202112080129

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Response to Reviewer 3 Comments

 

Point 1: The introduction is clear except for one key matter that weaves its way throughout this manuscript: What is the contribution of pathogenic fungi to ARD. In the Abstract (line 9) the authors ascribe ARD to soil fungi – “mainly caused by soil-borne fungal diseases”. In the Introduction (from line 33) the factors contributing to ARD are described as much more diverse. In line 75 the authors hypothesize that introduced microbes will suppress disease-causing fungi, thereby alleviating ARD. While tree growth parameters are significantly higher in the QSB-6 treatments, the fungal count data reported does not support a primary role for these fungo in ARD. To be considered for publication, these interrelated factors will need to be clarified.

 

Response 1: Thank you for your constructive comments. ARD has been attributed to a variety of biotic and abiotic causal factors, but current studies suggest that biotic factors such as fungi play a leading role in disease development. This has been widely demonstrated in other studies via soil pasteurization and the application of biocides (Garbeva et al., 2004). The previous research of our laboratory found that the relative abundance of F. oxysporum and F. solani was the largest difference after methyl bromide fumigation and replanted treatments in clay loam (Sheng, 2020). Quantitative analysis based on qPCR confirmed that the wide distribution of F. oxysporum in replanted soil around Bohai Gulf, and the isolated F. moniliforme (F. verticillioides), F. oxysporum, and F. proliferatum isolated from replanted apple soil around Bohai Gulf were highly pathogenic to seedlings of Malus hupehensis, which demonstrated that Fusarium was one of the main soil -borne pathogenic fungi that cause ARD in China (Liu, 2013; Yin, 2017; Wang, 2018; Duan, 2021). The discussion also mentioned that long-term continuous cropping leads to the transformation of the soil microbial community structure from “bacterial type” to “fungal type”, which destroys the soil microbial community structure, leading to an increase in harmful pathogens. The results of this experiment found that the inoculation strain QSB-6 can significantly reduce the number of soil fungi, increase the ratio of soil bacteria/fungi, and transform the soil to a "bacterial type" (Qin, 2017). The community structure of bacteria and fungi was also significantly different from that of continuous cropping soil. Based on the results of qPCR data, the abundance of soil Fusarium (F. moniliforme, F. solani, F. oxysporum, and F. proliferatum ) inoculated with the strain QSB-6 was also significantly reduced, and the content of phenolic acids in the soil was also significantly reduced. Previous studies have found that phlorizin can promote the growth of F. moniliforme (Yin, 2017). Based on the above results, it can be concluded that the inoculation strain QSB-6 can inhibit the reproduction of harmful Fusarium in the soil, thereby reducing ARD. Thank you very much!

 

Garbeva, P. V., Van Veen, J. A., and Van Elsas, J. D. (2004). Microbial diversity in soil: selection of microbial populations by plant and soil type and implications for disease suppressiveness. Annu. Rev. Phytopathol. 42, 243-270.

Sheng, Y.F.; Wang, H.Y.; Wang, M.; Li, H.; Xiang, L.; Pan, F.B.; Mao, Z.Q. Effects of soil texture on the growth of young apple trees and soil microbial community structure under replanted conditions. Horticultural Plant Journal 2020, 6(3), 123-131.

 

Wang, G.S.; Yin, C.M.; Pan, F.B.; Wang, X.B.; Xiang, L.; Wang, Y.F.; Mao, Z.Q. Analysis of the fungal community in apple replanted soil around Bohai Gulf. Horticultural Plant Journal 2018, 4(5), 175-181.

 

Yin, C.M.; Xiang, L.; Wang, G.S.; Wang, Y.F.; Shen, X.; Chen, X.S.; Mao, Z.Q. Phloridzin promotes the growth of Fusarium moniliforme (Fusarium verticillioides). Scientia Horticulturae 2017, 214, 187-194.

 

Duan, Y.N.; Chen, R.; Zhang, R.; Jiang, W.T.; Chen, X.S.; Yin, C.M.; Mao, Z.Q. Isolation, identification, and antibacterial mechanisms of Bacillus amyloliquefaciens QSB-6 and its effect on plant roots. Frontiers in microbiology 2021, 12.

 

Qin, S., Yeboah, S., Cao, L., Zhang, J., Shi, S., & Liu, Y. (2017). Breaking continuous potato cropping with legumes improves soil microbial communities, enzyme activities and tuber yield. PloS one, 12(5), e0175934.

 

Liu, Z. The Isolation and Identification of Fungi Pathogen from Apple Replant Disease in Bohai Bay Region and the Screening of the Antagonistic Trichoderma Strains. Master's Thesis. Shandong Agricultural University, China. 2013.

 

Point 2: The fumigation treatment clearly led to substantial growth increase of the trees, but the fungal counts and qPCR of specific fungo show that fumigation did not kill a large proportion of the fungi. This raises the question what the contribution of fungi are to suppression of apple tree growth – linking in with the point of the previous paragraph.

 

Response 2: Thank you for your constructive comments. Although chemical fumigation treatment can effectively kill most of the fungi in the soil, after seven months, the soil itself will recruit some fungi to form a new microbial community, and the number of fungi in the soil will slowly increase. Compared with the replanted soil, the number of fungi in LZ, QX, and TY orchards after fumigation treatment was reduced by 40.38%, 68.44% and 49.08%, respectively. The numbers of F. solani and F. oxysporum both reduced by about 50%, which was consistent with the results of Sheng (2020). Compared with the replanted soil, the number of fungi in sandy loam, loam and clay loam fumigated by methyl bromide was reduced by 43.50%, 38.94% and 63.91%, respectively. The number of F. solani and F. oxysporum also reduced significantly. In addition, the fungal composition of the soil after the fumigation treatment is obviously different from that of the replanted soil. In the fumigation treatment, the abundance of of Gp4, Ohtaekwangia, Opitutus, Mortierella and Synchytrium are higher, and the abundance of Fusarium is significantly reduced, which can effectively controlled cucumber Fusarium wilt disease Li , 2016). It shows that the fumigation treatment can improved the soil fungus community, reduced the abundance of the ARD-related pathogen Fusarium in the soil, and promote the growth of replanted young apple trees.Thank you very much!

