Exploring the Roles of Plant Growth-Promoting Rhizobacteria (PGPR) and Alternate Wetting and Drying (AWD) in Sustainable Rice Cultivation
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsAttached
Comments for author File: 
Comments.pdf
There is little logical progression between sections. Please improve it.
Author Response
The manuscript presents an important review on the potential synergy between PGPR and AWD irrigation in sustainable rice cultivation. However, the manuscript suffers from several major flaws that compromise its scientific quality.
Thank you for your valuable feedback. Please note that some sections highlighted above will be slightly different from those found in the revised manuscript. Regarding the grammatical errors, please be informed that the manuscript will be submitted to the MDPI English Editing service for language improvement once it has been approved to proceed toward publication.
My main concerns are as follows:
- Despite the title, the PGPR-AWD synergy is underexplored. Much of the text reviews PGPR and AWD separately, rather than analyzing their interactive effects. The supposed “synergy” between PGPR and AWD is not supported by in-depth analysis or systematic evidence. This is the main problem of this manuscript.
Noted. Based on the comment, it is considered that the title is revised. See Lines 2-4.
Exploring the Roles of Plant Growth-Promoting Rhizobacteria (PGPR) and Alternate Wetting-and-Drying (AWD) in Sustainable Rice Cultivation
- The authors propose the concept of a “System of Probiotics in Rice Intensification (SPRI)” without defining it rigorously or justifying its uniqueness. No operational A framework or case study is provided to support this term.
Noted. Please note the changes made and the explanation. See Lines 481-494.
The synergistic benefits observed from combining PGPR with AWD irrigation practices in rice cultivation have paved the way for a novel conceptual framework aimed at transforming sustainable rice production. Building upon the foundational principles of the SRI, which emphasize reduced water use, improved root health, and soil microbial activity, we introduce the System of Probiotics in Rice Intensification (SPRI). The core aim of SPRI is to create a synergistic ecosystem within the rice paddy that enhances nutrient cycling, improves plant health and resilience, optimizes water use, and ultimately leads to increased and sustainable rice yields while minimizing negative environmental impacts.
This section introduces the SPRI concept, a comprehensive exploration of its theoretical underpinnings, operational framework, and potential applications, along with supporting justification and case studies, is beyond the scope of the current manuscript. This forthcoming publication will provide a detailed definition, outline an operational framework, and present potential case studies to further elucidate and validate the SPRI concept.
- The manuscript lacks a discussion of contradictions, uncertainties, or challenges in PGPR–AWD integration (e.g., oxygen sensitivity of certain microbes, microbial washout during re-flooding, strain compatibility with field conditions).
Please refer to lines 261-267. A further expansion is on lines 417-436.
Oxygen sensitivity and Microbial washout during re-flooding sections
However, the dynamic shifts in oxygen and moisture regimes under AWD can pose physiological stress to some microbial communities, strictly those of the anaerobes or highly oxygen-sensitive PGPR strains. These abrupt transitions may impair microbial functions or reduce their population densities during re-flooding events, potentially limiting their full benefit to plant performance
Oxygen sensitivity, re-flooding, and Strain compatibility with field conditions
Despite limited direct studies on PGPR inoculation under AWD, existing literature suggests promising interactive effects. However, it is critical to acknowledge key implementation challenges. The strain compatibility with fluctuating soil environments remains a major barrier. Many commercial PGPR strains perform well under controlled environments but show inconsistent efficacy in open field conditions due to variations in soil physicochemical parameters, native microbial competition, or periodic soil drying and rewetting [116]. Additionally, microbial washout during re-flooding events can displace rhizobacteria from the root zone, particularly in soils with low organic matter content or poor structure [69]. This instability may reduce the long-term persistence and effectiveness of introduced beneficial strains.
Furthermore, however, maintaining stable microbial populations remains a challenge under AWD. The frequent changes under the AWD condition can cause psychological stress to some microbial communities, particularly the anaerobic strains [68]. Oxygen fluctuation during AWD cycles can alter microbial redox environments, disrupting enzymatic activity or metabolic pathways in sensitive microbial taxa [117, 118]. Certain nitrogen-fixing bacteria, such as those of the Azospirillum spp., may experience inhibited function during aerobic drying phases [119], while obligate aerobes may decline rapidly during re-flooding [69]. These contradictory effects emphasize the need for carefully selecting robust, field-compatible strains capable of enduring fluctuating water regimes during rice cultivation.
- When reporting on the plant growth-promoting effects of plant growth-promoting bacteria, researchers often tend to report the results of experiments that show an effect and not report the results of experiments that do not have an effect due to survivorship bias. This point should also be included in the Discussion.
The statement has been incorporated into the write-up; see Lines 342-344.
