Ecologically Based Management of Pineapple Mealybug Wilt: Controlling Dysmicoccus brevipes Mealybug Populations with Salicylic Acid Analogs and Plant Extracts
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript titled ¨ Controlling Mealybug (Dysmicoccus brevipes) population in Queen Victoria Pineapple (Ananas comosus) with Exogenous Application of Salicylic Acid, Acibenzolar-S-Methyl, and Methyl Salicylate: A Promising Ecological approach for Management of Pineapple Mealybug Wilt¨ presents a preliminary evaluation of six treatments, which include endogenous stress signaling compounds such as salicylic acid (SA) or its analogs and β-aminobutyric acid (BABA). The objective of this study was to test the effect of chemical priming stimuli on mealybug populations in plantlets of the Queen Victoria cultivar under controlled conditions (climate chamber and greenhouse). Although the authors explore an interesting ecological alternative for controlling an important pest and vector that limits pineapple production, additional information regarding the experimental design of the assays and statistical analysis should be addressed by the authors and applied to the document prior to publication.
Major modifications
The authors must support the biological basis of the assays to ensure validation. Specifically, they should address the following points: 1. Describe the age, stage, or size of the mealybugs used for plant or plantlet infestation. 2. Provide a rationale for choosing 45 days as the timepoint to evaluate and count the mealybugs, especially considering that the life cycle of these insects, as stated in the document, is also 45 days. Additionally, the authors should explain how the detection of PMWaVs was conducted—was it applied to the entire rearing mealybug population or to a subsample?
Regarding the experimental design and statistical analysis, the authors should consider the following: The experimental design in assay 1 showed clear differences in its establishment per year, and in 2019, it even demonstrated variations between cultivars. Concerning the results, data from the Queen Victoria cultivar exhibited visual differences between years in the assay and control. Was this statistically tested? How do the authors explain these differences, especially considering that the assay was conducted under controlled conditions? For this reason, this analysis should be performed and presented separately for each cultivar.
The discussion must be adjusted and oriented towards the results obtained for ASM and MeSA treatments in the QV cultivar because the results for AS were not consistent across the three assays. The statistical analysis of assay 1 should be reviewed, and in assays 2 and 3, the results were not statistically significant. MD2 was only evaluated in assay 1. The authors should address the differential results between SA and its analogs ASM and MeSA. Additionally, the information associated with triggering SAR should be presented more clearly as a hypothesis to be tested.
Minor modifications
Title
The title should be adjusted in order to include all treatments (general information) tested in this study.
Include the complete name of the disease: Pineapple Mealybug Wilt.
Abstract
Line 12. Parasitic complex that includes Pineapple Mealybug Wilt-associated Viruses (PMWaVs) and mealybugs (Dysmicoccus brevipes). Why is a parasitic complex? Vector-virus interaction is not parasitic especially in mealybugs which transmission is semipersistent. The use of complex is recommended.
Line 19. Either the Hawaiian hybrid MD-2 and the Queen Victoria tissue culture plants or traditional shoots were treated by direct or parental application on the soil of pineapple pots. The study only showed results in Queen Victoria for all assays, so adjustments are required.
Introduction
Line 32. Pineapple comosus. The scientific name of the species: pineapple (Ananas comosus).
Line 37. Use more precise term than ¨made up of¨.
Line 40. In this complex the presence of PMWaV2 should be included that is stated as requirement on the different reports from Sether et al.
Line 42. A set of virus-spreading organisms, such as mealybugs and ants. This statement should differentiate between mealybugs as vectors and ants as disseminators that transport mealybugs in the fields.
Line 49. primed distal tissue, tissue?. It means long-distance (systemic) defence signals or distal defence signals. Clarify the use of the word ¨tissue¨ in this context?
Line 55. Fusarium wilt is a disease, change for Fusarium sp., or in case for the Fusarium´s species.
Material and Methods
Line 64. Include the Celsius degree symbol and apply through the whole document.
Line 65. under 60 W Tarentula diodes. Under LED light of 60 W Tarentula diodes
Line 66. Plants grown in greenhouse conditions were, on average. Modified by Plants grown in the greenhouse under average conditions of ….
Line 73. Breeding?_ or rearing. Modify it through the document.
Line 76. PMWaV1, V2, and V3 were detected by real-time PCR multiplex. It’s confusing, it meant that the three virus species were detected. Include something like, the presence of PMWaV1-3 was tested by real-time PCR multiplex.
Line 78. For inoculation on pineapple plants. Inoculation is more used for pathogens, use infestation/ infested and apply through the document.
Line 82. Solutions for treatments were prepared as follows (General): 1 mM of salicylic acid (specific). Unify the text either in a general or a specific context.
Line 90. In the article, we named the treatments. In this study, we coded the treatments by
Line 93. It was quantified. Include pronoun.
Line 98. The experiment was realized in triplicate at day 1 and day 8 (Table 2). It means, the experiment was established in triplicate and evaluations were conducted at day 1 and day 8?
