Advancing Industrial Production of White Grifola frondosa: Liquid Inoculum Culture Parameter Optimization and Molecular Insights into Fruiting Body Development
, Jia-Yuan Wang 1, Shang-Shang Xiao 1, Su-Ya Liu 1, Bao-Yue Sun 1, Shou-Mian Li 1,3,4, Ming Li 1,3,4, Zhi-Qiang Wen 2,* and Xiao Li 1,3,4,*
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors-63-65 is it for G frondosa?, please add the species name
-102-110, as authors did literature studies in introduction in 2-3 paragraphs. Why authors still want to do optimization again that are repeated to almost 10 papers mentioned.? Authors should revise the objective by adding specific/uniqeness of this optimization
-107, Authors should also mentioned the target of objectives why RNA-Seq was conducted in this work. Which activities/phenotype that authors aim to study
-119, please add reference of this medium
-121, please add refernce
-152-153, authors should indicate criteria how to determine the stages of development? like duration or any phenotype?
-196, fig 1 which is 5 carbon? please recheck and revise it
-198, Glucose outperformed the others ... please revise this. As the statistical analysis, glucose is similar to other carbon sources
-205, based on fig1, there are 4 properties . So authors conclude this order based on which properties? futhermore, most stat say they are indifferent
-215, same comment of fig 1 to the fig 2, statistical analysis say all are not different.
-fig 3 same comment to fig 1 and 2
-fig 2-6, same comment to fig 1.
-332, please add reference to identify these DEGs
-347, please explain more about relative similarity, better describe criteria to justify to be similar of dramatic reprogramming
-fig 10b is too small to read
-fig 10c , authors should mark what are each cluster? is it in 10a?
-387, Drug metabolism ....i think cytochrome P450 is the housekeeping for oxidative pathway? how it is related to drug?
-3.6. please explain authors criteria to select these 3 genes for validation
-445,Notably, this work provides the first reported evidence for an optimal C/N ratio (10:1) (glucose:peptone) in liquid cultivation systems. .... many published papers even those reported in the introduction of this work also report optimization. So please revise this
Author Response
-63-65 is it for G frondosa?, please add the species name
Response: 63-65 is it for G. frondosa. As G. frondosa in 63-65 was not the first time appeared in this article, so we present it in the abbreviated form of the genus name.
-102-110, as authors did literature studies in introduction in 2-3 paragraphs. Why authors still want to do optimization again that are repeated to almost 10 papers mentioned.? Authors should revise the objective by adding specific/uniqeness of this optimization
Response: we have mentioned that no comprehensive study has systematically evaluated both mycelial yield and pellet quality—including size, density, and germination rate-in liquid cultures. Addressing this gap is essential for further optimizing industrial-scale production (Line 82-85). So we systematically optimized liquid culture conditions optimization including formula and culture conditions of liquid spawn medium, taking the mycelial biomass as the main index, combining the results of pellet diameter, pellet density, and germination time in this study.
As for the RNA-seq analysis, fruiting bodies development of G. frondosa are regulated by various factors, and the molecular regulatory mechanism of its growth and development has not been reported (Line 103-110) and only for mycelia and primordia development (94-102). So we conducted RNA-seq analysis across four distinct developmental stages of fruiting body differentiation.
-107, Authors should also mentioned the target of objectives why RNA-Seq was conducted in this work. Which activities/phenotype that authors aim to study
Response: Agreed. We have added at lines 103-110 and 115-118.
-119, please add reference of this medium
Response: Added at line 130.
-121, please add refernce
Response: Added at line 132.
-152-153, authors should indicate criteria how to determine the stages of development? like duration or any phenotype?
Response: Agreed. We have added at lines 162-165.
-196, fig 1 which is 5 carbon? please recheck and revise it
Response: Agreed. We have added at lines 205.
