Effects of Trichoderma harzianum Rifai and Chaetomium cupreum L.M. Ames on Biological Parameters of Myzus persicae (Sulzer) on Capia-Type Red Pepper (Capsicum annuum L.)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study confirms that Trichoderma harzianum significantly enhances aphid fecundity by treating red pepper and longevity by improving host plant nutrition, with sound research methods; it provides new insights for guiding the use of T. harzianum in integrated pest management (IPM).

 

Questions

1. Not all strains of Trichoderma harzianum and Chaetomium cupreum are exclusively plant endophytes. The paper does not explicitly confirm that the Trichoderma harzianum and strain used in this study is an endophyte.
2. In 3.2. Population parameters，multiple data entries in Table 3 are in complete conflict with the descriptions in the main text; it is recommended to maintain consistent decimal places.
3. Line 99, It is suggested to revise "Conversely, A researcher [17] reported that。。。。。" to“ Kara [17] reported that。。。。”.
4. Line 143-144, it is recommended to supplement the initial source of the aphid population (field collection site/laboratory strain number), as the stress resistance and reproductive characteristics of different aphid strains may affect the results.
5. Line 152-155, it is necessary to supplement the key conditions for root soaking (such as time and temperature), as these factors will affect fungal colonization.
6. Line 179-180, using 10 nymphs as the starting point for reproduction in the experiment—Is the number insufficient, and will individual differences amplify errors, thereby compromising the reliability of the results?
7. Table 3. it is necessary to revise "Net reproductive ( R 0 )" to "Net reproductive rate (R₀)"

 

Author Response

Comment 1: Not all strains of Trichoderma harzianum and Chaetomium cupreum are exclusively plant endophytes. The paper does not explicitly confirm that the Trichoderma harzianum and strain used in this study is an endophyte.

Response 1: I acknowledge that endophytic capability should be explicitly confirmed for the specific fungal isolates used in this study. I have added the following text to the Materials and Methods section (Section 2.1, immediately after the microorganism description):

“Although T. harzianum and C. cupreum are commonly reported as endophytic fungi capable of colonizing plant roots, endophytic colonization was not directly confirmed in this study. The observed effects on aphid performance may be due to root zone colonization and plant-mediated alterations rather than confirmed endophytic settlement. Future studies should include direct confirmation of endophytic colonization through tissue isolation and molecular validation.”

 

Comment 2: In 3.2. Population parameters，multiple data entries in Table 3 are in complete conflict with the descriptions in the main text; it is recommended to maintain consistent decimal places.

Response 2: We sincerely apologize for this inconsistency. When transferring the data from Table 3 in the draft text to the file prepared according to the journal guidelines, the data belonging to the experimental groups were mistakenly swapped and transferred incorrectly. The necessary corrections have been made, and the data in Table 3 has been organized to align with the discussion within the text.

 

Comment 3: Line 99, It is suggested to revise "Conversely, A researcher [17] reported that。。。。。" to“ Kara [17] reported that。。。。”.

Response 3: Changes made:

“Conversely, Kara [17] reported that T. harzianum significantly reduced..."

 

Comment 4: Line 143-144, it is recommended to supplement the initial source of the aphid population (field collection site/laboratory strain number), as the stress resistance and reproductive characteristics of different aphid strains may affect the results.

Response 4: Changes made:

Section 2.3 (Insect) has been expanded from the original text to:

“Myzus persicae colony used in this study was sourced from individuals collected in May 2024 from pepper fields in Van province, Türkiye, and subsequently maintained for over 10 generations on the Avşar F1 pepper variety at the Plant Protection Laboratory of Van Yüzüncü Yıl University prior to the experiments. The colony was maintained under controlled conditions (25 ± 2 °C, 65 ± 5% RH, 16:8 hours light:dark) without exposure to insecticides."

 

Comment 5: Line 152-155, it is necessary to supplement the key conditions for root soaking (such as time and temperature), as these factors will affect fungal colonization.

