Method for diagnosing the sulphur nutrition state of a plant

Abstract

The invention relates to a novel method for diagnosing the sulfur nutrition state of a plant by measuring the content of certain mineral nutrients in the leaves.

Claims

1. A method for diagnosing the sulfur nutrition state of a plant or plot, comprising the following steps: a) taking a leaf sample from the plant or taking a leaf sample representative of the plot; b) measuring the sulfur (S), chlorine (Cl) and phosphorus (P) content of the sample in mg/g; c) measuring the molybdenum (Mo) content of the sample in μg/g; d) calculating the Mo/S ratio and the (Cl+P)/S ratio; e) comparing with a reference Mo/S ratio and a reference (Cl+P)/S ratio of a plant or plot not suffering from sulfur deficiency, and/or with a reference Mo/S ratio and a reference (Cl+P)/S ratio of a plant or plot suffering from sulfur deficiency; and f) deducing the sulfur nutrition state of the plant or plot.

2. The method as claimed in claim 1, wherein the plant has been cultivated in an open field.

3. The method as claimed in claim 2, wherein: the plant or the plot is suffering from sulfur deficiency and the Mo/S ratio of the leaf sample is greater than the reference Mo/S ratio of a plant or of a plot suffering from sulfur deficiency; the plant or the plot is not suffering from sulfur deficiency and the Mo/S ratio of the leaf sample is less than the reference Mo/S ratio of a plant or of a plot not suffering from sulfur deficiency; or the plant or the plot has a risk of sulfur deficiency and the Mo/S ratio of the leaf sample is less than the reference Mo/S ratio of a plant or of a plot suffering from sulfur deficiency, and the Mo/S ratio of the leaf sample is greater than the reference Mo/S ratio of a plant or of a plot not suffering from sulfur deficiency.

4. The method as claimed in claim 2, wherein: the plant or the plot is suffering from sulfur deficiency and the Mo/S and (Cl+P)/S ratios of the leaf sample are respectively greater than the reference Mo/S and (Cl+P)/S ratios of a plant or of a plot suffering from sulfur deficiency, the plant or the plot is not suffering from sulfur deficiency and the Mo/S and (Cl+P)/S ratios of the leaf sample are respectively less than the reference Mo/S and (Cl+P)/S ratios of a plant or of a plot not suffering from sulfur deficiency, or the plant or the plot has a risk of S deficiency and the Mo/S and (Cl+P)/S ratios of the leaf sample are respectively less than the reference Mo/S and (Cl+P)/S ratios of a plant or of a plot suffering from S deficiency and respectively greater than the reference Mo/S and (Cl+P)/S ratios of a plant or of a plot not suffering from S deficiency.

5. The method as claimed in claim 2, wherein the plant or the plot is chosen from rapeseed, cabbage, tomato, corn, wheat, pea, globe amaranth, and barley.

6. The method as claimed in claim 2, wherein the plant or the plot analyzed for its sulfur nutrition state is from the same species as the plant or the plot not suffering from sulfur deficiency and/or as the plant or the plot suffering from sulfur deficiency.

7. The method as claimed in claim 2, wherein the leaf sample is dried and ground before steps b) and c).

8. The method as claimed in claim 2, wherein steps b) and c) are carried out by X-ray fluorescence spectrometry or by plasma mass spectrometry.

9. The method as claimed in claim 2, wherein steps b) and c) are carried out simultaneously.

10. The method as claimed in claim 2, wherein the plant or the plot is a plant of the species B. napus, B. oleracea, S. lycopersicum, Z. mays, T. aestivum, P. sativum, Arabidopsis thaliana, or Hordeum vulgare.

11. The method as claimed in claim 1, wherein: the plant or the plot is suffering from sulfur deficiency and the Mo/S ratio of the leaf sample is greater than the reference Mo/S ratio of a plant or of a plot suffering from sulfur deficiency; the plant or the plot is not suffering from sulfur deficiency and the Mo/S ratio of the leaf sample is less than the reference Mo/S ratio of a plant or of a plot not suffering from sulfur deficiency; or the plant or the plot has a risk of sulfur deficiency and the Mo/S ratio of the leaf sample is less than the reference Mo/S ratio of a plant or of a plot suffering from sulfur deficiency, and the Mo/S ratio of the leaf sample is greater than the reference Mo/S ratio of a plant or of a plot not suffering from sulfur deficiency.

12. The method as claimed in claim 1, wherein: the plant or the plot is suffering from sulfur deficiency and the Mo/S and (Cl+P)/S ratios of the leaf sample are respectively greater than the reference Mo/S and (Cl+P)/S ratios of a plant or of a plot suffering from sulfur deficiency; the plant or the plot is not suffering from sulfur deficiency and the Mo/S and (Cl+P)/S ratios of the leaf sample are respectively less than the reference Mo/S and (Cl+P)/S ratios of a plant or of a plot not suffering from sulfur deficiency; or the plant or the plot has a risk of S deficiency and the Mo/S and (Cl+P)/S ratios of the leaf sample are respectively less than the reference Mo/S and (Cl+P)/S ratios of a plant or of a plot suffering from S deficiency and respectively greater than the reference Mo/S and (Cl+P)/S ratios of a plant or of a plot not suffering from S deficiency.