-198, Glucose outperformed the others ... please revise this. As the statistical analysis, glucose is similar to other carbon sources
Response: glucose outperformed the others for that it produced higher pellet biomass (1.71 g/L), while maintaining favorable morphological characteristics, including a smaller diameter (0.85 mm) and highest density (400 pellets/mL).
-205, based on fig1, there are 4 properties . So authors conclude this order based on which properties? futhermore, most stat say they are indifferent
Response: Agreed. It has been deleted.
-215, same comment of fig 1 to the fig 2, statistical analysis say all are not different.
Response: Agreed. It has been deleted.
-fig 3 same comment to fig 1 and 2
Response: Agreed. It has been deleted.
-fig 2-6, same comment to fig 1.
Response: Agreed. It has been deleted.
-332, please add reference to identify these DEGs
Response: We have added reference to identify these DEGs at line 181-185.
-347, please explain more about relative similarity, better describe criteria to justify to be similar of dramatic reprogramming
Response: Thanks. The explanation has been added at line 334-335.
-fig 10b is too small to read
Response: Agreed. It has been changed.
-fig 10c, authors should mark what are each cluster? is it in 10a?
Response: Agreed. It has been added in line 348-349.
-387, Drug metabolism ....i think cytochrome P450 is the housekeeping for oxidative pathway? how it is related to drug?
Response: Agreed. It has been deleted.
-3.6. please explain authors criteria to select these 3 genes for validation
Response: Thanks for your suggestion. The explanation for selectting these 3 genes for validation has been added at line 389-399.
-445,Notably, this work provides the first reported evidence for an optimal C/N ratio (10:1) (glucose:peptone) in liquid cultivation systems. .... many published papers even those reported in the introduction of this work also report optimization. So please revise this
Response: While previous investigations have characterized carbon and nitrogen sources independently, the optimal C/N balance for liquid inoculum production remains unexplored. Notably, existing research has only established a 30:1 ratio (sucrose:peptone) as optimal for solid-substrate cultivation in Grifola frondosa (Sun et al., 2019). Through systematic optimization, we demonstrate for the first time that a significantly lower 10:1 C/N ratio (glucose:peptone) maximizes mycelial biomass production in liquid culture systems.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript represents an interesting attempt to understand the growth stages of Grifola frondosa via transcriptomic analysis that is gaining alot of attention. The research consists of two parts, viz. the optimisation of culture conditions of the inoculum, and transcriptomic analysis of fruiting bodies at different developmental stages. Standard methodologies were used. Overall, the manuscript is easy to understand and contains important findings for the mushroom field.
Main comment:
Both parts were carried out reasonably well, however, the linkage between the first and second parts have not been well-explained. The transcriptomic analysis can stand alone without the data on optimisation of media condition on inoculum. The justification for using liquid inoculum must be explained. Did the authors expect different results if a typical grain-based inoculum was used? I don't think so but I might be wrong. Please explain your rationale. Grain spawn is also more widely used in the industry as it is less susceptible to contamination.
I would suggest to remove the first part from this manuscript.
Other comments:
- The reason for choosing an albino strain for this study should be stated.
- Any molecular data to validate the species used? Please include in the supplementary info.
- Please describe the biological replicates used, are these different bags from the same cultivation batch or different batch?
- In the discussion, the authors may consider emphasizing the key findings of this study and how they contribute to bridging existing knowledge gaps in this area.
Author Response
The manuscript represents an interesting attempt to understand the growth stages of Grifola frondosa via transcriptomic analysis that is gaining a lot of attention. The research consists of two parts, viz. the optimisation of culture conditions of the inoculum, and transcriptomic analysis of fruiting bodies at different developmental stages. Standard methodologies were used. Overall, the manuscript is easy to understand and contains important findings for the mushroom field.
Response: Thanks for the positive comments. We have revised the Ms. carefully according to the suggestions one by one.
