Development of a Highly Sensitive Loop-Mediated Isothermal Amplification Incorporated with Flocculation of Carbon Particles for Rapid On-Site Diagnosis of Blood Disease Bacterium Banana

Round 1

Reviewer 1 Report

This manuscript shows that LAMP with specific primers has been developed to target bananas blood disease (BDB) which have been considered as the major obstacles to banana production. This newly developed technique has high sensitivity and specificity towards BDB. Field testing revealing that the LAMP-flocculation assay can effectively detect BDB at an early infection stage. Author also demonstrate that the LAMP and carbon flocculation assays are as reliable as PCR. The highly sensitive LAMP reaction, as a rapid diagnostic tool for banana disease BDB, can be used in diagnostic facilities with limited resources, which contributes to the banana industries and is critical for agricultural support services. Overall, the manuscript is well written and the results are easy to follow. Several aspects of the study need further interpretation or additional experimentation.

1. Figure 3. The results of the carbon flocculation assay which tested the LAMP assay sensitivity should be showed. And the picture is not neatly adjusted, please adjust it a little.
2. The photo formats in Figure 3, figure 4, Figure 5 and Figure 6 should be uniform.
3. The boxes of some pictures in Figure 6 and Figure 7 are missing. Please keep the standard uniform.
4. Figure 7. Lane M should also be shown in the diagram.
5. Image styles should be consistent and standardized, and Lane M of each graph should be displayed.

Author Response

1.  Figure 3. The results of the carbon flocculation assay which tested the LAMP assay sensitivity should be showed. And the picture is not neatly adjusted, please adjust it a little.

Response 1:       The results of the carbon flocculation assay have been included into Figure 3 and neatly adjusted.

2. The photo formats in Figure 3, figure 4, Figure 5 and Figure 6 should be uniform.

Response 2:       Figure 3, Figure 4, Figure 5 and Figure 6 have been edited as suggested by reviewer.

3.  The boxes of some pictures in Figure 6 and Figure 7 are missing. Please keep the standard uniform.

Response 3:       Figure 6 and Figure 7 have been edited.

4.  Figure 7. Lane M should also be shown in the diagram.

Response 4:       Lane M has been included in the gel picture in Figure 7.

Reviewer 2 Report

The manuscript presents the set up and validation of a rapid molecular-based method to detect blood disease of banana.

Some changes/correction are necessary

English needs to be improved to make the paper more readable.

Remove acronyms from the title

l39 causing lowering of yields and productivity

l55 ...disease are similar to Moko...

M&M

2.1 and 2.2  Insert the name of the isolates

2.6 be more detailed about the description of the pathogenic assays: wich is the inoculatio point? Are the plants incubated?

In M&M Ralstonia solanacearum and Fusarium oxysporum cubense are introduced, but it is not indicated in the following paragraph they utilization

Fig. 4 lowercase letter for solanacearum

l245 ...stalk at 5-day post inoculation...

The paragraph 3.4 is not clear. It needs to be rewritten to explain better the results

Please, in par 3.5 results must be illustrated and explained in statistical view. What does it means, were comparable??? It is not proper for a detecting method.

DISCUSSION must be rewritten with scientific rigour by explaining only the obtained results and eliminating redundant parts.

for example, sub-paragraph L326-334; L343-363 and Conclusion L391-405 are repetitions and redundant not necessary information that distracting attention from the main subject matter. Please remove all.

Rather, be consistent. for example L337 LAMP detects amount of??? what???

L338 Do primers have a detection limit???, Probably, the protocol!

However, please, explain in this paragraph the flocculation technique, how it happens, previous similar utilization. Explain the errors in the detection, L375-381

After these modifications and integration the paper could be considerate 

Author Response

1. English needs to be improved to make the paper more readable.

Response 1:    The manuscript has been proofread by London Proofreaders (Invoice No.: 362).

2. Remove acronyms from the title

Response 2:    The acronyms have been removed from the title.

3. L39 causing lowering of yields and productivity

Response 3:   The sentence has been edited as suggested by the reviewer (L41).

4. L55 ...disease are similar to Moko...

Response 4:    The sentence has been changed to “Banana blood disease exhibit similar symptoms to Moko banana disease……” (L58) according to the suggestion from Rev 3.