 

Sheng, Y.F.; Wang, H.Y.; Wang, M.; Li, H.; Xiang, L.; Pan, F.B.; Mao, Z.Q. Effects of soil texture on the growth of young apple trees and soil microbial community structure under replanted conditions. Horticultural Plant Journal 2020, 6(3), 123-131.

 

Li, R., Shen, Z., Sun, L., Zhang, R., Fu, L., Deng, X., & Shen, Q. (2016). Novel soil fumigation method for suppressing cucumber Fusarium wilt disease associated with soil microflora alterations. Applied Soil Ecology, 101, 28-36.

Point 3: The nature of the QSB-6 supplementation must be clarified. Was the cow dung and straw sterile or not when the culture was added? As this mixture was incubated warm for a day, non-sterile cow dung – straw microbiota could well have responded to the nutrients remaining in the bacterial culture, as well as to the culture, so that the mixture applied to soil before tree planting was either QSB-6 alone, or a diverse mixture of microorganisms.

 

Response 3: Thank you for your constructive comments. In Materials and Method 2.1, it has been mentioned that cow dung and wheat straw are fully sterilized when adding the inoculum. The bacterial density finally reached 5.0×10⁹ CFUg^-1_. The carrier can support the survival of strain QSB-6 while inhibiting the growth of target pathogens, thereby improving the performance of strain QSB-6 for plant disease control (Malusá, 2012; Wei, 2015). The raw materials (cow dung and wheat straw) in the formula are cheap and easy to obtain, and the fermentation level is high, which provides a good foundation for its large-scale industrial production (Smith, 1992; Vijayaraghavan, 2016). Thank you very much!

 

Smith, R. S. (1992). Legume inoculant formulation and application. Canadian journal of microbiology, 38(6), 485-492.

 

Wei, Z., Huang, J., Yang, C., Xu, Y., Shen, Q., and Chen, W. (2015). Screening of suitable carriers for Bacillus amyloliquefaciens strain QL-18 to enhance the biocontrol of tomato bacterial wilt. Crop Protection, 75, 96-103.

 

Malusá, E., Sas-Paszt, L., and Ciesielska, J. (2012). Technologies for beneficial microorganisms inocula used as biofertilizers. The scientific world journal, 2012.

 

Vijayaraghavan, P., Arun, A., Al-Dhabi, N. A., Vincent, S. G. P., Arasu, M. V., & Choi, K. C. (2016). Novel Bacillus subtilis IND19 cell factory for the simultaneous production of carboxy methyl cellulase and protease using cow dung substrate in solid-substrate fermentation. Biotechnology for biofuels, 9(1), 1-13.

 

Point 4: What was the amount of this mixture added to the soil before planting? If it was a high proportion, the chemical properties of the cow dung themselves would have contributed to growth. I. this case the experiment was not about the effect of QSB-6 alone, but the combined effect of bacteria and nutrients. In that case, there should have been a control of cow dung – straw addition without QSB-6 added.

 

Response 4: Thank you for your constructive comments. In Materials and Method 2.3, it has been mentioned that the amount of application for each young tree is controlled at 1kg. This experiment set up a blank carrier treatment without adding strain QSB-6, namely T1, to further verify the effect of strain QSB-6 on replanting young apple trees and the soil microbial environment. Thank you very much!

 

Point 5: There are multiple cases of questionable numbers. For example, line 104 reports bacterial count of 5.0 × 10⁹ CFU·g^-1, whereas Table 3 reports counts of ~ 5 x 10^6 per gram. The actinomycete count is ten time as high as the bacterial count (Table 3), but actinomycetes are but a part of the bacteria overall. While different culture media were used, and we never get all the bacteria to form colonies on agar, this is still of concern. The Y axis labels of Fig 4 range from 10^5 to 10^11. I do not see how there can be 10^ fungal cells (or even target genes) per gram of soil unless it is pure fungi.

 

Response 5: Thank you for your constructive comments. The Chinese bio-organic fertilizer production standard stipulating that the functional microorganism content should be greater than 2.0 × 10⁷ CFU g⁻¹ dry formulation (Emmert and Handelsman, 1999). The bacterial count reported in line 104 is 5.0 × 10⁹ CFU·g^-1, which is the content of the strain QSB-6 in the bacterial fertilizer, which has reached the production standard. The counting units in Table 3 have been checked and modified.  The CFU per gram of dry soil was used as the measurement unit for the bacterial, fungal, and actinomycetes populations. The data in Figure 4 is based on the plasmid DNA concentration and the transformed copy concentration. For the specific formula, see Materials and Method 2.4.6. It is used to measure the difference in the relative abundance of Fusarium among different treatments. The value does not represent the number of Fusarium spores present in the soil. Specific primers and standard curves have been added in the supplementary materials.

 

Emmert, E. A., and Handelsman, J. (1999). Biocontrol of plant disease: a (Gram-) positive perspective. FEMS Microbiology letters, 171(1), 1-9.

 

Point 6: The manuscript is clear, but the manuscript is full of grammatical errors.

 

Response 6: Thank you for your constructive comments. According to your suggestions, We have revised our manuscript by EditorBar Language Editing, Beijing for English writing. Thank you very much!

 

Point 7: Line 105 – 106: Some of these values are very low – much lower than those reported for the soil. Are there typographical errors here in the units?

 

Response 7: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 8: Line 134 – as the sol for DNA extraction was reportedly kept at -80℃, these samples at 4℃ were more likely used for culturable counts and not microbial structure.

 

Response 8: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 9: Line 194: Are you certain this was from MO BIO? They were bought out by Qiagen about a decade ago, so unless the kits were that old, they were obtained from Qiagen.

 

Response 9: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 10: Line 194: Please specify what gene was amplified by PCR.

 

Response 10: Thank you for your constructive comments. Specific primers and standard curves have been added in the supplementary materials. Thank you very much!

 

Point 11: Line 198: Please specify what plasmid was used.

