A Potassium Phosphite Solution as a Dual-Action Strategy Against Bean Anthracnose: Antifungal Activity and Defense Gene Priming

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 The manuscript enttled A Potassium Phosphite Solution as a Dual-Action Strategy Against Bean Anthracnose: Antifungal Activity and Defense Gene Priming described potassium phosphite solution could control the bean anthracnose. It is very intestering for control the bean disease in the future. I have some concerns for this manuscript:

(1) For the Introduction part, the author need to revise it and make it read comfortable. In other words, please change the order of the paragraph.

(2) The mechanism of the potassium phosphite solution for control the bean anthracnose need to perform more experiments.  In the current version, the author only vertify the solution display the strong inhbition to fungal growth and priming the defence gene expression. It is very simply. It author don't perform the filed experiment. We want to know more meachanims of the solution.

(3) The Discussion need to divide some part and add some title for this part.

 

Comments on the Quality of English Language

The English need to improve it.

Author Response

• Revisor 1

El manuscrito titulado "Una solución de fosfito de potasio como estrategia de doble acción contra la antracnosis del frijol: Actividad antifúngica y preparación de genes de defensa" describe que la solución de fosfito de potasio podría controlar la antracnosis del frijol. Resulta muy interesante para el control de esta enfermedad en el futuro. Tengo algunas inquietudes sobre este manuscrito:

• Comentario 1 : El autor debe revisar la introducción para que sea más fácil de leer. En otras palabras, cambie el orden de los párrafos.

Respuesta:   Se modificó la introducción y se reorganizaron los párrafos de la siguiente manera:

Phaseolus vulgaris es la leguminosa más cultivada para consumo directo en todo el mundo, ya que se considera una fuente de proteína dietética, particularmente en países en desarrollo [1]. Colletotrichum lindemuthianum (Sacc. & Magnus) LambScrib, el anamorfo de Glomerella cingulata , es el agente causal de la antracnosis en el frijol común ( P. vulgaris L.), una enfermedad devastadora que provoca la pérdida total del cultivo en condiciones favorables de alta humedad relativa (> 80%), lo que conlleva a graves pérdidas económicas [2,3]. C. lindemuthianum afecta a todos los órganos de la planta y los síntomas de la enfermedad incluyen lesiones necróticas en pecíolos, tallos, ramas y venas primarias y secundarias de las hojas. Aparecen cancros redondos hundidos en las vainas, lo que puede provocar malformación de las vainas, bajo número de semillas y muerte del tejido [4].

La demanda mundial de legumbres nutritivas, la adaptabilidad de los patógenos y el cambio climático hacen necesario controlar esta enfermedad a nivel mundial [3]. La estrategia más viable y rentable para el control de la antracnosis en frijol es el uso de cultivares resistentes a hongos. Sin embargo, debido a la alta variabilidad patogénica de C. lindemuthianum , esto es difícil de implementar [5]. Los fungicidas químicos fueron los primeros en controlar las enfermedades de las plantas; sin embargo, las especies de Colletotrichum muestran una pérdida de sensibilidad a los fungicidas, posiblemente debido al uso continuo [7]. Además, estos agentes químicos son tóxicos para el medio ambiente y la salud humana [7]. Entre las estrategias propuestas para el control de enfermedades, el priming es una técnica novedosa en la que la inducción de un estado fisiológico permite a una planta desplegar una respuesta de defensa más rápida y eficiente contra el estrés en comparación con una planta no preparada [8].

Recientemente, se han introducido productos derivados del fosfito como una alternativa para el manejo de enfermedades [9]. Estos productos se derivan de la neutralización del ácido fosforoso (H ₃ PO ₃ ) con una base, como el hidróxido de sodio, el hidróxido de potasio y el hidróxido de amonio. Una de las ventajas de los fosfitos es su doble acción. En altas concentraciones, actúan como un agente antifúngico al inhibir el crecimiento micelial fúngico y la germinación de las esporas fúngicas [10,7]. La exposición al fosfito en células fúngicas se ha relacionado con cambios en el metabolismo del fósforo y en compuestos que contienen este elemento, incluyendo la inhibición de reacciones cruciales de fosforilación en hongos [9,11]. Además, estos compuestos se han vinculado a alteraciones en la expresión génica fúngica asociada con la producción de proteínas de la pared celular y del citoesqueleto [12].