M&M

5. 2.1 and 2.2  Insert the name of the isolates

Response 5:  The name of the isolates and strains have been included as below:

                   Fusarium oxysporum f.sp. cubense (FocTR4) (L110)

                   Ralstonia syzygii subsp. celebesensis (Rsc) (GenBank No. CP019911.1) (L111)

                   Ralstonia solanacearum (Rs) strain PS107 (L113)

6. 2.6 be more detailed about the description of the pathogenic assays: which is the inoculation point? Are the plants incubated?

Response 6:  The protocol has been explained in more detail. The inoculated plants were placed in the greenhouse until the symptoms were observed after 5 days of inoculation. Leaves, stalk and midrib of the infected plants were collected. The samples were then subjected to the LAMP assay and carbon flocculation assay. (L216)

7. In M&M Ralstonia solanacearum and Fusarium oxysporum cubense are introduced, but it is not indicated in the following paragraph they utilization

Response 7:  The utilization of the Ralstonia solanacearum and Fusarium oxysporum cubense was further explained in 2.1. (L125)

8. Fig. 4 lowercase letter for solanacearum

Response 8:  The word of solanacearum has been removed from the picture in Fig.4. It has been mentioned in the caption of Fig. 4. (L274)

9. l245 ...stalk at 5-day post inoculation...

Response 9:  The sentence has been edited according to reviewer’s suggestion (L281).

10. The paragraph 3.4 is not clear. It needs to be rewritten to explain better the results

Response 10: Paragraph in section 3.4 has been rewritten to explain the results for easier understanding. (L 322–324)

11. Please, in par 3.5 results must be illustrated and explained in statistical view. What does it means, were comparable??? It is not proper for a detecting method.

Response 11:  Section 3.5 has been rewritten according to the reviewer’s comment. (L 351–357)

12               DISCUSSION must be rewritten with scientific rigour by explaining only the obtained results and eliminating redundant parts.

Response 12:  The redundant parts in Discussion has been removed according to the reviewer’s comment. (L 382-388)

13. For example, sub-paragraph L326-334; L343-363 and Conclusion L391-405 are repetitions and redundant not necessary information that distracting attention from the main subject matter. Please remove all.

Response 13: We have found that there are no repetitions and redundant information in the sub-paragraph L326-334; L343-363. In the sub-paragraph L326-334, we discussed the advantages of LAMP over PCR technique to use for early detection of BDB in bananas and the flocculation method as a readout technique to enhance the rapidity of detection of the amplified product. Meanwhile, information in the sub-paragraph L343-363, we discussed the weaknesses of gel electrophoresis and color-based methods that are not suitable to use on-site to visualize amplified products since both techniques rely on laboratory-based. We also highlighted the flocculation technique as the best method to apply in the field. In the Conclusion part (L391-405), We concluded the importance of the combination of LAMP and flocculation assays used for the detection of BDB of bananas on-site to strengthen our findings based on previous studies. We believe that our conclusion may help readers to understand well the whole of these techniques.  Therefore, we decided to not remove all these important facts from this discussion and conclusion.

14. Rather, be consistent. for example L337 LAMP detects amount of??? what???

Response 14:  The sentence has been edited according to the reviewer’s suggestion. (L413)

15. L338 Do primers have a detection limit???, Probably, the protocol!

Response 15:  The sentence has been edited according to the reviewer’s suggestion. (L414)

16. However, please, explain in this paragraph the flocculation technique, how it happens, previous similar utilization. Explain the errors in the detection, L375-381

Response 16: The explanation of the flocculation technique has been further discussed in the L463-470. Regarding the errors in the detection of the asymptomatic sample, we have performed 3 replicates for each control. The positive reaction of the asymptomatic sample in the healthy plot has been verified with the PCR technique which was presented in Figure 7a.

Reviewer 3 Report

This appears to be an important contribution.

Three issues should be addressed. 

First, clarify whether the Genbank isolate was the positive control in all assays. 

Second, for the positive response on an asymptomatic plant, verify whether symptoms eventually developed.  If not, this may be a false positive response rather than a successful early detection, and the Discussion should be revised accordingly.

Third, the designation of samples as P1, P2, etc. for both plots as well as for healthy plants is confusing.  If possible, a unique identifier for each plant sample should be used.

Some editorial suggestions include:

Line 17.  Delete "and".  Place a comma after BDB.

Line 21.  Delete "was".

Line 23. Insert "a" in front of "healthy"".

Line 30.  Insert "a" in front of "highly".

Line 46.  Use the lower case to spell "celebesensis".

