Results of the Single Release Efficacy of the Predatory Mite Neoseiulus californicus (McGregor) against the Two-Spotted Spider Mite (Tetranychus urticae Koch) on a Hop Plantation

Round 1

Reviewer 1 Report

Authors gave comparable effects of the predatory mite N. californicus and acaricides against two-spotted spider mite. Further, the study incorporates several relevant interactions (whether parameters, several evaluation dates, different sampling hights) which gives broaden view of the biological aspects in the hop protection.

There are some suggestions that should be taken into consideration:

Line 36 Reference (3), should start with number 1

Line 39 The reference Assis et al. should be indicated with a number

Line 77 In the table2; name O. nubilalis is not written correctly

The references list should be checked and a uniform style should apply.

Author Response

Authors gave comparable effects of the predatory mite N. californicus and acaricides against two-spotted spider mite. Further, the study incorporates several relevant interactions (whether parameters, several evaluation dates, different sampling hights) which gives broaden view of the biological aspects in the hop protection.

There are some suggestions that should be taken into consideration:

Line 36 Reference (3), should start with number 1

Response: Completed.

Line 39 The reference Assis et al. should be indicated with a number

Response: Completed.

Line 77 In the table2; name O. nubilalis is not written correctly

Response: Completed.

The references list should be checked and a uniform style should apply.

Response: Completed.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript “Results of the First Inundative Release of the Predatory Mite Neoseiulus Californicus (McGregor) Against The Two-Spotted Spider Mite (Tetranyus urticae Koch) on a hop plantation” concerns the use of a biological agent - living organism - to control spider mites in hop cultivation.

The authors declare that this is the first study on the use of Neoseiulus californicus in way of inundative application. This organism is quite commonly used to control of this spider mite, the flooding method could be interesting and innovative if used alone and not in combination with other agents - acaricides.

The manuscript certainly requires a thorough reconstruction both in the description of the research method, the available literature data, discussion. The aim of the study is slightly emphasized. Neoseiulus californicus was first called as Typhlodromus californicus, after it was moved to the genus Amblyseius and later to the genus Neoseiulus. Many papers and results were developed on this topic.

General comments.
My principal concern regards the lack of novelty about using this biological control, this organism has already been used both in the cultivation of hops and in the field. Only the use of the flooding method may be considered an interesting aspect. 

The design of experiment is poorly justified - why N. californicus was used together with with other acaricides? With fungicides and insecticides against Aphids it is obviously, but with acaricides… Method of biological control should be alternative to acaricides towards which pest developed resistance. Maybe better would be use these acaricides in lower doses and combine them with biological methods in order to more ecological treatments. Perhaps the flooding method would allow don’t use of other acaricides no longer.
There is no information about the economic side of such treatments.

Specific comments
Title should be rewritten - not the first,  but single release
Key words - should be rewritten - -words in the title should not be repeated in keywords, should there be an inoculative?
Introduction - The authors should clarify which is the novelty of the present study and extend the content of this part
The order of citations in text should be kept. The order of references in the List of references should be changed.
Method - how exactly was the harmfulness assessed - 5% it is 5% of the area cone with symptoms of feeding of the spider mites?
Results - The results from Fig 2 and Fig 4 would be more clearly presented in the table
Discussion - poor discussion. Compared to other studies using N. californicus with chemical products - this part should be more extended.

List of references should be extended, more novel of papers must be added.
Conclusions - no recommendation related with obtained results.

Only one year of study, the weather conditions are decisive for the effectiveness of the biological method. The research should be repeated in next years.

Author Response

Comments and Suggestions for Authors

The manuscript “Results of the First Inundative Release of the Predatory Mite Neoseiulus Californicus (McGregor) Against The Two-Spotted Spider Mite (Tetranyus urticae Koch) on a hop plantation” concerns the use of a biological agent - living organism - to control spider mites in hop cultivation.

