USE OF MICROORGANISMS AND CALCIUM FOR IMPROVED PLANT HELATH AND/OR RESILIENCE AGAINST PLANT PATHOGENS

Abstract

The present invention relates in general to the use of a kit or composition comprising at least one yeast and at least one bacterium, selected from the group consisting of lactobacillales, rhizobiales and bifidobacteriales, and/or comprising calcium for improving plant health, for improving plant resistance to plant pathogens, for preventing or reducing mycotoxin contamination of plant material, for improving plant resistance to a plant disease, for preventing perithecia formation of a plant pathogen on plant debris, for plant protection and/or as plant stimulant. The present invention also relates to corresponding compositions and kits and a method of applying such composition or kit to a living plant or plant debris.

Claims

1-11. (canceled)

12. A composition comprising at least two microorganisms and optionally calcium, wherein at least one of the microorganisms is a bacterium and at least one is S. cerevisiae, and wherein among the bacteria are lactobacillales, and wherein the composition does not comprise R. palustris.

13. A composition comprising at least the following bacteria: L. fermentum, L. casei, two different kinds of L. plantarum strains, and at least one S. cerevisiae strain, and optionally further comprising calcium.

14. The composition according to claim 12, wherein the composition comprises L. plantarum subsp. plantarum.

15. A method comprising the step of applying a composition or kit comprising: a) at least two different microorganisms, wherein the microorganisms are selected from bacteria and yeast, wherein at least one of the microorganisms is a bacterium and at least one is S. cerevisiae, and wherein the bacteria are selected from the group consisting of lactobacillales, rhizobiales and bifidobacteriales and optionally b) calcium, wherein the calcium is present in form of calcium chloride, calcium acetate, calcium citrate, calcium propionate, calcium carbonate, calcium lactate, in particular calcium chloride, calcium carbonate or calcium propionate, to a living plant and/or plant debris.

16. The method according to claim 15, wherein the composition or the components of the kit are applied to the ear or leaf of the living plant, preferably to the leaf.

17. The method according to claim 15, wherein the composition or the components of the kit are applied to plant debris.

18. The method according to claim 15, wherein the composition or the kit comprises calcium chloride and wherein calcium chloride is applied to the field comprising the living plant or plant debris in the range of about 0.3% to 5% (w/v), preferably 0.3 to % 0.9% (w/v).

19. The method according to claim 15, wherein the plant is selected from the group of small grain cereals, maize and grapevine, potato, sugar beet, onion, apple, oilseed rape and sunflower.

20. The method of claim 19, wherein the plant is selected from the group consisting of wheat, barley, oat, rye, triticale, maize, grapevine, in particular wherein the plant is selected from the group consisting of wheat, maize and grapevine.

21. The method according to claim 15, wherein the composition or kit comprises a bacterium selected from the group consisting of L. fermentum, L. casei, and L. plantarum.

22. The method according to claim 15, wherein the at least two different microorganisms are selected from the following microorganisms: L. fermentum, L. casei, L. plantarum, S. cerevisiae, R. palustris; Bifidobacterium bifidum, and B. animalis, preferably from L. fermentum, L. casei, L. plantarum, and S. cerevisiae.

23. The method according to claim 15, wherein the method improves plant health, improves plant resistance to plant pathogens, prevents or reduces mycotoxin contamination of plant material, improve plant resistance to a plant disease caused by a plant pathogen, prevents perithecia formation of a plant pathogen on plant debris, protects a plant and/or stimulates a plant.

24. The method according to claim 15, wherein the method is used for improving plant resistance to a plant pathogen, for improving plant resistance to a plant disease caused by a plant pathogen, and/or for preventing perithecia formation of a plant pathogen on plant debris, and wherein the plant pathogen is selected from the group consisting of Fusarium graminearum, Plasmopara viticola, Erysiphe necator, Phytophthora infestans, Cercospora beticola, Ramularia betae, Venturia inaequalis, Podosphaera leucotricha, Fusarium oxysporum and Sclerotinia sclerotiorum.

25. The method according to claim 15, wherein the pathogen is Fusarium graminearum and wherein the plant is wheat or maize.