 

Response 11: Thank you for your constructive comments. Specific primers and standard curves have been added in the supplementary materials. Four Fusarium (F. moniliforme, F. solani, F. oxysporum, and F. proliferatum) DNAs were used as templates, and specific primers were used for PCR amplification. The purified PCR products were connected to pMD18-T vector (TaKaRa Biotech, China), transformed into E.coli competent cells DH5α (TaKaRa Biotech, China). After sequencing verification, the plasmid was extracted according to the method of the plasmid extraction kit (TaKaRa Biotech, China). Measure its concentration, and carry out a gradient dilution with sterile water. The standard curves were generated by plotting the cycle threshold (Cq) values obtained for each specific DNA concentration versus the log of the initial concentration of species DNA.Thank you very much!

 

Point 12: Line 208: If you used 27f and 1492r then you amplified almost the entire 16S gene and not just one of its nine variable regions.

 

Response 12: Thank you for your constructive comments. The primers used in this study were used for the amplification of most eubacterial 16SrRNAs (Weisburg, 1991). Thank you very much!

 

Weisburg, W. G., Barns, S. M., Pelletier, D. A., & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of bacteriology, 173(2), 697-703.

 

Point 13: Line 227: As you report tRFLP data, why does it state here that the samples were sequenced? Do I assume that fragment lengths were determined using the same equipment used for Sanger sequencing?

 

Response 13: Thank you for your constructive comments. According to your suggestions, the method has been re-described. Thank you very much!

 

Point 14: Line 252: “became significant” – No, data are either significantly different or not. Please rephrase all your results in term of what treatments led to differences that were statistically different. You report ANOVA results, so this is a matter of revising the text.

 

Response 14: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 15: Figure 1: While the data in Table 2 report that trees in TY soil were larger, they look smaller in the figure. This may the result of how photographs were taken – from what distance or with what magnification. Unless some size marker can be added, I suggest removing this figure.

 

Response 15: Thank you for your constructive comments. The picture has been re-adjusted and modified. The picture was the result of artificial shooting with Canon PowerShotG16 camera. In order to ensure that the trees are displayed completely in the picture, the shooting distance is random, but each picture uses the same plant height measuring rule as a measuring tool. Can it be used as a size marker? If not, I can accept to delete this picture. Thank you very much!

 

Point 16: Section 3.2: Focus on the ratios of bacteria:fungi and actinomycetes:fungi as this is likely a critical part of why the trees grew better.

 

Response 16: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 17: Table 3: Actinomycetes cannot be more than bacteria overall. Please check yoru raw data again.

 

Response 17: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised, and corrected the counting unit. Thank you very much!

 

Point 18: Figure 3: Please explain in the figure legend the scale used for red to blue. Was this linear or logarithmic? Also, the names of the samples are abbreviated from your other way of naming samples, so please add a key.

 

Response 18: Thank you for your constructive comments. According to your suggestions, Figure 3 has been annotated in detail. Thank you very much!

 

Point 19:  Please check the unites for the four graphs – 10^5 to 10^11.

 

Response 19: Thank you for your constructive comments. According to your suggestions, I have recalculated the data, there is no error in the data unit, and the order of magnitude can refer to other articles in this laboratory (see below). Thank you very much!

 

Sheng, Y.F.; Wang, H.Y.; Wang, M.; Li, H.; Xiang, L.; Pan, F.B.; Mao, Z.Q. Effects of soil texture on the growth of young apple trees and soil microbial community structure under replanted conditions. Horticultural Plant Journal 2020, 6(3), 123-131.

 

Duan, Y.N.; Chen, R.; Zhang, R.; Jiang, W.T.; Chen, X.S.; Yin, C.M.; Mao, Z.Q. Isolation, identification, and antibacterial mechanisms of Bacillus amyloliquefaciens QSB-6 and its effect on plant roots. Frontiers in microbiology 2021, 12.

 

Point 20: Please define the approach taken to develop the cluster analyses in the figure legend.

 

Response 20: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 21: Line 448: “destroys” is a very strong word. It is more likely that there are shifts in the community composition, but a total destruction would require autoclaving, strong radiation or some other very aggressive treatment.

 

Response 21: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 22: Line 450: “flora” refers to plants. I do know that the word flora was used a lot in the past to refer to bacteria, and this is probably because in the early days of microbiology in Europe, non-medical microbiology was studied in botany departments. The preferred term in the more recent literature is “microbiota”.

 

Response 22: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 23: Line 458: The term “bacteriostatic” is not appropriate here. While these bacteria may have bacteriostatic properties as well, suppression of fungal growth would be fungistatic.

 

Response 23: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

 

 

 

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript by Duan et al. outlines analysis of apple tree establishment and associated soil chemical and microbial composition when planted in soil treated by the biocontrol strain B. amyloliquefaciens QSB-6 versus fumigation or fertilizer addition. The Experiments appear well designed and the effect of QSB-6 on apple tree growth is interesting. However, before I am able to evaluate the conclusions drawn, a number of questions should be clarified, outlined below:

 

The introduction is clear except for one key matter that weaves its way throughout this manuscript: What is the contribution of pathogenic fungi to ARD. In the Abstract (line 9) the authors ascribe ARD to soil fungi – “mainly caused by soil-borne fungal diseases”. In the Introduction (from line 33) the factors contributing to ARD are described as much more diverse. In line 75 the authors hypothesize that introduced microbes will suppress disease-causing fungi, thereby alleviating ARD. While tree growth parameters are significantly higher in the QSB-6 treatments, the fungal count data reported does not support a primary role for these fungo in ARD. To be considered for publication, these interrelated factors will need to be clarified.

 

The fumigation treatment clearly led to substantial growth increase of the trees, but the fungal counts and qPCR of specific fungo show that fumigation did not kill a large proportion of the fungi. This raises the question what the contribution of fungi are to suppression of apple tree growth – linking in with the point of the previous paragraph.

 

The nature of the QSB-6 supplementation must be clarified. Was the cow dung and straw sterile or not when the culture was added? As this mixture was incubated warm for a day, non-sterile cow dung – straw microbiota could well have responded to the nutrients remaining in the bacterial culture, as well as to the culture, so that the mixture applied to soil before tree planting was either QSB-6 alone, or a diverse mixture of microorganisms.