The authors declare that this is the first study on the use of Neoseiulus californicus in way of inundative application. This organism is quite commonly used to control of this spider mite, the flooding method could be interesting and innovative if used alone and not in combination with other agents - acaricides.

Response: We appreciate the given comment. We agree that the beneficial organism that was used in our research is commonly used in different studies, which we have listed at the end of this response (they are not cited in the paper). After a detailed review of the literature, we found 2 references that reported the use of N. californicus in hop production (i.e., Vostrel et al., Barber et al.). We also found several reports investigating the synergy between acaricides and N. californicus. Our study did not investigate the synergy between acaricides and biological control. When the biological control agent was applied, we used no synthetic insecticides; however, we used fungicides, which do not have a negative impact on beneficial organisms. We have found these data in the IOBC database (Jansen 2013), as cited in the manuscript.

Silva et al. (2019): Impact of vineyard agrochemicals against Panonychus ulmi (Acari: Tetranychidae) and its natural enemy, Neoseiulus californicus (Acari: Phytoseiidae) in Brazil. Crop Protection. 123:5-11.

Akyazi and Libourd, (2019). Biological control of twospotted spider mite (Trombidiformes: Tetranychidae) with the predatory mite Neoseiulus californicus (Mesostigmata: Phytoseiidae) in blackberries. Florida Entomologist. 102: 373-381.

Fraulo and Liburd, 2007. Biological control of twospotted spider mite, Tetranychus urticae with predatory mite, Neoseiulus californicus in strawberries. Experimental and Applied Acarology. 43: 109-119.

Ibrahim et al. (2005). Biological control of citrus brown mite Eutetranychus orientalis using predatory mite, Neoseiulus californicus (McGregor) (Acari:Tetranychidae & Phytoseiidae) on citrus trees. Egyptian Journal of Agricultural Research. 83: 131-139.

Takano-Lee and Hoodle, 2001. Biological control of Oligonychus perseae (Acari: Tetranychidae) on avocado: IV. Evaluating the efficacy of a modified mistblower to mechanically dispense Neoseiulus californicus (Acari: Phytoseiidae). International Journal of Acarology. 27: 157-169.

The manuscript certainly requires a thorough reconstruction both in the description of the research method, the available literature data, discussion. The aim of the study is slightly emphasized. Neoseiulus californicus was first called as Typhlodromus californicus, after it was moved to the genus Amblyseius and later to the genus Neoseiulus. Many papers and results were developed on this topic.

Response: We appreciate the given comment. According to Tixier (2018), there were only 2 studies (Vostrel et al., Barber et al) performed regarding the use of Neoseiulus californicus as a biocontrol agent before 2012. We have added some additional information in the Introduction and Materials and methods. As mentioned previously (in previous responses), several studies have been found; however, they are not directly associated with hop production. The use of Neoseiulus californicus is mainly related to Citrus sp. Papers in which the use of Neoseiulus californicus in hop cultivation is reported were added to the references list.

Tixier (2018). Predatory mites (Acari: Phytoseiidae) in Agro-Ecosystems and conservation biological control: a review and explorative approach for forecasting plant-predatory mite interactions and mite dispersal.

General comments.
My principal concern regards the lack of novelty about using this biological control, this organism has already been used both in the cultivation of hops and in the field. Only the use of the flooding method may be considered an interesting aspect. 

Response: We have taken into consideration your comment. We agree that the use of the flooding method would be interesting; however, our aim was to investigate the efficiency of the single release of the predator, as this aspect was not investigated before.

The design of experiment is poorly justified - why N. californicus was used together with with other acaricides? With fungicides and insecticides against Aphids it is obviously, but with acaricides… Method of biological control should be alternative to acaricides towards which pest developed resistance. Maybe better would be use these acaricides in lower doses and combine them with biological methods in order to more ecological treatments. Perhaps the flooding method would allow don’t use of other acaricides no longer.
There is no information about the economic side of such treatments.

