Review Reports - Induction of Defense Responses in Pinus sylvestris Seedlings by Methyl Jasmonate and Response to Heterobasidion annosum and Lophodermium seditiosum Inoculation

Round 1

Reviewer 1 Report

Dear authors.

You have obtained very interesting experimental results - congratulations! The following questions are worth considering in the discussion: Is it known how durable MeJa-induced plant resistance is to the pathogens tested? Do you think this mechanism might be similar to the priming phenomenon? It is known how the resistance is induced by the application of Trichoderma fungi and how the stress is studied by the expression of HSP genes (Hot Shock Proteins). Would it also make sense in your case to determine stress using this method? Why was TLP chosen? (https://doi.org/10.1007/s10658-020-02162-y)

In the case of H. annosum infestation, some researchers believe that the size of the vessels makes a difference, the wider the easier the fungus grows, could this have made a difference in your case? Could MeJa have affected their reduction in size and slower penetration of the fungus into the tissue?

And how would the fungal metabolites themselves behave, could spraying them stimulate resistance responses, including MeJa synthesis?

For future research, I suggest studies on terpenes, sterols, and phenolic compounds, e.g., with GCMS? Photosynthetic efficiency, on the other hand, can be measured by chlorophyll fluorescence. The mere presence of pathogens in the soil that attack fine roots (e.g. Phytophthora) can also induce shoot resistance to infection by, for example, Hymenoscyphus fraxineus (doi:10.3390/f9080442).

Has L. pinastri also been found in infected needles, which then show characteristic transverse lines between the pycnidia? It is considered a saprotroph and not a needle pathogen, if it were not present this would support this hypothesis. Recently, a dangerous needle pathogen near Latvia (e.g. in Lithuania and Poland) seems to be Lecanosticta acicola (https://doi.org/10.1111/efp.12589), which could cause the damage described in the article (apart from P. seditiosum).

L74 - use normal fonts for "spp." instead Italic (check througth the whole manuscript).

Author Response

Dear authors.

To our knowledge, this is the first report on the effect of MeJA application on resistance to H. annosum and L seditiosum. The durability of the effect in this and other studies (on other conifer and pathogen species) is discussed in the discussion (Lines 553-583).

Do you think this mechanism might be similar to the priming phenomenon? It is known how the resistance is induced by the application of Trichoderma fungi and how the stress is studied by the expression of HSP genes (Hot Shock Proteins). Would it also make sense in your case to determine stress using this method? Why was TLP chosen? (https://doi.org/10.1007/s10658-020-02162-y)

And how would the fungal metabolites themselves behave, could spraying them stimulate resistance responses, including MeJa synthesis?

Priming of defence responses is a part of induced resistance. In this study, we were interested in the response of MeJA treated scots pine seedlings to inoculation by two fungal pathogens. TLP was used as a marker of induction of defence responses, as it is a gene that is directly involved in defence responses, and has been shown by our laboratory and other studies to have a negative impact on a range of fungal pathogens.

Thank you for the interesting research direction. We did not assess the effect of anatomical parameters in resistance, however, this is a direction of further investigation. Similarly with the use of fungal metabolites to induce defence responses. However, the discussion was ammended to include these suggestions for further study directions.

Recently, a dangerous needle pathogen near Latvia (e.g. in Lithuania and Poland) seems to be Lecanosticta acicola (https://doi.org/10.1111/efp.12589), which could cause the damage described in the article (apart from P. seditiosum).

Only the presence of the fungal species used for inoculation was tested for in this study. Given that the plants were growing in outdoor conditions prior to inoculation, there is a possibility that other fungi were present in the plants. However, as these were not tested for, we refrain from speculation on other possible pathogens present in the seedlings.

Regarding Lecanosticta acicula – the forest pathology laboratory in our institute tested one Scots pine stand that was suspected to be infected with Lecanosticta acicula, but this infection was not confirmed (A Zaļuma, unpublished).

L74 - use normal fonts for "spp." instead Italic (check througth the whole manuscript). - This was corrected throughout the manuscript

Reviewer 2 Report

This manuscript describes results from 3 experiments involving methyl jasmonate and inoculation by two pathogens of Scots pine. The paper has some useful findings that will be of interest to other researchers and forest managers. I provide a few general comments, then specific suggestions below. While some editorial suggestions are made, they are by no means comprehensive and the paper should have a thorough English edit. In summary, I believe some clarification will greatly strengthen the value of the paper.

