New Antifungal Compound, 6-Pentyl-α-Pyrone, against the Maize Late Wilt Pathogen, Magnaporthiopsis maydis

Round 1

Reviewer 1 Report

The manuscript is about the use of 6-Pentyl-α-Pyrone as an antifungal agent against the maize late wilt disease caused by Magnaporthiopsis maydis.

The manuscript has practical relevance with interesting applications, and it is well designed and written. However, there are some points, which require the authors attention.

1. I think this work contains some repeated experiments previously reported by the authors. I recommend focusing only on the newly added experiments (the antifungal potential of 6-pp) and avoiding repeated ones.

2. There is extensive use of self-citation by the authors. There are 19 self-citation references in this manuscript. It is recommended to contribute to the publication of more broadly and scientifically based references.

3. The length of the manuscript is very long and renders the study difficult to follow. There are some repeated parts in the introduction, methods, and references. I recommend shortening the over-whole manuscript to be easy to follow.

4. It is not clear why the authors used late cultivation of the maize cultivar (please provide the exact cultivation time). Was it in the authors intention from the beginning or what. This needs to be explained, as the late cultivation did not provide the optimal conditions for disease development. This will interfere with the obtained results, as the growth development of the maize plants was normal, and the disease symptoms were minor during the growth period.

5. The discussion is poor, and the authors should focus on the protective role of the tested treatments under field conditions. There is no mention of the 6-pp structure-activity relationship (SAR).

Author Response

Responses to Reviewer 1’s comments

We thank the reviewer for investing substantial efforts, which undoubtedly contribute to this manuscript. The remarks and suggestions improved this paper’s scientific soundness and accuracy. Your contribution is greatly appreciated.

The manuscript is about the use of 6-Pentyl-α-Pyrone as an antifungal agent against the maize late wilt disease caused by Magnaporthiopsis maydis. The manuscript has practical relevance with interesting applications, and it is well designed and written. However, there are some points, which require the authors' attention.

Reply: Thank you for the positive evaluation of our manuscript. All your remarks and suggestions were addressed carefully and thoroughly, as detailed below.

I think this work contains some repeated experiments previously reported by the authors. I recommend focusing only on the newly added experiments (the antifungal potential of 6-pp) and avoiding repeated ones.

Reply: The reviewer is correct. Some data regarding the application of Trichoderma asperellum (P1 strain) were collected during an extensive experiment that scanned biological control solutions in the field (Degani et al. J. Fungi 2021, 7, 444. https://doi.org/10.3390/jof7060444). Yet, these data were not afforded much attention in our previous report, and we believe it is essential to present them in the current context to get a better focus and clear picture that provides complete accumulated knowledge on the topic. It is significant to demonstrate that whereas the P1 strain has a limited ability to provide a bio-shield against the late wilt pathogen during a full growing season, its secreted metabolites, particularly the 6-PP active ingredient, could provide solid protection. We hope this explanation addresses your concern. If not, we could make any changes required.

There is extensive use of self-citation by the authors. There are 19 self-citation references in this manuscript. It is recommended to contribute to the publication of more broadly and scientifically based references.

Reply: The number of self-citations was reduced to 15, and 19 new references were added, so the overall reference number is now 84, and the self-citation ratio is 18% (well within the acceptable self-citation ratio according to the MDPI instructions). We believe that all remaining self-citations are necessary to present the most updated relevant information and to describe the methods properly.

The length of the manuscript is very long and renders the study difficult to follow. There are some repeated parts in the Introduction, Methods, and references. I recommend shortening the over-whole manuscript to be easy to follow.

Reply: We carefully and thoroughly overviewed the Introduction and the Materials and Methods sections. We eliminated any repetitive or unnecessary information and edited other parts so that the text would be as clear and focused as possible. Some basic information regarding late wilt disease and the Magnaporthiopsis maydis pathogen was left unchanged. Since the disease is considered to be exotic and unfamiliar in most parts of the world, it is essential to present this information to the manuscript’s readers so they could understand the importance of the work and the challenges we face.

It is not clear why the authors used late cultivation of the maize cultivar (please provide the exact cultivation time). Was it in the authors' intention from the beginning or what. This needs to be explained, as the late cultivation did not provide the optimal conditions for disease development. This will interfere with the obtained results, as the growth development of the maize plants was normal, and the disease symptoms were minor during the growth period.