Response: We appreciate your comment. We described in detail in the manuscript and also in Tables 1 and 2 that no acaricides and insecticides were used in the Biological Control treatment, while we used fungicides with no harmful effect on N. californicus for disease control (according to Jansen, 2013 - IOBC database). Our aim was to compare the efficicacy of the biological control method with that of chemicals (acaricides).

We have conducted an approximate cost analysis of using acaricides compared with the biological control. All data are presented in Table 3. Costs are calculated per treatment.

* the prices of synthetic acaricides are calculated according to the price of one liter or kg of preparation per hectare (Nissorun 10 WP = 99.29 EUR / kg; Vertimec Pro = 119.98 EUR / l)

Specific comments
Title should be rewritten - not the first,  but single release

Response: We agree with the given comment. The title was rewritten as ”Results of the Single Release Efficacy of the Predatory Mite Neoseiulus californicus (McGregor) Against the Two-Spotted Spider Mite (Tetranychus urticae Koch) on a Hop Plantation”

Key words - should be rewritten - -words in the title should not be repeated in keywords, should there be an inoculative?

Response: Done.

Introduction - The authors should clarify which is the novelty of the present study and extend the content of this part

Response: Done. Please see the text in the last paragraph.

The order of citations in text should be kept. The order of references in the List of references should be changed.

Response: Corrected as instructed.

Method - how exactly was the harmfulness assessed - 5% it is 5% of the area cone with symptoms of feeding of the spider mites?

Response: Yes, 5 % is 5 % of the area cone with symptoms of feeding. This has been mentioned as “visibly changed colour of a cone” (see sub-chapter 2.3. Assessment of Damage on Hop Cones).

Results - The results from Fig 2 and Fig 4 would be more clearly presented in the table

Response: Corrected as instructed. Figures 2 and 4 were presented in Tables.

Discussion - poor discussion. Compared to other studies using N. californicus with chemical products - this part should be more extended.

Response: Corrected as instructed.

List of references should be extended, more novel of papers must be added.

Response: Corrected as instructed.

Conclusions - no recommendation related with obtained results.

Response: Corrected as instructed.

Only one year of study, the weather conditions are decisive for the effectiveness of the biological method. The research should be repeated in next years.

Response: The results will be published as a short communication to overcome the problems mentioned by the reviewer.

Author Response File: Author Response.pdf

Reviewer 3 Report

Ms. Ref. Title: Results of the first inundative release of the predatory mite Neoseiulus californicus (McGregor) against the two-spotted spider Mite (Tetranychus  urticae Koch) on a hop plantation  submitted in Applied Sciences

The authors conduct a study on the control of a mite pest (Tetranychus urticae - TSSM). This species is highly polyphagous known as a pest for different crops and able to inhibit horticultural or fruit production and sometimes the vitality of plants. The worldwide spread and the difficult to control with the use of chemical means above all when the species is at high density on crops, make interesting biological control with predator as mite predators. TSSM feeding activity decreases the photosynthetic ability of the leaves and causes direct mechanical damage to the hop cones above all, when temperature rise during season. Neoseiulus californicus in the control of TSSM is widely used especially in greenhouse crops while for hops the information on control is limited, so the authors' study is interesting.

However, the study has some critical issues.

The predatory mite has long been known to be an active predator on TSSM in hops and various study and research experiences are reported in the bibliography even if inundative release of predator is not reported in the bibliography to my knowledge.

The authors managed the aphid or lepidopteran infestations in plots using Imidacloprid or Lambda-cyhalotrin but made no indication how they avoid the side effects of the active ingredient on N. californicus. Several bibliographic references and the producers' companies report a strong incidence of this ingredient with declines of up to 75% of predator. Detrimental effects on the predatory mite for these active ingredients is above all for lambda-cyhalotrin that was used for example in August in full activity of N. californicus. As the authors, explain all this?

