USE OF ALGINATES OLIGOMERS TO IMPROVE PLANT PROTECTION AGAINST PATHOGENS

Abstract

The invention concerns the use of alginate oligomers in sequence α(1-4)Guluronane and/or β(1-4)Mannuronane having a DP greater than 30, as an active ingredient of biocontrol products in plants, and that may be combined with another molecules such as phytohormones, against cryptogamic diseases of plants, in particular of vine (Vitis vinifera), or to protect other species against aggressors such as pathogenic microbes or insects.

Claims

1. Use of alginate oligomers in sequence α(1-4)Guluronane and/or β(1-4)Mannuronane having a DP greater than 30, as active ingredient, to enhance plant protection against pathogens.

2. Use according to claim 1, wherein the alginate oligomers are combined with a phytohormone.

3. Use according to claim 2, wherein the phytohormone is chosen from salicylate and its derivatives, jasmonate and its derivatives, pipecolic acid, and beta amino butyric acid.

4. Use according to claim 3 wherein the phytohormone derivatives are chosen from methyl salicylate, methyl glucose salicylate, methyl jasmonate, and jasmonate isoleucine.

5. Use according to claim 1, wherein the alginate oligomers in sequence are 1,4 β-D-mannuronane and/or 1,4 α-L-guluronane oligomers.

6. Use according to claim 1, wherein pathogens are chosen from fungi, oomycetes, bacteria, virus.

7. Use according to claim 6, wherein the oomycete is Plasmopara viticola.

8. Use according to claim 1, wherein the plant is vine.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0018] FIGS. 1A and 1B represents the sporulation and the protection induced by alginate oligomers, having a degree of polymerization higher than 30, against downy mildew (P. viticola) of the vine (Vitis vinifera). Plants were sprayed with A1 (DP 491), A2 (DP 228), and A3 (DP 355) at 1 g/L, 2 g/L, and 4 g/L, or water (negative control). The first and second leaves from the top of plants were inoculated with P. viticola (10.sup.4 sporangia mL.sup.−1) at 48 hpt. At 6 dpi, photographs of sporulating leaf disks were analyzed by the Visilog software. ANOVA was used for data analyses and Tukey's HSD test was used for lettering at 0.05% threshold. Conditions with different letters are significantly different.

[0019] FIGS. 2A and 2B represent the infection index and the protection induced by alginate oligomers having a degree of polymerization higher than 30, against powdery mildew (E. necator) of the vine (Vitis vinifera). Plants were sprayed with A1 (DP 491), A2 (DP 228), and A3 (DP 355) at 1 g/L, 2 g/L, and 4 g/L, or water (negative control). The first and second leaves from the top of plants were inoculated with E. necator (10.sup.5 conidies.mL.sup.−1) at 48 hpt. At 15 dpi, symptoms were quantified by using a score scale from 0 (healthy leaf) to 3 (large spot). ANOVA was used for data analyses and LSD Fischer's test was used for lettering at 0.05% threshold. Conditions with different letters are significantly different.

[0020] FIG. 3 represents the protection induced by alginate oligomers having a degree of polymerization higher than 30 against Zymoseptoria tritici of the wheat (Vitis vinifera). ANOVA was used for data analyses and LSD Fischer's test was used for lettering at 0.05% threshold. Conditions with different letters are significantly different.

[0021] FIG. 4 represents Scanning Electron Microscopy (SEM) observations of leaf surfaces after treatment with alginates which act as a physical barrier. Top line of pictures show leaf treated with adjuvant as negative control and the lower line show leaf treated with the alginate ALG/M (5 g/L).

[0022] FIG. 5 represents the vine protection (Vitis vinifera) against downy milddew (P. viticola) induced by alginate oligomers used in combination with pipecolic acid, a regulator of plant immunity. M indicates the combination with the alginate A1 1 g/L and acid pipecolic 0.1 mM.

[0023] FIG. 6 represents the expression level of the PR2 gene encoding 1,3 β-D-glucanase in the vine under different conditions.