Reply: Thank you for this important remark. Favorable conditions for the disease outbreak are always preferred. Still, conducting field experiments is a complex operation, depending on many factors (availability of the field, labor, budget, equipment, etc.), therefore they cannot always be performed under ideal conditions. Such practical reasons led to the late season 2020 experiment. Still, studying the pathogenesis in such a situation can provide vital information, as has happened here, and could contribute to the accumulative scientific knowledge of the disease.

We revised the relevant paragraph in the Materials and Methods section to better explain this (lines 214-218): “The 2020 experiment was conducted in autumn for practical reasons (mainly the availability of the field). Thus, due to the late season, the average meteorological parameters quantified during the experimental duration (relatively low temperature, radiation and evaporation, and high precipitation) were not optimal for the LWD burst.”

We also discuss this issue in the Discussion section (lines 560-566): “In our study, the field conditions were not optimal for LWD development, leading to weak dehydration symptoms appearing at the harvest. Indeed, this season was rainier and colder, and these conditions were probably the cause of the low yields and a minor outbreak of LWD. To support this, early sowing of corn in Egypt reduced LWD [75], while late summer planting reduced disease severity in India [27]. It is possible that the full potential of the T. asperellum-based biological control was not manifested under these conditions.”

The Discussion is poor, and the authors should focus on the protective role of the tested treatments under field conditions. There is no mention of the 6-pp structure-activity relationship (SAR).

Reply: We agree and have edited and improved the Discussion section according to your suggestions and remarks. As you can see, the section was reorganized, enriched, updated, and is now focused more on the results:

The following paragraphs were added:

• (lines 546-552): “In recent years, biopesticides have gained significant attention as an alternative to environmentally problematic chemical pesticides [54]. Understanding the roles of endo-phytic community assemblages and their impact on plant health would be a major contribution to sustainable integrated agriculture. Trichoderma species are used widely in agriculture to stimulate plant growth and control different pathogens affecting crops, serving as valuable tools for sustainable food production [74]. Still, the action mechanism of this bio-protective species is poorly understood.”

 

• (Lines 557-566): “Obvious differences exist between the growth room and the field conditions that may explain the results presented here. As a start, the field’s unstable conditions may have a significant impact on the results. In our study, the field conditions were not optimal for LWD development, leading to weak dehydration symptoms appearing at the harvest. Indeed, this season was rainier and colder, and these conditions were probably the cause of low yields and a minor outbreak of LWD. To support this, early sowing of corn in Egypt reduced LWD [75], while late summer planting reduced disease severity in India [27]. It is possible that the full potential of the T. asperellum-based biological control was not manifested under these conditions.”

 

• (Lines 567-578): “To support this line of thinking, inspecting similar research results published recently by Estévez-Geffriaud (2021) [76] provides evidence that T. asperellum (strain T34 applied as seed treatment) positively affected maize productivity and health under late wilt disease stress. Regardless of the water regime, T34 improved kernel P and C content, kernel dry weight and number. Under drought, this Trichoderma treatment improved leaf relative water content, water use efficiency, PSII maximum efficiency, and photosynthesis. Moreover, T34 reduced LWD incidence by up to 13% and enhanced the yield per plant in LWD moderate tolerant and resistant maize genotypes. Still, no yield improvement was measured in susceptible lines [76]. It may be inferred from Estévez-Geffriaud and other studies (for example [33]) that the Trichoderma species, the specific strain, the plant’s LWD susceptibility degree, and environmental conditions could all affect the success of the biological treatment.”

 

• (Lines 579-585): “It should also be considered that T. asperellum protection is mainly affected during the sprouting stage, which is regarded as the pathogen entry and establishment phase [21,23]. Later in the season, this initial protection may lose effectiveness if the pathogen finds a way to establish itself in the plant’s vascular system and disrupt its development. If such a scenario is the case, integrated biological and chemical protection may assist in overcoming the pathogen’s establishment and provide us with the benefit of low fungicides usage with a live protective barrier [54].”