 

What was the amount of this mixture added to the soil before planting? If it was a high proportion, the chemical properties of the cow dung themselves would have contributed to growth. I. this case the experiment was not about the effect of QSB-6 alone, but the combined effect of bacteria and nutrients. In that case, there should have been a control of cow dung – straw addition without QSB-6 added.

 

There are multiple cases of questionable numbers. For example, line 104 reports bacterial count of 5 x 10^9 per gram, whereas Table 3 reports counts of ~ 5 x 10^6 per gram. The actinomycete count is ten time as high as the bacterial count (Table 3), but actinomycetes are but a part of the bacteria overall. While different culture media were used, and we never get all the bacteria to form colonies on agar, this is still of concern. The Y axis labels of Fig 4 range from 10^5 to 10^11. I do not see how there can be 10^ fungal cells (or even target genes) per gram of soil unless it is pure fungi.

 

Specific points:

1. The manuscript is clear, but the manuscript is full of grammatical errors.
2. Line 105 – 106: Some of these values are very low – much lower than those reported for the soil. Are there typographical errors here in the units?
3. Line 134 – as the sol for DNA extraction was reportedly kept at -80C, these samples at 4C were more likely used for culturable counts and not microbial structure.
4. Line 194: Are you certain this was from MO BIO? They were bought out by Qiagen about a decade ago, so unless the kits were that old, they were obtained from Qiagen.
5. Line 194: Please specify what gene was amplified by PCR.
6. Line 198: Please specify what plasmid was used.
7. Line 208: If you used 27f and 1492r then you amplified almost the entire 16S gene and not just one of its nine variable regions.
8. Line 227: As you report tRFLP data, why does it state here that the samples were sequenced? Do I assume that fragment lengths were determined using the same equipment used for Sanger sequencing?
9. Line 252: “became significant” – No, data are either significantly different or not. Please rephrase all your results in term of what treatments led to differences that were statistically different. You report ANOVA results, so this is a matter of revising the text.
10. Figure 1: While the data in Table 2 report that trees in TY soil were larger, they look smaller in the figure. This may the result of how photographs were taken – from what distance or with what magnification. Unless some size marker can be added, I suggest removing this figure.
11. Section 3.2: Focus on the ratios of bacteria:fungi and actinomycetes:fungi as this is likely a critical part of why the trees grew better.
12. Table 3: Actinomycetes cannot be more than bacteria overall. Please check yoru raw data again.
13. Figure 3: Please explain in the figure legend the scale used for red to blue. Was this linear or logarithmic? Also, the names of the samples are abbreviated from your other way of naming samples, so please add a key.
14. 4: Please check the unites for the four graphs – 10^5 to 10^11
15. 5: Please define the approach taken to develop the cluster analyses in the figure legend.
16. Line 448: “destroys” is a very strong word. It is more likely that there are shifts in the community composition, but a total destruction would require autoclaving, strong radiation or some other very aggressive treatment.
17. Line 450: “flora” refers to plants. I do know that the word flora was used a lot in the past to refer to bacteria, and this is probably because in the early days of microbiology in Europe, non-medical microbiology was studied in botany departments. The preferred term in the more recent literature is “microbiota”.
18. Line 458: The term “bacteriostatic” is not appropriate here. While these bacteria may have bacteriostatic properties as well, suppression of fungal growth would be fungistatic.

 

 

 

 

 

Author Response

Response to Reviewer 3 Comments

 

 

 

Point 1: The introduction is clear except for one key matter that weaves its way throughout this manuscript: What is the contribution of pathogenic fungi to ARD. In the Abstract (line 9) the authors ascribe ARD to soil fungi – “mainly caused by soil-borne fungal diseases”. In the Introduction (from line 33) the factors contributing to ARD are described as much more diverse. In line 75 the authors hypothesize that introduced microbes will suppress disease-causing fungi, thereby alleviating ARD. While tree growth parameters are significantly higher in the QSB-6 treatments, the fungal count data reported does not support a primary role for these fungo in ARD. To be considered for publication, these interrelated factors will need to be clarified.

 

Response 1: Thank you for your constructive comments. ARD has been attributed to a variety of biotic and abiotic causal factors, but current studies suggest that biotic factors such as fungi play a leading role in disease development. This has been widely demonstrated in other studies via soil pasteurization and the application of biocides (Garbeva et al., 2004). The previous research of our laboratory found that the relative abundance of F. oxysporum and F. solani was the largest difference after methyl bromide fumigation and replanted treatments in clay loam (Sheng, 2020). Quantitative analysis based on qPCR confirmed that the wide distribution of F. oxysporum in replanted soil around Bohai Gulf, and the isolated F. moniliforme (F. verticillioides), F. oxysporum, and F. proliferatum isolated from replanted apple soil around Bohai Gulf were highly pathogenic to seedlings of Malus hupehensis, which demonstrated that Fusarium was one of the main soil -borne pathogenic fungi that cause ARD in China (Liu, 2013; Yin, 2017; Wang, 2018; Duan, 2021). The discussion also mentioned that long-term continuous cropping leads to the transformation of the soil microbial community structure from “bacterial type” to “fungal type”, which destroys the soil microbial community structure, leading to an increase in harmful pathogens. The results of this experiment found that the inoculation strain QSB-6 can significantly reduce the number of soil fungi, increase the ratio of soil bacteria/fungi, and transform the soil to a "bacterial type" (Qin, 2017). The community structure of bacteria and fungi was also significantly different from that of continuous cropping soil. Based on the results of qPCR data, the abundance of soil Fusarium (F. moniliforme, F. solani, F. oxysporum, and F. proliferatum ) inoculated with the strain QSB-6 was also significantly reduced, and the content of phenolic acids in the soil was also significantly reduced. Previous studies have found that phlorizin can promote the growth of F. moniliforme (Yin, 2017). Based on the above results, it can be concluded that the inoculation strain QSB-6 can inhibit the reproduction of harmful Fusarium in the soil, thereby reducing ARD. Thank you very much!