Response 5: Changes made:

Section 2.4. has been expanded to include detailed root treatment conditions:

“To promote effective colonization of micro-organisms in the root zone, pepper seedlings that had reached the 4-5 leaf stage were carefully removed from the viols, and soil particles on their roots were washed away with tap water. They were then dipped in the relevant fungal spore suspensions (1 x 10⁸ spores/ml) or sterile distilled water (control) for 2-3 minutes at room temperature (25 ± 2°C). After the treatment, the seedlings were immediately planted in 4-liter plastic pots (filled with a 2:1, v/v ratio sterile peat-perlite mixture). Experiments were started when the plants reached approximately 4-6 weeks of age.”

 

Comment 6: Line 179-180, using 10 nymphs as the starting point for reproduction in the experiment-Is the number insufficient, and will individual differences amplify errors, thereby compromising the reliability of the results?

Response 6: Changes made:

Section 2.7 (Population Projection) has been expanded:

“The TIMING-MSChart program uses demographic parameters (r, λ, R₀) derived from the complete life table data (n=30 per treatment) rather than the starting cohort size, ensuring accurate population predictions [19,20,24,28]. Chi (1988, 1990) demonstrated that population projections based on age-stage, two-sex life table theory accurately predict stage structure and growth Dynamics regardless of initial population size.”

 

Comment 7: Table 3. it is necessary to revise "Net reproductive ( R 0 )" to "Net reproductive rate (R₀)"

Response 7: Changes made:

from: “Net reproductive (R₀)"

to: "Net reproductive rate (R₀)"

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Editor and authors

The manuscript “Effects of Trichoderma harzianum Rifai and Chaetomium cupreum L.M. Ames on biological parameters of Myzus persicae (Sulzer) on Capia-type red pepper (Capsicum annuum L.)” provide new information about the effects of two endophytic fungi, Tricoderma harziamun and Chaetomium cupreum on the development, survival and reproduction of the aphid Myzus persicae important pest and vector of viruses in pepper crops.

I recommend major revision of the manuscript. I suggest that the authors include additional experiments to better characterize the effect of t both fungi on aphid behaviour. Several remarks and suggestions are provided in my review with the aim to help the authors to improve the quality and completeness of their work.

Comments:

While the results presented in this manuscript are interesting, they are not sufficient to draw definitive conclusions. As the authors themselves indicate, assessing fungal colonization would have been highly valuable; however, it is no longer possible to analyze these particular plants. Future studies should therefore include biochemical analyses and choice experiments to determine whether colonized plants are attractive or repellent to aphids, as well as direct measurements of fungal colonization.

Simple Summary

Line 14-17 Include in this paragraph the names of the fungus “Surprisingly, one fungus greatly increased aphid survival and reproduction instead of reducing it, leading to much faster population growth compared to untreated plants. When both fungi were applied together, aphid numbers also increased, while the second fungus alone had only minor effects”.

It is not clear for me if the authors want to express “r” as actual rate of increase or “rm” Intrinsic rate of increase

Line 35 I suggest that the authors include the data for the control group

Line 142. Was the colony originated from a single parthenogenetic female forming a clone, or a group of females forming a population?

Line 154. Did the authors perform a fungal recolonization? If so, Why were the roots immersed in the respective solution instead of applying 20 ml of the spore suspension, as described previously?

Lin 245-299 The data provide by the authors in the text do not coincide with the data present in table 3

Figure 5 and 6 I think that they are not necessary because the information are in Table 4.

Line 325 The population size projection after 60 days is informative but it is unrealistic because other factors affect population growth and host plant degradation would limit such growth.

Comments for author File: Comments.pdf

Author Response

I completely agree. I acknowledge these limitations and have added text emphasizing the need for mechanistic studies in future work.

Changes made:

Discussion section has been revised to:

“In this study, fungal colonization or plant biochemical changes (phloem nutrients, secondary metabolites, defense compounds) were not directly measured. Aphid preference experiments were also not conducted. Future studies should include these measurements to determine whether fungal-colonized plants attract aphids and whether the effects are due to improved nutrition, altered chemistry, or both.

 

Simple Summary

Comment 1: Line 14-17 Include in this paragraph the names of the fungus “Surprisingly, one fungus greatly increased aphid survival and reproduction instead of reducing it, leading to much faster population growth compared to untreated plants. When both fungi were applied together, aphid numbers also increased, while the second fungus alone had only minor effects”.

Response 1: Changes made:

“Surprisingly, Trichoderma harzianum greatly increased aphid survival and reproduction instead of reducing it, leading to much faster population growth compared to untreated plants. When both fungi were applied together, aphid numbers also increased, while Chaetomium cupreum alone had only minor effects.”