Line 48.  Insert "is" in front of "also.

Line 50.  Incomplete sentence.  Delete "Whereas in" and capitalize "Long".

Line 52-53.  Be careful to distinguish the disease versus the causal agent.  Replace with "The BDB pathogen, R. syzygii, is very closely related to Ralstonia solanacearum and belongs to phylotype IV within the R. solanacearum group due to its resemblance to the R. solanacearum strains from Indonesia 11,12".

Line 55.  Replace "Symptoms of banana blood disease exhibit similar characteristics to Moko..." with "Banana blood disease exhibits similar symptoms to Moko...".

Lines 59-60. Replace " become necrotic" with " necrosis".

Line 82.  Rewrite " La Mer and Healy" as " Healy and La Mer".

Line 86.  Clarify the common name.  Rewrite " banana disease (BDB)" as "blood disease of banana (BDB)".

Line 100.  Incomplete sentence.  Replace "Whereas" with "The".

Line 102.  I recommend citing the source for the medium used (I assume this is: Kelman, A. 1954.  Phytopathology 64:693-695).  If this was from a commercial preparation, cite the supplier.

Line 103.  Insert "the" in front of "Foc".

Line 109.  Clarify "plain agar".  Is this water agar?

Line 111.  Hyphenate compound adjectives, e.g. "2-ml", 1.5-mL tube (line 120).

Line 118.  Insert "a" in front of "leaf".

Line 111 abbreviates milliliter as "ml", but line 120 uses the upper case "mL".  Either might be acceptable, but be consistent with the Journal's preferred format.  Do likewise for microliter.

Line 160.  Delete "which".

Line 164.  Delete "was".

Line 185.  Replace "were" with "was".  Replace "the" with "a".

Lines 186-187.  Replace "A completely randomised block design (RCBD) with four replicates was im-plied as the experimental model" with "The experiment was arranged as a completely randomised block design (RCBD) with four replicates".

Line 190.  Unless the inoculations were repeated for five days, replace " after 5 days of inoculation" with "5 days after inoculation".

Line 202.  Delete "Whereas" and capitalize "Samples".

Line 206.  Replace "has been" with "was".

 Line 209.  Replace primers" with "primer".

Line 218.  These amounts appear to be the mass, not concentration.  Replace "by the concentration of"  with "with".

Line 223.  Delete "concentration of".

Line 225.  Replace "Concentrations" with "Amounts".

Figure 4.  The figure and caption might be more clear if the photos were identified as "4A" and "4B".  The caption should also identify the "+ve" treatment.

Lines 244-254.  Revise this paragraph to read "The LAMP assay and carbon flocculation assay were performed on the samples of leaves, leave midrib, and stalk 5 days post-inoculation.  The LAMP products of leaves (D), leaf midrib (P), stalk (T), and a positive control (R. syzygii subsp. celebesensis, GenBank No. CP019911.1) were separated on an agarose gel for confirmation of DNA amplification in the positive LAMP reactions (Figure 5).  However, no amplification was observed in the negative control (no template). Likewise, the carbon flocculation assay showed that the DNA particles of D, P, T, and the positive control were flocculated and settled on the bottom of the tube within 20 seconds, thus revealing successful amplified long DNA in LAMP. However, charcoal in the negative control remained suspended, displaying an unsuccessful amplified LAMP assay."

Lines 247 and 258.  It may be useful to clarify in the Methods that the Genbank accession was used as the positive control throughout, in which case this need not be repeated in the results.  If this was not the case, clarify what the positive control was in each experiment.

Line 261.  Replace "readout" with "results".

Line 263.  Replace "leave midrib" with "leaf midrib".

Line 265.  It may be helpful to clarify that the "B" samples refer to "stem".  That abbreviation had not appeared in the text before.

Line 267.  Revise and condense to read "... flocculation assay (Figure 6).  A positive.."

Line 270.  Replace "flocculation" with "flocculated".

Lines 273-276.  Revise sentence structure to read "Meanwhile, a positive reaction was detected in eight out of 11 sets of samples (B, T, and D) from the infected plot at Taman Pertanian Universiti in the carbon flocculation assay, indicating that approximately 73 % of the plot was infected by BDB.".

