Screening and Evaluation of Rice to Assess Antibiosis and Antixenosis Resistance to White-Backed Planthopper (Sogatella furcifera)

Comments 1: The lack of correlations between some of the measured parameters is striking and, based on the information provided, seems to be counter-intuitive at times. I think this may stem from a lack of more profound interpretation of bioassay results. For example, Figure 1A might suggest that PTB33 is highly susceptible, whereas Figure 1B suggests it may be highly resistant. Some help in interpreting these trends is warranted. The usual interpretation is that salivary sheaths indicate frequent probing due to an inability to detect phloem tubes. So the large honeydew area might also include xylem wastes. Furthermore, some detailed studies with BPH have shown that short term bioassays such as honeydew, sheath staining and EPGs all assess the insects immediate or short term responses during the establishment phase. After a relatively short time, 1 or 2 days, the feeding improves as planthoppers adapt to a novel host (i.e., not TN1). 

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we added what we described in the discussion section, Lines 388-403.

Comments 2: The EPG information, although detailed, is very difficult to follow. There is perhaps too much happening in Figures 2, 3 and 4. Furthermore, the 1-way ANOVA is incorrect and likely leads to greater chances of type I errors. Because the different waveforms are not independent, then a 2-way ANOVA is more correct. The detection of significant differences based on genotypes might also be questioned as to what is biologically significant.

Response 2: We thank the reviewer for the valuable comments regarding the clarity, statistical analysis, and biological interpretation of the EPG data. Regarding the complexity of the EPG results, we acknowledge that the original presentation of Figures 2–4 may have been difficult to follow, so we have simplified them. Figure layouts and legends were also revised to improve clarity and logical structure. We agree that the initial use of one-way ANOVA was not optimal given the interdependence of EPG waveform variables.

Comments 3: Please indicate the units in Table 1. Instar development stages are also unlikely to be independent and I would suggest applying a multivariate analysis, repeated measure, or 2-way ANOVA to the data. Please also explain how missing data were handled. The mortality of individuals is apparent and likely affected the results, for example, RD49 has a DT of 0.0 for adults, is this correct? the test insects probably died here (is that possible?), if so, the correct entry for the analysis and table would be missing data. It is important to check this because development times are the main variable explaining the results of the PCA analysis (Figure 9). Indeed RD49 appears to be the most resistant genotype based on many of the tests. 

Response 3: We thank the reviewer for these important comments. We have checked that Table 1 has been revised to clearly indicate the units (days) for all developmental parameters.

The instar developmental stages were therefore reanalyzed using a one-way ANOVA with rice genotype (the objective was to compare the rice genotype) as a fixed factor, and the Results section has been updated accordingly.

Regarding missing data, individuals who died before completing a given developmental stage were treated as missing values and excluded from developmental time calculations for that stage. The previously reported value of 0.0 for adult developmental time in RD49 reflected the absence of surviving adults and has now been corrected to missing data (NA) in both the table and analyses.

Comments 4: For Figure 8, how were multiple comparison degrees of freedom handled? Figure 8 is very nice, but I would suggest removing the non significant symbols and keep all symbols pointing the right direction. This would highlight what seems to be best correlated. Also, why are some symbols 'squashed'?

Response 4: We thank the reviewer for the positive comment on Figure 8 and for the helpful suggestions regarding its presentation and statistical clarity.

To address potential ambiguity, we have expanded the figure legend to clearly distinguish significant and non-significant correlations and to explain the directionality of all symbols. The shape and narrowness of the ellipses reflect the strength of the correlation, with narrower ellipses representing stronger correlations approaching ±1, and more circular shapes indicating weaker relationships in Figure 8.

Comments 5: The discussion could be better organized to relate plant features with insect responses. Currently the discussion is a bit disjointed. Some recent, useful studies on WBPH virulence and interactions with rice could be added to support the discussion, including some detailed bioassays that use some of the same varieties.

Response 5: We thank the reviewer for this helpful suggestion. We added the discussion in Line 533-550.

Comments 6: The materials and methods should include references for all the methods that have been described elsewhere.

Response 6: We thank the reviewer for this important comment. The Materials and Methods section has been carefully revised to include appropriate references for all experimental procedures that have been described previously in the Materials and Methods section.

Comments 1: I have reviewed this manuscript carefully. The author used the honeydew drop method, electrical penetration mapping technology and growth rate analysis to evaluate the feeding behavior of white-backed planthopper; conducted investigations on the physiological and chemical properties of rice; and analyzed the expression of resistance-related genes to select resistant varieties in a targeted manner to promote sustainable pest management. There are some questions to be answered by the author. My main comments are as follows: Major concerns:

In Figure 1, the area experiment of Honeydew shows that the area of PTB33 is the largest, while that of TN1 is smaller. Is this normal? As far as I know, the TN1 variety is susceptible to white-backed planthopper, while the PTB33 variety is highly resistant to it.

Response 1: We thank the reviewer for the careful evaluation of our manuscript and for the insightful comments. We address each major concern as follows.

First, regarding Figure 1, we agree that TN1 is a well-known susceptible variety, whereas PTB33 is highly resistant to WBPH. The larger honeydew area observed on PTB33 may not indicate susceptibility but rather reflects compensatory feeding behavior. Honeydew excretion does not exclusively represent phloem sap intake and may include xylem-derived excretions when insects experience difficulty in establishing sustained phloem feeding. In resistant varieties such as PTB33, WBPH often exhibit increased probing activity and disrupted feeding, resulting in larger honeydew deposits despite poor feeding efficiency. This interpretation has now been clarified in the revised Discussion.