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsLine88-91. Define the principles behind AWD in terms of numbers such as ranges or percentage that clearly shows the advantage of the approach. As this is a review article, numbers are crucial to support the advantage laid out in this manuscript.
Line 144. In figure 1, please include a scale bar as reference to 1 cm. This will provide a better visual cue for analyzing the ratio and arbitrary sizes of the two plant systems.
Line 150-151. Include viruses and algae as part of the group of microorganisms here, specially for aquatic-land ecosystem interface like rice paddy fields.
Line 163. Please include the endophytic fungal group as well in this section. They are critical in the microbial ecosystem.
Line 207-and this entire section. The common viable and culturable species of microorganisms were the classical focus of microbiologists, but it is also needed to mention a huge unculturable microbes (dark matter microbiology), that is still poorly understood. Since Figure 2 presented these group members, this must be incorporated in this review article.
Figure 2. The data presented here was obtained from which publication? This must be clearly indicated in the figure title.
Line 225. Please include the effect of antimicrobial compounds and other signaling molecules that affects the survival, competition, growth of PGRP and the host plant.
Line 232-233. Please provide direct object of the clause “pathogenic attack”. The grammar and syntax is broken
Line 250. Glucose in its natural form, do not contain any phosphorus atom. Please clarify this erroneous statement.
Line 251. The vital processes doesn’t “contain” phosphorus. Please revise into the correct context.
Line 264 – 269. Please provide bonafide species bacteria that positively affect (measured and published) towards phytohormones, exopolysaccharide (exopolymeric molecules), and halotolerance (osmoregulators) effect on plants/rice. Do this for all relevant sections just like how explicit examples were presented in section 3.3 of the manuscript (starting at line 309).
Figure 3. The abbreviated words need to be mentioned with its full meaning, specially those that are not mentioned in the text of the manuscript. E.g. SOD, CAT, POX, etc. This can be accomplished by defining these abbreviations in the figure title.
Author Response
Thank you for your valuable feedback. Please note that some sections highlighted below will be slightly different from those found in the revised manuscript. Regarding the grammatical errors, please be informed that the manuscript will be submitted to the MDPI English Editing service for language improvement once it has been approved to proceed toward publication.
1. Line88-91. Define the principles behind AWD in terms of numbers such as ranges or percentage that clearly shows the advantage of the approach. As this is a review article, numbers are crucial to support the advantage laid out in this manuscript.
Thank you and noted. I have revised the highlighted paragraph and included actual research findings from an article titled Alternate wetting and drying: A water-saving and eco-friendly rice production system by Ishfaq et al., (2020). The revised paragraph is now on Lines 88 to 95.
The key principles of Alternate Wetting and Drying (AWD) involve a shift from continuous flooding to controlled cycles of flooding and drying. This practice offers significant advantages, primarily in reducing water consumption while maintaining or even enhancing rice yields. For instance, Ishfaq et al. [16]. demonstrated the substantial water savings associated with AWD, noting that conventional rice production typically requires 3 to 5 times more water than other cereal crops. Their findings further revealed that implementing AWD can reduce water usage by a significant range of 25% to 70% compared to traditional continuous flooding, all without compromising rice yields.
2. Line 144. In figure 1, please include a scale bar as reference to 1 cm. This will provide a better visual cue for analyzing the ratio and arbitrary sizes of the two plant systems.
Thank you. I have revised the image while at the same time adding in the scale value. See line 147.
3. Line 150-151. Include viruses and algae as part of the group of microorganisms here, specially for aquatic-land ecosystem interface like rice paddy fields.
I have included the algae and viruses in the group of soil microorganisms and briefly expanded on their role in soil ecology and agriculture. See Lines 192-216.
Algae are eukaryotic organisms, although some are prokaryotic (cyanobacteria, often referred to as blue-green algae). They are photosynthetic, autotrophic organisms that can be either unicellular or multicellular and are primarily found in moist soil surfaces or waterlogged environments. They can improve soil structure through the secretion of extracellular polysaccharides that bind soil particles. Their presence enhances soil aeration and water retention, which can benefit crop growth, especially in degraded soils. The presence and type of algal communities can indicate the ecological status of a soil or water body. However, under certain conditions, excessive algal growth in aquatic systems can lead to eutrophication, depleting oxygen levels, and harming other aquatic life. Planting seeds in such low gas exchange conditions can hinder seed emergence.
