Rhizosphere Acidification as the Main Trait Characterizing the Differential In Vitro Tolerance to Iron Chlorosis in Interspecific Pyrus Hybrids

Round 1

Reviewer 1 Report

In this study, the authors investigated the main trait differential of interspecific Pyrus hybrids in vitro tolerance to iron chlorosis. The study focused on physiological responses of different interspecific Pyrus hybrids under in vitro culture conditions simulating lime induced chlorosis. However, there are some problems in this manuscript.

In the Introduction, the line 95-96: I suggest you add some description of the parents of the hybrid. What characteristics of these varieties are adapted to iron-deficiency conditions？Why did you choose a cross line of these varieties? Do they have a consistent phenotype? If there are differences in their stress resistance mechanisms then whether the differences in their hybrids are comparable？

In the Materials and methods, line 118-120, please add a table for these descriptions, along with parental information. Provide some photos of experimental results, including culture, treatment, symptom etc. Can experimental treatments (10 or 20mM NaHCO₃) induce chlorosis in plants? There is no data on roots and plant iron nutrition.

In the Results, data for different hybrids should be listed separately, rather than assuming they are not different in the line chart. We can't see the difference between different hybrids in the picture you provided. In addition, the pictures need to be beautified. Please redescribe the data to reflect, as far as possible, the variation of the different hybrids directly over time.

In addition, in the References, line 389-390 and line 388, it is close but not completely correct. Please check the format.

Author Response

Thanks for your comments and appreciation of the work done.

With the objective of giving an answer to the problems you commented in your review:

In the Introduction, the line 95-96: I suggest you add some description of the parents of the hybrid. What characteristics of these varieties are adapted to iron-deficiency conditions？Why did you choose a cross line of these varieties? Do they have a consistent phenotype? If there are differences in their stress resistance mechanisms then whether the differences in their hybrids are comparable?

From line 93 to 101 the following has been added

of their incompatibility with some varieties and their susceptibility to calcareous soils. Pyrus rootstocks have a better tolerance to these soils, are fire blight tolerant and compatible with pear varieties. A breeding programme using interspecific hybridization between ‘Pyriam’ and diploïds Pyrus species (Simard M.H. et al. 2011) has been developed. A part of this programme (Simard M.H. 2009) dealing with higher tolerance to global warming is done in collaboration with IRTA and is under development to improve the tolerance to lime induced chlorosis in Spain (Asin et al., 2011; Dolcet-Sanjuan et al., 1992). Four mediterranean species belonging to INRAE Angers genetics ressources have been choosen for their tolerance to droughtness, hot summer and to iron chlorosis (Lombard, P.B. and Westwood, M.N., 1987).

In the Materials and methods, line 118-120, please add a table for these descriptions, along with parental information. Provide some photos of experimental results, including culture, treatment, symptom etc. Can experimental treatments (10 or 20mM NaHCO₃) induce chlorosis in plants? There is no data on roots and plant iron nutrition.

from line 125 to 129  There was a mistake  writing OH111 instead of OH11 so I have change in Pyriam : it is its real international name of its COV.

Two clones (coded 74 and 170) were ‘Pyriam’ x Pa hybrids, eight clones (coded 66, 101, 103, 105, 109, 123, 129, and 162) were ‘Pyriam’  x Pap hybrids, seven clones (coded 25, 36, 38, 45, 66, 87 and 88) were ‘Pyriam’ x Pcc hybrids, and three clones (coded 55, 58 and 111) were ‘Pyriam’ x Pe hybrids. In addition, a clone (coded 12) derived from open pollinated P. communis “Williams” (Pc) was included.

Figure 1 has been added, to give more detail on the experimental conditions and the differencial chlorosis symptoms of some clones.

In the Results, data for different hybrids should be listed separately, rather than assuming they are not different in the line chart. We can't see the difference between different hybrids in the picture you provided. In addition, the pictures need to be beautified. Please redescribe the data to reflect, as far as possible, the variation of the different hybrids directly over time.

As indicated in the text of the chard, when statistical significance is not found among clones, they are shown as a line in the chard. Besides, showing all individual hybrids would make difficult to see differences.

In addition, in the References, line 389-390 and line 388, it is close but not completely correct. Please check the format.

The volum has been added to the reference.

from line 439 to 440, and additional reference has been added

Simard M.H., Guisnel R., Demilly D., Billy B., Honoré D. and Daguin F., 2011. Is dwarfing in pear rootstocks due to aneuploïd genetic structures? XIth ISHS Pear Symposium Patagonia (Argentina), 23-26 Nov. 2010. Acta Hort. 909,1,59-66.

Reviewer 2 Report

I have some problems with clarity of descriptions,  in the Material and methods and Result parts.

As I understand the authors examined the effects for 20 clones -hybrids of 4 Prunus species with higher iron-deficiency tolerance and one clone- P. communis “Williams” (Pcw), when different  amounts of CaHCO3 in medium with  2 μM Fe3+DTPA were applied

Why did the authors name similarity in changes in chlorophyl content between of hybrids when they are presented as one group, how it has been compared? (Fig 1A). In the Fig. 1A they compared 4 hybrids with Pcw clone, when in the Fig 1B the model of comparison is different: one of the resistant to Fe-deficiency - hybrid Pcc was compared with the rest of hybrids together with Pcw clone. System of comparisons is not uniform. Why the authors decided for such statistical comparisons? For example, how many replicates of the samples have been obtained  in the first group- created by 20 clones and the second- created by one clone in the Fig. 1A? It is a question about the relevancy of such comparison. For me each hybrid should be treated as a group for greater transparency.