 

Garbeva, P. V., Van Veen, J. A., and Van Elsas, J. D. (2004). Microbial diversity in soil: selection of microbial populations by plant and soil type and implications for disease suppressiveness. Annu. Rev. Phytopathol. 42, 243-270.

Sheng, Y.F.; Wang, H.Y.; Wang, M.; Li, H.; Xiang, L.; Pan, F.B.; Mao, Z.Q. Effects of soil texture on the growth of young apple trees and soil microbial community structure under replanted conditions. Horticultural Plant Journal 2020, 6(3), 123-131.

 

Wang, G.S.; Yin, C.M.; Pan, F.B.; Wang, X.B.; Xiang, L.; Wang, Y.F.; Mao, Z.Q. Analysis of the fungal community in apple replanted soil around Bohai Gulf. Horticultural Plant Journal 2018, 4(5), 175-181.

 

Yin, C.M.; Xiang, L.; Wang, G.S.; Wang, Y.F.; Shen, X.; Chen, X.S.; Mao, Z.Q. Phloridzin promotes the growth of Fusarium moniliforme (Fusarium verticillioides). Scientia Horticulturae 2017, 214, 187-194.

 

Duan, Y.N.; Chen, R.; Zhang, R.; Jiang, W.T.; Chen, X.S.; Yin, C.M.; Mao, Z.Q. Isolation, identification, and antibacterial mechanisms of Bacillus amyloliquefaciens QSB-6 and its effect on plant roots. Frontiers in microbiology 2021, 12.

 

Qin, S., Yeboah, S., Cao, L., Zhang, J., Shi, S., & Liu, Y. (2017). Breaking continuous potato cropping with legumes improves soil microbial communities, enzyme activities and tuber yield. PloS one, 12(5), e0175934.

 

Liu, Z. The Isolation and Identification of Fungi Pathogen from Apple Replant Disease in Bohai Bay Region and the Screening of the Antagonistic Trichoderma Strains. Master's Thesis. Shandong Agricultural University, China. 2013.

 

Point 2: The fumigation treatment clearly led to substantial growth increase of the trees, but the fungal counts and qPCR of specific fungo show that fumigation did not kill a large proportion of the fungi. This raises the question what the contribution of fungi are to suppression of apple tree growth – linking in with the point of the previous paragraph.

 

Response 2: Thank you for your constructive comments. Although chemical fumigation treatment can effectively kill most of the fungi in the soil, after seven months, the soil itself will recruit some fungi to form a new microbial community, and the number of fungi in the soil will slowly increase. Compared with the replanted soil, the number of fungi in LZ, QX, and TY orchards after fumigation treatment was reduced by 40.38%, 68.44% and 49.08%, respectively. The numbers of F. solani and F. oxysporum both reduced by about 50%, which was consistent with the results of Sheng (2020). Compared with the replanted soil, the number of fungi in sandy loam, loam and clay loam fumigated by methyl bromide was reduced by 43.50%, 38.94% and 63.91%, respectively. The number of F. solani and F. oxysporum also reduced significantly. In addition, the fungal composition of the soil after the fumigation treatment is obviously different from that of the replanted soil. In the fumigation treatment, the abundance of of Gp4, Ohtaekwangia, Opitutus, Mortierella and Synchytrium are higher, and the abundance of Fusarium is significantly reduced, which can effectively controlled cucumber Fusarium wilt disease Li , 2016). It shows that the fumigation treatment can improved the soil fungus community, reduced the abundance of the ARD-related pathogen Fusarium in the soil, and promote the growth of replanted young apple trees.Thank you very much!

 

Sheng, Y.F.; Wang, H.Y.; Wang, M.; Li, H.; Xiang, L.; Pan, F.B.; Mao, Z.Q. Effects of soil texture on the growth of young apple trees and soil microbial community structure under replanted conditions. Horticultural Plant Journal 2020, 6(3), 123-131.

 

Li, R., Shen, Z., Sun, L., Zhang, R., Fu, L., Deng, X., & Shen, Q. (2016). Novel soil fumigation method for suppressing cucumber Fusarium wilt disease associated with soil microflora alterations. Applied Soil Ecology, 101, 28-36.

Point 3: The nature of the QSB-6 supplementation must be clarified. Was the cow dung and straw sterile or not when the culture was added? As this mixture was incubated warm for a day, non-sterile cow dung – straw microbiota could well have responded to the nutrients remaining in the bacterial culture, as well as to the culture, so that the mixture applied to soil before tree planting was either QSB-6 alone, or a diverse mixture of microorganisms.

 

Response 3: Thank you for your constructive comments. In Materials and Method 2.1, it has been mentioned that cow dung and wheat straw are fully sterilized when adding the inoculum. The bacterial density finally reached 5.0×10⁹ CFUg^-1_. The carrier can support the survival of strain QSB-6 while inhibiting the growth of target pathogens, thereby improving the performance of strain QSB-6 for plant disease control (Malusá, 2012; Wei, 2015). The raw materials (cow dung and wheat straw) in the formula are cheap and easy to obtain, and the fermentation level is high, which provides a good foundation for its large-scale industrial production (Smith, 1992; Vijayaraghavan, 2016). Thank you very much!

 

Smith, R. S. (1992). Legume inoculant formulation and application. Canadian journal of microbiology, 38(6), 485-492.

 

Wei, Z., Huang, J., Yang, C., Xu, Y., Shen, Q., and Chen, W. (2015). Screening of suitable carriers for Bacillus amyloliquefaciens strain QL-18 to enhance the biocontrol of tomato bacterial wilt. Crop Protection, 75, 96-103.

 

Malusá, E., Sas-Paszt, L., and Ciesielska, J. (2012). Technologies for beneficial microorganisms inocula used as biofertilizers. The scientific world journal, 2012.

 

Vijayaraghavan, P., Arun, A., Al-Dhabi, N. A., Vincent, S. G. P., Arasu, M. V., & Choi, K. C. (2016). Novel Bacillus subtilis IND19 cell factory for the simultaneous production of carboxy methyl cellulase and protease using cow dung substrate in solid-substrate fermentation. Biotechnology for biofuels, 9(1), 1-13.