EXAMPLES

Example 1: Protection Induced by Alginate Oligomers Against Downy Mildew (P. Viticola) and Powdery Mildew (Erysiphe Necator) of the Vine (Vitis Vinifera)

[0024] 1. Plant Material

[0025] Grapevine (V. vinifera cv. Marselan) herbaceous cuttings were grown in individual pots (10×7×7 cm) containing a mixture of peat and perlite (4:1, vol/vol) in a greenhouse at 25±4 and 18±7° C. (day and night, respectively) until they developed 6-8 leaves. Artificial illumination was supplemented when the natural light was less than 200 μmol. m..sup.−2 s.sup.−1 during the 16 h light period. Plants were watered with a fertilization solution (Topfert2 Plantin. France).

[0026] 2. Treatment with Alginates

[0027] Alginates A1, A2, A3 with a degree of polymerization (DP) of 491, 228, 355 respectively were used. They were prepared at 1, 2 and 4 g/L in ultrapure water and applied to both the upper and lower faces of leaves until the point of run-off using a manual sprayer. Plants were maintained in the greenhouse in the conditions described above. Ultrapure water was used as negative control and sprayed in the same manner.

[0028] 3. Plant Inoculation and Disease Assessment

For Downy Mildew Disease:

[0029] A P. viticola isolate was maintained in the greenhouse on cv. Marselan plants as previously described (Trouvelot et al., 2008) [13]. Sporangia were collected from sporulating leaves using a brush and suspended in distilled water at a concentration of 10.sup.4 sporangia.mL.sup.−1. Inoculation was performed 48 hours post-treatment (hpt) with alginates by spraying the freshly prepared downy mildew suspension onto the lower face of the leaf. Plants were then placed overnight in a humid chamber (relative humidity of 100%), and then transferred back to the greenhouse, in the conditions described above.

[0030] Briefly, 6 days post-inoculation (dpi), leaf disks were punched out and placed with the abaxial side uppermost, on a moist Whatman paper, in a closed plastic box. This system was left overnight in darkness and saturated relative humidity to trigger sporulation. Disease intensity was assessed by measuring the leaf area covered by the pathogen sporulation using a “macro” developed for the image analysis Visilog 6.9 software (Noesis, France; Kim Khiook et al., 2013) [14] and the disease reduction rate is calculated as follows: (1−(treated sporulating area/control sporulating area))×100. Forty-eight disks from 4 plants were used per condition. Two independent experiments were performed.

[0031] As shown in FIG. 1A, whatever the concentration of alginates used, A1, A2 and A3 having a DP higher than 30 greatly reduce the sporulation of P. viticola on leaves of vine plants grown in greenhouse. At 4 g/L, these alginates thus confer a strong phyto-protection about 90% (FIG. 1B).

For Powdery Mildew Disease:

[0032] Alginates treatment for powdery mildew were also performed 48 hours before inoculation. A concentration of 10.sup.5 conidies.mL.sup.−1 was used to inoculate the lower and upper faces of leaves. Inoculated plants were then kept in greenhouse with 25/18° C. day/night temperatures and an 18-h photoperiod. Alginates effects were evaluated 15 day after inoculation by visually assigning scores to leaf symptoms (0=healthy leaf; 1=some points; 2=non-contiguous spots; 3=large spots, contiguous or not). Experiment was carried out once.

[0033] FIG. 2A shows that the alginates confer effective protection against powdery mildew disease. At 4 g/L, this protection can reach 75% about (FIG. 2B).

Example 2: Protection Induced by Alginate Oligomers Against Septoria (Zymoseptoria Tritici) of Wheat (Triticum Aestivum)

[0034] 1. Plant Material and Treatment with Alginates

[0035] Wheat (Triticum aestivum cv. Alixan) plants have been cultivated under greenhouse until the third leaf of the plants was fully developed (for 4 weeks about). Treatments with alginates A1 (DP 491), A2 (DP 228), and A3 (355) were carried out 48-hours before inoculation on leaves by using a manual sprayer. Distilled water with 0.05% of Tween 20 has been used as negative control and solvent for alginate preparations. There were 12 plants per pot and 3 pots per modality.

2. Plant Inoculation and Disease Assessment

[0036] Forty-eight hours after treatment plants were inoculated with a suspensions of Z. tritici at 10.sup.6 spores.mL.sup.−1 (using a similar sprayed) and then kept in greenhouse at 16 h/8 h day/night photoperiod and at 20° C. for 4 weeks, until symptoms appear on the leaves.