Lines 297-302.  Revise to read "Validation of both developed diagnostic methods has been performed by PCR. DNA from the samples (P1, P2, P3, P4, P5, P6, P7, P8, P10, and P11) from the infected plot of Taman Pertanian Universiti were extracted and analyzed using PCR (Figure 7). The results of PCR were comparable with the results of LAMP and carbon flocculation assays, thus proving that the LAMP and carbon flocculation assays are as reliable as PCR due to comparable results from the amplification of all samples.".

Figure 7.  The photo of P6 appears to have been cropped as the T lane is incomplete.  Is a more complete photo available?

Lines 311-313. Clarify whether "bacterial wilt" is the same as BDB, as the use of "R. solanacearum" is confusing.

Lines 326-329.  These sentences appear to be redundant.  Can this be condensed?

Line 360.  Replace "had" with "has been".  Correct the spelling of "implemented".

Line 370.  Delete "was".

Line 372.  Replace "after five days of inoculation" with "five days after inoculation".  Delete "Whereas", and capitalize "In."

Line 373.  Delete "was".

Line 375.  Insert "though" before "no".

Line 377.  Condense to read "eight out of 11 sets of infected samples".

Line 379.  Delete "is".

Line 380.  Insert "was" in front of "able".

Pages 12-14.  In most journals, the key words in titles of journal articles are not capitalized, but this is done inconsistently here. Please use a consistent format.  

Lines 435, and 443-444.  Please provide the complete citations.

Line 448.  There appears to e an extra space after "History".

Line 449.  Insert the journal name and volume number after "2009".  Acta Hortic. 828, 

Line 485 (and throughout).  Italicize Latin names in titles if they were italicized in the original.  Do not capitalize "pv." or species and subspecies epithets.

Lines 487-489.  This appears to be an incomplete citation.  Add the rest of the title, as well as Journal name: "Plant Pathology".

Author Response

1. First, clarify whether the Genbank isolate was the positive control in all assays.

Response 1:  Ralstonia syzygii subsp. celebesensis (Rsc) (GenBank No. CP019911.1) was used as positive control in all the assays in this study. It has been added in section 2.1. (L126)

2. Second, for the positive response on an asymptomatic plant, verify whether symptoms eventually developed. If not, this may be a false positive response rather than a successful early detection, and the Discussion should be revised accordingly.

Response 2:  We have performed 3 replicates for each asymptomatic sample. The positive reaction of the asymptomatic sample in the healthy plot has been verified with the PCR technique which was presented in Figure 7a.

3. Third, the designation of samples as P1, P2, etc. for both plots as well as for healthy plants is confusing. If possible, a unique identifier for each plant sample should be used.

Response 3 :  The P1 and P2 for healthy plants have been changed to H1 and H2 in Figure 6 in order to avoid the confusion. (L328 and L458)

Line 17.  Delete "and".  Place a comma after BDB.

Response: “and” has been deleted (L 17)

Line 21.  Delete "was".

Response:  “was” has been deleted (L 21)        

Line 23. Insert "a" in front of "healthy"".

Response:  “a” has been added (L 23)  

Line 30.  Insert "a" in front of "highly".

Response: “a” has been added (L 30)

Line 46.  Use the lower case to spell "celebesensis".

Response: The word of "Celebesensis" has been changed to "celebesensis". (L48)

Line 48.  Insert "is" in front of "also.

Response: “is” has been added in front of “also” (L 50)

Line 50.  Incomplete sentence.  Delete "Whereas in" and capitalize "Long".

Response: The sentence has been edited according to reviewer’s comment (L 53).

Line 52-53.  Be careful to distinguish the disease versus the causal agent.  Replace with "The BDB pathogen, R. syzygii, is very closely related to Ralstonia solanacearum and belongs to phylotype IV within the R. solanacearum group due to its resemblance to the R. solanacearum strains from Indonesia 11,12".

Response: The sentence has been edited according to reviewer’s comment (L 55 - 56).

Line 55.  Replace "Symptoms of banana blood disease exhibit similar characteristics to Moko..." with "Banana blood disease exhibits similar symptoms to Moko...".

Response: The sentence has been edited according to reviewer’s comment (L 58).

Lines 59-60. Replace " become necrotic" with " necrosis".

Response: “become necrotic” has been replaced with “necrosis” (L 63).

Line 82.  Rewrite " La Mer and Healy" as " Healy and La Mer".

Response: " La Mer and Healy" has been replaced with " Healy and La Mer" (L 90).

Line 86.  Clarify the common name.  Rewrite " banana disease (BDB)" as "blood disease of banana (BDB)".