 

Point 4: What was the amount of this mixture added to the soil before planting? If it was a high proportion, the chemical properties of the cow dung themselves would have contributed to growth. I. this case the experiment was not about the effect of QSB-6 alone, but the combined effect of bacteria and nutrients. In that case, there should have been a control of cow dung – straw addition without QSB-6 added.

 

Response 4: Thank you for your constructive comments. In Materials and Method 2.3, it has been mentioned that the amount of application for each young tree is controlled at 1kg. This experiment set up a blank carrier treatment without adding strain QSB-6, namely T1, to further verify the effect of strain QSB-6 on replanting young apple trees and the soil microbial environment. Thank you very much!

 

Point 5: There are multiple cases of questionable numbers. For example, line 104 reports bacterial count of 5.0 × 10⁹ CFU·g^-1, whereas Table 3 reports counts of ~ 5 x 10^6 per gram. The actinomycete count is ten time as high as the bacterial count (Table 3), but actinomycetes are but a part of the bacteria overall. While different culture media were used, and we never get all the bacteria to form colonies on agar, this is still of concern. The Y axis labels of Fig 4 range from 10^5 to 10^11. I do not see how there can be 10^ fungal cells (or even target genes) per gram of soil unless it is pure fungi.

 

Response 5: Thank you for your constructive comments. The Chinese bio-organic fertilizer production standard stipulating that the functional microorganism content should be greater than 2.0 × 10⁷ CFU g⁻¹ dry formulation (Emmert and Handelsman, 1999). The bacterial count reported in line 104 is 5.0 × 10⁹ CFU·g^-1, which is the content of the strain QSB-6 in the bacterial fertilizer, which has reached the production standard. The counting units in Table 3 have been checked and modified.  The CFU per gram of dry soil was used as the measurement unit for the bacterial, fungal, and actinomycetes populations. The data in Figure 4 is based on the plasmid DNA concentration and the transformed copy concentration. For the specific formula, see Materials and Method 2.4.6. It is used to measure the difference in the relative abundance of Fusarium among different treatments. The value does not represent the number of Fusarium spores present in the soil. Specific primers and standard curves have been added in the supplementary materials.

 

Emmert, E. A., and Handelsman, J. (1999). Biocontrol of plant disease: a (Gram-) positive perspective. FEMS Microbiology letters, 171(1), 1-9.

 

Point 6: The manuscript is clear, but the manuscript is full of grammatical errors.

 

Response 6: Thank you for your constructive comments. According to your suggestions, We have revised our manuscript by EditorBar Language Editing, Beijing for English writing. Thank you very much!

 

Point 7: Line 105 – 106: Some of these values are very low – much lower than those reported for the soil. Are there typographical errors here in the units?

 

Response 7: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 8: Line 134 – as the sol for DNA extraction was reportedly kept at -80℃, these samples at 4℃ were more likely used for culturable counts and not microbial structure.

 

Response 8: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 9: Line 194: Are you certain this was from MO BIO? They were bought out by Qiagen about a decade ago, so unless the kits were that old, they were obtained from Qiagen.

 

Response 9: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 10: Line 194: Please specify what gene was amplified by PCR.

 

Response 10: Thank you for your constructive comments. Specific primers and standard curves have been added in the supplementary materials. Thank you very much!

 

Point 11: Line 198: Please specify what plasmid was used.

 

Response 11: Thank you for your constructive comments. Specific primers and standard curves have been added in the supplementary materials. Four Fusarium (F. moniliforme, F. solani, F. oxysporum, and F. proliferatum) DNAs were used as templates, and specific primers were used for PCR amplification. The purified PCR products were connected to pMD18-T vector (TaKaRa Biotech, China), transformed into E.coli competent cells DH5α (TaKaRa Biotech, China). After sequencing verification, the plasmid was extracted according to the method of the plasmid extraction kit (TaKaRa Biotech, China). Measure its concentration, and carry out a gradient dilution with sterile water. The standard curves were generated by plotting the cycle threshold (Cq) values obtained for each specific DNA concentration versus the log of the initial concentration of species DNA.Thank you very much!

 

Point 12: Line 208: If you used 27f and 1492r then you amplified almost the entire 16S gene and not just one of its nine variable regions.

 

Response 12: Thank you for your constructive comments. The primers used in this study were used for the amplification of most eubacterial 16SrRNAs (Weisburg, 1991). Thank you very much!

 

Weisburg, W. G., Barns, S. M., Pelletier, D. A., & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of bacteriology, 173(2), 697-703.

 

Point 13: Line 227: As you report tRFLP data, why does it state here that the samples were sequenced? Do I assume that fragment lengths were determined using the same equipment used for Sanger sequencing?

 

Response 13: Thank you for your constructive comments. According to your suggestions, the method has been re-described. Thank you very much!

 

Point 14: Line 252: “became significant” – No, data are either significantly different or not. Please rephrase all your results in term of what treatments led to differences that were statistically different. You report ANOVA results, so this is a matter of revising the text.

 

Response 14: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 15: Figure 1: While the data in Table 2 report that trees in TY soil were larger, they look smaller in the figure. This may the result of how photographs were taken – from what distance or with what magnification. Unless some size marker can be added, I suggest removing this figure.

 

Response 15: Thank you for your constructive comments. The picture has been re-adjusted and modified. The picture was the result of artificial shooting with Canon PowerShotG16 camera. In order to ensure that the trees are displayed completely in the picture, the shooting distance is random, but each picture uses the same plant height measuring rule as a measuring tool. Can it be used as a size marker? If not, I can accept to delete this picture. Thank you very much!

 

Point 16: Section 3.2: Focus on the ratios of bacteria:fungi and actinomycetes:fungi as this is likely a critical part of why the trees grew better.

 

Response 16: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 17: Table 3: Actinomycetes cannot be more than bacteria overall. Please check yoru raw data again.

 

Response 17: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised, and corrected the counting unit. Thank you very much!

 

Point 18: Figure 3: Please explain in the figure legend the scale used for red to blue. Was this linear or logarithmic? Also, the names of the samples are abbreviated from your other way of naming samples, so please add a key.