13. The method as claimed in claim 1, wherein the plant or the plot is chosen from rapeseed, cabbage, tomato, corn, wheat, pea, globe amaranth, and barley.

14. The method as claimed in claim 1, wherein the plant or the plot analyzed for its sulfur nutrition state is from the same species as the plant or the plot not suffering from sulfur deficiency and/or as the plant or the plot suffering from sulfur deficiency.

15. The method as claimed in claim 1, wherein the leaf sample is dried and ground before steps b) and c).

16. The method as claimed in claim 1, wherein steps b) and c) are carried out by X-ray fluorescence spectrometry or by plasma mass spectrometry.

17. The method as claimed in claim 1, wherein steps b) and c) are carried out simultaneously.

18. The method as claimed in claim 1, wherein the plant or the plot is a plant of the species B. napus, B. oleracea, S. lycopersicum, Z. mays, T. aestivum, P. sativum, Arabidopsis thaliana, or Hordeum vulgare.

19. A method for adjusting the sulfur fertilization of a plant or of a plot, comprising the following successive steps: A) carrying out the method of diagnosis as claimed in claim 1 on the plant or the plot; and B) adding a sulfur-containing fertilizer if a sulfur deficiency or a risk of sulfur deficiency is detected during step A).

20. The method as claimed in claim 19, wherein a sulfur deficiency is detected when: the Mo/S ratio of the leaf sample is greater than the reference Mo/S ratio of a plant or of a plot suffering from sulfur deficiency; and the Mo/S and (Cl+P)/S ratios of the leaf sample are respectively greater than the reference Mo/S and (Cl+P)/S ratios of a plant or of a plot suffering from sulfur deficiency.

21. The method as claimed in claim 19, wherein a risk of sulfur deficiency is detected when: the Mo/S ratio of the leaf sample is (i) greater than the reference Mo/S ratio of a plant or of a plot not suffering from sulfur deficiency and (ii) less than the reference Mo/S ratio of a plant or of a plot suffering from sulfur deficiency; and the Mo/S and (Cl+P)/S ratios of the leaf sample are respectively (i) greater than the reference Mo/S and (Cl+P)/S ratios of a plant or of a plot not suffering from sulfur deficiency and (ii) less than the reference Mo/S and (Cl+P)/S ratios of a plant or of a plot suffering from sulfur deficiency.

Description

FIGURE LEGENDS

(1) FIG. 1: graph which represents the relative change (expressed as % relative to a plant not suffering from S deficiency) of the amount of several mineral nutrients (N, P, K, S, Ca, Mg, Fe, Cu, Zn, Mn, Mo, B and Na) in a B. napus plant suffering from S deficiency (i.e. without provision of sulfur). The plants were cultivated in a greenhouse and under hydroponic conditions. The amount of each element in a plant corresponds to the content of each element multiplied by the dry biomass of the whole plant.

(2) FIG. 2: graphs which represent: (A) the change in the amount of S in a B. napus plant suffering from S deficiency (−S) and in a B. napus plant not suffering from S deficiency (+S); (B) the change in the amount of Mo in a B. napus plant suffering from S deficiency (−S) and in a B. napus plant not suffering from S deficiency (+S); and (C) the change in the Mo/S ratio in a B. napus plant suffering from S deficiency (−S) and in a B. napus plant not suffering from S deficiency (+S). The plants were cultivated in a greenhouse and under hydroponic conditions. The amount of each element in a plant corresponds to the content of each element multiplied by the dry biomass of the whole plant.

(3) FIG. 3: graphs which represent the change in the S content (A and B), in the Mo content (C and D) and in the Mo/S ratio (E and F) in: a sample of leaf emerged before the application of a sulfur deficiency (A, C and E), and a sample of new leaf appearing during the sulfur deficiency (B, D and F)
of B. napus plants suffering from S deficiency (−S) and of B. napus plants not suffering from S deficiency (+S). The plants were cultivated in a greenhouse and under hydroponic conditions.

(4) FIG. 4: graph which represents the S content (A, B and C), the Mo content (D, E, F) and the Mo/S ratio (G, H and I) of the leaves in samples of leaves from B. napus cultivated under open field conditions (i.e. +/−S fertilization and +/−N fertilization, expressed in kg/ha). The leaf samples were harvested 15 days (A, D, G) or 47 days (B, C, E, F, H, I) after a sulfur fertilization.