26. The method according to claim 15, wherein the composition or kit comprises L. plantarum subsp. plantarum.

27. The method of claim 23, wherein the plant disease comprises fusarium head blight, fusarium ear rot, downy mildew, powdery mildew, Potato late blight, Cercospora leaf spot, Ramularia leaf spot, apple scab, basal root rot and Sclerotinia stem or head rot; in particular fusarium head blight or fusarium ear rot.

Description

EXAMPLES

[0062] In the following, specific examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figure and the examples below. All such modifications fall within the scope of the appended claims.

TABLE-US-00001 TABLE 1 Overview of components used Components used Chemical/Microorganisms Component A Calcium chloride Component B Lactiplantibacillus plantarum subsp. plantarum (formerly known as Lactobacillus plantarum), Lacticaseibacillus casei (formerly known as Lactobacillus casei), Limosilactobacillus fermentum (formerly known as Lactobacillus fermentum), Saccharomyces cerevisiae Component C Lactiplantibacillus plantarum subsp. plantarum (formerly known as Lactobacillus plantarum), Lacticaseibacillus casei (formerly known as Lactobacillus casei), Limosilactobacillus fermentum (formerly known as Lactobacillus fermentum), Saccharomyces cerevisiae, Bifidobacterium bifidum, Bifidobacterium animalis, Rhodopseudomonas palustris Component D Calcium carbonate from natural resources Component E Propionic acid, 99% Component F Magnesium chloride Component G Sodium silicium oxide

Example 1: Effect of Microorganisms and Calcium on Living Cereal Plants (Wheat)

[0063] 1.1 Small Scale Field Experiment Season 1

[0064] Two winter wheat varieties (“Lennox” and “Capo”) and 2 spring wheat varieties (“Trappe” and “Kronjet”) were sown in 1 m.sup.2 plots (96 plots/variety). Experimental design in the first small scale field season were completely randomized blocks with 3 replications, respectively. In these experiments the complete plot (1 m.sup.2) was treated with the variants of interest.

[0065] A particular technique for artificial inoculation to mimic the natural Fusarium infection process as closely as possible was used: the so-called kernel-spawn method F. graminearum colonized maize kernels were distributed on the soil surface between the wheat plants about 3 weeks before anthesis (ca. 15 gr/m.sup.2). On the kernels perithecia are produced which eject the ascospores in the air. These spores infected the wheat ears, leading to a constant infection pressure over a longer period of time and thus mimicking closely a natural infection process. To support infection, the complete experiment was mist-irrigated to provide sufficient humidity for disease initiation.

[0066] Table 2 summarizes all prototypes used in Example 1.1. Experiment variant W1 contains microbial species (see Table 1). Variants W2-W4 deal with the cations Ca.sup.2+, Mg.sup.2+ and Si.sup.2+. They were applied on the ear only. The inventors deliberately choose a high concentration of the cations to make sure to see effects (if present), however not too high in order to prevent phytotoxic reactions caused by excessive cation concentrations.

TABLE-US-00002 TABLE 2 Summary of the set-up of the first small scale field experiment Microbial Calcium Other Prototype Component Concentration Component Concentration Components Concentration Control — — — — — — W1 Component C 5 l/ha — W2 — Component A 3% — W3 — — Component 10 kg/ha F W4 — — Component 10 kg/ha G PPP Folicur 1.5 l/ha

[0067] After about 50% heading of wheat ears and short before flowering (about 2-3 days) the ears were treated. In this case the test substances can act via induction of SIR. But also direct interaction with the plant pathogen is possible due to physical contact with the test organisms/calcium. Other mechanisms of antagonism can be tested in this way, including direct inhibition of the pathogen or competition for nutrients. To this end a hand sprayer was used to apply 100 ml/plot of the suspension/solution. For each wheat genotype the inventors used 3 replications of each treatment and 10 control plots. All treatments were completely randomized within each wheat genotype. During the flowering period of the wheat varieties the experiment was mist irrigated every second day for about 20 hours with water pulses of 20 seconds repeated every 20 minutes to promote Fusarium infection. The inoculum is continuously produced in form of ascospores originating on perithecia which develop on the Fusarium (Gibberella zeae) colonized kernels distributed on the soils surface.

