Lignin–Chitosan Nanocarriers for the Delivery of Bioactive Natural Products against Wood-Decay Phytopathogens

Round 1

Reviewer 1 Report

The paper by Sanchez-Hernandez et al states the preparation of lignin-chitosan based nanocarriers and their application in planta.

The general concept is interesting, as lignin nanocarriers have been successfully used in planta and chitosan is compatible with their synthesis and has additional influence on plant stimulation.

However, I think, a lot of improvement is needed before publication.

First of all, I cannot find any proof given by the authors that they actually prepared any lignin/chitosan nanocarriers. The EM picture clearly shows inorganic crystals but no nanocarriers. The EM images need to be taken out before publication! I recommend purification of the dispersion before taking EM images and use low beam intensity to visualize. Also AFM might be a better option.

Further: the loading of biobased drugs and oils is certainly interesting, however, the concept of nanocarriers remains unclear - why not simply emulsifying them using chitosan and a surfactant? Why does one need a nanocarrier? Nanocarrier only make sense if they can open up selectively. This has not been shown here. Injection the emulsion of the plant oils would result in the same effect as the lignin-enriched samples (which might or might not contain any nanocarriers).

The authors must prove before publication the formation of nanocarriers - also the high amount of chitosan ver likely results not in the formation of chitosan but only the reaction with lignin-MA - crosslinking needs a certain amount of NH2:MA groups - if the amount of one is too high, no crosslinking occurs.

growth studies of pathogens: if the dispersion was not purified after the reaction, an antifungal effect is obvious - you put in antifungal substances - a control is missing with the antifungal aloe and/ or a fungal strain that does dot degrade lignin to prove that nanocarrier mediated delivery has taken place.

Solubility: Kraft lignin is insoluble in water. So is it after modification with methacrylic acid groups - the authors state that they dissolve lignin methacrylate in water - how is this possible? It might be at most suspendable ? please explain.

what happens to the plastic piece that is injected into the stem of the plant? it remains inside of the plant? But it certainly will not be suitable to use it again for injection - the surrounding tissue will change and decay. I suggest a different way of application.

 

In general, I believe the topic is timely and important however the data provided with this study are far off being publishable and I vote strongly against publication in this state - control experiments need to be conducted, dispersion properties and colloidal properties in general need optimization. Also the synthesis of the nanocarriers needs to be discussed. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript “Lignin-Chitosan Nanocarriers for the Delivery of Bioactive Natural Products against Wood-Decay Phytopathogens” reported a synthesized nanocarrier for encapsulating the extracts from Rubia tinctorum, Silybum marianum, Equisetum arvense and Urtica dioica to assay in vitro against Neofusicoccum parvum. This research is meaningful, and the results are interesting. However, there are many problems should be addressed before its possible acceptance.

1. The first sentence of Abstract mentioned that “nanomaterials capable of encapsulating drugs and releasing them when subjected to certain external stimuli”. What is the relation of this work with external stimuli?
2. The full name of MIC should be provided in Abstract.
3. The last paragraph of Introduction should be rewritten. It should show the attractiveness of this work and its potential applications.
4. The detailed characterizations of encapsulation efficiency should be provided, including the equations used.
5. The molecular weight distribution of the obtained COS should be provided.
6. In the section of thermal gravimetry (TGA) and differential scanning calorimetry (DSC) analyses, why the condition of N₂:O₂ (4:1) was selected.
7. The preparations and characterizations of methacrylated lignin (ML) and chitosan oligomers (COS) should be supplemented in detail.
8. The number of 3.4.1. Antibacterial Activity should be checked.
9. The nanoparticle size distribution and zeta potential as well as the stability of this system should be added.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

“Lignin−Chitosan Nanocarriers for the Delivery of Bioactive Natural Products against Wood-Decay Phytopathogens” presents a very interesting and promising method for combating pathogens of woody crops using natural compounds. The research was well-planned and thoroughly performed, both in vitro and in vivo. The results are described clearly and well-discussed. I would only suggest adding an explanation of what exactly was measured and where using a Handy Plant Efficiency Analyzer (lines 270-273).

Author Response

“Lignin−Chitosan Nanocarriers for the Delivery of Bioactive Natural Products against Wood-Decay Phytopathogens” presents a very interesting and promising method for combating pathogens of woody crops using natural compounds. The research was well-planned and thoroughly performed, both in vitro and in vivo. The results are described clearly and well-discussed.

 

Q1. I would only suggest adding an explanation of what exactly was measured and where using a Handy Plant Efficiency Analyzer (lines 270-273).

Response: An explanation has been provided, as requested by the Reviewer. The revised text now reads: “Measurements of fast chlorophyll fluorescence induction kinetics were carried out using a continuous excitation chlorophyll fluorimeter (model Handy PEA+, Hansatech Instruments, Pentney, Norfolk, UK), on a bimonthly basis throughout the vegetative period (from late May till the beginning of September). Five measurements were taken on each arm of the 20 plants under study, to investigate subtle differences in the fluorescence signature of samples, which could be indicative of stress factors affecting the photosynthetic efficiency of the plant. The ratio of the variable fluorescence (Fv) over the maximum fluorescence value (Fm) was chosen as an indicator of the maximum quantum efficiency of Photosystem II, being a sensitive indication of plant photosynthetic performance. Presented as a ratio between 0 and 1, healthy samples typically achieve a maximum Fv/Fm value of approx. 0.85, while lower values suggest that the plant has been exposed to some type of biotic or abiotic stress factor. [...]"