Miscanthus sinensis ‘Gracillimus’ Shows Strong Submergence Tolerance Implying Its Potential Utilization in Construction of Ecological Ditches

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors:

The title should be corrected {what is meant by vegetation filter construction? What do you mean by VDDs? Resources related to 3 plant species under test were not observed! Where are plant seeds obtained from? What is meant by 8 kg of medium? Why is the volume of water not known? What is the experimental design used? What were the control conditions of the treatments? What is the meaning of all these grasses being treated with T4 to enhance their growth? Why is recovery growth included? Researchers have not been able to provide NPSP conditions, which has been the most important research challenge! What was the advance and delay of planting in the greenhouse and the flooding conditions? What were the control conditions of the treatments in the 2 considered cases? How can the changes in the measured traits be attributed to the conditions of the treatments? Other factors such as fertilizing, irrigation, etc. may have affected the trait values!

Author Response

We appreciate your good and constructive suggestions and comments. We have made major revisions to our manuscript accordingly. The details are listed below point by point. The red parts are your comments and questions.

1. The title should be corrected what is meant by vegetation filter construction?

Ok, thank you for your fundamental suggestions. We have made an exact presentation on this point in the revised manuscript.

2. What do you mean by VDDs?

Thank you for your careful examination and fundamental suggestions. VDDs refer to drainage ditches planted with herbaceous plants for ecological management and water quality improvement. Research shows that varying submergence durations impact the growth, physiological traits, and nitrogen accumulation of three grass species. The results guide species selection and layout planning for ecological ditches. This point has been presented in the revised manuscript.

3. Resources related to 3 plant species under test were not observed! Where are plant seeds obtained from?

The main species involved are Miscanthus sinensis 'Gracillimus', Elytrigia repens (L.) Nevski 'Jingcao No. 2', and Pennisetum alopecuroides 'Ziguang'. Miscanthus sinensis 'Gracillimus' was propagated not through seeds but through rhizomes, and it is a variety independently bred by the Beijing Academy of Agricultural and Forestry Sciences.Elytrigia repens (L.) Nevski 'Jingcao No. 2' was also propagated through rhizomes and is a nationally approved variety independently bred by the Grass and Flower Research Institute of the Beijing Academy of Agricultural and Forestry Sciences. Pennisetum alopecuroides 'Ziguang' was similarly propagated through rhizomes. All these varieties are independently bred, with the genetic material sourced from the same institution.

4. What is meant by 8 kg of medium?

We feel so sorry for the unclear presentation of these points. This culture medium means it can hold a weight of eight kilograms. We have added all these details in the revised version.

5. Why is the volume of water not known?

Thank you for your careful examination and fundamental suggestions. In some cases, experiments may use a standardized volume of water (for example, based on the container's volume or calculated using water flow and velocity), and it may not be necessary to explicitly record the volume of water for each experiment. Generally, more focus is placed on variables like plant growth, pollutant removal efficiency, and other factors, while the water volume acts as an indirect influencing factor.

6. What is the experimental design used?

Thank you for your good questions. In this study, a greenhouse simulation experiment was conducted to investigate the effects of different submergence durations (T1 to T7) on the growth characteristics, physiological indicators, and nitrogen accumulation of three grass species. Each treatment had 3 replicates, each pot contained two plants as one replicate. The control group (T1) had no submergence treatment. The experiment was carried out in large PVC containers, with water levels maintained using tap water. Data from the experiment were analyzed using one-way analysis of variance (ANOVA), with statistical analysis performed using SPSS software. A significance level of P < 0.05 or 0.01 was applied. The experiment aims to provide scientific guidance for the selection and layout of plant species in ecological channels in China.

7. What were the control conditions of the treatments?

Thank you for your insightful comments and suggestions. The control group (T1) was not subjected to any flooding treatment. In other words, the experimental plants in the T1 group were not affected by submergence time and served as a baseline for comparing the effects of different flooding durations on plant growth and nitrogen accumulation. By comparing the results with the control group, the researchers could isolate and analyze the specific impacts of flooding on the plants. The control group provided a natural, untreated state, allowing for a clear observation of the differences between the treatment groups and the plant's natural growth conditions.

8. What is the meaning of all these grasses being treated with T4 to enhance their growth?

In this experiment, the purpose of T4 treatment was to evaluate whether different grass species could resume growth after a certain period of submergence, and promote their growth by restoring the growth phase. This treatment helps to simulate the short-term flooding situations that grass species may experience in the actual environment, especially for those that will enable recovery after flooding. The addition of the T4 treatment means that these grass species have undergone a submergence phase, allowing them time to "recover" growth, thus observing differences in the ability of grass species to recover after submergence.

