Endophyte Bacillus velezensis Isolated from Citrus spp. Controls Streptomycin-Resistant Xanthomonas citri subsp. citri That Causes Citrus Bacterial Canker
Guixia Hao
Round 1
Reviewer 1 Report
The authors investigated the antagonistic activities of 66 endophytic bacteria isolated from nine citrus cultivars to control streptomycin-resistant Xcc. They found the suspension of EB-39 exhibited the highest antibacterial activity against three wild-type and six streptomycin-resistant Xcc strains, with the inhibition zones between 39.47 ± 1.6 and 45.31 ± 1.6 mm. The ethyl acetate extract of EB-39 also controlled both wild-type and streptomycin-resistant Xccstrains, with the inhibition zones between 29.28 ± 0.6 and 33.88 ± 1.3 mm. Scanning electron microscopy indicated the ethyl acetate extract of EB-39 induced membrane damage and lysis. In vivo experiments using the detached leaves of a susceptible Citrus species showed that EB-39 significantly reduced the incidence of canker on the infected leaves by 38%.In addition, the authors identified the EB-39 as Bacillus velezensisby BLAST hoomolgy analysis on the 16S rRNA gene sequence.
These results suggest that isolated EB-39 is a novel biocontrol agent against CBC caused by wild-type and streptomycin-resistantXcc strains. However, there are some major concerns about their conclusions:
1. more experiments are needed to confirm that EB-39 is Bacillus velezensis. For example, the authors should use standard or commercial cells of Bacillus velezensis to perform the same experiments that have been done on their EB-39 endophyte. These results will confirm if EB-39 is Bacillus velezensis and provide more insights about EB-39.
2. The SEM figures showthe ethyl acetate extract of EB-39 induced cell lysis. But the mechanism is not clear. More experiments are required to test if, as the authors claimed, EB-39 releases antibacterial compounds to break the cell wall.
3. For MIC assay, it is not clear what concentration (protein concentration or chemical concentration) of EB-39 was monitored.
Some minor concerns:
1. In Figure 3B, it isnot clear what a, b, and c mean.
2. Check the legend for other figures as well.
Author Response
Reviewer #1
Comments and Suggestions for Authors
The authors investigated the antagonistic activities of 66 endophytic bacteria isolated from nine citrus cultivars to control streptomycin-resistant Xcc. They found the suspension of EB-39 exhibited the highest antibacterial activity against three wild-type and six streptomycin-resistant Xcc strains, with the inhibition zones between 39.47 ± 1.6 and 45.31 ± 1.6 mm. The ethyl acetate extract of EB-39 also controlled both wild-type and streptomycin-resistant Xcc strains, with the inhibition zones between 29.28 ± 0.6 and 33.88 ± 1.3 mm. Scanning electron microscopy indicated the ethyl acetate extract of EB-39 induced membrane damage and lysis. In vivo experiments using the detached leaves of a susceptible Citrus species showed that EB-39 significantly reduced the incidence of canker on the infected leaves by 38%. In addition, the authors identified the EB-39 as Bacillus velezensis by BLAST homology analysis on the 16S rRNA gene sequence.
These results suggest that isolated EB-39 is a novel biocontrol agent against CBC caused by wild-type and streptomycin-resistant Xcc strains. However, there are some major concerns about their conclusions:
More experiments are needed to confirm that EB-39 is Bacillus velezensis. For example, the authors should use standard or commercial cells of Bacillus velezensis to perform the same experiments that have been done on their EB-39 endophyte. These results will confirm if EB-39 is Bacillus velezensis and provide more insights about EB-39.
Response: We appreciate the reviewer for the kind suggestion. Actually, in our unpublished data, we got the several Bacillus velezensis isolates from the Rural Development Agriculture (RDA) from Korea government. The isolates included standard B. velezensis, therefore we could confirm our B. velezensis is a novel endophyte controlling wilde-type and also streptomycin-resistant Xcc strains. We got the similar data for the usage of the several B. velezensis, and are preparing several papers using the data.
Furthermore, to identify the differences in the antibacterial capacity for the several B. velezensis isolates, we are planning to sequence the whole genome and do RNA sequencing in order to get insights about the genes in EB-39.
In addition to our study, there are several papers indicate the antimicrobial activity of B. velezensis as follows;
Huang et al. (2017) Bacillus velezensis strain HYEB5-6 as a potential biocontrol agent against anthracnose on Euonymus japonicas. Biocontrol Science and Technology 27: 636–653. Identified the Bacillus velezensis by 16s rRNA sequence.
Lim et al. (2017) Diffusible and volatile antifungal compounds produced by an antagonistic Bacillus velezensis G341 against various phytopathogenic fungi. Plant Pathol. J. 33 : 488-498. Identified the Bacillus velezensis by 16s rRNA and gyrA sequences.
The SEM figures show the ethyl acetate extract of EB-39 induced cell lysis. But the mechanism is not clear. More experiments are required to test if, as the authors claimed, EB-39 releases antibacterial compounds to break the cell wall.