 

Response 18: Thank you for your constructive comments. According to your suggestions, Figure 3 has been annotated in detail. Thank you very much!

 

Point 19:  Please check the unites for the four graphs – 10^5 to 10^11.

 

Response 19: Thank you for your constructive comments. According to your suggestions, I have recalculated the data, there is no error in the data unit, and the order of magnitude can refer to other articles in this laboratory (see below). Thank you very much!

 

Sheng, Y.F.; Wang, H.Y.; Wang, M.; Li, H.; Xiang, L.; Pan, F.B.; Mao, Z.Q. Effects of soil texture on the growth of young apple trees and soil microbial community structure under replanted conditions. Horticultural Plant Journal 2020, 6(3), 123-131.

 

Duan, Y.N.; Chen, R.; Zhang, R.; Jiang, W.T.; Chen, X.S.; Yin, C.M.; Mao, Z.Q. Isolation, identification, and antibacterial mechanisms of Bacillus amyloliquefaciens QSB-6 and its effect on plant roots. Frontiers in microbiology 2021, 12.

 

Point 20: Please define the approach taken to develop the cluster analyses in the figure legend.

 

Response 20: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 21: Line 448: “destroys” is a very strong word. It is more likely that there are shifts in the community composition, but a total destruction would require autoclaving, strong radiation or some other very aggressive treatment.

 

Response 21: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 22: Line 450: “flora” refers to plants. I do know that the word flora was used a lot in the past to refer to bacteria, and this is probably because in the early days of microbiology in Europe, non-medical microbiology was studied in botany departments. The preferred term in the more recent literature is “microbiota”.

 

Response 22: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 23: Line 458: The term “bacteriostatic” is not appropriate here. While these bacteria may have bacteriostatic properties as well, suppression of fungal growth would be fungistatic.

 

Response 23: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

 

 

 

 

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Editing date	2021-12-14
Title	Effects of Bacillus amyloliquefaciens QSB-6 on the growth of replanted apple trees and soil microbial environment
Corresponding author	Zhiquan Mao
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We are truly grateful to you critical comments and thoughtful suggestions on our manuscript, comprehensive the comments of reviewer # 1 and 2, we have improved and revised some of the content in the manuscript, and checked the full text. Please review again during your busy schedule. Thank you very much! The specific changes are as follows:

 

Response to Reviewer 1 Comments

 

 

Point 1: The term “bacterial fertilizer” should be reformulated since evidence of plant growth promotion by Bacillus amyloliquefaciens QSB-6 is not provided by this study.

Bacterial name missing “verify the effects of QSB-6”

Response 1: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 2: This study is based on the assumption that “Fusarium was one of the main soil-borne pathogenic fungi that cause ARD” (line 44), therefore most if the study is oriented towards this fungus. Nevertheless, a number of studies reports that the role of Fusarium towards plants is not so strict, but rather may also be associated with good soil health in permanent tree crops, for example:

 

Bacon, C.W., Yates, I.E., 2006. Endophytic Root Colonization by Fusarium Species: Histology, Plant Interactions, and Toxicity BT  - Microbial Root Endophytes, in: Schulz, B.J.E., Boyle, C.J.C., Sieber, T.N. (Eds.), . Springer Berlin Heidelberg, Berlin, Heidelberg, pp. 133–152. doi:10.1007/3-540-33526-9_8

 

Therefore this concept should be reformulated in the manuscript.

 

Response 2:  Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 3: Three orchards have been tested in this study, but data on the effect of the Bacillus QSB-6 strain treatment on the growth of apple plants have been analysed separately per each site (results in paragraph 3.1), in this way it is difficult to draw conclusions on the effect of strain QSB-6.

 

Response 3: Thank you for your constructive comments. In this experiment, three orchards with different textures (loam, sandy loam and clay loam) were selected to evaluate the biocontrol effect of strain QSB-6, and it was found that strain QSB-6 had the most significant effect on the growth promotion of replanted apple young trees under the condition of loam soil (TY), followed by sandy loam (QX) and clay loam (LZ). This was consistent with the results of previous studies (Sheng, 2020). The occurrence of Apple replant disease was the most serious under clay loam conditions, followed by sandy loam and loam. However, strain QSB-6 also exerts a good biocontrol effect under the clay loam condition. The overall growth of the replanted apple saplings treated with T2 was better than that with T1(Figure 1). From this, it can be concluded that the QSB-6 bacterial fertilizer treatment can promote the growth of replanted young apple trees. Thank you very much!

 

Sheng, Y.F.; Wang, H.Y.; Wang, M.; Li, H.; Xiang, L.; Pan, F.B.; Mao, Z.Q. Effects of soil texture on the growth of young apple trees and soil microbial community structure under replanted conditions. Horticultural Plant Journal 2020, 6(3), 123-131.

Point 4: In Data analysis paragraph it is reported that “Comparisons were made using the least significant difference (LSD) test” (line 231), while the note in Table 2 reports that “Numbers followed by the same letter were not significantly different based on Duncan Multiple Range Test”. It is not clear which statistical test was performed to compare plant growth parameters. Furthermore, in Table 2, T1 and T2 treatment groups share the same letter in almost all conditions, meaning that the differences between the groups are not statistically significant, how can the authors say that the bacterial fertilizer treatment (T2) significantly promoted plant growth if it did not differ from control (T1)??

Response 4: Thank you for your constructive comments. According to your suggestion, the data has been analyzed. In Table 2, the plant height and ground diameter of the T1 and T2 treatments of the LZ orchard were also different, but they were not significant, and there were significant differences in the number of branches and the average branch length. Figure 1 showed that the overall growth of the replanted apple saplings treated with T2 was better than that with T1. The insignificant difference between LZ orchard treatments may be related to the high severity of continuous cropping under clay loam conditions (Sheng, 2020) and the slower growth and reproduction of strain QSB-6. It can also be seen from the results in Table 3 that the number of bacteria in the LZ orchard treated with T2 was significantly lower than that in the QX and TY orchards. There were significant differences between the T1 and T2 treatments of QX and TY orchards. Thank you very much!

 

Point 5: Authors state that “strain QSB-6 can reproduce normally after inoculating the soil” (line 362) but no specific viability test was performed.