General comments:

There was a good statement of the aim of the study, although it needs to be clear right in the purpose, that young seedlings were tested and why that is relevant. There were no stated objectives – the text after the purpose statement was essentially a list of methods. This does not allow the reader to determine if the objectives were met or not.

There is a lack of specific information on numbers of seedlings, needles and discs sampled and processed throughout the methods. More specific examples of this follow. In some cases these numbers showed up later in the results, but should be clearly stated in the methods.

The description of statistical analysis methods is inadequate. As an example, how were categories of symptom expression in experiment 3 compared? According to the single sentence on statistical methods, both parametric and non-parametric methods were used – why and when?

There is inconsistency in how “lesion” is used compared to length or growth of infection. A lesion is a symptom that can be caused by fungal infection or physical damage. This inconsistency is problematic, especially where total length of necrosis is used as a measure of longitudinal growth of the fungus. The length of necrosis would be significantly affected by the inoculation method, and unless I missed it, this experiment did not include performing the inoculation with “blanks” or uncolonized sapwood pieces. This is a significant problem as the extent of lesion formation due to the inoculation method alone cannot be determined.

Specific comments:

45, “on” should be “by” or “of”
70 the fungus does not remain viable in soil. It remains viable in roots in soil.
71, change to “….Resistance or relative tolerance of trees to root rot…”
72, genetic determination, and the well-developed inoculation methods are two totally different topics and should be in different sentences.

l.86 and 87, it should be more clear that this refers to the age of seedlings when planted

91-101, here is where a specific statement of objectives, and not a summary of methods used or steps taken, should be included.
141, add “seedlings” after “control” to read “…22 control seedlings…”
211, inadequate description of statistical analyses.
214, here is an example of where more information is needed. Were all of the trees sampled? Were only symptomatic trees sampled? How many needles?
236, RNA extracted from all seedlings?
259-262, reword as repetitive: “One week after treatment, at the time of inoculation with H. annosum, all seedlings appeared healthy. After six weeks almost all MeJA treated ramets were partly wilted, and all control ramets appeared healthy.” Delete next sentence.
260-274, here is another section where more information is needed on actual numbers. Too much use of “many, some” etc.
269, how was “infection frequency” determined?

l.270-271, as described above, total necrosis length may not be the same as longitudinal growth of fungi.

278-297, awkwardly written, I had to read it several times to understand.
326, how many samples? Avoid “some”
326-327, not a good presumption to make
333-334, alternatively, the inoculation didn’t take.
338, avoid “in most cases”
347, delete “treatment when”
384, numbers needed
388, delete “were detected”
389, 390, why are these numbers a range (+)? If this is an infection frequency it should be a Yes infected or No not infected, with no range. If this is the result of analysis of multiple needle samples from each sampled seedling, it could be a range. This is where the lack of information on how many samples were taken and processed, is causing confusion.
394-397, information here repeats the table and could be deleted.
429, results here are suspect due to confusion of lesion length with infection length.
430, “small number of tested individuals” – the actual number should be in the methods.
555-567, I am wondering about the spatial arrangement of the seedlings outside, and if only a clump of them were infested by the sawfly and the weevil, and this same clump became part of the control trees, rather than the treated trees, by chance.

Figure 3, x axis difficult to read

Figure 4. Legend insufficient to understand what the figure is showing.

Table 2. first column, instead of (number) shouldn’t this be (percent)?

Author Response

General comments:

There was a good statement of the aim of the study, although it needs to be clear right in the purpose, that young seedlings were tested and why that is relevant. There were no stated objectives – the text after the purpose statement was essentially a list of methods. This does not allow the reader to determine if the objectives were met or not.

The last paragraph of the introduction was ammended to make the aims and objectives more explicit.

There is a lack of specific information on numbers of seedlings, needles and discs sampled and processed throughout the methods. More specific examples of this follow. In some cases these numbers showed up later in the results, but should be clearly stated in the methods.

The methods section has been ammended with specific number of analysed samples.

The description of statistical analysis methods is inadequate. As an example, how were categories of symptom expression in experiment 3 compared? According to the single sentence on statistical methods, both parametric and non-parametric methods were used – why and when?