Response: " banana disease (BDB)" has been replaced with "blood disease of banana (BDB)" (L96).

Line 100.  Incomplete sentence.  Replace "Whereas" with "The".

Response: “Whereas” has been replaced with “The” (L113).

Line 102.  I recommend citing the source for the medium used (I assume this is: Kelman, A. 1954.  Phytopathology 64:693-695).  If this was from a commercial preparation, cite the supplier.

Response:  The citation has been included in section 2.1 (L 115).

Line 103.  Insert "the" in front of "Foc".

Response: “the” has been added in front of “Foc” (L 116).

Line 109.  Clarify "plain agar".  Is this water agar?

Response: “plain agar” has been replaced with PDA media (L122).

Line 111.  Hyphenate compound adjectives, e.g. "2-ml", 1.5-mL tube (line 120).

Response: 2 mL and 1.5 mL were changed to 2-mL and 1.5-mL (L 124 and L 137).

Line 118.  Insert "a" in front of "leaf".

Response: The sentence has been edited as suggested by the proofreader (L 134).

Line 111 abbreviates milliliter as "ml", but line 120 uses the upper case "mL".  Either might be acceptable, but be consistent with the Journal's preferred format.  Do likewise for microliter.

Response: All the mililiter dan microliter have been standardized to ‘mL’ and ‘µL’.

Line 160.  Delete "which".

Response: “Which” has been deleted (L184).

Line 164.  Delete "was".

Response: “was” has been deleted. (L 188)

Line 185.  Replace "were" with "was".  Replace "the" with "a".

Response: “were” has been replaced with “was” and “the” replaced with “a”. (L 211)

Lines 186-187.  Replace "A completely randomised block design (RCBD) with four replicates was im-plied as the experimental model" with "The experiment was arranged as a completely randomised block design (RCBD) with four replicates".

Response: The sentence has been replaced with “The experiment was arranged as a completely randomised block design (RCBD) with four replicates". (L 212)

Line 190.  Unless the inoculations were repeated for five days, replace " after 5 days of inoculation" with "5 days after inoculation".

Response: “after 5 days of inoculation” has been replaced with “5 days after inoculation”. (L217)

Line 202.  Delete "Whereas" and capitalize "Samples".

Response: “Whereas” has been deleted. (L 232)

Line 206.  Replace "has been" with "was".

Response: “has been” has been replaced with “was”. (L 235)

Line 209.  Replace primers" with "primer".

Response: “primers” has been replaced with “primer”. (L239)

Line 218.  These amounts appear to be the mass, not concentration.  Replace "by the concentration of"  with "with".

Response: “by the concentration of” has been replaced with “with”. (L247)

Line 223.  Delete "concentration of".

Response: “concentration of” has been deleted. (L254)

Line 225.  Replace "Concentrations" with "Amounts".

Response: “Concentration” has been replaced with “Amounts”. (L255)

Figure 4.  The figure and caption might be more clear if the photos were identified as "4A" and "4B".  The caption should also identify the "+ve" treatment.

Response: The figure caption has been added in “above” and “below” for easier understanding. +ve control also has been added in to the caption. (L273-275)

Lines 244-254.  Revise this paragraph to read "The LAMP assay and carbon flocculation assay were performed on the samples of leaves, leave midrib, and stalk 5 days post-inoculation.  The LAMP products of leaves (D), leaf midrib (P), stalk (T), and a positive control (R. syzygii subsp. celebesensis, GenBank No. CP019911.1) were separated on an agarose gel for confirmation of DNA amplification in the positive LAMP reactions (Figure 5).  However, no amplification was observed in the negative control (no template). Likewise, the carbon flocculation assay showed that the DNA particles of D, P, T, and the positive control were flocculated and settled on the bottom of the tube within 20 seconds, thus revealing successful amplified long DNA in LAMP. However, charcoal in the negative control remained suspended, displaying an unsuccessful amplified LAMP assay."

Response: The paragraph has been replaced according to the reviewer’s suggestion. (L280-289).

Lines 247 and 258.  It may be useful to clarify in the Methods that the Genbank accession was used as the positive control throughout, in which case this need not be repeated in the results.  If this was not the case, clarify what the positive control was in each experiment.

Response: Using R. syzygii subsp. celebesensis (GenBank No. CP019911.1) as the positive control in this study has been explained in the methodology. (L 126)

Line 261.  Replace "readout" with "results".