Viruses are distinct from fungi, nematodes, protozoa, microarthropods, and algae as they are not considered cellular organisms. They are acellular entities that require a living host to replicate. Viruses in agricultural systems can be both influential and detrimental. In soil ecosystems, they primarily infect bacteria (bacteriophages), fungi, and protozoa, thus playing a crucial role in regulating microbial populations and contributing to nutrient cycling through microbial turnover. By lysing their microbial hosts, viruses release cellular contents into the soil, which can be mineralized and made available for plant uptake. It is often referred to as the "viral shunt," as it influences the structure and function of microbial communities and soil biogeochemical processes. However, viruses are also significant plant pathogens, capable of infecting a wide range of crops, causing diseases that can severely reduce plant growth and yield.
3. Line 163. Please include the endophytic fungal group as well in this section. They are critical in the microbial ecosystem.
I have inserted a description of endophytic fungi. See Lines 165-168.
Endophytic fungi reside within plant tissues without causing apparent disease and can confer various benefits to their host, including enhanced tolerance to biotic and abiotic stresses, improved nutrient uptake, and even direct promotion of plant growth.
4. Line 207-and this entire section. The common viable and culturable species of microorganisms were the classical focus of microbiologists, but it is also needed to mention a huge unculturable microbes (dark matter microbiology), that is still poorly understood. Since Figure 2 presented these group members, this must be incorporated in this review article.
Thank you. I have made mention of the uncultured microbes as suggested. See Lines 219-223.
While significant progress has been made in understanding the roles of culturable bacteria, it's important to acknowledge the vast and largely unexplored realm of unculturable microbes, often referred to as 'dark matter microbiology.' This substantial portion of the bacterial community, which remains poorly understood due to limitations in current cultivation techniques, likely plays critical but yet-to-be-defined roles in soil ecosystems [1].
5. Line 225. Please include the effect of antimicrobial compounds and other signaling molecules that affect the survival, competition, and growth of PGRP and the host plant.
Thank you. I have included the effect of antimicrobial compounds and other signaling molecules that affect the survival, competition, and growth of PGRP and the host plant. See Lines 249-256.
These microorganisms play a critical role in the soil food web. They contribute to soil fertility by transforming nutrients, decomposing organic matter, and suppressing plant pathogens [52, 53]. Notably, Plant Growth-Promoting Rhizobacteria (PGPR) within this diverse community engage in complex interactions mediated by a variety of antimicrobial compounds and other signaling molecules. These compounds influence the survival, competition, and growth of both the PGPR themselves and the host plant. For instance, signaling molecules facilitate the establishment of beneficial symbiotic relationships, while antimicrobial compounds can suppress harmful pathogens, creating a more favorable environment for plant growth. Beyond direct effects, these microorganisms also promote plant growth, improving plant health and productivity [35]. They are essential components of soil health, influencing soil fertility, plant health, and crop productivity [12, 54]. Their presence in the soil ecosystem can significantly impact crop growth and development, making them an important factor in sustainable agriculture. The AWD cultivation method creates a conducive condition for diverse communities of microbes to proliferate and form complex interactions in the rhizosphere.
6. Line 232-233. Please provide direct object of the clause “pathogenic attack”. The grammar and syntax is broken
Thank you. I have revised the sentence accordingly. See Lines 274-275.
Additionally, these crops are highly susceptible to pests and to pathogenic attacks by fungi, bacteria, and viruses.
7. Line 250. Glucose in its natural form, do not contain any phosphorus atom. Please clarify this erroneous statement.
Thank you for the suggestion. I have revised the sentence to avoid any misconceptions. See lines 298-300.
The second most demanded plant nutrient is P. It is a fundamental component in adenosine 5'-triphosphate (ATP), phosphorylated sugars (intermediates in glucose metabolism), nucleic acids, and other vital biological processes [62].
8. Line 251. The vital processes doesn’t “contain” phosphorus. Please revise into the correct context.
Thank you for the suggestion. I have revised the sentence to avoid any misconceptions. 298-300.
9. Line 264 – 269. Please provide bonafide species bacteria that positively affect (measured and published) towards phytohormones, exopolysaccharide (exopolymeric molecules), and halotolerance (osmoregulators) effect on plants/rice. Do this for all relevant sections just like how explicit examples were presented in section 3.3 of the manuscript (starting at line 309).
Noted. I have provided examples of bacteria species that influence plant growth and survival. See lines 309-327.