 

• (Lines 597-605): “One of the advances in using Trichoderma-based protection against LWD is that these species likely maintain a symbiotic lifestyle inside the host plant organs that may provide a season-long defense. Also, the Trichoderma species can trigger the maize disease resistance response systemically through the biosynthetic induction of salicylic acid, jasmonic acid, brassinolide, reactive oxygen species, and defense enzymes [78]. Trichoderma asperellum significantly inhibited the growth of F. verticillioides in an in vitro competition assay [79] and showed a control efficiency of 50% against Fusarium graminearum under field conditions [80]. These examples imply that field biocontrol application of T. asperellum may offer broad pathogen protection.”

 

The following paragraph was rewritten and expanded:

• (Lines 619-630): “Fungi are incredible producers of a huge range of xenobiotics, including some well-known compounds that inhibit the growth of other fungi. Secondary metabolites are pivotal defensive microorganism-pathogen interactions during host invasion and establishment, as well as the plant microbiome defense response. These include antifungals, mycotoxins, lactams/lactones, and other extracellular components. Such a compound is 6-PP [81]. This metabolite was identified as one of the key bioactive compounds of several Trichoderma species [62], with a strong relationship found between 6-PP biosynthesis and biocontrol ability. 6-PP inhibits mycelial growth, spore germination and pigmentation of tested plant pathogenic fungi [63]. 6-PP also reduces the production of the mycotoxin deoxynivalenol (DON) by F. graminearum and fusaric acid by F. moniliforme [64]. Our study results support the importance of this molecule and are opening up a new research direction that could lead to a new LWD bio-pesticide product.”

The following paragraphs were added:

• (Lines 631-639): “Significant knowledge gaps still exist regarding the action mechanism of 6-PP. The compound is oxygenated and thus was proposed to be part of the oxylipin family [82,83]. This suggests that independent of their source, similar oxylipin-like structure compounds may provoke strong chemotropic responses in Trichoderma– and possibly other fungal species – and hence may establish an interspecies signaling mechanism. In mycoparasitism, it was implied [84] that the plant oxylipin recruits the mycoparasite towards roots affected by fungal plant pathogens – or already as a precautionary measure. At the same time, the fungal oxylipin 6-PP induces morphological adjustments in Trichoderma needed to launch a localized mycoparasitic attack.”

 

• (Lines 640-645): “The 6-PP mass release at this stage would not only have a repressive antifungal action but might also act as a highly secreted signal in the regions where the colony contacts the prey fungus to promote hyphal redirection and hyphal growth as part of the mycoparasitism response. Other secondary metabolites produced by  T. asperellum are likely involved during these events to refine and support the chemoattraction process and the mycoparasitism attack [84].”

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Authors

 

Please find my comments below.

Page 3 line 135: From where the 6-pentyl-α-pyrone (6-PP) purified active has been obtained? Dos the author purified it or was it purchased from a company? Please clarify.

Page 4 line 162: Does the author do the pathogensity test for M. maydis isolates used in this experiment?

Page 5 line 214: Trichoderma asperellum scientific name?

Page 11 line 449: The manuscript is on the Magnaporthiopsis maydis pathogen. What is the aim to test the 6-PP on other pathogens?

Page 11 line 452: Figure 3. In the Bioassay, there is no mention of this test on the methodology. 2.5. 6-pentyl-α-pyrone evaluation in a net house (Page 7 line 278). Also, you need to mention the consternation of the secreted metabolites of Trichoderma asperellum used in this test. Also, you need to test the pure 6-PP active ingredient on the fungi.

Page 12 line 468:  Figure 4. Disease symptoms look like blight symptoms, not wilt.

Page 14 line 506: Figure 6. The infected picture is not showing clear wilting symptoms. Provide another clear picture of the symptoms or remove Figure 6.

Page 15 line 522: Figure 7. All abbreviation needs to be clarified.

Page 15 line 522: Why is non-infected control still showing a peak of M. maydis DNA in 40 days? Please explain.

 

 

Author Response

Responses to Reviewer 2’s comments

We would like to express our sincere appreciation to the reviewer for the essential and helpful advice. The time and effort invested are greatly appreciated and certainly contributed to the manuscript and improved it. Thank you. All your remarks and suggestions were addressed carefully and thoroughly, as detailed below.

Page 3, line 135: From where the 6-pentyl-α-pyrone (6-PP) purified active has been obtained? Does the author purified it or was it purchased from a company? Please clarify.

