PONE-D-20-23665

Morus alba: phenotyping for Meloidogyne javanica, biological nematicides assessment and study of these relationships with yield and quality of leaves, cocoon and health of the silkworm.

PLOS ONE

Dear Dr. Machado,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: No

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Partly

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2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Partly

Reviewer #4: Partly

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3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

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(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript deals with multiple objectives: a) evaluation of the damages caused by nematodes to the mulberry, b) evaluation of biocontrol agents against nematodes in mulberry, c) evaluation of mulberry leaves from treated plants for feeding silkworms. Some parts of the research could be interesting. However, I cannot understand why silkworms could be damaged when fed with leaves harvested from plants treated with biocontrol agents to the roots 75 days before. Preliminary evidence (or literature) for this should be provided as a rationale for the research conducted here.

It should be noted that the manuscript lacks Results and Discussion. it must be a mistake during the upload.

The abstract must contain the results and the main findings, while the introduction to the problem must be shortened. The following sentences are in contrast one to another: "... Meloidogyne javanica is one of the most important nematode species parasitizing mulberry. To define management strategies, it is important to know if the crop species is damaged by the parasitism of the nematode ...". I would not speak about "disease" caused by a nematode, but damage. The Introduction section contains several interesting information; however, it should be better focused on the topic of the manuscript, and other information should be removed to avoid confusion in the readers. English must be revised by a mother tongue with background in the field of this research, as it is unacceptable in the present form.

Other minor comments are the following.

L77: these sentences are in contrast: "...from the second day after infection, but only in long term experiments,..."

L97: arenaria

L111: list the chemical nematicides registered for application on mulberry in Brazil and, possibly, elsewhere.

L122: the biological control agents Pseudomonas fluorescens and Trichoderma viridae must be introduced. Why did you use them?

L126: you must introduce Artemisia nilagirica extract. Why did you use it?

L186: report the manufacturer

L209: report the formula

L214: Bartlett's test, Shapiro-Wilk's test, and Box-Cox method precede ANOVA, but not the contrary.

L225: abbreviate the genus name after the first occurrence

L230: describe the method for inoculation. Report the dosage per pot.

L364: 5S rRNA is an unusual housekeeping gene: report citation. What is "another constitutive gene"?

Reviewer #2: I have read and reviewed the manuscript “Morus alba: phenotyping for Meloidogyne javanica, biological nematicides assessment and study of these relationships with yield and quality of leaves, cocoon and health of the silkworm” By da Silva et al. The article explains the necessity of being able to grow mulberry in order to cultivate silkworms for commercial purposes. Part of the problem is the susceptibility to parasitic nematodes which interferes with cultivation. Furthermore, the current nematicides also have a negative impact on cultivation.

Major points. The Results and Discussion sections are missing. Consequently, I am recommending a rejection. Furthermore, the writing was poor. The work needs a professional editing, but after the experiments have been done and the completed manuscript written. I have highlighted a few minor points regarding the writing, but every sentence had poor structure.

Minor points:

Introduction:

Sericulture involves the crop of mulberry leaves (Morus alba) [note: please put the genus and species name before leaves].

the silkworm cultivation and the spinning. [note: please put genus and species of the silkworm]

note: always italicize genus and species.

Mulberry is a perennial species with lifespan estimated in about 20 years, easy to

grow and with high rusticity, being cropped in Brazil since 1930. [note: Mulberry is a perennial species with lifespan estimated in about 20 years. Mulberry is easy to

grow and has high rusticity, being cropped in Brazil since 1930.]

In this country, its cultivation stretches between August to May [4] and each tree produces near of four kilograms of leaves per year [7]. [note: In Brazil, mulberry cultivation occurs between August to May [4]. Each tree produces nearly four kilograms of leaves per year [7].

Several Meloidogyne species had been reported parasitizing mulberry worldwide. [note: Several Meloidogyne species have been reported parasitizing mulberry worldwide].

In another study with mulberry, using different population densities of M. Arenaria race 2 [note: M. arenaria].

encasulation? [note: encapsulation?].

Note: I could continue with the grammatical errors.

Materials:

Seedlings will be produced from cuttings taken from mature plants, with buds developed, collected from the germplasm collection maintained by Instituto de Desenvolvimento Rural do Paraná – IDR – IAPAR/ EMATER, located at the municipality of Londrina, Paraná State, Brazil. [note: Seedlings have been produced from cuttings {the work has already been done}.

Seedlings of the three mulberry cultivars, after acclimatization, will be tested

against [note: Seedlings of the three mulberry cultivars, after acclimatization, have been tested

against]

Note: I could continue with the grammatical errors.