[0068] Table 3 represents data on reduction of FHB symptoms as assessed 21 days after anthesis. All main ears in the plot (from 96 to 225 ears) were evaluated for FHB symptoms and the percentage of diseased ears was calculated. Disease incidence (diseased ears) in the control was set at 100% and the data of the treatments are expressed as percentage of the untreated control. For example, for the fungicide treatment (PPP) “Folicur®” (active ingredient: Tebuconazole, Bayer Crop Science) the mean symptom level relative to the control over all genotypes was 32% (data not shown): this represents a reduction of the symptoms of 68% as compared to the control. ANOVA analyses were done with disease incidence data.

TABLE-US-00003 TABLE 3 Summary of the results of the first small scale field experiment. Genotype/ Prototype Tissue Reduction of Symptoms Variety used treated (Disease incidence %) Capo, Lennox, Control no treatment — Trappe, Kronjet PPP ear  68*** W1 ear  23** W2 ear 29* W3 ear ns W4 ear ns ***p ≤ 0.001(highly significant); **p ≤ 0.01; *p ≤ 0.05; +, p ≤ 0.10; ns, p > 0.10 (not significant)

[0069] Results are summarized as follows (see Table 3): [0070] 1) The fungicide Folicur® (PPP) showed the largest reduction in symptoms: a reduction of 68% as compared to the control treatment. [0071] 2) The microbial treatment variant W1 reduced symptoms by 23% [0072] 3) The calcium containing variant W2 led to a significant reduction of the FHB symptoms with 29% [0073] 4) Silicium and magnesium had no influence on FHB symptom reduction, even when applied at high concentrations.

[0074] 1.2 Small Scale Field Experiment Season 2 In a second small scale field season, the most effective treatments of the first field experiment that included calcium and microbial components were again investigated, now in combination, along with new prototypes treatments listed in Table 4. Methods of testing and analysis were identical as described in the first experiment with following exceptions:

[0075] In the second season, only half of the plot (3 rows) was treated and the second half of the plot was not treated, functioning as a direct control for the prototypes under investigation (5 replications). Due to challenging weather conditions during ear emergence, only one variety, “Trappe”, could be finally assessed for FHB symptoms (Table 5)

TABLE-US-00004 TABLE 4 Summary of the set-up of the second field experiment. Microbial Calcium Other Prototype Component Concentration Component Concentration Components Concentration Control — — — — — — W6 Component B 1 l/ha Component A 0.9% — W12 Lacticaseibacillus 3 l/ha — casei 1 (single strain) W13 Limosilactobacillus 3 l/ha — fermentum 1 (single strain) W14 Limosilactobacillus 3 l/ha — fermentum 2 (single strain) W15 Limosilactobacillus 3 l/ha — fermentum 3 (single strain) W16 Lactiplantibacillus 3 l/ha — plantarum subsp. plantarum 1 (single strain) W17 Lactiplantibacillus 3 l/ha — plantarum subsp. plantarum 2 (single strain) W18 Lactiplantibacillus 3 l/ha — plantarum subsp. plantarum 3 (single strain) W19 Lactiplantibacillus 3 l/ha — plantarum subsp. plantarum 4 (single strain) W20 Saccharomyces 3 l/ha — cerevisiae 1 (single strain) W21 Saccharomyces 3 l/ha — cerevisiae 2 (single strain) W22 Saccharomyces 3 l/ha — cerevisiae 3 (single strain) W23 Saccharomyces 3 l/ha — cerevisiae 4 (single strain) W25 — Component D 3 kg/ha PPP Folicur ® 1.5 l/ha

[0076] In this experiment, a new composition with reduced number of microbial strains was used in combination with calcium (W6). Furthermore, single strains were analysed for potential effects on FHB reduction (W12-23)(see Table 4). On the one hand, all single strains that are part of microbial component B were screened. Furthermore, additional single strains from the same genus were purchased from public repositories were assessed for potential effects on symptom reduction in FHB. Finally, a further calcium variant, calcium carbonate (CaCO.sub.3) from natural sources was also assayed for symptom reduction (W25).