(5) FIG. 5: graph which represents the SO.sub.4.sup.2− content (A, B and C), the (Cl.sup.−+NO.sub.3.sup.−+PO.sub.4.sup.3−) content (D, E and F) and the (Cl.sup.−+NO.sub.3.sup.−+PO.sub.4.sup.3−)/SO.sub.4.sup.2− ratio (G, H and I) of leaf samples in samples of leaf from B. napus cultivated under open field conditions (i.e. +/−S fertilization and +/−N fertilization, expressed in kg/ha). The leaf samples were harvested 15 days (A, D, G) or 47 days (B, C, E, F, H, I) after a sulfur fertilization.

(6) FIG. 6: graphs which show a correlation between the SO.sub.4.sup.2−, PO.sub.4.sup.3−, Cl.sup.− and NO.sub.3.sup.− content and the S, P, Cl and N content respectively. The graphs show a correlation between the SO.sub.4.sup.2−, PO.sub.4.sup.3−, Cl.sup.− and S, P, Cl content respectively (A, B and C), but an absence of correlation between NO.sub.3.sup.− and N (D).

(7) FIG. 7 graph which shows a correlation between the (Cl.sup.−+NO.sup.3−+PO.sub.4.sup.3−)/SO.sub.4.sup.2− ratio and the (Cl+P)/S ratio.

EXAMPLES

Example 1

Preparation of the Leaf Samples for the Laboratory Tests on B. napus

(8) Seeds of B. napus L. variety Boheme were germinated in demineralized water on perlite for 7 days in the dark and then 5 days in natural light. Just after the appearance of the first leaves, the sowings were placed in a greenhouse under hydroponic conditions, between October and December, at a temperature of 20° C. during the day and 15° C. at night.

(9) The natural light was supplemented with sodium lamps (Master Greenpower T400W, Philips, Amsterdam, The Netherlands) (350 μmol.Math.m.sup.−2.Math.s.sup.−1 of photosynthetically active radiation) for 16 hours a day.

(10) The nutritive solution for the sowings contained: 3.75 mM of KNO.sub.3, 0.5 mM of MgSO.sub.4, 0.5 mM of CaCl.sub.2, 0.25 mM of KH.sub.2PO.sub.4, 0.2 mM of EDTA-2NaFe, 14 μM of H.sub.3BO.sub.3, 5 μM of MnSO.sub.4, 3 μM of ZnSO.sub.4, 0.7 μM of CuSO.sub.4, 0.7 μM of (NH.sub.4).sub.6Mo.sub.7O.sub.24, 0.1 μM of CoCl.sub.2, 0.04 μM of NiCl.sub.2 and buffered at pH 6.6 with 0.91 mM of CaCO.sub.3. This solution was regularly renewed as a function of the disappearance of the NO.sub.3.sup.− in the solution in order to maintain optimal nourishment conditions. For this purpose, the amount of NO.sub.3.sup.− in the nutritive solution was measured using strips provided for this purpose (Merck Millipore, Darmstadt, Germany).

(11) After 4 weeks of growth, the plants were separated into two batches fed with a nutritive solution suitable for optionally inducing a sulfur deficiency while at the same time maintaining one and the same concentration for the other elements of the nutritive solution: Batch 1: Control plants (+S) cultivated with 508.7 μM of SO.sub.4.sup.2−, Batch 2: Sulfur-deprived plants (−S) cultivated with 8.7 μM of SO.sub.4.sup.2− (the presence of S is linked to the provision of SO.sub.4.sup.2− of the trace elements of the nutritive solution, this amount is negligible).

(12) These suitable nutritive solutions were also regularly renewed as a function of the disappearance of the NO.sub.3.sup.− in the nutritive solution (approximately every two days at the end of the experiment).

(13) The leaves present on day 0 (D0) were identified and labeled, and were considered to be “leaves emerged” before the application of the sulfur deficiency, while the leaves which appeared on the subsequent days were harvested separately and identified as “leaves appearing” after the application of the sulfur deficiency.

(14) Four independent samples were taken on day 0 (D0), on day 1, 2, 3, 7, 13, 21 and on day 28 (D28). Day 0 corresponds to the day starting from which the plants were fed with a suitable nutritive solution. The four samples taken on each of the days each correspond to four control plants (+S) or four sulfur-deprived plants (−S).

(15) The whole plants were then frozen in liquid nitrogen and stored at −80° C. for subsequent analysis. For each of the samples, a subsample of leaf was freeze-dried and ground to fine powder using a ball mill (MM400, Retsch, Haan, Germany). The powder thus obtained was used for measuring the content of the various mineral nutrients.