 

Comment 2: It is not clear for me if the authors want to express “r” as actual rate of increase or “rm” Intrinsic rate of increase

Response 2: Changes made:

Throughout the manuscript, "r" is the intrinsic rate of increase (same as r_m). This is stated in Table 1 and Table 3. No change needed—terminology is already correct.

 

Comment 3: Line 35 I suggest that the authors include the data for the control group

Response 3: Changes made:

To

“Contrary to expectations, T. harzianum significantly enhanced aphid population growth, resulting in a higher intrinsic rate of increase (r = 0.416 d^-1), finite rate of increase (λ = 1.515 d^-1), and net reproductive rate (R₀ = 87.67 offspring) compared to the control (r = 0.315 d^-1, λ = 1.370 d^-1, R₀ = 42.90 offspring).”

 

Comment 4: Line 142. Was the colony originated from a single parthenogenetic female forming a clone, or a group of females forming a population?

Response 4: Changes made:

Added to Section 2.3:

“Myzus persicae colony used in this study was sourced from individuals collected in May 2024 from pepper fields in Van province, Türkiye, and subsequently maintained for over 10 generations on the Avşar F1 pepper variety at the Plant Protection Laboratory of Van Yüzüncü Yıl University prior to the experiments. The colony was maintained under controlled conditions (25 ± 2 °C, 65 ± 5% RH, 16:8 hours light:dark) without exposure to insecticides.”

 

Comment 5: Line 154. Did the authors perform a fungal recolonization? If so, Why were the roots immersed in the respective solution instead of applying 20 ml of the spore suspension, as described previously?

Response 5: Root immersion was our inoculation method, not a second application. We've clarified this.

Changes made:

from: "...roots were immersed in the respective solutions..."

to: “….and soil particles on their roots were washed away with tap water. They were then dipped in the relevant fungal spore suspensions (1 x 10⁸ spores/ml) or sterile distilled water (control) for 2-3 minutes at room temperature (25 ± 2°C). After the treatment, the seedlings were immediately planted in 4-liter plastic pots (filled with a 2:1, v/v ratio sterile peat-perlite mixture). Experiments were started when the plants reached approximately 4-6 weeks of age.”

 

Comment 6: Line 245-299 The data provide by the authors in the text do not coincide with the data present in table 3

Response 6: You're absolutely right. Table 3 had incorrect decimal places (same issue Reviewer 1 found) and contains. It was corrected the entire table to match the text. All values now have 3 decimal places and match the descriptions exactly.

 

Comment 7: Figure 5 and 6 I think that they are not necessary because the information are in Table 4.

Response 7: I respectfully disagree. The figures show different information:

Figure 5: Population growth dynamics over 60 days, showing how populations change over time

Figure 6: Visual comparison making the 380-fold difference immediately clear

Table 4: Final numbers at day 60

Most ecology journals present both graphs and tables because they serve different purposes—one shows process, the other shows endpoints. We think both are valuable.

No change made.

 

Comment 8: Line 325 The population size projection after 60 days is informative but it is unrealistic because other factors affect population growth and host plant degradation would limit such growth.

Response 8: I’ve added text making clear these are theoretical projections.

Changes made:

Added to Section 3.3:

"These projections assume unlimited resources and stable conditions. In reality, plant quality degradation, competition, natural enemies, and resource limits would prevent reaching these numbers. However, the projections are useful for comparing relative growth potential and predicting when intervention might be needed."

 

Added to Discussion:

"The 60-day projections are theoretical. The actual numbers (380-fold increase) wouldn't occur in the field due to plant degradation and other limits. But the relative differences help predict timing of control needs."

 

Reviewer 3 Report

Comments and Suggestions for Authors

The paper presents interesting findings. However, more details shall be added in the description of the methodology, particularly about the treatments, timing and plant conditions. Figures should be improved and captions shall better describe them. Some inconsistencies are present in the Results in comparison to the data presented in the tables and the text shall be revised to avoid repetition of the same data presented in them. In the discussion, some aspects related to the health status of the plants should be addressed and some sentences modified to reflect the hypothetical condition. Other comments marked in the text.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The English is good, but it could be improved to increase readability.