Por el contrario, los fosfitos en bajas concentraciones actúan como fertilizantes y/o bioestimulantes, lo que aumenta la absorción y asimilación de nutrientes, mejora la calidad de los productos e induce respuestas de defensa ante estreses bióticos y abióticos [13,7]. La aplicación de fosfitos induce diversos mecanismos de defensa de las plantas, incluso en ausencia de patógenos [10,9,11], y aumenta la actividad de señalización de la resistencia sistémica adquirida (SAR) [7].

Por lo tanto, el estudio de la actividad antifúngica de un producto derivado del fosfito de potasio (H ₃ PO ₃ ) sobre C. lindemunthianum y su efecto como inductor de resistencia a enfermedades en plántulas de frijol podría contribuir a la comprensión del uso de este producto en el manejo de la antracnosis.

 

• Comentario 2 : El mecanismo de la solución de fosfito de potasio para controlar la antracnosis del frijol requiere más experimentos. En la versión actual, el autor solo verifica que la solución muestra una fuerte inhibición del crecimiento fúngico y estimula la expresión de genes de defensa. Es muy simple. El autor no realizó el experimento de campo. Queremos conocer más mecanismos de la solución.

Respuesta : El trabajo demostró un efecto antifúngico evaluado in vitro , inhibiendo el crecimiento, el porcentaje de esporulación y el desarrollo de sus hifas. Sin embargo, es el primer trabajo que demuestra el efecto del fosfito in vitro y la inducción de defensa contra Colletotrichum lindemuthianum , expresando algunos genes candidatos de defensa en plántulas tratadas e infectadas con el patógeno.

Es importante señalar que todos los estudios sobre el nivel de expresión o inducción de defensas en plantas infectadas tratadas con soluciones a base de fosfito se han centrado principalmente en patógenos como los oomicetos, y ninguno se ha centrado en el nivel de expresión de genes de defensa en plantas de frijol infectadas con C. lindemuthianum.

Como perspectiva, y para dar continuidad, se evaluará el efecto Phi en plantas de frijol en diferentes etapas fisiológicas y productivas mediante un enfoque transcriptómico diferencial. Sin embargo, este no fue el objetivo de nuestro trabajo actual. El enfoque transcriptómico mostrará las diferencias entre plantas tratadas y no tratadas de forma más completa. De esta manera, validaremos la expresión de algunos genes mediante qRT-PCR y confirmaremos si los expresados ​​en este trabajo presentan el mismo comportamiento.

• Comentario 3 La discusión necesita dividirse en alguna parte y agregar algún título para esta parte.

Respuesta : Seguimos la recomendación y en el manuscrito podrás encontrar los títulos de cada sección.

Línea 262: Sección 4.1. Efecto antifúngico de la solución de fosfito de potasio sobre C. lindemuthianum

Línea 286: Sección 4.2. Reducción de la antracnosis del frijol por efecto de la solución de fosfito de potasio.

Línea 323: Sección 4.3. Inducción de la expresión de genes de defensa mediante solución de fosfito de potasio.

Reviewer 2 Report

Comments and Suggestions for Authors

Title. A Potassium Phosphite Solution as a Dual-Action Strategy Against Bean Anthracnose: Antifungal Activity and Defense Gene Priming

Manuscript ID: horticulturae-3483107

This study investigates the antifungal activity of a potassium phosphite solution against the bean anthracnose pathogen, Colletotrichum lindemuthianum. Results indicated that the solution effectively reduced mycelial growth and germination rates of the fungus. Additionally, it demonstrated a priming effect in Sutagao bean plants, leading to decreased disease severity and increased expression of defense-related genes (PR1, PR3, PR4, and POD). The findings suggest that potassium phosphite can be an effective component in managing bean anthracnose. by viewing the article at this stage, I recommend a Minor revision

Comments and suggestions.

In the abstract, clarify whether the reduction in mycelial growth (42%) and germination (48%) is statistically significant.