Comments 2: The survival rate experiment results of the white-backed planthopper in Figure 5 show that the survival rate of the white-backed planthopper is the highest in KDML105, and relatively lower in ASD7. This indicates that KDML105 is a variety that is susceptible to the white-backed planthopper genes. But why is UBN03078-80-354-7 used instead of KDML105 in the analysis of the changes in plant hormone-related metabolites after the invasion of white-backed planthoppers?

Response 2: We thank the reviewer. Second, concerning the survival results in Figure 5, KDML105 indeed showed higher WBPH survival and was classified as a susceptible genotype. However, UBN03078-80-354-7 was selected for plant hormone and metabolite profiling because it represents a clearly contrasting susceptible line with stable and consistent physiological responses under WBPH infestation. This line was selected based on combined evidence from survival, developmental performance, and feeding behavior assays rather than survival rate alone. The rationale for this selection has now been explicitly stated in the Methods and Discussion sections.

Comments 3: In Figure 10, the expression levels of some genes are marked as significant, while those of others are not. Why is that?

Response 3: We thank the reviewer for the valuable comments regarding the clarity. In Figure 10, not all genes showed statistically significant expression changes because WBPH resistance is regulated by specific signaling pathways rather than by uniform activation of all defense-related genes. Only genes that showed consistent, biologically meaningful responses to infestation reached statistical significance. This point has been clarified in Figure 10. Asterisks indicate significant differences between infested and control plants within the same genotype (P ≤ 0.05; P ≤ 0.01; Student’s t-test).

Comments 4: The rice seedlings that the white-backed planthopper feeds on should be specifically marked with the exact period, such as how many days old the seedlings are, or whether they are in the seedling stage or the tillering stage of the rice plants?

Response 4: We thank the reviewer for these important comments. We have mentioned the rice stage in the Materials and Methods section. Unless stated otherwise, plants were used at the tillering stage (25–30 days after sowing); panicle-stage assays were repeated at heading to test tissue/stage specificity. Lines 548-550.

Comments 5: Figure 10 shows the expression levels of 6 genes. Why these 6 genes were chosen and what were the basis for the selection? It should not have been a random and blind process.

Response 5: We thank the reviewer. the six genes analyzed in Figure 10 were not selected randomly. They were chosen based on prior evidence of their involvement in salicylic acid (SA)- and jasmonic acid (JA)-responsive defense pathways and their reported roles in rice resistance to sap-sucking insects. The criteria and rationale for gene selection are now clearly explained in the revised Methods section, with appropriate references.

Comments 6:

Minor concerns:

1. Figure 5: These colors and shapes are too similar. It is recommended that the author change some distinguishable colors and shapes to facilitate the drawing.
2. The reference formats are inconsistent. It is hoped that the author will carefully check and maintain one format.

Response 6: We appreciated the Reviewer's specific details. Figure 5 by applying a set of clearly distinguishable colors and marker shapes for each treatment to improve visual clarity and readability.

All references have now been thoroughly checked and reformatted to ensure full consistency with the journal’s reference style. In-text citations and the reference list have been standardized accordingly.

Comment 1: The author made some modifications to certain parts. However, in Figure 1, the area test results of honey juice show that the area of PTB33 is the largest, while the area of TN1 is smaller. This issue doesn't have practical significance just by looking at the statistical data. It is suggested that the author provide pictures of "Areas of honeydew deposition". There are many researchers who have published related articles online. You can refer to them. Additionally, conducting the "Seedling response" experiment would also be helpful, as this would provide specific pictures and make the experimental results more convincing

Response 1: We sincerely thank the reviewer for this constructive and insightful comment.

Regarding the honeydew area results in Figure 1, we agree that statistical differences alone may not fully demonstrate biological relevance. As correctly pointed out, the apparent inconsistency between PTB33 (resistant) and TN1 (susceptible) emphasizes the necessity of deeper parameter evaluation rather than relying solely on honeydew area as a single resistance indicator. The results from Figure 1 prompted a comprehensive multivariate analysis to identify which parameters are most suitable for screening for resistance to Sogatella furcifera. Specifically, we performed Principal Component Analysis (PCA) to evaluate the relative contribution of feeding behavior, honeydew excretion, growth performance, and associated traits to cultivar differentiation. The PCA results demonstrated that not all measured parameters contributed equally to resistance discrimination. In particular, the honeydew area alone was not a reliable standalone indicator for white-backed planthopper resistance, as certain cultivars exhibited variability in excretion patterns that did not consistently correspond with survival or developmental performance. This finding highlights the importance of integrating multiple parameters rather than relying on a single phenotypic assay.

To address the reviewer’s concern regarding visualization, we have now included representative images of honeydew deposition areas as a Supplementary Figure (Figure S1). These images provide visual confirmation of the quantitative measurements and improve the transparency and interpretability of the results.

With respect to the suggested “Seedling response” experiment, we agree that visual symptom assessment can strengthen biological interpretation. In the revised manuscript, we have clarified that resistance evaluation in this study focused primarily on antixenosis- and antibiosis-related parameters quantified through feeding behavior and performance assays.