Example 2

Measurement of the Mineral Nutrient Content by Mass Spectrometry

(16) a) Measurement of the Sulfur (S) and Nitrogen (N) Content

(17) In order to measure the sulfur (S) and nitrogen (N) content, 4 mg of solids (SC) of powder were placed in tin capsules. The amount of S or N was determined with a continuous flow isotope ratio mass spectrometer (Nu Instruments, Wrexham, United Kingdom) coupled to a C/N/S analyzer (EA3000, Euro Vector, Milan, Italy). The total S content (S.sub.tot) or the total N content (N.sub.tot) at a time “t” was measured as follows:
S.sub.tot(or N.sub.tot)=[% S.sub.t(or N.sub.t)×SC.sub.t]×100

(18) b) Measurement of the Content of the Other Mineral Nutrients

(19) The measurement of the P, K, Ca, Mg, Fe, Cu, Zn, Mo, Mn, B, Ni and Na content in the leaf samples was carried out with a high-resolution plasma mass spectrometer (HR ICP-MS, Thermo Scientific, Element 2™, Bremen, Germany) after digestion of the powder prepared according to example 1 by an add and microwave treatment (Multiwave ECO, Anton Paar, Ies Ulis, France) using 800 μl of HNO.sub.3 (Thermo Fischer, Illkirch, France), 200 μl of H.sub.2O.sub.2 (SCP Science, Quebec, Canada) and 1 ml of Milli-Q water for 40 mg of solids (SC). Each leaf sample was supplemented with two internal standards, gallium and rhodium (SCP Science, Quebec, Canada), at a final concentration of 10 and 2 μg.Math.l.sup.−1, respectively, and diluted with Milli-Q water in order to obtain a solution containing 2.0% (v/v) of nitric acid. This solution was then filtered on a 40 μm Teflon filtration system (Courtage Analyses Services, Mont-Saint-Aignan, France). The mineral nutrient content was measured using standard curves prepared and verified with a certified reference of lemon tree leaves (CRM NCS ZC73018, Sylab, Metz, France). The mineral nutrient content was then calculated using the same formula as for S and N.

Example 3

Impact of a Sulfur Deficiency on the Mineral Nutrient Content

(20) The content of several mineral nutrients was measured for samples prepared according to example 1, after 21 days of sulfur deficiency (D21). The data are presented in the form of mean±standard deviation for the four samples of 4 plants. All the data were analyzed using the Student's test (Excel software) and marked with one or more asterisks when the difference is significant between the (+S) and (−S) samples (*P<0.05, **P<0.01, ***P<0.001). The results are presented in FIG. 1.

(21) As expected, after 21 days of S deficiency, the absorption of S is decreased by −100.7±0.90% compared with the (+S) plants. The absorption of other mineral nutrients was also decreased: N (−8.7±1.06%), K (−20.2±0.88%), Ca (−22.0±2.41%) and Na (−23.4±1.85%). The adsorption of B is, for its part, more greatly decreased (−52.9±2.82%). Conversely, the S deficiency greatly increases the absorption of Mo (+197.0±10.73%). The absorption of P, Mg, Fe, Cu, Zn and Mn is not affected by the S deficiency.

Example 4

Change in the Mo/S Ratio During an S Deficiency

(22) The S and Mo content of various tissues of the plants (leaves, roots) was measured for samples prepared according to example 1, on day 0 (D0), on day 1 (D1), 2, 3, 7, 13, 21 and on day 28 (D28). FIG. 2 presents the S or Mo content and the Mo/S ratio in the form of mean±standard deviation in four plants. All the data were analyzed using the Student's test (Excel software) and marked with one or more asterisks when the difference is significant between the (+S) and (−S) samples (*P<0.05, **P<0.01, ***P<0.001). The results are presented in FIG. 2.

(23) The amount of S of the sulfur-deprived (−S) plants remained at a basal level (FIG. 2A) indicating an absence of absorption of S. The amount of S of the control (+S) plants constantly increases over time and is significantly higher than for the sulfur-deprived (−S) plants starting from D3.

(24) Conversely, the amount of Mo of the sulfur-deprived (−S) plants and of the control (+S) plants increases over time, but to a higher level for the sulfur-deprived (−S) plants (FIG. 2B). For example, the amount of Mo is 2.23 times greater for the sulfur-deprived (−S) plants compared with the control (+S) plants on D28. As early as the first day, the amount of Mo of the sulfur-deprived (−S) plants is 28% greater than for the control (+S) plants. This difference becomes much more marked starting from D3.

(25) Consequently, the Mo/S ratio in the sulfur-deprived (−S) plants increases significantly starting from D1, and is approximately 21 times greater on D28 (FIG. 2C). The Mo/S ratio of the control (+S) plants does not increase over time.

(26) Conclusion: The Mo/S ratio makes it possible to significantly distinguish a plant suffering from sulfur deficiency and a plant not suffering from sulfur deficiency.

(27) Example 5

Change in the Mo/S Ratio During an S Deficiency for Various Types of Leaf

(28) The S and Mo content was measured for samples prepared according to example 1 on day 0 (D0), on day 1, 2, 3, 7, 13, 21 and on day 28 (D28), by distinguishing the “leaves emerged” before the sulfur deficiency and the “new leaves” appearing during the sulfur deficiency.