Response: The ethyl acetate extract of EB-39 induced cell lysis, indicating the antimicrobial compounds that disrupt the cell envelope, consequently cell contents was leaked and cell debris or fibrous material was observed. We are in the process of separating and identifying the active compounds with the help of an expert majoring in natural compounds. In addition, we are conducting and analyzing the data of RNA sequencing from the cells cultured in two different culturing media. EB-39 exerted different antibacterial activity against Xcc when it was grown in the two different media, therefore, we are in the process of analyzing the RNA sequencing data from the EB-39 grown in the two different media to identify the active compounds.
After isolating the active compounds from EB-39, we will use the active compounds to identify whether they can induce cell lysis on Xcc. We also added one sentence as “The antimicrobial compounds might destabilize Xcc cell membranes, leading to leaking of cell contents and leaving of cell debris.” (Lines 400-401).
For MIC assay, it is not clear what concentration (protein concentration or chemical concentration) of EB-39 was monitored.
Response: We used various concentrations of ethyl acetate extract of EB-39 (ranging from 23.4–1,500 µg mL-1) to determine the values of MIC and MBC analysis (mentioned in the manuscript, Line-203-204 and also the supplementary figure S3). To determine the MIC value, two-fold dilution method is used, and the MIC values of EB-39 ethyl acetate extract were ranged from 46.8 to 93.7 µg mL-1 (Table 3) (Line-322).
Some minor concerns:
In Figure 3B, it is not clear what a, b, and c mean.
Response: We rewrote the figure legends as the reviewer had suggested.
Check the legend for other figures as well.
Response: We appreciate the reviewers for careful reviewing our manuscript. We checked all the figures legends and revised them correctly.
Reviewer 2 Report
Muhammad and coworkers investigated the antagonistic activities of entophytic bacteria from citrus and they identified one of them, EB-39, exhibited the highest antibacterial activity against the wild-type and streptomycin-resistant Xcc strains. Further they demonstrated that the ethyl acetate extract of EB-39 induced membrane damage and lysis of Xcc strains. They also showed that EB-39 significantly reduced citrus canker using detached leaf assay. This work is interesting and may contributes to control citrus canker in the long run. The experiments were well designed and the results were clearly explained. In addition to show EB-39 inhibition of Xcc strains growth, they also demonstrated the membrane were disrupted from the chemicals produced by this endophyte. Some minor points to consider are included below: Line 52, suggest delete “many varieties of ”. It is kind of confusion to list citrus varieties with other plant species. Line 126, Table 1, do authors know the listed citrus varieties are resistant or susceptible? If yes, please list in table 1. In addition, Line 257-258: the three promising endophytes were isolated from which citrus variety? Please provide the information. Line 129: I would not call “in vivo” for detached leaf assay. Just state detached leaf assay. I would think in vivo as in planta assay. Line 166: why killed with chloroform? Please explain. Line 216: why use methanol instead of ethyl acetate treatment as control? Same for line 310. explain Line 229: In vivo, suggest change to “Detached leaf” Line 243: tested change to “scored” Line 347: no scale bar in figure 4 Figure 5: It will be good to determine the bacterial number in different infiltrated zones. You can see how Xcc strains are inhibited and also the endophyte multiplication. By the way, how this endophyte get into plant? Through stomata or seed? As you mentioned in line 430 for spray treatment. Line 386: streptomycin-resistant mutant strains
Author Response
Reviewer # 2
Comments and Suggestions for Authors
Muhammad and coworkers investigated the antagonistic activities of entophytic bacteria from citrus and they identified one of them, EB-39, exhibited the highest antibacterial activity against the wild-type and streptomycin-resistant Xcc strains. Further they demonstrated that the ethyl acetate extract of EB-39 induced membrane damage and lysis of Xcc strains. They also showed that EB-39 significantly reduced citrus canker using detached leaf assay. This work is interesting and may contributes to control citrus canker in the long run. The experiments were well designed and the results were clearly explained. In addition to show EB-39 inhibition of Xcc strains growth, they also demonstrated the membrane were disrupted from the chemicals produced by this endophyte.
Some minor points to consider are included below:
Line 52, suggest delete “many varieties of”. It is kind of confusion to list citrus varieties with other plant species.
Response: We appreciate the reviewer for the kind suggestion. We deleted “many varieties of” from the sentence (Line-52).
Line 126, Table 1, do authors know the listed citrus varieties are resistant or susceptible? If yes, please list in table 1.
Response: We used nine (9) different varieties for endophyte isolation. But for the experiments of pathogenicity and disease suppression ability of EB-39 against X. citri subsp. citri (XccW2) on one variety of citrus (Hwangkeum hyang), which is very susceptible to Xanthomonas and easy to determine the effect of extract on the capacity for disease control. We did not check the levels of susceptibility of the citrus varieties by ourselves, therefore, we did not include the resistant or susceptible information for the citrus varieties.
Line 257-258: The three promising endophytes were isolated from which citrus variety? Please provide the information.