9. Why is recovery growth included?

Thank you for your questions. The introduction of recovery growth in this experiment was designed to assess the resilience of grass species to undergo flooded treatment. Specifically, this stage is of great significance for the experiment. Whether different grass species can resume their growth after experiencing flooding reflects their flooding tolerance and growth toughness. In the actual ecological environment, grass species are often faced with intermittent flooding rather than persistent flooding. By introducing a recovery growth phase, the experiment was able to simulate this natural hydrological condition to help assess the adaptation and rate of recovery of grass species after changes in water levels. Some grass species may show strong growth capacity during the recovery period after flooding, which will be very helpful for future applications in ecological restoration. Therefore, by assessing recovery growth, researchers can screen out those grass species that can still grow healthily in extreme water conditions.

10. Researchers have not been able to provide NPSP conditions, which has been the most important research challenge!

This study was not conducted in real field conditions primarily due to the numerous uncertainties present in such environments, such as sudden droughts, damage from small animals, and the effects of pesticides, which make it difficult to clarify the impact of flooding stress as a single factor on the three plant species. Therefore, we conducted simulated experiments in a greenhouse. Conducting experiments in actual field conditions would be more meaningful for future practical applications.

11. What was the advance and delay of planting in the greenhouse and the flooding conditions?
    
    1. Planting schedule:

 In late autumn 2017, grass seedlings of the grass species were acquired from the Xiaotangshan basement of our institute. Seedlings were grown in a greenhouse for two months and then transplanted into plastic containers containing approximately 8 kg of the medium. These plants were placed under flooded conditions of varying duration during the rainy season in July.

2. Flooding conditions:

Including 0 days (as control, T1), 1 day (T2), 3 days (T3), 5 days (T4), 7 days (T5), 9 days (T6), and 11 days (T7). Triplicate samples were prepared for each treatment for each grass species, each consisting of two plants in a container. Before the treatment, the container containing the herbs was placed in larger PVC boxes, and tap water was added to the boxes to ensure that the water level remained 10 cm above the top of the herb. During the experiment, the temperature range was maintained at 20 - 30℃ and the humidity was maintained between 40% and 60%.

12. What were the control conditions of the treatments in the 2 considered cases?

For the experiments, plants treated with T1 were designated as controls. This means that these plants did not undergo any flooding treatment and they grew under normal conditions as a benchmark for experiments to compare the growth of other plants treated with different flooding times. The experiment set up different flooding durations, including 0 (control), 1,3,5,7,9, and 11 days, as indicated by T1, T2, T3, T4, T5, T6, and T7, respectively. These two conditions provide the basic contrasting data for the experiment to assess the effects of different flooding times on plant growth, physiological properties, and nitrogen accumulation.

13. How can the changes in the measured traits be attributed to the conditions of the treatments?
    
    1. Biomass accumulation: Pen and Ely biomass increased aboveground after temporary flooding treatment (e.g., T2), but decreased significantly after prolonged flooding (e.g., T7). The biomass of Mis was less affected by water flooding and showed a strong tolerance. 2. Photosynthesis and chlorophyll content: Under the condition of flooding, the chlorophyll content of all grass species decreased compared with the control group, and with the extension of flooding time, the decreasing trend of chlorophyll content was more obvious, which affected photosynthesis. 3. Root activity: flooding with T6 significantly inhibited the root activity of the three grass species. Among them, Mis showed a strong tolerance with a smaller decrease in root activity. 4. Nitrogen assimilation: Pen nitrogen assimilation decreased significantly with the extension of flooding time, Mis did not change significantly, and Ely increased first and then decreased. These changes reflect the different responses of different grass species to flooding stress, with Mis showing the strongest tolerance. These results are of great significance for selecting grass species suitable for ecological ditch construction and provide a scientific basis for optimizing ecological ditch management and enhancing their ecological benefits.
    
    
14. Other factors such as fertilizing, irrigation, etc. may have affected the trait values!

Thank you for your careful inspection and your basic advice. Yes, other factors, such as fertilization, irrigation, etc., may also affect the values of plant traits. In agricultural production and plant growth research, fertilization and irrigation are two important management measures, that can significantly affect plant growth status, physiological characteristics, and nitrogen accumulation, and we added the main information in the modified "Introduction section" section. If you have any other good suggestions or questions, please feel free to inform us and we will try our best to make corrections.

 

 

 

 

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, please find 24 comments of me in the attached annotated PDF version of your manuscript. Thank you. Kind regards.

Comments for author File: Comments.pdf

Author Response

Thank you very much for your suggestions. I have made revisions to address your comments, which are highlighted in red in the article.