Results: There is no Results section

Discussion: There is no discussion section

Reviewer #3: Review comments PONE-D-20-23665

Lines 157-162, the objectives of research seem to be overlapping. The authors need to clarify it.

In the “Phenotyping of mulberry (Morus alba) to Meloidogyne spp” section, the authors need to provide validations why eggs are harvested at “90 days from inoculation” to calculate reproduction factor (RF)? The authors also need to show how the RF is calculated in this section first.

Line 258, is “Ninety DAI” the correct date here?

The experiments in the two sections “Evaluation of biologicalnematicides in the control of Meloidogyne javanica in mulberry cultivar Miura” and “Evaluation of the impacts of the biological nematicides in the yield and quality of mulberry leaves cultivar Miura” can be combined to save time and efforts.

Lines 364-365, the authors need to specify “or another constitutive gene.”

Reviewer #4: The authors want to address the following questions: 1. If mulberry cultivar is damaged by RKN. 2. What is the efficiency of biological nematicides. 3. Whether biological nematicides would improve the quality of leaves or change the nutrient composition, thus change the silkworm production, finally affect the silk industry? In another words, whether biological nematicides have any impact on silk industry.

They designed corresponding experiment to answer those questions.

The first experiment is to phenotyping mulberry cultivar, which I found the word phenotyping a little confusing. They basically want to look at whether three mulberry cultivars are susceptible/resistant to four RKN species. I have some questions about the method here, mostly the reproduce factor (RF) they are going to use. Based on their method, I would say 99% if not 100% of the RF would be greater than 1, which could potentially increase false positives when doing analysis. Also one of the RKN species they will chose is not introduced in the introduction, if in Brazil mostly M. incognita and M. javanica were found, why chose other two that were not reported yet? If they all found in Brazil, then they should be introduced first. They plan to inoculate 1000 eggs per plant, but for their next experiment, they will do 2000 eggs instead, I would suggest keep the inoculation the same.

The second experiment is to evaluate the effect of three biological nematicides on M. Javanica, the method seems standard, I donot have much comment here.

The final series of experiment is supposed to answer the third question (the biggest). They chose two biological nematicides to continue, analyses the nutrition of the leaves, histological analysis of intestinal and glandular tissue of silkworm, and qPCR on couple of the genes that are involved in silk production. The methods itself seem to be standard, but my concern is whether those experiments will generate data to answer the question. For example, I would assume not only two genes involved in silk production. Is it enough to characterize couple genes through qPCR to answer the expression pattern change?

line77 where did you get this? the second day the nematode probably have not sucked nutrient from the plants yet. I think this sentence needs to be edited more carefully, at least cite the paper if it is true.

Line 78 For yield loss, it is very hard to accurately determine.

Line 85-95 those two paragraphs could be condensed and merged

Line 111-114 Any papers to support?

Line 129-134 those two active components are used mostly for insecticides, any citations on biological nematicides effect?

Line 135-143 if there is more background on RKN infecting mulberry cultivars that would be great

line148 Are those four RKN species all reported in affecting mulberry, there is one species that is not introduced anywhere.

lines 150-151 what kind of interaction?

Line158-160 why evaluate 3 and then study two later, what is the criteria for selecting those two?

Line 171 I not sure histological analyzes by itself can support the conclusion on this aspect

line 203 2ml might be too much water, 1ml is a good volume.

Lines 211 any literature for these standards? Since you harvest after 90 days, I suspect 99% of the value will be greater than 1, and that has nothing to do with resistant cultivar, because you have at least 3 to 4 generations. Even on resistant cultivar, the RKN still reproduce, just not as many as susceptible.

Line243 why one-way

Line 264 not very clear which methods you are referring to.

Lines 268-269 please describe more specifically, which are the variables that you will put into multivariate analyzes. Is this typical for nutrient analysis?

Line290 again, the analysis should be more specific

Lines 328 are you using the same sample as previous experiment? How to collect the sample for qPCR. If you use previous sample, my concern is whether the tissue if fresh enough to ensure that no environmental factors interferer with the RNA expression.

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Reviewer #1: Yes: Giovanni Bubici

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Morus alba: host reaction for Meloidogyne javanica, biological nematicides assessment and study of these relationships with yield and quality of leaves, cocoon and health of the silkworm.

PONE-D-20-23665R1

Dear Dr. Machado,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Adler R. Dillman, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for addressing the reviewers concerns in the revision. The revision is significantly improved.

There are a few minor textual suggestions I would make that I think would improve clarity.

For example, "encasulation," which you have used on line 194 to mean "reach the pupal stage," is not a word, to my knowledge. I only find reference to it in other papers as a typo where the word "encapsulation," is meant. However, in the context used on line 194 "encapsulation" does not mean pupation.

Line 27: change "damages" to damage.

Reviewers' comments: