Distinct effects of phyllosphere and rhizosphere microbes on invader Ageratina adenophora during its early life stages

Reviewer #1 (Public Review):

Summary:

The work by Zeng et al. comprehensively explored the differences in the effects of leaf and soil microbes on the seed germination, seedling survival, and seedling growth of an invasive forb, Ageratina Adenophora, and found evidence of stronger effects of leaf microbes on Ageratina compared with soil microbes, which were negative for seed germination and seedling survival but positive for seedling growth. By further DNA sequencing and fungal strain cultivation, the authors were able to identify some of the key microbial guilds that may facilitate such negative and positive feedback.

Strengths:

(1) The theoretic framework is well-established.

(2) Relating the direction of plant-microbe feedback to certain microbial guilds is always hard, but the authors have done a great job of identifying and interpreting such relationships.

Weaknesses:

(1) In the G0 and G21 inoculation experiments, allelopathic effects from leaf litters had not been accounted for, while these two experiments happened to be the ones where negative feedback was detected.

(2) The authors did not compare the fungal strains accumulated in dead seedlings to those accumulated in live seedlings to prove that the live seedlings indeed accumulated lower abundances of the strains that were identified to increase seedling mortality.

(3) The data of seed germination and seedling mortality could have been analyzed in the same manner as that of seedling growth, which makes the whole result section more coherent. I don't understand why the authors had not calculated the response index (RI) for germination/mortality rate and conducted analyses on the correlation between these RIs with microbial compositions.

(4) The language of the manuscript could be improved to increase clarity.

Reviewer #2 (Public Review):

Summary:

The study provides strong evidence that leaf microbes mediate self-limitation at an early life stage. It highlights the importance of leaf microbes in population establishment and community dynamics.

The authors conducted three experiments to test their hypothesis, elucidating the effects of leaf and soil microbial communities on the seedling growth of A. adenophora at different stages, screening potential microbial sources associated with seed germination and seedling performance, and identifying the fungus related to seedling mortality. The conclusions are justified by their results. Overall, the paper is well-structured, providing clear and comprehensive information.

Author Response

Provisional Response to Public Reviews

Reviewer #1 (Public Review):

Summary:

The work by Zeng et al. comprehensively explored the differences in the effects of leaf and soil microbes on the seed germination, seedling survival, and seedling growth of an invasive forb, Ageratina adenophora, and found evidence of stronger effects of leaf microbes on Ageratina compared with soil microbes, which were negative for seed germination and seedling survival but positive for seedling growth. By further DNA sequencing and fungal strain cultivation, the authors were able to identify some of the key microbial guilds that may facilitate such negative and positive feedback.

Thank you very much for your assessment.

Strengths:

(1) The theoretic framework is well-established.

(2) Relating the direction of plant-microbe feedback to certain microbial guilds is always hard, but the authors have done a great job of identifying and interpreting such relationships.

Thank you very much for your assessment.

Weaknesses:

(1) In the G0 and G21 inoculation experiments, allelopathic effects from leaf litters had not been accounted for, while these two experiments happened to be the ones where negative feedback was detected.

We did not directly test the allelopathic effects. However, our inoculation of sterile litter or soil indicated the potential allelopathic role in germination and seedling mortality. Interestingly, such allelopathic effects are elicited by leaf litter not by soil, which include delaying germination time (see Fig. 1a) and killing some seedlings (see Fig. 1c). Nonetheless, microbial effects are significantly more adverse than allelopathic (also see Fig. 1e). We will discuss this point in the resubmitted version.

(2) The authors did not compare the fungal strains accumulated in dead seedlings to those accumulated in live seedlings to prove that the live seedlings indeed accumulated lower abundances of the strains that were identified to increase seedling mortality.

Thanks for your concerns. We have not isolated fungi from healthy seedlings to make a comparative study. However, our team work previously found that the seedling-killing Allophoma strains obtained in this study had the same ITS genes as the leaf endophyte and leaf spot pathogen Allophoma associated with mature A. adenophora individual; some seedling-killing Alternaria also occur in healthy seedlings inoculated by leaf litter. We thus assumed that these seedling-killing fungi, e.g., Allophoma and Alternaria, likely exist in A. adenophora mature individual by a lifestyle switch from endophytic to pathogenic, and these fungi can kill seedling only at very early life stage of A. adenophora.

Thus, we discussed this point as: “We did not isolate fungi from healthy seedlings in this study. However, a previous report revealed that the dominant genera in healthy seedlings inoculated with leaf litter were Didymella and Alternaria (Kai Fang et al., 2019). Based on these results, these fungal genera likely exist in A. adenophora by a lifestyle switch from endophytic to pathogenic. The virulence of these strains for seedling survival under certain conditions may play an essential role in limiting the population density of A. adenophora monocultures.” See Lines 416-435.

Here, we also will consider adding more sentences to discuss your concerns in the resubmitted version as: “It is worth to explore the dynamic of these strains along with seedling development and to determine if these strains kill seedling only at very early stage.”

(3) The data of seed germination and seedling mortality could have been analyzed in the same manner as that of seedling growth, which makes the whole result section more coherent. I don't understand why the authors had not calculated the response index (RI) for germination/mortality rate and conducted analyses on the correlation between these RIs with microbial compositions.

Thanks so much. Response index (RI) was calculated as: (variablenon-sterile–variablesterile)/variablesterile)). Because mortality rates of some sterile groups were zero values, it is impossible to calculate their RIs. Relatively, microbes rarely affect seed germination time (GT) and rate (GR) (see Fig. 1a,b). Therefore, we preferred to make a direct comparison of their difference between non-sterile and sterile treatments (see also Figure S2), and we also conducted a correlation by these values with microbial compositions rather than by RIs (see Fig. 4).

We will emphasis this point in the Materials and Methods when resubmit our revision.

(4) The language of the manuscript could be improved to increase clarity.

We will improve this in the resubmitted version.

Reviewer #2 (Public Review):

Summary:

The study provides strong evidence that leaf microbes mediate self-limitation at an early life stage. It highlights the importance of leaf microbes in population establishment and community dynamics.

Thank you very much for your assessment.

The authors conducted three experiments to test their hypothesis, elucidating the effects of leaf and soil microbial communities on the seedling growth of A. adenophora at different stages, screening potential microbial sources associated with seed germination and seedling performance, and identifying the fungus related to seedling mortality. The conclusions are justified by their results. Overall, the paper is well-structured, providing clear and comprehensive information.

Thank you very much for your assessment.