The phrase "fungicidal effect" should be revised to "antifungal activity" unless actual fungicide-like properties are demonstrated.

The introduction mentions phosphates as fungicides and defense inducers but does not explain their mode of action well. Adding a brief explanation would strengthen this section.

The transition between fungicidal activity and priming could be smoother. Consider introducing the concept of "dual function" earlier in the introduction.

 The manuscript mentions Line 100 that the conidial suspension was adjusted to 1 × 10⁷ conidia mL⁻¹. Was this based on prior optimization? If so, provide a reference.

The reference genes should be validated for stability across treatments. Are technical replicates used for qPCR? If so, how many?

Figure 1 Consider providing a higher-resolution image.

Gene expression analysis PR1 expression decreases at 72 hpi. Does this suggest transient induction? Consider discussing whether the increased PR gene expression correlates with observed disease reduction.

Line 274. The discussion compares this study’s 17% disease reduction with others that reported 56–81%. However, it does not address why this difference occurred. Was it due to cultivar differences, application timing, or pathogen variability?

The discussion should expand on how PR gene activation contributes to resistance. Are these genes involved in SAR (systemic acquired resistance)?

Make conclusions more specific instead of stating that phosphates can be included in anthracnose management, specify their potential as an alternative or complementary strategy.

Some citations e.g 16, 17, 24, could be replaced with more recent studies to enhance credibility.

Author Response

Reviewer 2

This study investigates the antifungal activity of a potassium phosphite solution against the bean anthracnose pathogen, Colletotrichum lindemuthianum. Results indicated that the solution effectively reduced mycelial growth and germination rates of the fungus. Additionally, it demonstrated a priming effect in Sutagao bean plants, leading to decreased disease severity and increased expression of defense-related genes (PR1, PR3, PR4, and POD). The findings suggest that potassium phosphite can be an effective component in managing bean anthracnose. by viewing the article at this stage, I recommend a Minor revision

• Comment 1: In the abstract, clarify whether the reduction in mycelial growth (42%) and germination (48%) is statistically significant.

Response: In the abstract, line 19 to 22 it was modified: The results showed a significant statistical reduction in the antifungal effect of potassium phosphite solution on C. lindemuthianum, reducing mycelial growth by 42% and germination by 48% at a dose of 5 mL L^-1.

 

• Comment 2: The phrase "fungicidal effect" should be revised to "antifungal activity" unless actual fungicide-like properties are demonstrated.

Response:

In the abstract, lines 19 to 22: The results showed a significant statistical reduction in the antifungal effect of potassium phosphite solution on C. lindemuthianum, reducing mycelial growth by 42% and germination by 48% at a dose of 5 mL L^-1.

In the Introduction, lines 65 to 68: Therefore, the study of the antifungal activity of a product derived from potassium phosphite (H₃PO₃) on C. lindemunthianum and its effect as an inducer of disease resistance in bean seedlings could contribute to the understanding of the use of this product in the management of anthracnose.

In the Discussion, page 9, lines 263 to 265: Various studies have demonstrated the efficacy of phosphite base solutions similar to the product evaluated in this study (28% P2O5, and 26% K2O) in controlling anthracnose caused by Colletotrichum species, as well as their antifungal activity in in vitro evaluations.

In the Conclusions, lines 394 to 395: The potassium phosphite solution (P₂O₅ 28% and K₂O 26%) had direct antifungal activity against C. lindemuthianum. It reduced mycelial growth by 42% and germination by 48% at a dose of 5 mL L^-1.

 

• Comment 3: The introduction mentions phosphates as fungicides and defense inducers but does not explain their mode of action well. Adding a brief explanation would strengthen this section.

Response: Lines 50-64

Recently, phosphite-derived products have been introduced as an alternative to disease management [9]. These products are derived from the neutralization of phosphorous acid (H₃PO₃) with a base, such as sodium hydroxide, potassium hydroxide, and ammonium hydroxide. One of the advantages of phosphites is their dual action. In high concentrations, they act as an antifungal agent by inhibiting fungal mycelial growth and germination of fungal spores [10,7]. Phosphite exposure in fungal cells has been related to changes in phosphorus metabolism and in compounds containing this element, including the inhibition of crucial phosphorylation reactions in fungi [9,11]. Additionally, these compounds have been linked to alterations in fungal gene expression associated with the production of cell wall and cytoskeleton proteins [12].