Line12-30. The abstract is not concise and report different information that we can found on M&M (For example the date of predator release or TSSM monitoring). It is desirable to substantially modify the abstract and report with the right emphasis a summary, in a certain number of words, of the fundamental aspects of the MS examined.

Line 28 change “work” with study.

Line 60-78. In the M&M it is not clear how plot was separated or how blocks were chosen within the hop orchard. How many rows or plants for tratments? Are TSSM uniformly distributed in hop field? In general, TSSM shows aggregated distribution pattern in different crops.

Line 79. In the paragraph, the authors counted the mobile forms and TSSM eggs. It is not clear why even the mobile forms of N. californicus were not counted. This monitoring is very important in order to relate the interaction between the predator and the prey.

Line In tab 2 use English term for active ingredient for example “Imidacloprid” instead of “Imidakloprid”.

Line 108-112. The paragraph of the statistical analysis adopted is missing for assessing data. For example, can the data be used through parametric analysis? Have tests for normality been conducted? What tests have been adopted?

Line 116. dF means degrees of freedom! Why don’t write simply df or DF.

Line 132. In Figure 2, a greater density of eggs is evident in the upper part of the plant and is in line with the preference that the pest has in attacking the plant in growing shoots. This result is not well considered in the discussion of the results.

Line 204. The authors correctly report the climatic trends of the study area but for the study it is not clear what are the relationships of climate with the effects on the population of N. calicornicus or of TSSM.

Line 214-216. Remove “This section may be divided by subheadings…”

Line 218. Better native not autochthonous.

Author Response

Reviewer 3

Comments and Suggestions for Authors

Ms. Ref. Title: Results of the first inundative release of the predatory mite Neoseiulus californicus (McGregor) against the two-spotted spider Mite (Tetranychus urticae Koch) on a hop plantation submitted in Applied Sciences

The authors conduct a study on the control of a mite pest (Tetranychus urticae - TSSM). This species is highly polyphagous known as a pest for different crops and able to inhibit horticultural or fruit production and sometimes the vitality of plants. The worldwide spread and the difficult to control with the use of chemical means above all when the species is at high density on crops, make interesting biological control with predator as mite predators. TSSM feeding activity decreases the photosynthetic ability of the leaves and causes direct mechanical damage to the hop cones above all, when temperature rise during season. Neoseiulus californicus in the control of TSSM is widely used especially in greenhouse crops while for hops the information on control is limited, so the authors' study is interesting.

However, the study has some critical issues.

The predatory mite has long been known to be an active predator on TSSM in hops and various study and research experiences are reported in the bibliography even if inundative release of predator is not reported in the bibliography to my knowledge.

Response: Thank you for your valuable comment. Reviewer 2 had a similar opinion, and the corrected version of the article contains two previously non-cited articles (Vostrel et al., Barber et al.), while four papers regarding the use of the same predatory mite in other (non-hop) crops are not cited in the text.

The authors managed the aphid or lepidopteran infestations in plots using Imidacloprid or Lambda-cyhalotrin but made no indication how they avoid the side effects of the active ingredient on N. californicus. Several bibliographic references and the producers' companies report a strong incidence of this ingredient with declines of up to 75% of predator. Detrimental effects on the predatory mite for these active ingredients is above all for lambda-cyhalotrin that was used for example in August in full activity of N. californicus. As the authors, explain all this?

Response: In the treatment “Biological control”, no synthetic insecticides were used. The insecticides were used only in the ”chemical” treatments, i.e., “Control”, “Acaricide 1” and “Acaricide 2”. This was not indicated in the first version of the paper, but it is mentioned in the corrected version. Please see lines 69-81 in the manuscript.

Line12-30. The abstract is not concise and report different information that we can found on M&M (For example the date of predator release or TSSM monitoring). It is desirable to substantially modify the abstract and report with the right emphasis a summary, in a certain number of words, of the fundamental aspects of the MS examined.

Response: Corrected as instructed. Abstract has been modified. Please see lines 12-31 in manuscript.