TABLE-US-00005 TABLE 5 Summary of the results of the second field experiment. Genotype/ Prototype Tissue Reduction of Symptoms Variety used treated (Disease incidence %) Trappe Control no treatment — PPP ear  56** W6 ear  28*** W12 ear ns W13 ear ns W14 ear ns W15 ear ns W16 ear ns W17 ear ns W18 ear ns W19 ear ns W20 ear ns W21 ear ns W22 ear ns W23 ear ns W25 ear 13*

[0077] As a result, no significant reduction of symptoms could be observed when applying single microbial strains (W12-W23) (see Table 5). Calcium carbonate alone reduced FHB symptoms by 13%, while the combination of the microbial component B together with CaCl.sub.2 reduced symptoms by 28%. Overall infection pressure was high in this field trial, since also the chemical PPP Folicur® reduced FHB symptoms by no more than 58%.

[0078] 1.3 Greenhouse Experiment 1 Two wheat varieties (“Remus”, spring wheat, susceptible for Fusarium head blight (FHB), and “Capo”, winter wheat and medium resistant) were sown in pots (10 plants/pot filled with 7 L of substrate) in the greenhouse. The substrate was a mixture of compost, peat and sand. A mineral fertilizer was applied at tillering. For each treatment 4 pots were randomly selected and treated in an identical way. The 4 pots were regarded as a single entry and placed and evaluated together (as a quadratic unit consisting of 4 neighbouring pots) in the greenhouse. In the experiments 3 to 4 replications (units of four pots each) were used.

[0079] Spore suspensions of the plant pathogen F. graminearum were produced in mung bean broth with the bubble breeding method and small aliquots were frozen at −80° C. until use. Final spore suspensions contained either 20.000 (low concentration to be used on “Remus”) or 50.000 (high concentration for application on “Capo”) Fusarium macroconidia/mL.

[0080] In order to be able to treat the ears, the amount of the product required for the area (0.038 m.sup.2/pot) was suspended in the 20 mL water. The test organisms/cations were applied by spraying the suspension on the ears after heading but at least 2-3 days before flowering. Also part of the leaf canopy was wetted (especially flag leaf). This was done for each pot individually. With this strategy, a combination of several mechanisms of antagonists can be tested including induction of systemic induced resistance (SIR) but also direct antagonism such as competition for nutrients as well as direct inhibition of the pathogen (calcium).

[0081] At flowering the ears in each pot were treated with a Fusarium spore suspension. To this end, 20 mL of the spore suspension in low or high concentration was applied with a hand sprayer on the flowering ears in each pot. Subsequently the ears were covered with a plastic bag for 24 or 48 hours to ensure sufficient humidity for infection. During the experiment temperature in the greenhouse was 18/20° C. (night/day) and the plants were daily illuminated for 16 hours.

TABLE-US-00006 TABLE 6 Summary of the set-up of the first greenhouse experiment Microbial Other Prototype Component Concentration Calcium Component Concentration Components Control — — — — — W5 Component 1 l/ha Component A 1.5% — B W6 Component 1 l/ha Component A 0.9% — B W7 Component 1 l/ha Component D, 25 gr + 5 l/ha — B Component E 57 ml ad 1 l W8 Component 1 l/ha Component D, 25 gr + 10 l/ha — B Component E 57 ml ad 1 l

[0082] In the first greenhouse experiment, the combination of microbial component B and CaCl.sub.2 were tested at different CaCl.sub.2 concentrations (W5-6) (see Table 6). Furthermore, additional calcium variants were tested, which included CaCO.sub.3 in a mixture with propionic acid, yielding calcium propionate from natural sources (W7-8). The experiment further included an untreated control for reference.

[0083] At 7, 11, 15 and 18/19 days after inoculation the 2 disease parameters were assessed: Disease Incidence (% diseased ears) and Disease Severity (% diseased spikelets of the diseased ears only). Thereafter Disease Intensity (% of diseased spikelets over all ears) was calculated. In the end, the % reduction of symptoms was calculated for each disease parameter (compared to the control treatment). ANOVA analyses were performed for statistical analysis.