In contrast, phosphites in low concentrations act as fertilizers and/or biostimulants, which increase the absorption and assimilation of nutrients, improve the quality of products and induce defense response to biotic and abiotic stresses [13,7]. The application of phosphites induces various plant defense mechanisms, even in the absence of pathogens [10,9,11], and increases systemic acquired resistance (SAR) signaling activities [7].

• Comment 4: The transition between fungicidal activity and priming could be smoother. Consider introducing the concept of "dual function" earlier in the introduction.

Response: In the introduction, the dual function of phosphites was introduced.

Lines 50-64

Recently, phosphite-derived products have been introduced as an alternative to disease management [9]. These products are derived from the neutralization of phosphorous acid (H₃PO₃) with a base, such as sodium hydroxide, potassium hydroxide, and ammonium hydroxide. One of the advantages of phosphites is their dual action. In high concentrations, they act as an antifungal agent by inhibiting fungal mycelial growth and germination of fungal spores [10,7]. Phosphite exposure in fungal cells has been related to changes in phosphorus metabolism and in compounds containing this element, including the inhibition of crucial phosphorylation reactions in fungi [9,11]. Additionally, these compounds have been linked to alterations in fungal gene expression associated with the production of cell wall and cytoskeleton proteins [12].

In contrast, phosphites in low concentrations act as fertilizers and/or biostimulants, which increase the absorption and assimilation of nutrients, improve the quality of products and induce defense response to biotic and abiotic stresses [13,7]. The application of phosphites induces various plant defense mechanisms, even in the absence of pathogens [10,9,11], and increases systemic acquired resistance (SAR) signaling activities [7].

 

• Comment 5: The manuscript mentions Line 100 that the conidial suspension was adjusted to 1 × 10⁷ conidia mL⁻¹. Was this based on prior optimization? If so, provide a reference.

Response: In the line 11, the reference [17] was added. This article used the same C. lindemuthianum inoculum concentration to infect bean seedlings.

Romero, G., González, S., Royero, W., & González, A. (2024). Morphological and transcriptional analysis of Colletotrichum lindemuthianum race 7 during early stages of infection in common bean. Genetics and molecular biology, 47(1), e20220263. https://doi.org/10.1590/1678-4685-GMB-2022-0263

 

• Comment 6: The reference genes should be validated for stability across treatments. Are technical replicates used for qPCR? If so, how many?

Response: In the first paragraph of section 2.5 Effect of potassium phosphite solution on defense genes expression we added the information:

Line 139 to 142: To determine changes on defense gene expression by application of potassium phosphite in all treatments, the RNA was extracted from three leaves of bean seedlings at 0, 24, 48, 72, and 96 hours post-inoculation (hpi) (three biological replicates) using a CTAB-based protocol with LiCl.

• At the end of the section 2.5, we complete the information:

Line 165 to 167: For the differential expression analysis of the defense genes, qPCR was performed using the relative quantification method with three cDNA replicates for each treatment at 24, 48, 72, and 96 hpi (three technical replicates).

 

• Comment 7: Figure 1 Consider providing a higher-resolution image.

Response: The size and resolution of the image were modified.

• Comment 8: Gene expression analysis PR1 expression decreases at 72 hpi. Does this suggest transient induction? Consider discussing whether the increased PR gene expression correlates with observed disease reduction.

Response: Lines 352-362: The PR1 gene in bean plants infected with C. lindemuthianum has been identified as a key marker that is expressed during the early stages of infection in both susceptible and resistant hosts. However, its expression is stronger and occurs earlier in resistant plants, reaching a peak expression up to 72 hours post-inoculation (hpi) [32]. In this study, transient PR1 expression was detected in susceptible Sutagao bean plants treated with phosphite. This transient response correlated with severity scores, as potassium phosphite-treated plants exhibited symptoms, albeit less severe than untreated plants, with a 17% reduction in severity. This suggests that the resistance induced was neither robust nor long lasting. A higher dose or multiple applications (e.g. at seed, transplant or other vegetative stage) may be required to enhance the priming effect to achieve a more sustained and effective defense response.