Main comment:
Both parts were carried out reasonably well, however, the linkage between the first and second parts have not been well-explained. The transcriptomic analysis can stand alone without the data on optimisation of media condition on inoculum. The justification for using liquid inoculum must be explained. Did the authors expect different results if a typical grain-based inoculum was used? I don't think so but I might be wrong. Please explain your rationale. Grain spawn is also more widely used in the industry as it is less susceptible to contamination.
I would suggest to remove the first part from this manuscript.
Response: Thanks for your suggestion. The industrial cultivation of G. frondosa (maitake mushroom) has been rapidly developing. In this industrialized production system, liquid inoculum plays a crucial role due to its short production cycle, uniform mycelial age, and high compatibility with mechanized processes (lines 55-60). Currently, most cultivated G. frondosa strains exhibit gray or dark-gray coloration (line 43). However, we successfully isolated an albino strain during cultivation through tissue separation, obtaining a white-pigmented G. frondosa variant (lines 103-104). The industrial cultivation parameters (substrate packaging, fruiting induction, environmental controls) of white strain Gr0001+3 have been studied (Huang et al., 2018). While the liquid culture requirements and developmental biology of white-pigmented strains remain unexplored. This discovery prompted our investigation into optimal liquid culture conditions for this white strain. And we systematically optimized liquid culture conditions optimization including formula and culture conditions of liquid spawn medium, taking the mycelial biomass as the main index, combining the results of pellet diameter, pellet density, and germination time which was the most comprehensive evaluation methodology.
Furthermore, given the limited research on fruiting body development mechanisms in G. frondosa (lines 96-102), we cultivated the white strain using our newly optimized liquid culture protocol. We systematically collected samples at four distinct developmental stages of fruiting body formation for RNA-seq analysis. In this study, combining strain enhancement with developmental biology insights to advance industrialized production of premium-quality maitake mushrooms (lines 118-119)-two inextricably linked dimensions essential for optimizing industrial G. frondosa production systems.
Other comments:
- The reason for choosing an albino strain for this study should be stated.
Response: Agreed. It has been added in lines 103-109.
- Any molecular data to validate the species used? Please include in the supplementary info.
Response: Thanks for your suggestion. It has been added at line 103-109. This strain Gr0001+3 has been achieved before and its industrial cultivation parameters (substrate packaging, fruiting induction, environmental controls) have been studied.
- Please describe the biological replicates used, are these different bags from the same cultivation batch or different batch?
Response: Thanks. Added at line 166.
- In the discussion, the authors may consider emphasizing the key findings of this study and how they contribute to bridging existing knowledge gaps in this area.
Response: Thanks. We have improved the discussion at lines 434-439, 454-455, 463-471, 475-486.
Round 2
Reviewer 1 Report
Comments and Suggestions for Authorsaccept
Author Response
The Comments and Suggestions for Authors: accept. And the English could be improved to more clearly express the research.
Response: We appreciate the acceptance of our Ms. for publication. The Ms. has undergone professional English language editing through MDPI Author Services to ensure clarity and accuracy.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have provided satisfactory responses to the reviewer’s queries and have amended the manuscript where necessary. The revised version is now much clearer.
However, there remains one section that still requires the authors’ attention.
Figure 8: Please include a description explaining how to differentiate the key stages in mushroom development. The current representation, based solely on photographs, appears rather subjective. I suggest adding descriptions of the primordia or fruiting body at each stage, along with an approximate timeline.
Author Response
The authors have provided satisfactory responses to the reviewer’s queries and have amended the manuscript where necessary. The revised version is now much clearer.
Response: Thanks for the positive comments. We have revised the Ms. carefully according to the suggestions one by one.
However, there remains one section that still requires the authors’ attention.
Figure 8: Please include a description explaining how to differentiate the key stages in mushroom development. The current representation, based solely on photographs, appears rather subjective. I suggest adding descriptions of the primordia or fruiting body at each stage, along with an approximate timeline.
Response: Thanks for your suggestion. We have added the description of the primordia or fruiting body at each stage, along with an approximate timeline at lines 164-167, 293-297, 300-304.