TABLE-US-00007 TABLE 7 Summary of results of the first greenhouse experiment Reduction of Symptoms (%) Genotype/ Prototype Disease Disease Disease Variety used incidence % severity % intensity % Remus Control — — — W5 33.9* 18.2ns 45.7** W6 61.6** 30.5* 72.4*** W7 ns ns 32.7* W8 ns ns ns

[0084] As a result, only one of the calcium propionate-containing prototypes (W8) in combination with component B had no statistically significant effect on symptom reduction of FHB. All other prototypes led to reduction of disease intensity. Overall, the variants including calcium chloride (W5-6), performed better in reduction of disease symptoms. However, a reduction of CaCl.sub.2 input from 5 l/ha to 3 l/ha as shown with prototype W6, led to an even improved outcome and reduced disease intensity by 72.4% as compared to W5 with a reduction of 45.7%.

[0085] 1.4 Green House Experiment 2

[0086] This experiment was performed and evaluated in the same way as the first greenhouse experiment (see 1.3) with the exemption that in this experiment, two spring wheat varieties, Capo and Remus, were included.

TABLE-US-00008 TABLE 8 Summary of the set-up of the second greenhouse experiment Microbial Calcium Other Prototype Component Concentration Component Concentration Components Concentration Control — — — — — — W6 Component 1 l/ha Component A 0.9% — B W9 Component 1 l/ha Component D 3 kg/ha — B (CaCO3) W10 Component 1 l/ha Component D, 7.5 l/ha — B 120 gr + Component E 220 ml ad 1l W11 Component 1 l/ha Component D, 5 l/ha — B 120 gr + Component E 220 ml ad 1l PPP Folicur ® 1.5 l/ha

[0087] In this experiment, microbial component B was again combined with different calcium variants including CaCl.sub.2 (W6), CaCO.sub.3 (W9) or calcium propionate (W10-11) as wells to the fungicide Folicur (see Table 8).

TABLE-US-00009 TABLE 9 Summary of results of the second greenhouse experiment Reduction of Symptoms (%) Genotype/ Prototype Disease Disease Disease Variety used incidence % severity % intensity % Remus, Control — — — Capo PPP 55.3* 61.5**  82.4** W6 26.9* 17.6* 39.7* W9 ns ns 26.5* W10 9.3+ ns 21*   W11 ns ns ns

[0088] Similarly, as in the first greenhouse experiment, the combination of microbial component B with different calcium variants showed effects (see Table 9), with CaCl.sub.2 yielding highest reduction (W6, 39.7%), CaCO.sub.3 alone (W9) a lesser pronounced reduction of 26.5% and CaCO.sub.3 plus propionic acid (W10) either a mild reduction of 21% (W10) or no significant reduction (W11). The fungicide Folicur reduced symptoms by 82.4%.

[0089] 1.5 Green House Experiment 3

[0090] The third greenhouse experiment was essentially performed and evaluated in the same way as the first greenhouse experiment (see 1.3) with the exemption that in this experiment the winter wheat variety Capo was used only.

TABLE-US-00010 TABLE 10 Summary of the set-up of the third greenhouse experiment Microbial Calcium Other Prototype Component Concentration Component Concentration Components Concentration Control — — — — — — W6 Component B 1 l/ha Component A 0.9% — W9 Component B 1 l/ha Component D 3 kg/ha — W10 Component B 1 l/ha Component D, 7.5 l/ha — 120 gr + Component E 220 ml ad 1l W24 — Component D 1.5 kg/ha PPP Folicur ® 1.5 l/ha

[0091] In the second greenhouse experiment, microbial component B was again combined with different calcium variants including CaCl.sub.2 (W6), CaCO.sub.3 (W9, W24) or calcium propionate (W10) as wells to the fungicide Folicur (see Table 10).