(29) It was shown that, regardless of the type of leaf analyzed, the S deficiency decreases the S contents but increases the Mo contents (FIGS. 3A-F). Consequently, the Mo/S ratio increases over time in the leaves of the plants suffering from sulfur deficiency (FIGS. 3E-F), this being regardless of the type of leaf analyzed.

(30) Conclusion: The Mo/S ratio of the leaves is increased very early on during a sulfur deficiency, regardless of the type of leaf.

Example 6

Change in the Mo/S Ratio after an S Fertilization and Impact of N Fertilization

(31) A field of rapeseed (B. napus), was divided into three distinct plots, each having a different S fertilization level, of 36, 12 or 0 kg S ha.sup.−1.

(32) Each of the plots was subdivided into three, each subdivision having a different N fertilization level (0, 65 or 125 kg N ha.sup.−1).

(33) Leaf samples were taken in each of the subdivisions. The S and Mo content was measured for each sample at 15 and 47 days after S fertilization.

(34) The S content was significantly reduced under the S deficiency condition, independently of the N fertilization level (65 or 125 kg N ha.sup.−1) (FIG. 4A-C). The highest Mo content was observed in the plants having received N fertilization without or with little S fertilization (0 or 12 kg S ha.sup.−1) (FIG. 4D-F). The Mo/S ratio made it possible to differentiate the plots as a function of their S fertilization level 0, 12 and 36 kg S ha.sup.−1 (FIG. 4G-I). Indeed, the plants with N fertilization (65 or 125 kg N ha.sup.−1) and without S fertilization had the highest Mo/S ratio, whereas the plants with S fertilization at 36 kg S ha.sup.−1 had the lowest Mo/S ratio. The plants with S fertilization at 12 kg S ha.sup.−1 had an intermediate Mo/S ratio.

(35) Interactions were observed between the N fertilization and the S fertilization. It may be assumed that the level of growth of the plants was significantly reduced in the absence of N fertilization. For example, 15 days after the S fertilization, the biomass of the mature leaves was 5.03±0.38 g SC leaf.sup.−1 in the non-N-fertilized plot, whereas the biomass in the N-fertilized plot with 65 kg N ha.sup.−1 was 6.70±0.01 g SC leaf.sup.−1 (p<0.01). This decrease in the growth of the non-N-fertilized plants had a direct impact on the S content given that the S requirements of the non-N-fertilized plants were not as great as for the N-fertilized plants. Indeed, the N-fertilized plants consumed more S to ensure their growth. Thus, the Mo/S ratio was significantly lower (FIG. 4G-I) in the absence of N fertilization, which reflects a lower S requirement for the non-N-fertilized plants.

(36) Likewise, it was observed that the Mo/S ratio was higher for the plants with the highest N fertilization level (i.e. 125 kg N ha.sup.−1) independently of the S fertilization level, which also reflects the higher S requirement of the N-fertilized plants.

(37) Conclusion: This example therefore shows that the Mo/S ratio of the leaves can also be used for plants cultivated under open field conditions. The Mo/S ratio can give early and long-lasting discrimination of the plants having variable S availabilities (provided in this example by an S fertilization).

Example 7

Reproducibility on Various Plant Species

(38) The Mo/S ratio was calculated on samples of leaf from various plant species: B. oleracea, T. aestivum, Z. mays, P. sativum and S. lycopersicum, under growth conditions in a greenhouse and during which the provision of sulfur was eliminated or reduced.

(39) For each of the species, under conditions of S deficiency, the S content was significantly reduced and the Mo content was significantly increased (Table 1, below).

(40) TABLE-US-00001 Number of days S (mg .Math. g.sup.-1 SC) Mo (ug .Math. g.sup.-1 SC) [Mol]:[S]Ratio Species of treatment +S −S +S −S +S −S B. napus 0 10.33 ± 0.46  154.57 ± 10.16 149.75 ± 7.34   1 10.30 ± 0.39  9.70 ± 0.56    175.35 ± 6.87  237.01 ± 7.700*** 170.23 ± 1.12  247.40 ± 18.67**  21 15.25 ± 0.39  2.33 ± 0.14*** 308.49 ± 14.02 617.82 ± 40.08*** 202.21 ± 6.52  2660.38 ± 91.28***  B. oleracea 20 4.83 ± 0.46 1.90 ± 0.04***  4.02 ± 0.21 5.03 ± 0.16**  8.44 ± 0.49 26.53 ± 1.16*** T. aestivum 2 3.60 ± 0.18 3.45 ± 0.22    90.17 ± 9.43 201.48 ± 7.72***  249.52 ± 18.17 584.45 ± 24.95*** 8 3.47 ± 0.06 3.12 ± 0.00**  85.14 ± 1.23 719.90 ± 36.62*** 245.32 ± 5.56  2306.22 ± 110.52*** 24 3.37 ± 0.02 1.85 ± 0.05*** 79.83 ± 2.08 977.11 ± 32.63*** 236.67 ± 5.58  5285.33 ± 236.13*** Z. mays 0 2.82 ± 0.10 22.62 ± 0.85 80.18 ± 1.54  5 3.05 ± 0.03 2.11 ± 0.13*** 24.67 ± 0.48 468.16 ± 28.15*** 80.86 ± 0.82 2224.10 ± 72.16***  18 2.39 ± 0.05 0.95 ± 0.04*** 27.95 ± 1.65 562.63 ± 34.46*** 116.67 ± 5.06  5934.41 ± 137.28*** P. sativum 0 2.75 ± 0.09 20.48 ± 4.22 73.76 ± 12.95 19 2.73 ± 0.10 2.20 ± 0.14*    9.43 ± 0.50 12.46 ± 0.34**  34.49 ± 0.67 57.63 ± 5.58**  S. lycopersicum 65 10.10 ± 0.71  1.83 ± 0.27***  2.86 ± 0.03 5.09 ± 0.82*   2.88 ± 0.21 27.90 ± 1.96***