The methods section describing the statistical analysis has been updated. In experiment 3, the numbers of individuals in each visual infection category were not statistically compared “A Mann – Whitney tests (non-parametric test – infected/non-infected) was used to compare H. annosum and L. seditiosum infection frequency and t-tests (parametric test – fungal growth in mm) was used to compare mean longitudinal H. annosum growth. Both tests were performed using R software (R 3.6.2.)”

There is inconsistency in how “lesion” is used compared to length or growth of infection. A lesion is a symptom that can be caused by fungal infection or physical damage. This inconsistency is problematic, especially where total length of necrosis is used as a measure of longitudinal growth of the fungus. The length of necrosis would be significantly affected by the inoculation method, and unless I missed it, this experiment did not include performing the inoculation with “blanks” or uncolonized sapwood pieces. This is a significant problem as the extent of lesion formation due to the inoculation method alone cannot be determined.

The text was reworded to avoid using the term ‘lesions’ with regard to the spread of H. annosum in the stems of inoculated seedlings. As stated in the materials and methods section, the extent of H. annosum growth in stems was evaluated by microscopic observation of conidiophores or positive PCR results. Therefore, the results and discussion section were changed to have more precise wording in this regard.

Specific comments:

45, “on” should be “by” or “of” – corrected “on” to “of”
70 the fungus does not remain viable in soil. It remains viable in roots in soil – added “in roots” .
72, change to “….Resistance or relative tolerance of trees to root rot…” – added “ trees to”
71-73, genetic determination, and the well-developed inoculation methods are two totally different topics and should be in different sentences. - separated sentence – “Resistance or relative tolerance of trees to root rot is genetically determined [30-33]. Furthermore, an artificial inoculation methods are well developed [31,34].”
87, it should be more clear that this refers to the age of seedlings when planted – added “when planting”

91-101, here is where a specific statement of objectives, and not a summary of methods used or steps taken, should be included. – The final paragraph of the introduction was ammended to state the aims more explicitly.

142, add “seedlings” after “control” to read “…22 control seedlings…” – added “seedlings”
175, deleted “and necrosis”
207-209, added sentence “60 needles (10 needles from a selection of each cassette) from the seedlings were once a month sampled and analyzed with L. seditiosum specific primers. After the first positive PCR results, all seedlings were analyzed –“.
209-211, moved sentence “symptomatic needles (approx. 5 - 6) from the top of the seedling were collected and the presence of L. seditiosum was verified by PCR using species-specific primers [48].”
215-217, inadequate description of statistical analyses – changed sentence “A Mann – Whitney tests was used to compare H. annosum and L. seditiosum infection frequency and t-tests was used to compare mean longitudinal H. annosum growth. Both tests were performed using R software (R 3.6.2.)”
220-222, here is an example of where more information is needed. Were all of the trees sampled? Were only symptomatic trees sampled? How many needles?- changed sentence with additional info “Total DNA was extracted from 71 individuals (770 stem fragments) and from 162 seedlings (approx. 700 needles) using a CTAB method [49]. A total number of 180 needles were periodically sampled to analyzed with L. seditiosum specific primers.”
244-246, RNA extracted from all seedlings? Change sentence and added missing info “RNA was extracted from seven MeJA treated ramets and three control ramets us-ing a CTAB buffer-based method [51] modified by Rubio-Pina and Zapata-Petrez [52]. From each ramet were taken two pine needles.”
269-271, reword as repetitive: “One week after treatment, at the time of inoculation with H. annosum, all seedlings appeared healthy. After six weeks almost all MeJA treated ramets were partly wilted, and all control ramets appeared healthy.” Delete next sentence. – change sentences to “One week after treatment, at the time of inoculation with H. annosum, all seedlings appeared healthy. After six weeks almost all MeJA treated ramets were partly wilted, and all control ramets appeared healthy.
271, Deleted sentence “All controls and MeJA treated ramets were inoculated with H. annosum one week after treatment”
269-283, here is another section where more information is needed on actual numbers. Too much use of “many, some” etc. – 270, added amount of wilted ramets “13 of 15” and deleted “almost all” ,275, - added info about ramets “four of” and deleted “some”
277, how was “infection frequency” determined? Changed to “there were no significant differences in the proportion of infected individuals between controls (71%) and MeJA treated (64%) ramets”
.279-280, as described above, total necrosis length may not be the same as longitudinal growth of fungi. – change sentence “as determined by total length of all discs with detected conidiophores”
280-283, awkwardly written, I had to read it several times to understand. The sentence was rewritten to make it more understandable – “In the first four time points (0 -50 h) of MeJA treatment, only small increases of TLP gene expression were observed (less than 20 fold), but 14 days after MeJA treatment (and, respectively, one week after inoculation with H. annosum) large increases in TLP gene expression were observed in needles, even up to a 193-fold increase compared to expression before treatment (p < 0.05) (Figure 3)”
335, how many samples? Avoid “some” – the text was modified to make it more precise and understandable.
332, not a good presumption to make – add “presumably” - presumably was added to the text.
342-344, alternatively, the inoculation didn’t take – the sentences was reworded to explicitly state that this refers to negative PCR test results from the inoculation site, while discs above and below the inoculation site were positive for H.annosum PCR tests - “Presumably, the failure to detect H. annosum by PCR at the inoculation site, while discs above and below the inoculation point were positive, was due to the presence of PCR-inhibiting substances and poor-quality DNA degraded by wounding, fungal inoculation and flame sterilization.”
346-347, avoid “in most cases” – deleted “in most cases”
356, delete “treatment when” – deleted “treatment when”
393, numbers needed – added numbers “60”
394 – deleted “periodically”, added – “once a month”
397, delete “were detected” – deleted “were detected”
389, 390, why are these numbers a range (+)? If this is an infection frequency it should be a Yes infected or No not infected, with no range. If this is the result of analysis of multiple needle samples from each sampled seedling, it could be a range. This is where the lack of information on how many samples were taken and processed, is causing confusion. – this text was changed to reflect the number of seedlings where L. seditiosum was detected by PCR. In addition, Table 2 was ammended to present the obtained results more clearly.
209-211, added sentence “After the first positive PCR results, all seedlings were analyzed - symptomatic needles (approx. 5 - 6) from the top of the seedling were collected and the presence of L. seditiosum was verified by PCR using species-specific primers [48].”