TABLE-US-00011 TABLE 11 Summary of results of the third greenhouse experiment Reduction of Symptoms (%) Genotype/ Prototype Disease Disease Disease Variety used incidence % severity % intensity % Capo Control — — — PPP 29.2+ ns 54.6* W6 25.7+ 26.3+ 47.9* W9 ns 22.5+ ns W10 ns 21.8+ 27.6+ W24 ns ns ns

[0092] Similarly, as in the first and second greenhouse experiment, the combination of microbial component B with different calcium variants showed effects, with CaCl.sub.2 yielding highest reduction of disease intensity (W6, 47.9%), reaching almost the level of PPP (54,6) (see Table 11). In this experiment, CaCO.sub.3 alone (W9) only reduced disease severity significantly, but not overall intensity, a lower concentration of CaCO.sub.3 alone had no significant effect (W24). CaCO.sub.3 plus propionic acid (W10) again showed reduction of disease intensity by 27.6%.

[0093] 1.6 Large Scale Field Experiment Season 1

[0094] The goal of these experiments was to test promising components under real practical conditions. This means that the farmers applied the prototypes with their own equipment. The farms were located in the 3 important climatic regions in Austria: The North-Alpine region (wet and warm), the Pannonicum (dry and hot) and the Illyricum (wet and hot). This approach gives a good overview of the effectiveness of the prototype under different environmental conditions. The fields selected for the tests were normal fields used for farming. Previous crop was maize in all cases, which leads to a higher probability of Fusarium infection in wheat in the following season.

[0095] Well-repeated experiments with proper statistical design came to use. This included replications (typically 3 to 4) and care was taken to have respective controls (=no treatment) in vicinity of the treated variants (Prototype W6). The prototype was applied at the prescribed concentration (in L/ha) on the ears at 50% heading or shortly thereafter, but not later than 2-3 days before flowering. Fusarium infection occurred under natural conditions, hence no artificial infection using Fusarium spores was done. Infection pressure varied between locations according to the different climatic condition during flowering, ranging from low to high overall Fusarium infection rates.

[0096] Two to three weeks after flowering FHB disease was assessed. To this end up to 500 ears for each entry were visually evaluated for typical FHB symptoms including water-soaked spots and spreading of the disease (wilted spikelets or ear segments). Each ear was classified being diseased or healthy and the Disease Incidence (DI=percentage of diseased ears) was calculated. The percentage of reduction of symptoms was calculated (in comparison to the control).

TABLE-US-00012 TABLE 12 Summary of results from large scale field trials Prototype Tissue Reduction of Location treated treated Symptoms (in %) Location 1 (low Control no treatment — infection pressure) W6 ear 49.7* Control no treatment — Location 2 (low- W6 ear 45.7* medium infection pressure) Location 3 (high Control no treatment — infection pressure) W6 ear 22.8**

[0097] The results (see Table 12) show that, compared to the untreated control, the combination of microbial and calcium containing components as used in Prototype W6 lead to reduction of FHB symptoms in all locations. Depending on the climatic conditions and infection pressure in the field, the FHB symptoms were reduced by 49.7% and 45.7%, respectively in locations with low to medium infection pressure. At high Fusarium infection pressure, still a reduction of 22.8% of FHB symptoms could be observed. Overall, the results obtained with prototype W6 corresponded reasonably well over all different test systems and genotypes used, from the greenhouse to the large-scale field.

[0098] 1.7 Large Scale Field Experiment Season 2

[0099] In a second large scale field season, the treatments of the first field experiment that included calcium and microbial components were again investigated. Methods of testing and analysis were identical as described in the first experiment with following exceptions: [0100] Only one site in Upper-Austria was included in the analysis, due to heavy droughts in other parts of the country where no infection with Fusarium could be observed. [0101] This time, the trial included 2 treatment variants: (1) a single treatment as described in 1.6 and (2) a dual treatment at emergence of the flag leaf (around BBCH 37-39) and at 50% ear emergence (around BBCH 55) or shortly thereafter. [0102] To this end 250 ears for each entry were visually evaluated for typical FHB symptoms including water-soaked spots and spreading of the disease (wilted spikelets or ear segments).