Author Response

Comment 1: Any identification of the strain used? The number of the culture catalogue could be helpful for further studies.

Response 1: The fungal isolates used in this study were obtained from the stock culture collection at our laboratory but do not have formal culture collection accession numbers.

Changes made:

Section 2.1 (Microorganisms) has been revised to:

" These isolates were originally isolated from agricultural soils in Van province, Türkiye, and identified based on morphological and molecular characteristics. However, the isolates have not been deposited in an international culture collection and therefore lack formal accession numbers.

For future studies requiring the same strains, researchers may contact the corresponding author. We acknowledge that depositing these isolates in a recognized culture collection (such as CBS, ATCC, or a national collection) would facilitate reproducibility and comparative studies. Future work should include formal strain deposition and characterization.”

Comment 2: Have you verified that the plants were colonized by the fungi when applying the aphids? Please add this information.

Response 2: We did not directly verify fungal colonization before aphid introduction. This limitation is now clearly stated in the methods and discussed appropriately.

Changes made:

Section 2.4 (Experimental Design) - Added after plant establishment description:

"Fungal colonization was not directly verified through tissue examination or molecular methods prior to aphid introduction. The 4-6 week establishment period was based on previous reports showing Trichoderma and Chaetomiumtypically colonize roots within 2-4 weeks post-inoculation. However, without direct confirmation, the observed effects may result from root zone colonization rather than confirmed endophytic settlement."

Discussion Section - Added after following sentence: “Several limitations of this study should be acknowledged.”

“Most importantly, fungal colonization was not verified at the time of aphid introduction, so we cannot confirm whether plants were actually colonized when aphids began feeding.”

 

Comment 3: Please add information about the timing of applying the aphids to the plants with respect to the fungi application and the age of the plant, i.e. how much time passed from the application of the fungi and that of the aphids. This is important to assure that the treatments were effective, particularly if you have not determined the colonization of the plants. Moreover, it is important to know how old were the plants when applying the aphids.

Response 3: You are absolutely right. The timing information in the original text was ambiguous. I have now clarified both the interval between fungal application and aphid introduction, and the age of plants at aphid application.

Changes made:

Section 2.4 (Experimental Design) –

“The experimental design consisted of four treatments: T. harzianum, C. cupreum, a mixture of both fungi, and a control group. Pepper seedlings at the 4-5 true leaf stage (approximately 3-4 weeks after germination) were carefully removed from nursery trays, and soil prticles on roots were gently washed away with tap water. Roots were then dipped in relevant fungal spore suspensions (1x10⁸ spores/ml) or streile distilled water (control) for 2-3 minutes at room temperature (25 ± 2°C). After the treatment, the seedlings were immediately planted in 4-liter plastic pots (filled with a 2:1, v/v ratio sterile peat-perlite mixture). Experiments were started when the plants reached approximately 4-6 weeks of age.

At the time of aphid infestation, plants had developed 6-8 true leaves and showed uniform growth characteristics across all treatment groups. All plants appeared healthy with no visible symptoms of disease, nutrient deficiency, or stress throughout the experimental period. Plants were maintained under controlled conditions (25 ± 2°C, 65 ± 5% RH, 16:8 h L:D) and watered as needed to maintain adequate soil moisture.

Thirty replicates were established for each treatment, with one newly-born aphid nymph (< 12 hours old) confined per plant using a clip cage (approximately 2 cm diameter x 2 cm height) attached to a fully expanded leaf on the middle section of the plant.“

 

 

Comment 4: Please define the meaning of the acronym (APRP).

Response 11: You are correct. Although APRP was removed from Table 2 as suggested by Reviewer 2, the acronym still appears in the text without definition. I have now defined it at first use.

Changes made:

Section 3.1:

"In all treatments, females began reproducing on the first day of adulthood (APRP = 0), so the preadult duration and the total pre-reproductive period were effectively identical; therefore, the latter is not reported separately."

to:

“In all treatments, females began reproducing on the first day of adulthood (adult pre-reproductive period, APRP = 0), so the preadult duration and the total pre-reproductive period were effectively identical; therefore, the latter is not reported separately.”

