Detection of Root-Knot Nematode Meloidogyne luci Infestation of Potato Tubers Using Hyperspectral Remote Sensing and Real-Time PCR Molecular Methods
Round 1
Reviewer 1 Report
Proposed study described two innovative methods for detection of root gall nematode pest in potato tubers. Study was performed accurately and described precisely. Non destructive methods for detection of latent infestation with hyperspectral imaging would have a great impact on efficient control of spreading of the M. fallax and “ethiopica” group of root gall nematodes through planting material of potato.
Species differentiation by remote sensation method should be tested with other species of root-gall nematodes in next studies.
Author Response
Reviewer 1: We agree that other RKN species have to be included in further remote sensing studies. In fact, this is one of the goals of our current international project NemDetect (grant GP/EFSA/ALPHA/2018/02).
Reviewer 2 Report
Dear authors
Since real-time PCR is not species specific, what does this mean for the practitioner? Is the mere identification of a genus a sufficient signal to take action, e.g. select and destroy infested potato tubers? Do the different species of root-knot nematodes differ in terms of damage and hence economic losses? Are there species whose damage we can accept and the losses they cause? If so, species analysis would be very important. If, on the other hand, the mere detection of the genus Meloidogyne means that the farmer must take countermeasures, then diagnostics at the current level are quite sufficient. In this case, further biodiversity studies would only have a scientific aspect to increase our knowledge. In forestry, false positives are a problem. If we detect the quarantine species Bursaphelenchus xilophilus, the consequence is the destruction of seedlings or trees, so if we get a positive result for a related other species, we have a "false alarm" with negative consequences. Is there a similar risk with potatoes and their pests?Can hyperspectral imaging-based methods be less effective in detecting root knot nematodes in other potato varieties because of the shape of the tubers or the content of, for example, starch or secondary metabolites (e.g. alkaloids)? How do you rate such risks ... as low or very likely?
Author Response
Reviewer 2:
Answer to question 1 (molecular methods): The genus Meloidogyne spp. (root-knot nematodes) comprises more than 90 nominal species, four species have been recognised as quarantine pests by EPPO. Two quarantine pest species, M. chitwoody and M. fallax, are serious potato pests and drastic measures (e.g. destruction of plant material and sterilization of all equipment) are needed to prevent their spread. The real-time PCR approach developed here, enables to detect group-specific of three RKN species, namely M. luci, M. ethiopica and M. inornata. M. luci and M. ethiopica are both potato pests, besides both species are listed on the EPPO alert list of quarantine organisms. The information on M. inornata is so scarce however, as a sister species has the potential to cause similar damage on potato. No species specific molecular method to identify these species is available at the moment. It is therefore convenient to use such method in practice, to potentially detect and stop spreading of these pests. Anyway, we strongly agree with the reviewer that infested potato with any RKN species having great damage potential should be controlled, especially in seed potato production.
Answer to question 2 (hyperspectral imaging): Spectral signatures of plants are the effect of complex interactions between light and plant tissues, and viewing geometry. While we do not expect tuber shape to play a major role in detection accuracy, since viewing geometry can be accounted for in pre-processing, tuber chemical composition can affect detection reliability. The exact effect of nematode infestations of tuber chemical composition, e.g. which compounds or their amounts change, is currently unknown, and will be the subject of future research. The results of this study indicate that nematodes elicit specific changes in spectral signatures (e.g. the peak-dip-peak series at 1776, 1841, and 1890 nm, respectively). These might be used as a fingerprinting method to quickly detect infestations. But currently there is not enough available data and published research to confirm this. If these effects are truly specific, at least for RKNs, then the influence of potato variety on detection accuracy would be low. On the other hand, if these changes are not specific enough to enable the development of a fingerprinting method, then new classification models for each nematode species-potato variety would have to be developed. In fact, this is one of the goals of our current international project NemDetect (grant GP/EFSA/ALPHA/2018/02). With the currently available information, we would provide a conservative estimate, and rate these risks as moderately likely.
Reviewer 3 Report
In general, the title is interesting, but still, some notes must be fixed.
I wished you have found a way to detect nematodes before harvesting time; no worry, still a good title.
Comments have posted inside the pdf file,
Comments for author File: 
Comments.pdf
Author Response
Reviewer 3: The manuscript has been proofread by an English editor, and is written is UK English. Since the Editor of Remote sensing confirmed, that this UK English style is acceptable, we have chosen to keep this grammar style. The last paragraph of the introduction has been restructured and rewritten, and now clearly states the aims and hypotheses of this study. In line 224 the statement that the smoothing window was symmetrical is correct (the number of smoothing points to the left and right of each band was the same (i.e. 7). Smoothing window width is then calculated as: #pts-left + #pts-right + 1=15. An asymmetric window would not have the same number of points in both directions of each band). In line 258, comment: The possibility of early detection of RKN infestations on above-ground (i.e. pre-harvest) parts of plants using hyperspectral imaging has been proven in tomato plants (Susič, N. et al. 2018, 10.1016/j.snb.2018.06.121). Early detection of RKN infestations in potatoes are currently under study in the international project NemDetect (grant GP/EFSA/ALPHA/2018/02). In line 451 the term "a symptomatic tuber" is correct. The method is sensitive enough to reliably detect one symptomatic peel out of a hundred. The statement in lines 454-456 is correct. A female with an egg mass attached to it was added to uninfested potato peels. The reviewer’s suggested correction changes the meaning of this sentence, i.e. that the female with egg mass was attached to uninfested potato peels, which is not the case.