401 – added info “and only 28% of MeJA treated”
402 - information here repeats the table and could be deleted – deleted “, 41 seedlings were scored as 1 (0-5%) 31 as 2 (6-35%) and 5 seedlings as 5 (96-100%) (were wilted). 62 (72%) MeJA treated seedlings had no yellow or brown spots, and of the remaining seedlings, 13 were assessed as grade 1, three as grade 2, two as grade 3, and five as grade 5 (Table 2).”

435, results here are suspect due to confusion of lesion length with infection length. – as mentioned previously, the use of the word ‘lesions’ was not correct, and this has been changed throughout the text. The length of fungal growth within stems was assessed – either by microscopic observation of conidiophores, or by PCR. This has been added to the methods section.
436, “small number of tested individuals” – the actual number should be in the methods. The number of treated and inoculated individuals in Experiment 2 are given in Table 1. The text was appended to include this information in the discussion section.
561-562, I am wondering about the spatial arrangement of the seedlings outside, and if only a clump of them were infested by the sawfly and the weevil, and this same clump became part of the control trees, rather than the treated trees, by chance. – As mentioned in the text, this was an unplanned stress event and therefore experimental conditions were not optimal. However, N. sertifer eggs were found on MeJA individuals, but no damage was seen. As stated in lines 577-578 (original version) “On MeJA treated trees, needles with N. sertifer eggs started browning and development of larvae was delayed or suspended.”

Figure 3, x axis difficult to read - this figure is now included as supplementary material along with the real time PCR data

Figure 4. Legend insufficient to understand what the figure is showing.

Legend changed to “Comparison of the sensitivity of Taq and FIREPol DNA polymerases in detecting H. annosum in DNA extracted from stem discs of two seedlings. Circles indicate DNA samples from stem discs at the inoculation points, lanes to the left and right contain PCR products from DNA samples extracted from discs below and above the inoculation point, respectively.”

Table 2. first column, instead of (number) shouldn’t this be (percent)?

Number deleted, and table text reworded to be more understandable.

Round 2

Reviewer 2 Report

The authors have addressed the main concerns I raised in the first version with respect to lack of specific numbers (of samples for example) and confusion between lesion and infection extent.