Biological assay in controlled conditions
Line 102. MD2 hybrid instead hybrid MD2. Apply through the whole document.
Line 103. Assays were conducted in 2019 and 2022, and not in the period 2019-2022, which might be mistaken for including 2021.
Line 110-111. Plants without mealybugs were used to control mealybug contamination inside culture chambers or greenhouses. It’s not clear, it means monitoring?
Biological assays on pineapple offspring in greenhouse
Line 122. It is not clear. A total of 30ml of treatments and control. It is confusing (total amount used in all treatments and control). Modify it to an amount of 30 ml was used for each treatment and control.
Line 122. What was the age of the parental plants? It should be included in the text.
Line 123-124. According to the described methodology, the parental plants were vegetatively propagated? using the apical buds. Does this mean that plants were destroyed during the process? If so, where were the apical buds transferred? The authors should consider that for in vitro propagation, the establishment of shoots is part of the process, while vegetative propagation is conducted using suckers. Apply this distinction, when necessary, in the document.
Line 126. twice for SA and control. Experimental design is not clear, it means that 2 set x 5 plantlets = 10 plantlets per treatment, or 6 sets (twice =2x 3 sets) x 5 plantlets= 30 plantlets?
Line 139. determine the risk of harboring more than 25 mealybugs. How was established the number of 25 mealybugs as the limit to measure the risk?
Results
Line 167. Have been realized. Use past tense.
Line 171. Means were averaged in triplicate? Explain it?
Line 188. showing a 53% reduction in risk compared to the control?. The phrase must be adjusted; the control showed 53% according to Table 3.
Line 193. F. indica and H. lanceolatum. In italics, apply in whole document.
Line 200-201. In addition, except for three higher values, the trend looked like that of ASM and MeSA treatments (Figure 2). Rewrite the sentence.
Line 207. Figure 2. Include more contrast to the level of color to visualize the 3 sets.
Line 216. Applying treatments. Modify it to applied treatments.
Line 253. Finally. Adjust the connector, e.g. Based on that or considering that.
Discussion
Line 267. enzymatic markers, the SA. Include a period ¨enzymatic markers. The SA¨
Line 268. appeared to prime the plants for SAR. This must be stated as a hypothesis.
Line 280. No variety difference was observed in our study, but there may be different gene expressions between MD2 and QV.? The authors cannot make that statement because the mentioned cultivars were not tested in all experiments.
Line 301. The hypothesis of the priming of PRR and NLD or induced SAR seems to make more sense. This phrase should be connected to the pre- or post-text.
Line 302. Future studies should be confirmed? by an analysis of the molecular markers mentioned. Adjust this phrase.
Line 319. One showed, who showed?
Line 372. the mealybugs incidence. Modify it to the mealybug population.
Line 381. to reduce the incidence of mealybug populations. Modify it to reduce the mealybug population.
Comments on the Quality of English LanguageThe text should be edited by a native English speaker to improve its clarity.
Author Response
see uploaded file "reviewer1 ALS 27-01-2024"
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsReview of the manuscript entitled „Controlling Mealybug (Dysmicoccus brevipes) population in Queen Victoria Pineapple (Ananas comosus) with Exogenous Application of Salicylic Acid, Acibenzolar-S-Methyl, and Methyl Salicylate: A Promising Ecological approach for Management of Pineapple Wilt”. I think the paper is interesting. The research is consistently well planned and presented. I recommend that the paper be accepted for publication with the minor corrections presented below.
Specific comments:
1. The title of the paper is too long. It should indicate the most important result of the work, which should encourage the reader to read the work. I suggest changing the title to emphasize which experimental variants were effective against mealybugs.
2. L55-56. State the purpose of the work and the experimental conditions.
3. L58. Possible alternatives? Indicate what the proposed measures should be alternative to.
4. L64. What was the substrate for growing plants?
5. L64. 28 C 1 C? Please correct.
6. L66-67. e, 20 C at night/ 29 C during the day. Degrees of Celcius?
7. L69-70. in how many replicates were the SPAD measurements taken? How was the plant size measured? Was it the height of the plants? Was the study conducted in replicates?
8. L73. adapted from [30]... adapted from the method described by Pandey and Johnson [30].
9. L87. Essential oil?
10. L105. Treatments from Table 1?
11. L117. ...as previously described...In Table 1?
12. Fig. 1 What do the letters a and b in the diagram mean? What do the black dots mean? What do the vertical lines on the black dots mean? The abbreviation MD2 should be standardized (the authors use different versions of the notation). What does the abbreviation qv stand for?
13. Table 2: Indicate the value of n in the table.
14. L163. Which statistical test was used in the study?
15. L209. F. indica, H. lanceolatum are written in italics.
16. L236. p=6.833e-13?
17. Table 5. Give the value of (n). Size (cm) meant the height of the plants? Correct F. Indica; H. Lanceolatum to F. indica; H. lanceolatum.
Author Response
see uploaded file "reviewer2 ALS 27-01-2024"
Author Response File: Author Response.pdf