• Comment 9: Line 274. The discussion compares this study’s 17% disease reduction with others that reported 56–81%. However, it does not address why this difference occurred. Was it due to cultivar differences, application timing, or pathogen variability?

Response: In the manuscript, Lines 289-296, we explain this: The differences could be related to the bean material used and the concentration of the pathogen inoculum. The genetic and variety of bean plants used in this study (Sutagao cultivar, derived from the cross between Cabrera and G2333) differs from Perola [13], BRS Majestoso [25], and IPR Tangará [26], characteristics that determine resistance or susceptibility to the pathogen depending on its race. In addition, the inoculum concentration of C. lindemuthianum used to infect the plants in this study (1×10⁷ conidia mL^-1) was higher than 7x10⁵ conidia mL^-1 [13,25] and 1x10⁶ conidia mL-1 [26] used by other authors.

• Comment 10: The discussion should expand on how PR gene activation contributes to resistance. Are these genes involved in SAR (systemic acquired resistance)?

Response: Lines 329-337: In a transcriptomic analysis carried out at 6 hpi in bean seedlings susceptible and resistant to C. lindemuthianum and subjected to a previous stimulus with AS (salicylic acid) and MeJA (methyl jasmonate), 463 genes were differentially expressed [29]. Of these, 19 genes were up-regulated in the resistant genotype in response to stimulation with AS and 17 with MeJA. Ten of these were R genes, 11 genes encoding protein kinases, and ten genes belonging to the transcription factor family. This provides evidence for very early activation of signaling by a defense inducer, which promotes transcription and expression of defense responses in plants [29]. However, no other time points after infection were assessed to determine the duration and intensity of expression of these genes.

Response: In lines 387-3890 we completed the discussion about POD: At the transcriptomic level, genes encoding oxidoreductase activity were confirmed to be expressed in bean seedlings resistant to C. lindemuthianum [29]

 

• Comment 11: Make conclusions more specificinstead of stating that phosphates can be included in anthracnose management, specify their potential as an alternative or complementary strategy.

Response: The conclusions were modified as follows:

Lines 394-402: The potassium phosphite solution (P₂O₅ 28% and K₂O 26%) had direct antifungal activity against C. lindemuthianum. It reduced mycelial growth by 42% and germination by 48% at a dose of 5 mL L^-1. The effect of this compound on Sutagao bean plants stimulated the expression of PR1, PR3, PR4, and POD defense genes, but this increased when the plants were infected with C. lindemuthianum. The highest expression levels were observed for the chitinase genes (PR3 and PR4), which were maintained until 96 hpi, followed by the POD gene. These results demonstrate the dual activity of potassium phosphite as antifungal and priming inducer against bean anthracnose and its potential for inclusion in management programs.

• Comment 12: Some citations e.g 16, 17, 24, could be replaced with more recent studies to enhance credibility.

Response

Reference 16 is now the 19. However, it is important to maintain because it is the original standard system for evaluating anthracnose on bean.

Reference 17: refers to the original AUDPC equation. Reference 17 is now the 20.

Reference 24: It was deleted. There are more recent ones that support the idea

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

       The manuscript entitled "A Potassium Phosphite Solution as a Dual-Action Strategy Against Bean Anthracnose: Antifungal Activity and Defense Gene Priming" (Manuscript ID: horticulturae-3483107) has been reviewed. This paper has been proved the activity of potassium phosphite against Colletotrichum lindemuthianum, thereby providing a new strategy for control bean anthracnose. Additionally, the activity actions were also sought to reveal in this work. However, the experimental data were no enough to support the conclusions. Additional work should be done before publication. 

The problems were as follows:

1. In the section 3.1, the macroscopic and microscopic characteristics of C. lindemuthianum were present between the cultures grown with and without potassium phosphite solution. The results exhibited in Fig. 1. However, limited information was available. For better understanding of the effect of potassium phosphite on lindemuthianum, SEM analysis might to be necessary for the observation of hyphae and conidia ultrastructure.
2. In the section of 3.3, the expression levels of defense genes on bean plants were determined using qPCR method. Additionally, the activities of the defense enzymes related to the defense genes should be measured to verify the qPCR data.
3. In the Line 63, PDA should be given the full name of the medium first mention.
4. In the Line 98, The tumblers were then incubated in darkness at 24 °C for 21 days. In the Line 74 and 87, lindemuthianum was incubated at 25°C. The culture temperatures were different, why?