(41) The results show that the Mo/S ratio was significantly increased under the S deficiency condition. The value of the Mo/S ratio was specific for the plant species analyzed, with values ranging from 3 to 250 for the reference Mo/S ratio (i.e. control or +S plant) and from 27 to 5934 for the plants suffering from S deficiency (i.e. −S).

(42) Conclusion: Mo/S ratio is a reliable indicator for diagnosing the sulfur nutrition state independently of the plants species under consideration.

Example 8

Impact of the Sulfur Deficiency on the Cl and P Content and Advantage of the (Cl+P)/S Ratio

(43) a) Analysis of the SO.sub.4.sup.2−, PO.sub.4.sup.3−, Cl.sup.− and NO.sub.3.sup.− Ions

(44) The ions were extracted from 30 mg of a ground and freeze-dried sample of leaf of B. napus cultivated in an open field with various N and S fertilization levels according to the modes of example 6.

(45) The ions were extracted from the sample according to the steps below: (i) in a first step, the leaf sample was placed in 1.5 ml of a concentrated ethanol solution at 50%, (ii) the solution obtained was then incubated at 40° C. for 1 hour, (iii) the solution was then centrifuged at 12000 g for 20 min, (iv) the supernatant was collected, (v) the pellet was resuspended in 1.5 ml of water and then incubated for one hour at 95° C., (vi) the centrifugation (iii) and collection (iv) steps were repeated a second time and the two supernatants obtained in step (iv) were mixed.

(46) The supernatants were then evaporated under vacuum (Concentrator Evaporator RC 10.22, Jouan, Saint-Herblain, France). The dry residue was redissolved in 1.5 ml of ultra pure water and was filtered through a 45 μm filter.

(47) The anion content was then measured by HPLC with a conductivity detector (ICS3000, Thermo Scientific-Dionex, Villebon-sur-Yvette, France) using a running buffer containing 4.05 mM Na.sub.2CO.sub.3 and 1.26 mM NaHCO.sub.3 and through an analytical column (AS22 4*250 mm, Thermo Scientific-Dionex, Villebon-sur-Yvette, France).

(48) b) Analysis of the Mineral Nutrients S, P, Cl and N

(49) After drying and grinding, leaf samples were placed in a dish and the S, P and Cl content was determined by X-ray fluorescence spectrometry (Portable XRF S1 Titan 800, Bruker, Kalkar, Germany).

(50) The S, P and Cl content was determined using calibration curves obtained by a high-resolution plasma mass spectrometer (HR ICP-MS, Thermo Scientific, Element 2™, Bremen, Germany).

Example 9

Interest and Rationale of Taking into Account the (Cl+P)/S Ratio

(51) a) Interest of the (Cl.sup.−+NO.sup.3−+PO.sub.4.sup.3−)/SO.sub.4.sup.2− Ratio

(52) A field of rapeseed was divided into three distinct plots, each having been fertilized with S on D0 (i.e. first day of S fertilization) with various amounts of S, of 36, 12 or 0 kg S ha.sup.−1.

(53) The three plots were subdivided into three, each subdivision having a different N fertilization level, 0, 65 or 125 kg N ha.sup.−1.

(54) Leaf samples were taken in each of the subdivisions (i.e. in the 6 subdivisions). The SO.sub.4.sup.2−, Cl.sup.−, NO.sub.3.sup.− and PO.sub.4.sup.3− content and the (Cl.sup.−+NO.sub.3.sup.−+PO.sub.4.sup.3−)/SO.sub.4.sup.2− ratio were measured in the samples at 15 (D15) and 60 (D60) days after the S fertilization (or non-S-fertilization for the two subdivisions having an S fertilization level of 0 kg S ha.sup.−1).

