Co-Inoculation of Aflatoxigenic and Non-Aflatoxigenic Strains of Aspergillus flavus to Assess the Efficacy of Non-Aflatoxigenic Strains in Growth Inhibition and Aflatoxin B₁ Reduction

Round 1

Reviewer 1 Report

The manuscript needs a thorough revision of the English language. Some sentences are not clear.

The results of some experiments are debatable, the experiments in question need to be repeated or analysed differently.

The manuscript needs Major revision to be considered for publication.

The biggest concerns are listed below:

Line 50 – “In biological control, biocontrol” this sentence is redundant

In “materials and methods” chapter, specify, every time, if cultures were grown on the dark, light or a combination of both.

• In lines 92-93. The assumption “Moreover, c assumed to be zero, because, on day 1, the colony diameter was zero” was highly debatable. Even if the conidia were not germinated, each drop occupies a certain amount of surface when placed on the centre of the plate, producing a halo. The border of that halo should be considered as the starting point, and measures have to start from that point. When they become visible, larger drops will result in larger colonies than smaller drops, giving an erroneous interpretation of their growth if all were considered starting from zero.
• In line 101 “In co-inoculated treatment, 100 of each AF- and AF+ isolates” the unit of measurement is missing.
• Line 109 – the extraction of AfB1 was done in an inadequate volume of methanol.
• Looking at Figure 1, I wonder if you have been able to measure a single diameter, correctly. In every plate, conidia were spread everywhere. This experiment should be repeated, otherwise, chapter 3.1, should be removed.
• Figure 2 is not clear, this has to be better described in the caption.
• Figure 3 has been confused with figure 2.
• The calibration curve figure does not fit the description in the paragraph.
• Considering five Af- and five Af+ strains I will expect all the combination for the co-inoculation tests. Otherwise, the authors choose to perform only five pairs
• Figure 6 used a different unit of measurement compared to the text. It is better to only use ppb for they-axis.

Author Response

First reviewer’s comments

• Line 75 – the sentence has been rewritten and the redundancy was removed.
• Material and Methods – In this study, all the cultures had been grown in the dark, which is now mentioned in the specified sentence.
• Line 92-93 – Although, you raised an important point regarding the growth of Aspergillus flavus on 1^st day of incubation. However, the mycelial growth on 1^st day of incubation is negligible to include in the overall diametric growth rate of flavus. We have never come across any literature, in which the authors calculated the diametric growth rate of A. flavus in their studies. Generally, they started calculating growth rates from the 2^nd d of incubation, as halo could covers the media surafce but not the real mycelial growth. Therefore, we excluded the first-day insignificant growth of A. flavus in our study.
• Line 112-113 – the unit of the measurement has been included in co-inoculated treatment.
• Line 145-154 – the extraction of AFB₁ has been rewritten with few modifications.
• Figure 1 – While measuring the diametric growth rates of flavus, we also used the software called Image J, which precisely validated the colonies' diameters of aflatoxigenic and non-aflatoxigenic strains of A. flavus.
• Figure 2 – the caption has been modified according to the reviewer’s comment.
• Figure 3 – Figure 3 has been modified.
• Line 244 - The calibration curve has been modified to become fit with the paragraph description.
• In co-inoculation tests, we perform 5 pairs of AF^- and AF⁺
• Figure 6 – the unit of measurement has been changed into ppb.

Author Response File: Author Response.docx

Reviewer 2 Report

see comments in the uploaded manuscript

Comments for author File: Comments.pdf

Author Response

• Title – Word determines has been changed to assess.
• Line 80 – the new reference (Abbas et al., 2017) is added to the manuscript.
• Line 113 – recovery for aflatoxin B₁ has been added.
• Line 170-172 – the sentence has been changed according to the reviewer’s comment.
• Line 190-191 – The sentence has been changed into “ The diametric growth rates of AF⁺ and AF^- strains on MEA after a 7 d of incubation at various temperatures (25, 30, 35, and 40℃) is shown Figure 1.”
• Line 294 – the word onset has been removed.
• Line 304 – word probabilities has been changed into explanations.
• Line 315 – word revealed has changed into showed.
• Line 330 – the sentence has been modified.

Author Response File: Author Response.docx

Reviewer 3 Report

Cannot see the meaning of this research on “Introduction”  

Aflatoxin contamination in field corn is a well-known problem worldwide. However, aflatoxin in sweet corn usually is not a problem. The authors didn’t introduce either why choose sweet corn, or aflatoxin has any impact in Malaysia’s sweet corn, or why biocontrol is necessary for sweet corn.