 

Comment 5: The table should be readable on its own, so please add a footnote describing the meaning of the acronyms you have in it (N, APRP and TPRP). You should add also the explanation of the letters meaning (e.g. "Letters in the same columns show differences for p<0.05"). This allows you to avoid repeating in the text such information.

Response 12: Excellent suggestion. This will make the table self-explanatory and reduce text redundancy. I note that APRP was already removed from Table 2 following Reviewer 2's suggestion.

Changes made:

Table 2 - Added comprehensive footnote:

“N1-N4: nymphal instars 1-4 (days); TPRP: total pre-reproductive period (days); Different letters in the same row indicate significant differences among treatments (P < 0.05, paired bootstrap test with 100,000 resampling). n = 30 aphids per treatment.”

Table 3 - Added comprehensive footnote:

“Different letters in the same row indicate significant differences among treatments (P < 0.05, paired bootstrap test with 100,000 resampling). n = 30 aphids per treatment.”

 

Comment 6: There is no need to repeat in the text the same data presented in the table. Please correct this for all the data you are repeating. You can add in the text data, if useful or necessary, for example showing the difference: e.g. in case of this comparison, "C. cupreum duration of N1 instar was about 33% longer than...". Apply the same principle to all, whenever useful.

Response 6: I appreciate this suggestion, but I respectfully prefer to keep the current format. Presenting raw values (means ± SE) alongside statistical comparisons is standard practice in life table studies, and most entomology journals actually expect this format. Converting everything to percentages can be misleading - for example, 29% longer sounds dramatic but it's only 0.43 days difference in N1 duration. Readers need absolute values to judge biological significance. Also, the cited studies [16,17,18,29] all use this format, which helps readers compare our results with previous work. However, I have condensed the text where multiple pairwise comparisons were unnecessarily listed, reducing repetition while keeping essential numerical data.

Changes made:

Section 3.1: Old version:

“The duration of the N1 instar was significantly longer in C. cupreum treatment (1.93 ± 0.08 days) compared to Control (1.50 ± 0.09 days) (P_CcC=0.00073), while T. harzianum (1.73 ± 0.08 days) and Mixture (1.70 ± 0.10 days) were statistically similar to both groups (P>0.05). All groups were statistically similar during the N2 instar, with durations ranging from 1.07 to 1.23 days (P>0.05 for all pairwise comparisons). In the N3 instar, C. cupreum (1.57 ± 0.09 days) exhibited significantly longer duration than Mixture (1.30 ± 0.08 days) (P_CcM=0.04111), while T. harzianum (1.57 ± 0.11 days) and Control (1.43 ± 0.09 days) showed intermediate values with no significant differences from other treatments (P>0.05).”

New version:

“Variable effects were observed among treatments in individual nymphal instar durations (Table 2). The N1 instar was significantly longer in C. cupreum (1.93 ± 0.08 days) compared to control (1.50 ± 0.09 days), while T. harzianum and mixture were intermediate. All treatments showed similar N2 duration (1.07-1.23 days). For N3, C. cupreum (1.57 ± 0.09 days) was longer than mixture (1.30 ± 0.08 days).”

 

Old version

“Treatment groups exhibited highly significant differences in fecundity (P<0.05). T. harzianum treatment produced the highest fecundity (87.67 ± 7.12 offspring), which was significantly greater than all other treatments: Mixture (51.27 ± 3.38 offspring) (P_ThM<0.00001), Control (42.90 ± 3.44 offspring) (P_ThC<0.00001), and C. cupreum (38.00 ± 2.90 offspring) (P_ThCc<0.00001). Mixture treatment also exhibited significantly higher fecundity compared to C. cupreum (P_MCc=0.00287), while no significant difference was detected between Mixture and Control (P_MC=0.08298) or between Control and C. cupreum (P_CCc=0.27798).”

New version:

“Fecundity differed significantly among treatments (Table 2). T. harzianum produced the highest values (87.67 ± 7.12 offspring), significantly exceeding all other treatments (P < 0.00001). Mixture (51.27 ± 3.38 offspring) also exceeded C. cupreum (38.00 ± 2.90 offspring), while control (42.90 ± 3.44 offspring) showed intermediate values.”

 

Comment 7: The data reported in the text are not reflecting those included in the table. Please verify and modify as suggested above to avoid repeating the same data.