Line 28 change “work” with study.

Response: Done. Please see line 29.

Line 60-78. In the M&M it is not clear how plot was separated or how blocks were chosen within the hop orchard. How many rows or plants for tratments? Are TSSM uniformly distributed in hop field? In general, TSSM shows aggregated distribution pattern in different crops.

Response: The experiment included 0.54 ha (plot with a length of 100 m and a width of 54 m) of a 7.33 ha hop plantation. There were 135 hop plants in each treatment (plot with a length of 33 m and a width of 13.5 m). TSSM was uniformly distributed throughout the hop field. More information regarding the arrangement of the field experiment can be found in Chapter 2.1 Experimental Area and Use of Plant Protection Products. Additional explanation has been given. Please see lines 64-81.

Line 79. In the paragraph, the authors counted the mobile forms and TSSM eggs. It is not clear why even the mobile forms of N. californicus were not counted. This monitoring is very important in order to relate the interaction between the predator and the prey.

Response: We agree with your valuable comment; however, in the plan of the field experiment, we only provided a count of the pest that occurred in all four treatments, i.e., T. urticae. Please see line 92.

Line In tab 2 use English term for active ingredient for example “Imidacloprid” instead of “Imidakloprid”.

Response: Done. Corrected in Table 2.

Line 108-112. The paragraph of the statistical analysis adopted is missing for assessing data. For example, can the data be used through parametric analysis? Have tests for normality been conducted? What tests have been adopted?

Response: The analysis has been conducted according to our previous investigations. The data have been analysed using parametric analysis, and were tested for normality by conducting Shapiro-Wilk's test and visual inspection of their histograms and box plots showed data approximately normally distrubuted. The differences between the average numbers of eggs/mobile stages and damaged hop cones were calculated by analysis of variance (ANOVA) and the Student-Neuman-Keuls test of multiple comparisons. Please see lines 129-132.

Line 116. dF means degrees of freedom! Why don’t write simply df or DF.

Response: Done. dF has been replaced with df. Corrected through all manuscript. Not just line 116.

Line 132. In Figure 2, a greater density of eggs is evident in the upper part of the plant and is in line with the preference that the pest has in attacking the plant in growing shoots. This result is not well considered in the discussion of the results.

Response: Thank you for your valuable comment. We agree that we did not fully discuss the results in the first version. There was a typing mistake, and sampling height was not relavant to egg laying of two-spotted spider mites. We have added the statement that the number of two-spotted spider mite mobile stage was the highest on the top of the plants. According to our research, the number of mobile stages of two-spotted spider mites was influenced by light, as it was also detected. The highest number of two-spotted spider mites was detected on the top of the plants (4-6 m). Our finding, namely, that the number of eggs laid by two-spotted spider mites does not differ between different plant parts can be related to the fact that eggs from two-spotted spider mites are laid in lower plant parts on purpose because predators (N. californicus) usually feed on upper plant parts. Therefore, eggs from two-spotted spider mites may be left uneaten. Figure 2 has been transformed to Table 4. Please see line 161.

Actually, we cannot support this statement based on the statistical analysis. It has been explained in 3.1 subheading.

Line 204. The authors correctly report the climatic trends of the study area but for the study it is not clear what are the relationships of climate with the effects on the population of N. calicornicus or of TSSM.

Response: Thank you for you valuable comment. We have added additional explanation. On the day of the release of the predator, the average daily temperature reached almost 24°C, and 46 % R.h. was recorded. The first counting of two-spotted spider mites was performed at a daily temperature of 18.3°C and after 2 rainy days. We assume that the population of two-spotted spiders could be higher in dry conditions, as previous studies [18] reported that the T. urticae population is restrained mainly by rainfall in the open field conditions. A third counting of the two-spotted spider mite population was also performed after five rainy days. On the days of predator release (4th July) and cone collection (21st August), we recorded 20 days with R.h. values below 50 %. It is known that the tested predatory mite is not sensitive to low R.h. [19]. Please see lines 255-267 in the manuscript.