About experimental design:

1.       For co-inoculation of Aflatoxigenic and Non-aflatoxigenic Strains, why are the specific compositions AKR1- + AKR8+, AKR5- + ARV17+, AKL34- + ARV18+, AKL35- + ARV20+, AKL36- + ARV21+ applied? This design is not a statistical meaningful design.  

2.       Line 109: “100 of each AF- and AF+ isolates and the ratio of 50:50 percentage used on sweet corn kernels” is not clear.

Problems on results’ presentation:

1.       Line 143: “Figure 1. shows the diametric growth rates of AF+ and AF- isolates on MEA after a 7 d of incubation at various temperatures (25, 30, 35, and 40℃)”. However, the title for Figure 1 is “Hyphal expansion of AF- strains on MEA”, without AF+, which is not consistent with the result’s presenting. Which specific AF-   strain was used in this demonstration?

2.       Line 155: “Figure 2:  Diametric growth rates (mm/d) of AF+ and AF- strains on MEA incubated at various temperatures (25, 30, 35, 40℃) for 7 d”. No temperature is shown on the figure.  Are the Diametric growth rates of AF+ and AF- strains average of all AF+ or AF- strains or a specific strain?

3.       Line 164: for the Figure 3. Images of the AF+ and AF- isolates; (A) Grown on corn kernels in mixtures; (B, C) Grown on corn kernels separately; (D) Gown on PDA for comparing the growth on 7 d of incubation. Which strain is for B, which for C?

4.       Line 193: “AKR8-“?  

5.       Line 209: Figure 6. AFB1 production by AF+ strains. The label for X axis “Reduction in afb1 by non-aflatoxigenic strains” is neither clear or accurate. Each bar’s label should be specific AF+ or with co-inoculation AF- .  

6.       For “In Vitro Inoculation” of material and method, “Kernels have been incubated at 28°C and observed at 3, 5, and 7 d of incubation” (line 103-104). How many days were the kernels incubated for the results of Figure 5, Figure 6 and Table 2?

About conclusion:

“The current study provided in vitro evidence that the AKR5- and AKL34- biocontrol isolates successfully suppressed colony propagation, and subsequent AFB1 contamination by the AF+ isolates” is not accurate. How about AKL35- and AKR1-? What does “successfully suppressed” mean?

Author Response

• Introduction 41-72 – A paragraph regarding the incidence of AF in Malaysian food commodities and its outbreaks is added the introduction part.
• Experimental design – In our previous study, we have identified the five-strong aflatoxigenic strains and five non-aflatoxigenic strains, which have been chosen for co-inoculation treatments in the current study. Therefore, we tested the five non-aflatoxigenic strains against five strong aflatoxigenic strains to check their efficacy for growth inhibition and AFB₁
• Line 112-113, and 128-129 – 50 mL of each AF^- and AF⁺ isolates at 1:1 ratio was applied on sweet corn kernels in co-inoculation treatment.
• Results’ presentation
• Line 201 – Figure 1 caption is changed into diametric growth rates. Figure 1 shows the diametric growth rates of AKR5^- strain at four different temperatures (25, 30, 35, and 40°C) for 7 days of incubation.
• Line 204-208 – temperatures (25, 30, 35, 40°C) given now in Figure 2. Data are means of triplicates (n=3) with bars indicating a standard error (SE). Capital letters indicate a significant difference (p < 0.05) between AF⁺ strains and small letters indicate a significant difference (p < 0.05) between AF^- using Tukey's Honest Significant Difference (Tukey's HSD).
• Line 216-218 – (A) AKR5^- and ARV 18⁺ strains were grown on corn kernels in mixtures; (B, C) AKR5^- and ARV 18⁺ strains were grown on corn kernels separately following on 7 d of incubation.
• Line 193 – AKR8^- changed into AKR8⁺, since the strain was aflatoxigenic rather than non-aflatoxigenic.
• Line 263 – In Figure 6, each bar labelled with specific co-inoculated strains of AF⁺ and AF^-.
• Figure 3 – Captions for Figures 5, 6, and Table 2 updated by adding the days of incubation.
• Conclusion
• The words “successfully suppressed” have been changed into effectively inhibited and a sentence for AKR1^- and AKL35^- also written in the conclusion.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The English language needs to be improved. Pay attention to the verbs:

"AF are is characterized for their carcinogenicity..."  

or

"AFs are characterized for their carcinogenicity..." 