Reply: Thank you for this important remark. The 6-PP compound was purified and identified in our previous work (Degani et al. Biology 2021, 10, 897. https://doi.org/10.3390/biology10090897), but for the current research, it was obtained from Sigma-Aldrich (CAS number 27593-23-3, Buchs, Switzerland).

The missing information was added to the text (lines 130-133): “This three-year study examined the biocontrol potential of Trichoderma asperellum (P1) and its secreted metabolites, specifically the 6-pentyl-α-pyrone (6-PP, Sigma-Aldrich, CAS number 27593-23-3, Buchs, Switzerland) purified active ingredient, against the maize late wilt pathogen, Magnaporthiopsis maydis.”

Page 4, line 162: Does the author do the pathogensity test for M. maydis isolates used in this experiment?

Reply: Yes. All M. maydis isolates were examined in our lab for their aggressiveness towards LWD-susceptible maize hybrids. This work is currently under consideration for publication elsewhere. One of the M. maydis isolates designated Hm-2 (CBS 133165, deposited in the CBS-KNAW Fungal Biodiversity Center, Utrecht, The Netherlands) was studied extensively in our previous works (see, for example, Drori et al. Phytopathologia Mediterranea 2013 52(1), 16-29 https://www.jstor.org/stable/42685381). All isolates have moderate pathogenicity levels toward LWD-susceptible maize hybrids.

The following explanation was added to the text (lines 160-162): “The isolates were examined in our lab for their aggressiveness towards late wilt disease-susceptible maize hybrids and were found to have moderate pathogenicity levels (Ofir Degani, personal communication).”

Page 5, line 214: Trichoderma asperellum scientific name?

Reply: This is the scientific name. The fungus is sometimes also called “Trichoderma asperellum sensu lato” (see Samuels et al. Mycologia 2010, 102(4), 944-966.‏ https://doi.org/10.3852/09-243), but in many cases, the simple name form is used.

Page 11, line 449: The manuscript is on the Magnaporthiopsis maydis pathogen. What is the aim to test the 6-PP on other pathogens?

Reply: Thank you for this important remark. Indeed, our work focused on M. maydis and late wilt disease. Yet, as explained in the text (lines 451-453), late wilt disease is affected by other pathogens that are grouped together in the post-flowering stalk rot complex (PFSR, see Khokhar et al. Maydica 2014, 59(3), 226-242 https://journals-crea.4science.it/index.php/maydica/article/viewFile/1003/850).‏ Thus, it is important to demonstrate the impact of 6-PP compound on the other PFSR members, Fusarium verticillioides (the causal agent of stalk rot) and Macrophomina phaseolina (the charcoal rot agent). Since our data showed no such response (Figure 3), we dedicated our efforts to M. maydis, which had a strong reaction to 6-PP.

Page 11, line 452: Figure 3. In the Bioassay, there is no mention of this test on the methodology. 2.5. 6-pentyl-α-pyrone evaluation in a net house (Page 7 line 278). Also, you need to mention the consternation of the secreted metabolites of Trichoderma asperellum used in this test. Also, you need to test the pure 6-PP active ingredient on the fungi.

Reply: This is a correct remark. We apologize for this mistake. The following information was added to the Materials and Methods section (lines 268-281):

“2.5. Effect of Trichoderma asperellum-secreted metabolites in solid media cultures

The impact of T. asperellum-secreted metabolites on M. maydis, M. phaseolina, and F. verticillioides growth was evaluated in solid (PDA) growth media as previously described [56]. To prepare the T. asperellum’s secreted metabolites, five mycelial discs (6 mm) were removed from the margins of 2-4-day-old colonies and incubated for six days in 150 mL PDB in the dark at 150 rpm at a temperature of 28 ± 1°C. The growth medium was separated by Buchner funnel filtration through Whatman filter no. 3. The medium pH was adjusted to 5.1 ± 0.2 with NaOH and filtered again for sterilization using biofilter bottles (0.22-micron filter, BIOFIL 500 mL vacuum bottle filter, Indore, India). The Trichoderma cultures’ filtrate described above was used to prepare the PDA plates (instead of DDW). PDA powder was added to the filtrate according to the manufacturer’s instructions. After autoclave sterilization, the mixture was poured into Petri plates, which were seeded with a 6-mm-diameter M. maydis mycelial disc (from the margin of a fungus colony, see Section 2.2). The colonies were allowed to grow for six days and photographed.”