Response: We included one sentence in the manuscript as “The top three bacterial strains with antibacterial activity were EB-35, EB-39, and EB-44 isolated from Palsak mandarin, Dangyuja mandarin, and Hwangkeum hyang Citrus spp., respectively (Line-258-260).
Line 129: I would not call “in vivo” for detached leaf assay. Just state detached leaf assay. I would think in vivo as in planta assay.
Response: We appreciate the reviewer for the kind suggestion. We changed the “in vivo” words by “detached leaf assay” in the manuscript.
Line 166: Why killed with chloroform? Please explain.
Response: We tried to test the active compounds in the isolates, therefore, we should have killed all grown isolates, then added Xcc. The inhibition zone indicated the EB isolates contained antibacterial compounds in them; furthermore, killing EB isolates by chloroform could have the role of restricting the overgrowth of EB to confirm the activity of the EB metabolites on a certain time.
Line 216: Why use methanol instead of ethyl acetate treatment as control? Same for line 310. Explain.
Response: We used ethyl acetate during the metabolite extraction. After drying the ethyl acetate extract through rotary evaporation, the residues were collected in a tubular vial and dissolved in methanol. After drying the methanol in fume hood, the final concentration were made using methanol. As our extracted metabolites are dissolved in methanol, we used methanol as control.
There can be several advantages of using methanol for re-dissolving the metabolites instead of using ethyl acetate. Ethyl acetate can be used for the re-dissolving solvent, however, ethyl acetate is easily evaporated than methanol, and further isolation of metabolites using HPLC is not possible for the metabolites in ethyl acetate solution. When extracts are dissolved in methanol, then the metabolites in the extract can be analyzed using the HPLC column.
Line 229: In vivo, suggest change to “Detached leaf”.
Response: We appreciate the reviewer for the kind suggestion. We changed the “in vivo” words by “detached leaf assay” in the whole manuscript.
Line 243: Tested change to “scored”.
Response: We changed the word “tested” by “scored” (Line-245).
Line 347: No scale bar in figure 4.
Response: We added the scale bars in the Fig. 4. Actually in the bottom of the figures, there is a scale bar of 3 microM as dotted lines. We also included the scale bars in each figures in the Figure 4.
Figure 5: It will be good to determine the bacterial number in different infiltrated zones. You can see how Xcc strains are inhibited and also the endophyte multiplication.
Response: As the reviewer suggested, it would be great to determine the bacterial number in different infiltrated zones. When we conducted the experiments, we observed necrotic regions by the infiltration of Xcc, therefore, we did not count the number of bacterial cells. Instead we measured the disease lesion, so hope this experimental data would be enough for the submission of this manuscript.
Line 430: By the way, how this endophyte get into plant? Through stomata or seed? As you mentioned in line 430 for spray treatment.
Response: The endophyte can enter into the plant body by stomata, various wounds and roots. Santoyo et al. (Plant growth-promoting bacterial endophytes. Microbiological Research 183. 2016. 92–99) reported that the common mode of entry of endophytic bacteria into plant tissues is through primary and lateral root cracks, and diverse tissue wounds occurring as a result of plant growth. The endophytes may also enter into the plants through stomata, particularly on leaves and young stems.
Line 289: streptomycin-resistant mutant strains.
Response: As the reviewer suggested, we changed the “streptomycin-resistant mutant type strains” to “streptomycin-resistant mutant strains”.
Round 2
Reviewer 1 Report
For the major concenrs, the authors provided more information in their responses to review comments, but not enough to satisfy this reviewer without showing the data. If the authors had done similar experiments with standard B velezensis, they should show them in this paper not in future papers.
For the minor concerns, the authors edited the figure legends. However, it is still necessary to specify what a, b, and c represent, at least in one figure.
Author Response
Comments and Suggestions for Authors
Comments: For the major concerns, the authors provided more information in their responses to review comments, but not enough to satisfy this reviewer without showing the data. If the authors had done similar experiments with standard B velezensis, they should show them in this paper not in future papers.
Response:
We appreciate the reviewer for the kind suggestion. We added a figure (Figure 3C) in the manuscript and also changed the legends. We added two sentences as “We compared the antibacterial activity of EB-39 with a standard B. velezensis (KACC no. 17177) obtained from the Rural Development Agriculture (RDA), Republic of Korea. Compared with the extract of the standard B. velezensis, that of our newly isolated B. velezensis EB-39 had 21.1% and 18.0% higher antibacterial activity against XccW1 and XccM4 using 1 mg of ethyl acetate extract mL-1, respectively (Figure 3C). ” (Lines 302-307)
Comments: For the minor concerns, the authors edited the figure legends. However, it is still necessary to specify what a, b, and c represent, at least in one figure.
Response:
We appreciate the reviewers for careful reviewing our manuscript. We checked all the figures legends and revised them correctly. We also changed the different letters on bars which indicate significant differences at p < 0.05 by Duncan’s test. Instead of using “a, b, c’ on the bars, we changed the letters to “x, y, z” to avoid confusion.