[0037] Disease severity was determined 21 dpi by evaluating the percentage of the surface of infected leaves (3.sup.e leaf) bearing pycnidia. Pycnidia are the fructification organs of the fungus Z. tritici.

[0038] FIG. 3 shows effect induced by the alginates for one assay. As indicated, the alginates provide an effective protection (excepted A3 at 1 g/L) against Septoria disease. The alginates are effective when the percentage of lesion is low.

Example 3: Alginate Oligomers Act as Physical Barrier Against Pathogens

[0039] Forty-eight hours after the treatment with the alginate ALG/M (5 g/L) or by an adjuvant 0.05% (as negative control), leaves of vine were collected and the abaxial leaf surface was then characterized by cryo-scanning electron micrographs(cryo-SEMs) using a Hitachi (SU 8230) scanning electron microscope equipped with Quorum PP3000 T cryo attachment.

[0040] FIG. 4 shows, only in alginate condition, the presence of dried deposits which act as physical barriers around the sites of a pathogen attack (stomata in the case of P. viticola).

Example 4: Alginate Oligomers Used in Combination with a Regulator of Plant Immunity Improve Vine Protection Against Downy Mildew

[0041] Plant vines were treated with the alginate A1 (at 1 g/L), pipecolic acid (Pip; at 0.1 mM), or the combination of both molecules. Pipecolic acid is a small metabolite known to regulate plant immunity. The production of plant material, the treatment, inoculation and evaluation of disease steps were performed as explained before (c.f. Example 1 part 1 to 3).

[0042] The FIG. 5 shows the protection of vine induced by the two types of molecules applied alone or in combination against downy mildew (P. viticola). As indicated, the rate protection against P. viticola is around 35, 62 and 86% for Pip 0.1 mM, A1 and Pip 0.1 mM+A1 respectively. So, the combination between A1 and Pip 0.1 mM improve the rate protection in comparison with those obtained when molecules (A1 and Pip 0.1 mM) are applied alone.

Example 5: Pr2 Gene Expression Analyses by Qrt-PCR in the Vine (Vitis Vinifera) Treated by Alginates G/M

[0043] 1. Plant Material (as Described in Example 1)

[0044] 2. Treatments

[0045] The alginate ALG/M (from Laminaria japonica) was prepared at 5 mg.Math.mL.sup.−1 in distilled water with 0.05% surfactant and applied to both the upper and lower faces of leaves until the point of run-off using a manual sprayer. Plants were maintained in the greenhouse in the conditions described above. The sulphated laminarin PS3 (2.5 mg.Math.mL.sup.−1; positive control), water and the surfactant alone (negative controls) were sprayed in the same manner.

[0046] 3. Molecular Biology Experiments

[0047] Treated leaves from 3 plants per condition (water, surfactant S, PS3+S, ALGM+S, and ALG/M—treatment as described above) were collected at different time points [48, 72 and 96 hpt respectively)] and immediately frozen in liquid nitrogen. Total RNA was extracted from 80 mg of fine ground leaves with Purelink® Plant RNA Reagent (Ambion Life Technologies) according to the manufacturer's instructions, with an extra step with chloroform to obtain clear aqueous phase. DNA contaminations were removed with the DNA-Free™ DNA removal kit (Ambion Life Technologies) according to the manufacturers' instructions. The RNA yield and purity were determined by Nanodrop 2000 (Thermo Scientific), then checked on 1% agarose gel. Total RNA (1 mg) was used to synthesize cDNA using Superscript IV reverse transcriptase kit (Invitrogen Life technologies). qRT-PCR experiments were performed using the Absolute™ qPCR Sybr Green ROX mix (Thermo Scientific) as previously described by Gamm et al. (2011) [11].

[0048] Relative gene expression was determined with the comparative cycle threshold (Ct) method (2.sup.−ΔΔCt), where ΔΔCt=(Ct GI [treated sample]−Ct GI [control sample])−(Ct RG [treated sample]−Ct RG [control sample]). GI was the gene of interest PR2, and RG was the reference gene EF1 γ used as internal control for normalization (Dufour et al., 2013) [12]. Two independents experiments were performed.

[0049] As shown in FIG. 6, alginates did not increase the expression of the PR2 gene encoding 1,3 β-D-glucanase in the vine, like the negative surfactant (S) and water controls and unlike the positive PS3 control.

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