Line 214-216. Remove “This section may be divided by subheadings…”

Response: Done.

Line 218. Better native not autochthonous.

Response: »Autochthonous« was replaced with »indigenous«. Please see line 250.

Author Response File: Author Response.pdf

Reviewer 4 Report

The work takes into account an interesting topic that is also important in operational practice. However, the manuscript presents some conceptual difficulties that make it difficult to communicate the contents to the readers of the journal.

In this regard, I suggest a revision of the text by an expert capable of making the language more correct and fluent in English. Remembering that it is better to use an impersonal language in communicating the activities carried out avoiding repeatedly using "we have" but it is better to use an impersonal form such as "have been".

Moreover, it is necessary to decide whether the results obtained are illustrated in the text or in the graphs, it is useless to repeat the same concept twice.

For the above reasons, I recommend that you make an appropriate revision of the manuscript, which I would like to review in the new version.

Author Response

Comments and Suggestions for Authors

The work takes into account an interesting topic that is also important in operational practice. However, the manuscript presents some conceptual difficulties that make it difficult to communicate the contents to the readers of the journal.

In this regard, I suggest a revision of the text by an expert capable of making the language more correct and fluent in English. Remembering that it is better to use an impersonal language in communicating the activities carried out avoiding repeatedly using "we have" but it is better to use an impersonal form such as "have been".

Response: The manuscript was re-edited by American Journal Experts. The corresponding certificate is added as an attached file.

Moreover, it is necessary to decide whether the results obtained are illustrated in the text or in the graphs, it is useless to repeat the same concept twice.

Response: Done.

For the above reasons, I recommend that you make an appropriate revision of the manuscript, which I would like to review in the new version.

Response: Done.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Dear Authors, 

The manuscript has been revised and is partially open for publication. The manuscript has been revised and is partially open for publication. I suggest that the reader should be informed about the possibilities of other methods of safe pest control, including the text below, which may be a contribution to the continuation of research in the extended scope of experience. Unfortunately the manuscript covers only one year, this is a serious defect, research should continue.

The value of the manuscript can be increased by introducing into part of the text information about using not only biological agents to control of spider-mite , but also is possible to apply another environmentally friendly product, with wide range, e.g. containing spinosad which is compatible with acaricides (1), it is effective when is apply only alone, however its effectiveness can be influenced by different temperature(2).

1) Miller RJ, White WH, Davey RB, George JE, Perez de Leon A. Efficacy of spinosad against acaricide-resistant and -susceptible Rhipicephalus (Boophilus) microplus and acaricide-susceptible Amblyomma americanum and Dermacentor variabilis. J Med Entomol. 2011 Mar;48(2):358-65. doi: 10.1603/me08222. PMID: 21485374.

2) Jolanta Kowalska (2010) Spinosad effectively controls Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) in organic potato, Acta Agriculturae Scandinavica, Section B — Soil & Plant Science, 60:3, 283-286, DOI: 10.1080/09064710902934205

Author Response

Thank you for your valuable comments. The part of discussion is improved as instructed.

Reviewer 3 Report

The authors improved the structure of the manuscript in this new version and made changes requested by the reviewers. 

Author Response

Thank you for your valuable comment.

Reviewer 4 Report

I suggest a revision of the text by an expert capable of making the language more correct and fluent in English. Remembering that it is better to use an impersonal language in communicating the activities carried out avoiding repeatedly using "we have" but it is better to use an impersonal form such as "have been".

Moreover, it is necessary to decide whether the results obtained are illustrated in the text or in the graphs, it is useless to repeat the same concept twice.

Author Response

The language in the ms has been revised by experts from American Journal Experts (please see attached certificate). I believe that the results are presented (as figures and text) appropriately and that there is no unnecessary repetition of the results in the article.