The same comment is addressed to Fig 3A and 3B, Fig. 5A and 5B and Fig 7. Especially, that the authors wrote  in the line 340 that ”Pyrus communis Williams was the genotype used as control in the treatments …”.

The goal of this study the authors presented  in the statement: “Similar in vitro culture conditions, were used in the present work to first, study the differential physiological responses of Pyrus interspecific hybrids to lime-induced chlorosis, and second, simplify and shorten  the in vitro selection protocol for future selection of seedlings or clones.” But, of course it is difficult at this stage of the project to write about plant selection, especially seedlings.

It is better to extend the discussion also to other plants for better understanding the mechanisms analysed by the authors, adding some citations, like  V. D. Jolley, K. A. Cook, N. C. Hansen, W. B. Stevens. (1996) Plant physiological responses for genotypic evaluation of iron efficiency in strategy I and strategy II plants—A review. Journal of Plant Nutrition 19, 1241-1255 or Javier Abadía, Ana Álvarez-Fernández, Adamo D. Rombolaà, Manuel Sanz, Massimo Tagliavini & Anunciación Abadía (2004) Technologies for the diagnosis and remediation of Fe deficiency, Soil Science and Plant Nutrition, 50, 965-971.

Author Response

Thanks for your comments and appreciation of the work done.

With the objective of giving an answer to the problems you commented in your review:

As I understand the authors examined the effects for 20 clones -hybrids of 4 Prunus species with higher iron-deficiency tolerance and one clone- P. communis “Williams” (Pcw), when different  amounts of CaHCO3 in medium with  2 μM Fe3+DTPA were applied

NaHCO3 instead of CaHCO3

Why did the authors name similarity in changes in chlorophyl content between of hybrids when they are presented as one group, how it has been compared? (Fig 1A). In the Fig. 1A they compared 4 hybrids with Pcw clone, when in the Fig 1B the model of comparison is different: one of the resistant to Fe-deficiency - hybrid Pcc was compared with the rest of hybrids together with Pcw clone. System of comparisons is not uniform. Why the authors decided for such statistical comparisons? For example, how many replicates of the samples have been obtained  in the first group- created by 20 clones and the second- created by one clone in the Fig. 1A? It is a question about the relevancy of such comparison. For me each hybrid should be treated as a group for greater transparency.

When presented as a group, as indicated in the graphs legends, statistical significances were found between species and not between clones within the species. When two species are together is because no statistical significand was observed between them. The replicate of the samples are indicated in the materials and methods (line 198-202). Representing the results for each hybrid is showing too much data in the graph, and is not clarifying the message. When statistics indicates there is no difference among hybrids, is much cleared pooling them together.

The same comment is addressed to Fig 3A and 3B, Fig. 5A and 5B and Fig 7. Especially, that the authors wrote  in the line 340 that ”Pyrus communis Williams was the genotype used as control in the treatments …”.

The sentence has been removed from the paragraph, because we agree that it gives redundant information

The goal of this study the authors presented  in the statement: “Similar in vitro culture conditions, were used in the present work to first, study the differential physiological responses of Pyrus interspecific hybrids to lime-induced chlorosis, and second, simplify and shorten  the in vitro selection protocol for future selection of seedlings or clones.” But, of course it is difficult at this stage of the project to write about plant selection, especially seedlings.

The work started from seedlings which were cloned in vitro in order to select the best adapted to iron chlorosis. The work gives information about the approaches in which the Pyrus species are more relevant to overcome lime induced chlorosis. Of course, the future selected clone has to include other agronomic characters after been cloned, but the present work indicate that the acidification of the rhizosphere, specially for seedlings derived from Pcc, is and a good character to look for.

It is better to extend the discussion also to other plants for better understanding the mechanisms analysed by the authors, adding some citations, like. V. D. Jolley, K. A. Cook, N. C. Hansen, W. B. Stevens. (1996) Plant physiological responses for genotypic evaluation of iron efficiency in strategy I and strategy II plants—A review. Journal of Plant Nutrition 19, 1241-1255. Javier Abadía, Ana Álvarez-Fernández, Adamo D. Rombolaà, Manuel Sanz, Massimo Tagliavini & Anunciación Abadía (2004) Technologies for the diagnosis and remediation of Fe deficiency, Soil Science and Plant Nutrition, 50, 965-971.

Instead we have cited:

Römheld, V. and Marschner, H., 1986. Mobilization of iron in the rhizosphere of different plant species., p. 155-204. In: Tinker, A. and Läuchli, A. (eds.), Advances in Plant Nutrition. Praeger Scientific, New York, USA.

Gogorcena, Y., Abadía, J., and Abadía, A., 2004. A new technique for screening iron-efficient genotypes in peach rootstocks: elicitation of root ferric chelate reductase by manipulation of external iron concentrations. Journal of Plant Nutrition 27:1-15.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.