 

Response 5: Thank you for your constructive comments. In this experiment, microbial culture counts were carried out on the rhizosphere soil of different treatments using the dilution method of plate counting. It was found that the number of bacteria in the rhizosphere soil of the inoculated strain QSB-6 was significantly higher than that of other treatments, and the results of the previous potting treatments were consistent (Duan, 2021), and most of the bacterial colonies cultured on the plate were consistent with the morphology of strain QSB-6. A single colony with the same morphology was randomly picked and sequenced, and the similarity with the strain QSB-6 was found to be 100% (results not shown). so it was concluded that strain QSB-6 can reproduce normally after inoculating the soil, and the addition of the carrier also provided nutrients for strain QSB-6 to grow normally in the soil. Thank you very much!

Duan, Y.N.; Chen, R.; Zhang, R.; Jiang, W.T.; Chen, X.S.; Yin, C.M.; Mao, Z.Q. Isolation, identification, and antibacterial mechanisms of Bacillus amyloliquefaciens QSB-6 and its effect on plant roots. Frontiers in microbiology 2021, 12.

 

Point 6: Diversity indices described in paragraph 3.6 (lines 387-391) would benefit from bibliographic references.

 

Response 6: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 7: Data analysis of diversity indices should be revised, since in line 392 it is reported that “The diversity of soil fungi communities was significantly increased” , while in the abstract “QSB-6 can significantly reduce the diversity of soil fungi” line 23. Simpson’s index does not differ between T1 and T2 in fungi. Same for Shannon index of bacterial communities.

 

Response 7: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Simpson, Shannon, and Brillouin Index are all used to measure the diversity of microorganisms. There are limitations to using a single index to measure diversity.  Shannon index is closely related to richness and more sensitive to sparse species, while Simpson index is closely related to evenness and more sensitive to enriched species (Magurran, 1998; Xu, 2011).  No difference in Simpson index between T1 and T2 treatments can only indicate that the addition of organic carrier and bacterial fertilizer resulted in the similarity of the enriched species (the relative important value of newly added species is higher than 0.72) in the rhizosphere soil, while the Shannon and Brillouin Indexes of the T2 treatment were significantly lower than those of the T1 treatment. No difference in Shannon index between T1 and T2 treatments can only indicate that the addition of organic carrier and bacterial fertilizer results in the similarity of sparse species (the relative important value of newly added species is less than 0.72) in rhizosphere soil, while the Simpson and Brillouin Indexes of T2 treatment were significantly higher than those of T1 treatment. Thank you very much!

 

Magurran, A. E. (1988). Ecological diversity and its measurement. Princeton university press.

 

Xu, Q., Zhang, F., Xu, Z. Q., Jia, Y. L., & You, J. M. (2011). Some characteristics of Simpson index and the Shannon-Wiener index and their dilution effect. Pratacultural Science, 4, 527-531.

 

Point 8: The first part of the discussion (lines 417-433) should be moved to the introduction.

 

Response 8: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 9: It is stated that “This study found that the addition of strain QSB-6 can significantly reduced the content of the main phenolic acids in the soil” lines 482-483, but the significance of the results reported in figure 3 is not reported.

 

Response 9: Thank you for your constructive comments. According to your suggestions, the relevant data of Figure 3 has been added to the supplementary materials. Thank you very much!

 

Point 10: Which “pot study”? reference missing.

 

Response 10: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 11:Strain XNRB-3? Line 519

 

Response 11: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 12:English language should be revised.

 

Response 12: Thank you for your constructive comments. According to your suggestions, We have revised our manuscript by EditorBar Language Editing, Beijing for English writing. Thank you very much!

 

 

Response to Reviewer 2 Comments

 

 

 

Point 1:  The entire manuscript is poorly adapted to the TEMPLATE requirements of the Horticulture journal, please pay special attention to the References section, but also to the formatting of Tables and chapter titles and sub-headings. 


 

Response 1: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 2: Keywords should be in alphabetical order and should not repeat words appearing in the title of the manuscript.

 

Response 2: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 3: Tables 2, 3 and 4 are difficult to read, please add lines between LZ, QX and TY treatments (Tables 2 and 3) and between Fungi and Bacterial treatments (Table 4).

 

Response 3: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 4: In Tables 3 and 4 and Figures 2-5, abbreviations should be explained or referred to Table 2.

 

Response 4: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The manuscript has been improved.

 

Nevertheless, I get back to my previous comment on the role of Fusarium in ARD, I understand that the authors oriented their study towards Fusarium as major cause of ARD, but please remove reference 9 (Plant Soil (2012) 357:381–393) from line 49 since that study shows that Fusarium was present in soil in Italy, but that it was not correlated with plant disease.

 

Please add reference to sentence in Discussion "long-term continuous cropping leads to the transformation of the soil microbial community structure from a “bacterial type” to a “fungal type”, lines 457-8.

Author Response

Response to Reviewer 1 Comments

 

We are truly grateful to yours’ critical comments and thoughtful suggestions on our manuscript Effects of Bacillus amyloliquefaciens QSB-6 on the growth of replanted apple trees and the soil microbial environment (horticulturae-1491343). Those comments are all valuable for us. Based on these comments and suggestions, we have made careful modifications on the original manuscript, and the itemized response to reviewer’s comments is attached. We hope that these revisions are satisfactory and that the revised version will be acceptable for publication in Horticulturae.

 

Point 1: Nevertheless, I get back to my previous comment on the role of Fusarium in ARD, I understand that the authors oriented their study towards Fusarium as major cause of ARD, but please remove reference 9 (Plant Soil (2012) 357:381–393) from line 49 since that study shows that Fusarium was present in soil in Italy, but that it was not correlated with plant disease. 

 

Response 1: Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

 

Point 2: Please add reference to sentence in Discussion "long-term continuous cropping leads to the transformation of the soil microbial community structure from a “bacterial type” to a “fungal type”, lines 457-8.

 

Response 2:  Thank you for your constructive comments. According to your suggestions, the manuscript has been revised. Thank you very much!

Author Response File: Author Response.docx