You are probably aware that there are many ingredients in the growth medium extract (crude), and it is a highly complex task (involving significant HPLC and GC-MS analysis) to try to set their concentrations. Instead, we used the crude itself (100% without dilution) for preparing the PDA plates (see the description above).  

The pure 6-PP active ingredient was already tested in media plates against M. maydis (Degani et al. Biology 2021, 10, 897. https://doi.org/10.3390/biology10090897). Regarding inspecting 6-PP in a solid plate assay against the other PFSR members, Fusarium verticillioides and Macrophomina phaseolina, we think this aspect is important and worthy of a separate study that would examine several concentrations of the compound and further evaluation of its efficiency in protecting sprouts and mature plants.

Page 12, line 468: Figure 4. Disease symptoms look like blight symptoms, not wilt.

Reply: Indeed, wilt and blight symptoms can sometimes look similar. But these are in no doubt LWD typical dehydration symptoms. As the literature describes, the first symptom (appearing approximately 60 days after sowing) is moderate plant wilting progressing upwards. The symptoms include the development of light-green stripes on the leaves; the stripes become translucent, the leaves change their color to light-silver and yellow-brown, and the entire leaves roll inward from the edges (see Degani, O. J. Fungi 2021, 7, 989. https://doi.org/10.3390/jof7110989).

Page 14, line 506: Figure 6. The infected picture is not showing clear wilting symptoms. Provide another clear picture of the symptoms or remove Figure 6.

Reply: As suggested by the reviewer, the infected plants’ representative picture (in Figure 6) was replaced with one that better represents this treatment.

Page 15, line 522: Figure 7. All abbreviation needs to be clarified.

Reply: We carefully checked all the abbreviations to ensure that they are fully described.

Page 15, line 522: Why is non-infected control still showing a peak of M. maydis DNA in 40 days? Please explain.

Reply: The reviewer is correct; this should be better explained. As detailed in the growth room experiments (lines 178-182): “Two-liter pots were filled with commercial field peat soil from a location with a long record of LWD infection (Amir field, Mehogi-1 plot, coordinates: 33°09’59” N 35°36’5” E) [52,53]. Control was a similar peat soil from a nearby farm (Gadash experimental, coordinates: 33°10’48.6“N 35°35’11.6“E) with no record of LWD. If such an infestation occurred, it was supposed to be minor.”

The net house experiment was conducted under identical soil conditions. To clarify this point, we rewrote this explanation in the net house methodology description (lines 302-307): “Inoculation with the M. maydis was carried out as described for the growth room experiment, with the following changes: in the first inoculation step, 40 g of infected sterilized wheat grains were added to the upper 20 cm of the soil of each pot. Control was peat soil from the Gadash experimental farm having no record of LWD. If such an infestation existed, it was assumed to be very low.”

Thus, as revealed by the highly sensitive qPCR method, some very low levels of M. maydis DNA (near the method’s lower sensitivity threshold) could be identified in the control treatment. But such low levels were unable to evoke disease symptoms.

This explanation was added to the Results section (lines 519-522).

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors addressed all reviewer's comments, and the manuscript was improved.

 

Author Response

We want to express our sincere appreciation for your essential and helpful advice. The time and effort invested are greatly appreciated and certainly contributed to and improved the manuscript. Thank you.

Reviewer 2 Report

Dear Authors

The manuscript has been improved and it looks better.  Just one thing i mentioned in my previous comments, there is still un-appropriate self-citation in the manuscript, the first author Ofir Degani site himself 15 times. that need to be fixed.

Thanks

 

Author Response

Responses to Reviewer 2’s comments

We thank the reviewer for investing substantial efforts, which undoubtedly contribute to this manuscript. The remarks and suggestions improved this paper’s scientific soundness and accuracy. Your contribution is greatly appreciated.

The manuscript has been improved, and it looks better. Just one thing I mentioned in my previous comments, there is still un-appropriate self-citation in the manuscript, the first author Ofir Degani site himself 15 times. that need to be fixed.

Reply: The overall reference number in the manuscript is 84, and the self-citation ratio is 18% (well within the acceptable self-citation ratio according to the MDPI instructions). We believe all self-citations are necessary to present the most updated relevant information and describe the methods properly.