Author Response

• Reviewer 3

The manuscript entitled "A Potassium Phosphite Solution as a Dual-Action Strategy Against Bean Anthracnose: Antifungal Activity and Defense Gene Priming" (Manuscript ID: horticulturae-3483107) has been reviewed. This paper has been proved the activity of potassium phosphite against Colletotrichum lindemuthianum, thereby providing a new strategy for control bean anthracnose. Additionally, the activity actions were also sought to reveal in this work. However, the experimental data were no enough to support the conclusions. Additional work should be done before publication.

The problems were as follows:

• Comment 1: In the section 3.1, the macroscopic and microscopic characteristics of lindemuthianum were present between the cultures grown with and without potassium phosphite solution. The results exhibited in Fig. 1. However, limited information was available. For better understanding of the effect of potassium phosphite on lindemuthianum, SEM analysis might to be necessary for the observation of hyphae and conidia ultrastructure.

Response: There is no doubt that an SEM analysis would give us more information, but at this moment, we cannot access this type of microscopy. However, light microscopy was able to demonstrate the effect of Phosphites on the hyphal morphology.

• Comment 2: In the section of 3.3, the expression levels of defense genes on bean plants were determined using qPCR method. Additionally, the activities of the defense enzymes related to the defense genes should be measured to verify the qPCR data.

Response: It is important to evaluate the enzymatic activities of some defense enzymes detected by qPCR. However, this was not considered in this work.

• Comment 3: In the Line 73, PDA should be given the full name of the medium first mention.

Response: In line 72 PDA (Potato Dextrose Agar) was added after PDA

• Comment 4: In the Line 98, The tumblers were then incubated in darkness at 24 °C for 21 days. In the Line 74 and 87, lindemuthianum was incubated at 25°C. The culture temperatures were different, why?

Response: This is a mistake. The incubation temperature was 25°C. In line 97 it was corrected.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript "A Potassium Phosphite Solution as a Dual-Action Strategy Against Bean Anthracnose: Antifungal Activity and Defense Gene Priming" shows very interesting findings regarding the antifungal ability and activation of defense genes in the plant by Potassium Phosphite. The manuscript shows scientific quality and it is appreciated that it is concise and clear. Minor comments/suggestions were made in the PDF version of the attached manuscript.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The quality of English language is satisfactory, however, it can be substantially improved.

Author Response

• Reviewer 4

The manuscript "A Potassium Phosphite Solution as a Dual-Action Strategy Against Bean Anthracnose: Antifungal Activity and Defense Gene Priming" shows very interesting findings regarding the antifungal ability and activation of defense genes in the plant by Potassium Phosphite. The manuscript shows scientific quality and it is appreciated that it is concise and clear. Minor comments/suggestions were made in the PDF version of the attached manuscript.

peer-review-44308153.v1.pdf

Comments on the Quality of English Language

The quality of English language is satisfactory, however, it can be substantially improved.

Response: We will review the grammar style after changes will be accepted.

 

• Comment 1: In Line 93. Before this date, no changes were observed?

Response: Morphological evaluation was carried out 18 days after sowing, as this was when the C. lindemuthianum isolate obtained maximum growth in the medium without supplementation with potassium phosphite solution.

 

• Comment 2: In line 117: How did you make sure to spray 1 mL?

Response: Calibration of the airbrush nozzle and the volume to be used per plant was carried out before applying the phosphite solution to the plant leaves.

 

• Comment 3: Before the results section, I suggest integrating the statistical analysis section.

Response: The methodology is separated by sections and the respective statistical analysis is explained at the end of each one. We considered that by carrying out a single statistical analysis section, it would be necessary to go back to each experiment to understand it.

 

• Comment 4: replace inculation with inoculation in the figure 3.

Response: The replace was made.