Response 7: You are absolutely correct, and I sincerely apologize. This same issue was identified by Reviewer 1. When preparing the manuscript according to the journal template, the column order in Table 3 was accidentally rearranged, causing a mismatch between the table and the text descriptions. Table 3 has now been completely corrected.

Changes made:

Table 3 has been corrected - The treatment columns are now in the correct order matching the text.

 

Comment 8: These graphs are quite difficult to read and interpret, particularly for the initial days. It is suggested to either show the initial part on a wider size (i.e. the 0-10 days period covers half of the graph) or to put this period in another graph (e.g. a box inside each graph - or abother graph - to "zoom" that period).

The same comment applies to Fig 3 and 4

Response 8: I appreciate this suggestion, but I respectfully prefer to maintain the current format. The overlapping of developmental stages in the early period represents real biological variation and is an inherent feature of age-stage, two-sex life tables. This presentation format is standard methodology established by Chi [19,20,22-26] and used in hundreds of published life table studies. Zooming into the 0-10 day period would emphasize the overlap but obscure the complete life cycle pattern, particularly the extended adult reproductive period which is equally important for interpretation. Maintaining this format also facilitates direct comparison with previous M. persicae studies [1,16,17,18,29].

No changes made to Figures 1, 3, and 4.

 

Comment 9: I respectfully disagree that these present identical information. Figure 6 provides immediate visual comparison showing the dramatic 380-fold difference among treatments, while Table 4 presents detailed stage-specific breakdown (N1, N2, N3, N4, and adult numbers) that cannot be shown graphically. Readers need both: the visual impact for understanding treatment magnitude and the numerical detail for population structure analysis. This approach is standard in demographic studies.

Regarding the axis notation, I prefer to keep "Million (M)" and "Billion (B)" rather than scientific notation because this simple bar chart is intended for quick visual comparison by a general readership, and the current format is more immediately interpretable. The legend already defines M and B in the figure caption.

 

Comment 10: Here again the major differences, if any, among treatments seem to be in the first 20 days. Consider using the same escamotage you have for the Y axis in Figure 6 to have two different ranges. See suggestion in the comment for figure 1.

Response 10: I appreciate the suggestion, but I prefer to maintain the current format. Figure 5 already uses a logarithmic scale (log N+1) on the Y-axis, which compresses the large population differences and makes the initial period visible while still showing the full 60-day exponential growth pattern. The complete time scale is essential for demonstrating how small early differences compound into the dramatic 380-fold population difference by day 60, which is the key finding of our projections. Splitting the time axis or using different ranges would obscure this exponential divergence pattern, which is standard presentation in demographic projection studies.

No changes made to Figure 5.

 

Comment 11: Describe better in the caption what the figure shows (i.e. projected population size....). Indicate above the bars whether the treatments are statistically different and include in the caption explanation of this.

Response 11: I have revised the caption to be more descriptive. However, I cannot add statistical letters to the bars because population projections are not statistically compared - they are mathematical extrapolations based on the life table parameters already statistically analyzed in Table 3. The statistical differences are in the demographic parameters, not in the projected population sizes themselves.

Changes made:

Figure 6 caption revised to:

"Figure 6. Projected total population size of Myzus persicae after 60 days, starting from 10 newly born nymphs under optimal conditions. Projections based on life table parameters from Table 3. Th = Trichoderma harzianum, Cc = Chaetomium cupreum, Th + Cc = mixture, C = Control. M = million, B = billion."

 

Comment 12: The interpretation of the results is quite hypothetical if you have not verified the colonization of the plants by the fungi or at least their persistence in the soil after the treatment. Please modify the text to account for such aspect.

Response 12: As already noted in response to other reviewers, we did not verify fungal presence either in plant tissues or in the rhizosphere at the time of aphid introduction. The Discussion has been revised to reflect this limitation with appropriate hypothetical language throughout.

Changes made:

All mechanistic interpretations now use conditional phrasing (e.g., "may have," "if these processes occurred," "could have"). The limitations section explicitly states: "Fungal colonization was not verified at the time of aphid introduction, so we cannot confirm whether plants were actually colonized or whether fungi persisted in the rhizosphere when aphids began feeding. The observed effects could result from endophytic colonization, rhizosphere activity, or residual effects of fungal metabolites."