(55) The SO.sub.4.sup.2− content in the leaf samples was significantly reduced under the low S fertilization condition (FIG. 5a-c), independently of the N fertilization level. The decrease in the SO.sub.4.sup.2− content was compensated for by an increase in the Cl.sup.−, NO.sub.3.sup.− and PO.sub.4.sup.3− content (FIG. 5d-f). The lowest (Cl.sup.−+NO.sub.3.sup.−+PO.sub.4.sup.3−) content was measured in the leaf samples from the plants fertilized with the highest dose of S (i.e. 36 kg S ha.sup.−1). The leaf samples from the plants fertilized with N (65 or 125 kg N ha.sup.−1), without S fertilization, had the highest (Cl.sup.−+NO.sub.3.sup.−+PO.sub.4.sup.3−)/SO.sub.4.sup.2− ratio. The leaf samples from the plants fertilized with 36 kg S ha.sup.−1 of S had the lowest (Cl.sup.−+NO.sub.3.sup.−+PO.sub.4.sup.3−)/SO.sub.4.sup.2− ratio. The leaf samples from the plants fertilized with 12 kg S ha.sup.−1 of S had an intermediate (Cl.sup.−+NO.sub.3.sup.−+PO.sub.4.sup.3−)/SO.sub.4.sup.2− ratio. The (Cl.sup.−+NO.sub.3.sup.−+PO.sub.4.sup.3−)/SO.sub.4.sup.2− ratio therefore made it possible to differentiate the three plots as a function of their S fertilization level (36, 12 or 0 kg S ha.sup.−1) (FIG. 5G-I).

(56) Interactions were observed between the N fertilization and the S fertilization. It may be assumed that the level of growth of the plants was significantly reduced in the absence of N. For example, on D15, the biomass of the leaves was 5.03±0.38 g SC leaf.sup.−1 for the non-N-fertilized plants, whereas the biomass of the plants fertilized with 65 kg N ha.sup.−1 was 6.70±0.01 g SC leaf.sup.−1 (p<0.01). This decrease in the growth of the non-N-fertilized plants had a direct impact on the SO.sub.4.sup.2− content given that the SO.sub.4.sup.2− requirements of the non-N-fertilized plants were lower than for the N-fertilized plants. Indeed, the plants fertilized with N used more SO.sub.4.sup.2− to ensure their growth. For example, for one and the same S fertilization level, the SO.sub.4.sup.2− content in the leaf samples from the plants without N fertilization was significantly higher than (FIGS. 5A and C) or similar to (FIG. 5B) the content in the leaf samples from the plants receiving an N fertilization.

(57) However, the (Cl.sup.−+NO.sub.3.sup.−+PO.sub.4.sup.3−) content in the leaf samples from the plants with N fertilization and without S fertilization was unchanged compared with the (Cl.sup.−+NO.sub.3.sup.−+PO.sub.4.sup.3−) content of the leaf samples from the plants without N fertilization (FIG. 5d-f). Consequently, the (Cl.sup.−+NO.sub.3.sup.−+PO.sub.4.sup.3−)/SO.sub.4.sup.2− ratio was significantly decreased (FIGS. 5g and i) or was similar (FIG. 5h) in the absence of N fertilization, which reflects a lower S requirement.

(58) Similarly, the growth of the plants was further stimulated with an N fertilization of 125 kg N ha.sup.−1, which had the effect of increasing the S requirements and of reducing the SO.sub.4.sup.2− content in the leaves (p<0.05). This observation is illustrated in FIGS. 5b and c.

(59) It was also observed, by comparing FIGS. 6h and 6i, that the (Cl.sup.−+NO.sub.3.sup.−+PO.sub.4.sup.3−)/SO.sub.4.sup.2− ratio was increased for the plants having received the highest dose of N (i.e. 125 kg N ha.sup.−1), which reflects a higher S requirement for these plants.

(60) The (Cl.sup.−+NO.sub.3.sup.−+PO.sub.4.sup.3−)/SO.sub.4.sup.2− ratio was also calculated on leaf samples from other species cultivated in an open field (B. oleracea, T aestivum, Z. mays, M. truncatula, S. lycopersicum and B. napus) under controlled fertilization conditions, with S fertilization (+S) or sulfur deficiency (−S). The results are presented in Table 2 below.