• Line 75 – the sentence has been rewritten and the redundancy was removed.
• Material and Methods – In this study, all the cultures had been grown in the dark, which is now mentioned in the specified sentence.
• Line 92-93 – Although, you raised an important point regarding the growth of Aspergillus flavus on 1^st day of incubation. However, the mycelial growth on 1^st day of incubation is negligible to include in the overall diametric growth rate of flavus. We have never come across any literature, in which the authors calculated the diametric growth rate of A. flavus in their studies. Generally, they started calculating growth rates from the 2^nd d of incubation, as halo could covers the media surafce but not the real mycelial growth. Therefore, we excluded the first-day insignificant growth of A. flavus in our study.

It is ok to measure the diameter from the second day of incubation. But in chapter 2.3 you wrote "days 1, 3, 5, and 7".  So you did not exclude the first day.

• Line 112-113 – the unit of the measurement has been included in co-inoculated treatment.
• Line 145-154 – the extraction of AFB₁ has been rewritten with few modifications.
• Figure 1 – While measuring the diametric growth rates of flavus, we also used the software called Image J, which precisely validated the colonies' diameters of aflatoxigenic and non-aflatoxigenic strains of A. flavus.

Image J software is still never mentioned in the manuscript. Moreover, with that software, you probably measured the area of the plate occupied by the mycelium. Picture 1 clearly showed a non-circular growth, due to the spread of fungal spores that caused the growth of multiple colonies that interfere with the growth of the central colony. Under these circumstances, no diametric growth of the central inoculum could be measured.

• Figure 2 – the caption has been modified according to the reviewer’s comment.
• Figure 3 – Figure 3 has been modified.
• Line 244 - The calibration curve has been modified to become fit with the paragraph description.
• In co-inoculation tests, we perform 5 pairs of AF^- and AF⁺

In the manuscript, you wrote "In co-inoculated treatment, 50 mL of each AF^- and AF⁺ strains at a 1:1 ratio was applied to sweet corn kernels", but if you perform 5 pairs, you did not perform "each" combinations of AF^- and AF⁺ strains. You have to explain why you choose to perform only that "5 pairs", automatically excluding all the other combinations.

• Figure 6 – the unit of measurement has been changed into ppb.

Author Response

First reviewer’s comments

• The word AF has been changed into AFs in the manuscript wherever it is used as a plural.
• In chapter 2.3, the first day has been excluded from the sentence.
• Image J software has been added to the sentence now. That is software enables us to precisely measure the Plates’ area occupied by the mycelia, regardless of their shapes. Using this software, the mycelial areas either they are circular or non-circular have been measured simply by making dots around each colony and then the whole readings for strain were summed.
• In co-inoculation treatments, 5 pairs of AF^- and AF⁺ strains have been made, which is listed as below;

First pair: AKR1^- + AKR8⁺

Second pair: AKR5^- + ARV17⁺

Third pair: AKL34^- + ARV18⁺

Fourth pair: AKL35^- + ARV20⁺

Fifth pair: AKL36^- + ARV21⁺

The above combinations of aflatoxigenic and non-aflatoxigenic strains have been applied on sweet corn kernels separately to check the efficacy of non-aflatoxigenic strains in each combination in terms of growth inhibition and reduction in AFB₁ production.

Author Response File: Author Response.docx

Reviewer 3 Report

The author has corrected and clarified the problems in my first review. However, there are two problems still existing:

1. A big problem for the experimental design, which I mentioned in my first review, but didn’t address and correct yet: for co-inoculation of Aflatoxigenic and Non-aflatoxigenic Strains, why were the specific combinations of AKR1- + AKR8+, AKR5- + ARV17+, AKL34- + ARV18+, AKL35- + ARV20+, AKL36- + ARV21+ applied? This design has less randomness, which is not a statistical meaningful design.
2. This paper is about neither AF case investigation of epidemiology, nor AF toxicity, but AF B1 in sweet corn. Although little introduction was added to address why AF in sweet corn is a problem in Malaysia, too much unrelated basic knowledge were added in the introduction. For instance, lines from 56 to 69 are not necessary to this research.

Author Response

Third reviewer’s comments

1. In this research, our main objective was to find out the best potential biocontrol agents (AKR5^- and AKL34^-), which has been achieved. In the up-coming research, we will conduct laboratory as well as field trials in which we will test the efficacy of these potential biocontrol agents against all aflatoxin-producing Aspergillus flavus strains. Therefore, it could be the limitation of the current study that will be addressed in the upcoming research.
2. Lines 56 to 69 have been removed from the introduction part as suggested by the reviewer. Likewise, the references in the manuscript have been updated.

Author Response File: Author Response.docx