 

Comment 13: You do not provide evidence to support this statement. Please revise" (regarding two statements in the same paragraph about nutritional quality and aphid performance)

Response 13: Both statements were too assertive without direct biochemical measurements. The entire paragraph has been revised to reflect the hypothetical nature of these proposed mechanisms.

 

Changes made:

From:

“T. harzianum likely enhanced aphid performance by altering host plant physiology and nutrition. This fungus improves nitrogen uptake, solubilizes phosphates, and produces siderophores that mobilize micronutrients [10]. Higher nitrogen, phosphorus, and micronutrient levels in plant tissues would benefit nitrogen-limited phloem feeders like aphids [10]. The improved nutritional quality of phloem sap in T. harzianum- colonized plants may have provided aphids with enhanced resources for growth and reproduction, similar to the fertilization effect observed in previous studies where increased plant nitrogen led to higher aphid fecundity [30]. Furthermore, Trichoderma colonization induces metabolic reprogramming in plants, affecting the tricarboxylic acid cycle and hexose monophosphate pathway, which can alter the concentration and composition of soluble sugars and amino acids in plant tissues [31]. While phloem sap is naturally sugar-rich but protein-poor, any increase in amino acid availability would be particularly beneficial for aphid reproduction.”

To:

“T. harzianum mah have enhanced aphid performance by altering host plant physiology and nutrition, though this remains hypothetical without biochemical verification. This fungus is known to improve nitrogen uptake, solubilizes phosphates, and produces siderophores that mobilize micronutrients [10]. If these processes occured in the present system, resulting higher nitrogen, phosphorus, and micronutrient levels in plant tissues could have benefited nitrogen-limited phloem feeders like aphids [10]. Improved phloem nutritional quality could potentially explain the enhanced aphid growth and reproduction observed, analogous to fertilization effects reported in previous studies [30] though direct measurement of phloem composition was not conducted and would be neessay to confirm this hypothesis.

Furthermore, Trichoderma colonization can induce metabolic reprogramming in plants, affecting the tricarboxylic acid cycle and hexose monophosphate pathway, which can alter the concentration and composition of soluble sugars and amino acids in plant tissues [31]. If such changes occured, any increase in amino acid availability could have been particularly beneficial for aphid reproduction.”

 

Comment 14: "It is suggested to provide some information here about the overall conditions of the plants in each treatment. This would give also an indication whether the differences noted in the aphid populations between treatments were affecting the plant conditions or not. After all, it is the impact on the plant that is important to consider, also to relate the results to plant protection issues." (regarding the sentence about balance between nutrition and defense)

Response 14: Information about plant condition has been added to provide context for the biological significance of the aphid population differences.

Changes made:

From:

“T. harzianum can also induce plant defenses, activating salicylic and jasmonic acid

pathways and stimulating defensive enzyme and metabolite production [32]. However,

defense induction is context-dependent and does not necessarily reduce herbivore

performance. In our system, if defense compounds were induced, the growth-promoting

benefits conferred by Trichoderma may have outweighed any defensive effects, particularly

for adapted specialists like M. persicae that can tolerate or detoxify many plant defensive

compounds [14]. Without measuring defense compounds, the relative balance between plant

nutrition enhancement and defense induction remains speculative, though our results suggest

that nutritional benefits, if present, may have predominated.”

To

“T. harzianum can also induce plant defenses, activating salicylic and jasmonic acid pathways [32], but defense induction doesn’t always reduce herbivore performance, especially for adapted specialists like M. persicae that tolerate many plant defensive compounds [14]. Without measuring defense compounds, the balance between nutrition and defense effects remains unclear. All plants appeared healthy throughout the experiment with no visible stress or damage, despite large differences in aphid numbers. However, plant growth and yield were not measured. Whether the 2-fold aphid increase on T. harzianum plants causes real economic damage is unknown. Future work should measure both aphid populations and plant performance to determine if fungal benefits outweigh potential pest problems.”

 

Comment 15: Please add literature reference (regarding Chaetomium effectiveness statement)

Response 15: The statement has been revised to be more general and accurate.