(61) TABLE-US-00002 (Cl.sup.- + NO.sub.3.sup.- + PO.sub.4.sup.3-) [(Cl.sup.-] + [NO.sub.3.sup.-] + Number of days SO.sub.4.sup.2- (mg .Math. g.sup.-1 SC) content (mg .Math. g.sup.-1 SC) [PO.sub.4.sup.3-]):[SO.sub.4.sup.2-] Ratio Species of treatment +S −S +S −S +S −S B. napus 0 8.13 ± 0.61 56.79 ± 7.37  2.34 ± 0.28 3 8.73 ± 0.41 5.30 ± 0.48*** 63.65 ± 0.96 70.86 ± 5.68    2.44 ± 0.11 4.53 ± 0.39** 13 9.95 ± 0.17 2.68 ± 0.16*** 62.83 ± 5.86 86.42 ± 4.66**  2.11 ± 0.20 10.74 ± 0.17*** B. oleracea 135 7.05 ± 1.22 0.35 ± 0.04***  44.9 ± 6.85 61.87 ± 3.27*   6.60 ± 1.06 192.24 ± 40.89**  T. aestivum 0 3.03 ± 0.09 63.58 ± 0.80 21.02 ± 0.76 8 0.94 ± 0.05 1.15 ± 0.03**  59.34 ± 2.10 68.04 ± 0.84** 30.54 ± 0.53 59.60 ± 2.36*** 16 2.17 ± 0.06 0.50 ± 0.05*** 64.79 ± 1.29 70.19 ± 1.80*  30.01 ± 1.28 146.03 ± 15.81*** Z. mays 0 2.02 ± 0.49 74.74 ± 1.55 42.15 ± 7.16 5 3.44 ± 0.07 1.39 ± 0.08*** 78.31 ± 2.82 91.71 ± 1.28** 22.83 ± 1.09 66.49 ± 3.45*** 18 1.38 ± 0.05 0.27 ± 0.02*** 70.38 ± 3.46 103.28 ± 0.79*** 51.01 ± 2.19 384.67 ± 23.44*** S. lycopersicum 75 63.75 ± 4.79  50.39 ± 2.42*   68.84 ± 4.61 84.77 ± 2.44*   1.11 ± 0.15 1.69 ± 010**  M. truncatula 0 3.31 ± 0.16 25.32 ± 0.60  7.68 ± 0.21 8 3.60 ± 0.12 0.15 ± 0.03*** 25.40 ± 1.54 34.27 ± 3.44*   7.07 ± 0.48 231.78 ± 25.21*** 21 4.75 ± 0.18 0.26 ± 0.01*** 25.92 ± 1.94 34.90 ± 2.46*   5.49 ± 0.50 133.04 ± 12.48***

(62) The results show that the (Cl.sup.−+NO.sub.3.sup.−+PO.sub.4.sup.3−)/SO.sub.4.sup.2− ratio was significantly increased under the S deficiency condition regardless of the species. The values for the (Cl.sup.−+NO.sup.3−+PO.sub.4.sup.3−)/SO.sub.4.sup.2− ratio are specific for the plant species analyzed.

(63) Conclusion: The decrease in the SO.sub.4.sup.2− content is compensated by an increase in the (Cl.sup.−+NO.sub.3.sup.−+PO.sub.4.sup.3−) content, and therefore an increase in the (Cl.sup.−+NO.sub.3.sup.−+PO.sub.4.sup.3−)/SO.sub.4.sup.2− ratio. Consequently, the (Cl.sup.−+NO.sub.3.sup.−+PO.sub.4.sup.3−)/SO.sub.4.sup.2− ratio is an indicator which makes it possible to diagnose the sulfur nutrition state of a plant, independently of the plant species under consideration.

(64) b) Validation of the (Cl+P)/S Ratio

(65) In order to facilitate the implementation of the invention, a simplification of the calculation of the (Cl.sup.−+NO.sub.3.sup.−+PO.sub.4.sup.3−)/SO.sub.4.sup.2− ratio was sought.

(66) Firstly, it was shown, on the rapeseed leaf samples, that the content of Cl.sup.−, NO.sub.3.sup.−, PO.sub.4.sup.3− and SO.sub.4.sup.2− ions correlated with the Cl, N, P and S content, respectively.

(67) FIG. 6 shows a correlation between the content of Cl.sup.−, NO.sub.3.sup.−, PO.sub.4.sup.3− and SO.sub.4.sup.2− ions (measured by HPLC) and the Cl, N, P and S content (measured by X-ray fluorescence spectrometry). The Cl.sup.−, PO.sub.4.sup.3− and SO.sub.4.sup.2− content correlates strongly with the Cl, P and S content (FIG. 6a-c) with correlation coefficients of 0.97, 0.70 and 0.91, respectively.

(68) Conversely, no correlation between the NO.sub.3.sup.− content and the N content was observed (FIG. 6d).

(69) Subsequently, it was shown that the (Cl.sup.−+NO.sub.3.sup.−+PO.sub.4.sup.3−)/SO.sub.4.sup.2− ratio correlated significantly with the (Cl+P)/S ratio (FIG. 7), with a correlation coefficient of 0.94. FIGS. 8b and 8c show that this correlation is independent of the developmental stage of the plant.

(70) Conclusion: The (Cl+P)/S ratio is a reliable indicator which is easy to implement for diagnosing the sulfur nutrition state of a plant.