Response: “readout” has been replaced with “results”. (L 308)

Line 263.  Replace "leave midrib" with "leaf midrib".

Response: “leave midrib” has been replaced with “leaf midrib”. (L 310)

Line 265.  It may be helpful to clarify that the "B" samples refer to "stem".  That abbreviation had not appeared in the text before.

Response: Abbreviations of “B, T and D” have been clarified in the text. (L 313)

Line 267.  Revise and condense to read "... flocculation assay (Figure 6).  A positive.."

Response: The sentence has been edited according to reviewer’s suggestion. (L 315-316)

Line 270.  Replace "flocculation" with "flocculated".

Response: “flocculation” has been replaced with “flocculated”. (L 318)

Lines 273-276.  Revise sentence structure to read "Meanwhile, a positive reaction was detected in eight out of 11 sets of samples (B, T, and D) from the infected plot at Taman Pertanian Universiti in the carbon flocculation assay, indicating that approximately 73 % of the plot was infected by BDB.".

Response: The sentence has been edited according to the reviewer’s comment. (L322-324)

Lines 297-302.  Revise to read "Validation of both developed diagnostic methods has been performed by PCR. DNA from the samples (P1, P2, P3, P4, P5, P6, P7, P8, P10, and P11) from the infected plot of Taman Pertanian Universiti were extracted and analyzed using PCR (Figure 7). The results of PCR were comparable with the results of LAMP and carbon flocculation assays, thus proving that the LAMP and carbon flocculation assays are as reliable as PCR due to comparable results from the amplification of all samples.".

Response:  The sentence has been edited according to the reviewer’s suggestion. (L 351 – 357)

Figure 7.  The photo of P6 appears to have been cropped as the T lane is incomplete.  Is a more complete photo available?

Response: Figure 7 has been replaced with an improved picture. (L 365)

Lines 311-313. Clarify whether "bacterial wilt" is the same as BDB, as the use of "R. solanacearum" is confusing.

Response: Symptoms of BDB are very similar to “bacterial wilt” caused by R.solanacearum. Therefore, BDB always referred to as bacterial wilt although it’s not. It has been further explained in the text. (L 375)

Lines 326-329.  These sentences appear to be redundant.  Can this be condensed?

Response: The sentences have been condensed into one sentence. (L 378-385)

Line 360.  Replace "had" with "has been".  Correct the spelling of "implemented".

Response: “had” has been replaced with “has been”. Spelling of “implemented” has been corrected. (L439)

Line 370.  Delete "was".

Response: “was” has been deleted. (L 450)

Line 372.  Replace "after five days of inoculation" with "five days after inoculation".  Delete "Whereas", and capitalize "In."

Response: “after five days of inoculation” has been replaced with “five days after inoculation” and “Whereas” has been deleted. (L 452)

Line 373.  Delete "was".

Response: “was” has been deleted. (L 453)

Line 375.  Insert "though" before "no".

Response: “though” has been inserted before “no”. (L 456)

Line 377.  Condense to read "eight out of 11 sets of infected samples".

Response: The sentence has been edited according to reviewer’s comment. (L 457)

Line 379.  Delete "is".

Response: “is” has been deleted. (L 460)

Line 380.  Insert "was" in front of "able".

Response: The sentence has been revised according to Proofreader’s comment. (L 461)

Pages 12-14.  In most journals, the key words in titles of journal articles are not capitalized, but this is done inconsistently here. Please use a consistent format. 

Response: All the key words in titles of journal articles in Reference have been capitalized. (page 16-18)

Lines 435, and 443-444.  Please provide the complete citations.

Response: Complete citations have been added into the Reference (L 545). Citation 8 has been removed from the text and Reference list. (L 405)

Line 448.  There appears to e an extra space after "History".

Response: The extra space has been deleted. (L 548)

Line 449.  Insert the journal name and volume number after "2009".  Acta Hortic. 828,

Response: Journal name and volume number have been added to the reference. (L 549)

Line 485 (and throughout).  Italicize Latin names in titles if they were italicized in the original.  Do not capitalize "pv." or species and subspecies epithets.

Response: The mistake has been edited. (L 587)

Lines 487-489.  This appears to be an incomplete citation.  Add the rest of the title, as well as Journal name: "Plant Pathology".

Response: The complete title and the journal name have been included in the citation. (L 591 – 592)

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The reviwer requirements have been addressed