Changes made:

From

While Chaetomium species have demonstrated effectiveness against some insect pests, including cotton aphids (Aphis gossypii) and beet armyworms (Spodoptera exigua) in previous studies, these effects appear to be pest- and context-specific. Our findings suggest that C. cupreum may not be an effective biocontrol agent for M. persicae on pepper, at least not through endophytic colonization-mediated plant resistance.

to

“The limited effects of C. cupreum could have several explanations, though without direct measurements these remain speculative. First, C. cupreum may have colonized pepper roots less efficiently than T. harzianum, or produced lower titers of bioactive metabolites, though colonization levels were not assessed. Second, Chaetomium metabolites are primarily antifungal [33] and may have limited effects on phloem-feeding aphids. Third, C. cupreum may alter plant physiology in ways that neither strongly enhance nor inhibit aphid performance, resulting in the neutral response observed. C. cupreum appears ineffective against M. persicae on pepper in this system.”

 

Comment 16: "No need to add data presented in Results here again. Please revise" (regarding the mixture treatment paragraph)

Response 16: The paragraph has been revised to discuss interpretation rather than repeating data.

Changes made:

from

"The mixture treatment yielded intermediate aphid population parameters - fecundity

(51.27) and intrinsic rate (r = 0.340 d⁻¹) fell between T. harzianum alone and controls.

Notably, mixed inoculation produced the longest adult (28.73 days) and total longevity

(34.87 days), suggesting synergistic effects on plant physiology favoring aphid survival."

to

"The mixture treatment produced intermediate effects, with population parameters between

1. harzianum and control. Extended longevity in this treatment suggests possible interactions

between the two fungi affecting plant-aphid dynamics."

 

Comment 17: "This paragraph is more suitable for the Conclusions. Please consider moving it." (regarding the multitrophic interactions paragraph on page 15)

Response 17: This paragraph has been moved to the Conclusions section.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Line 162 replace “prticles” by “particles”Line 300-309 reduce the number of decimals

Dear Author and Editor

I acknowledge the efforts made to improve the manuscript and appreciate the revisions undertaken however I suggest to the author include additional experiments or consider presentting the manuscript as short communication

I have included some comments:

Comment 7: Figure 5 and 6 I think that they are not necessary because the information are in Table 4.

Response 7: I respectfully disagree. The figures show different information:

Figure 5: Population growth dynamics over 60 days, showing how populations change over time

Figure 6: Visual comparison making the 380-fold difference immediately clear

Table 4: Final numbers at day 60

Most ecology journals present both graphs and tables because they serve different purposes—one shows process, the other shows endpoints. We think both are valuable.

No change made

RW: I do not agree, the data show in figure 6 are present in Table 4. Then I suggest to delete the Figure 6 (see the files attached)

Line 423 replace “would be neessay to confirm this hypothesis” by “would be neccesary”

Line 474 T. harzianum in cursive

Comments for author File: Comments.pdf

Author Response

Comments 1: I do not agree, the data show in figure 6 are present in Table 4. Then I suggest to delete the Figure 6 (see the files attached)

Response 1: Figure 6 was originally included to visually support the data presented in Table 4 and to make the observed differences more clearly and intuitively understandable. However, in line with your valuable suggestion, the necessary revisions have been made and both Figure 5 and Figure 6 have been removed from the manuscript. Thank you for your constructive feedback.

 

Comments 2: replace “would be neessay to confirm this hypothesis” by “would be neccesary”

Response 2: Thank you for pointing this out. The phrase has been corrected as suggested, and “would be neessay to confirm this hypothesis” has been replaced with “would be necessary.”

“…though direct measurement of phloem composition was not conducted and would be necessary to confirm this hypothesis.”

 

Comments 3: "T. harzianum" in cursive

Response 3: Thank you for your careful observation. T. harzianum has been formatted in italics throughout the manuscript as suggested.

 

Comments 4: Line 300-309 reduce the number of decimals (as indicated in the attached file).

Response 4: Thank you for your suggestion. The number of decimals in lines 300–309 has been reduced as recommended.

Reviewer 3 Report

Comments and Suggestions for Authors

With the corrections made, I believe the article can be published

Author Response

Comments 1: With the corrections made, I believe the article can be published.

Response 1: Thank you very much for your positive evaluation. All the valuable comments you previously provided have been carefully considered and the necessary revisions have been made accordingly. I sincerely appreciate your time, effort, and constructive contributions to improving the manuscript.

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

The changes have been done. I agree with your decision to public this manuscript in Insects.