Microbes can mitigate stress on crops through several pathways. For instance, Trichoderma spp. (primarily fungi) and Bacillus spp. suppress plant pathogens by producing antimicrobial compounds [40, 49]. Certain beneficial microbes can also infiltrate plant tissues, stimulating the deposition of lignin and other cell wall components [64], thereby enhancing the crop's structural integrity to withstand pest attacks and strong winds, preventing lodging and grain loss. Furthermore, specific bacterial species are well-documented for their positive effects on plant physiology. For example, Azospirillum brasilense is known to produce auxins and other growth-promoting substances that enhance root development and nutrient uptake [2, 3]. Similarly, certain Bacillus strains, such as B. subtilis, have been shown to produce cytokinins, further contributing to plant growth and development [4, 5]. Regarding water stress, the AWD practice encourages limited water use, and this can be complemented by the action of exopolysaccharide (EPS)-producing bacteria. For instance, B. amyloliquefaciens has been reported to produce significant amounts of EPS, which can improve soil water retention and enhance water availability to plant roots during dry periods [6]. In saline soils, halotolerant bacteria play a crucial role. Pseudomonas putida, for example, can produce osmoprotectants like proline and glycine betaine, which help rice plants maintain osmotic balance and tolerate ionic stress [7, 8]. These specific examples highlight the diverse mechanisms by which well-characterized bacterial species contribute to plant resilience under various stress conditions.
10. Figure 3. The abbreviated words need to be mentioned with its full meaning, specially those that are not mentioned in the text of the manuscript. E.g. SOD, CAT, POX, etc. This can be accomplished by defining these abbreviations in the figure title.
Noted, edited the caption by including the abbreviations. I also revised Figure 3. See lines 356-360.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe review is well-structured and clearly written, with a logical flow from introducing the issue to exploring PGPR in AWD systems and potential research directions. Writing is accessible and does a good job of synthesising complex scientific concepts.
Below are a few suggestions:
- Grammatically incomplete and unclear sentences in a few places.
- The potential drawbacks or limitations of using PGPR are not addressed.
- The text implies broad benefits from PGPR without acknowledging that their effectiveness may vary with soil type, climate, rice variety, and existing microbial communities.
- You use both "rice paddies" and "rice cultivation"—ensure consistency unless the distinction is deliberate.
Author Response
Thank you for your valuable feedback. Please note that some sections highlighted below will be slightly different from those found in the revised manuscript. Regarding the grammatical errors, please be informed that the manuscript will be submitted to the MDPI English Editing service for language improvement once it has been approved to proceed toward publication.
The review is well-structured and clearly written, with a logical flow from introducing the issue to exploring PGPR in AWD systems and potential research directions. Writing is accessible and does a good job of synthesising complex scientific concepts.
Below are a few suggestions:
1. Grammatically incomplete and unclear sentences in a few places.
As mentioned above, after reviewing, the manuscript will be processed through MDPI's English Editing Services to correct any grammatical errors.
2. The potential drawbacks or limitations of using PGPR are not addressed.
Noted. Please refer to lines 396-415.
Although PGPR offers numerous benefits in the field of agriculture and perhaps other industries, its application may have certain limitations and challenges that must not be overlooked. A major concern is the specificity of interaction between PGPR and host plants. As seen in leguminous crops, whereby they interact with only certain bacterial species, shows that different crops often require specific strains of PGPR. Notably, a strain that works well for one plant may not be effective for another. Furthermore, he performance of PGPR can be heavily influenced by environmental factors such as soil type, temperature, and moisture levels (Chaitanya Kumar Jha et al., 2011; Kannan et al., 2015; Reddy, 2014). There is also a risk that some PGPR, especially when applied excessively or inappropriately, could cause harm. Certain strains may produce toxic compounds or lead to plant infections if they penetrate plant tissues (Kannan et al., 2015). Beyond plant health, PGPR use can also raise ecological concerns. Introducing non-native bacterial strains into a new environment may disrupt existing microbial communities or interfere with natural soil nutrient cycling (Malacrinò et al., 2020). It is important to recognize that while PGPR can enhance nutrient bioavailability and support plant growth, they do not represent a complete substitution for conventional practices such as chemical fertilization. Synthetic fertilizers offer a more rapid and often more consistent supply of macronutrients and micronutrients, which the nutrient mobilization and cycling activities of PGPR may not always fully and immediately replicate [5]. Hence, opening options to integration practices that can lead to an economically and environmentally sustainable farming approach.
3. The text implies broad benefits from PGPR without acknowledging that their effectiveness may vary with soil type, climate, rice variety, and existing microbial communities.
Noted. I have updated the manuscript accordingly (refer to the input made above).
4. You use both "rice paddies" and "rice cultivation"—ensure consistency unless the distinction is deliberate. Noted. I have edited where necessary (line 369). We consider using both based on the context mentioned in the article.
Noted. I have edited where necessary (line 369). We consider using both based on the context mentioned in the article.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsI believe it can be accepted
Author Response
Thank you for your feedback and contribution towards improving the manuscript.
Reviewer 2 Report
Comments and Suggestions for AuthorsAccept in the current revised form. All comments and suggestions were addressed properly
Author Response
Thank you for your contributions towards helping us improve the manuscript.