TABLE-US-00013 TABLE 13 Summary of the set-up and results of the large scale field experiment in season 2 Prototype Tissue Reduction of Location treated treated Symptoms (in %) Location with high Control no treatment — infection pressure W6 - single ear 30.6+ W6 - double Flag leaf, ear 68.0***

[0103] In the second season, the results of the large-scale field trial are summarized in table 13 and show again a reduction of symptoms of around 30% even at high infection pressure with a single application of prototype W6. A dual application of prototype W6 lead to a highly significant reduction of symptoms of 68%. Again, these data are consistent with what could be observed in prior trials and under different testing conditions.

Example 2: Effect of Microorganisms and Calcium on Perithecia Production on Crop Debris

[0104] Reduction of primary inoculum of the fungus is one of the most important strategies in control of Fusarium head blight disease. F. graminearum survives saprophytically on crop residues during the winter. Ascospores (sexual spores) of Gibberella zeae (=perfect form of F. graminearum) which are formed within asci in the fungal fruiting bodies (perithecium) serve as the primary inoculum in spring. Ascospores are forcibly discharged from the perithecium, land on susceptible parts of the plant and start infection. Therefore, inhibition of perithecia production on the Fusarium contaminated crop debris of cereals and maize crops of the past season results in reduced infection of the new crops.

[0105] Therefore, the inventors aimed at identifying means to inhibit perithecial production on crop debris.

[0106] For the field experiment, maize stalks were first cut into 7 cm long pieces and then each piece was longitudinally cut in half. The halves of three stalk pieces were placed in a 10 cm autoclavable plastic mesh bag and the corresponding half of the same pieces were placed in a separate bag labelled in a way to easily recognize the two corresponding halves of the same stem piece for experiment evaluation. One bag was used for antagonist treatment and the other one served as control. 12 bags containing a total number of 36 stalk pieces (36 replications) was used for each treatment or control. The pieces in the bags were immersed in distilled water overnight, and after decanting the water, placed in aluminium trays and subsequently autoclaved. The bags were then immersed for 3 min in a conidial suspension (3×10.sup.4 spores/ml) of a strong perithecia producing G. zeae strain. The inoculated pieces were incubated at 22° C. in darkness for 48 h.

TABLE-US-00014 TABLE 14 Summary of variants assayed in the perithecia assay on maize stalks Microbial Concentration Calcium Concentration Other Concentration Control — — — — — — Prototype M1 — Component A 33% Prototype M2 Component B undiluted — PPP Folicur ® 1.5 l/ha

[0107] For treatment, one of the two corresponding bags was sprayed with the respective prototype variants (see Table 14) until runoff and the second bag was only sprayed with sterile distilled water. The bags were left overnight in the laboratory at room temperature (RT) and were next day transferred to the wheat or maize field and placed randomly on the soil surface between the wheat or maize plants. After 3-4 weeks, the bags were evaluated and the number of perithecia in a total area of 1 cm 2 of the maize stalk surface was counted.

[0108] The percentage of perithecia reduction in treatments was then calculated by comparison with the control. ANOVA analyses were performed for statistical analysis.

TABLE-US-00015 TABLE 15 Results perithecia assay on maize stalks Prototype Tissue Reduction of used treated Perithecia (%) Control maize stalks — Chemical PPP maize stalks 48.74*** M1 maize stalks 69.79*** M2 maize stalks 60.09**

[0109] As a result, M1 containing calcium reduced the perithecia numbers on the maize stalks in the field about 70% and the microbial composition M2 inhibited the perithecia formation by 60% (see Table 15). The commercial chemical fungicide PPP reduced the perithecia numbers only about 49%.

Example 3: Effect of Microorganisms and Calcium on Other Diseases in Agricultural Crops

[0110] 3.1 Downey Mildew in Grapevine

[0111] The inventors also observed that the microorganisms and calcium, alone or in combination, reduced disease pressure of other diseases on other crops. Respective results were obtained for downey mildew (Plasmopara viticola, “Peronospora”) and powdery mildew (Erysiphe necator; “Oidium”) on grapevines and Fusarium oxysporum in onions.

[0112] Exemplary tests for reduction of Peronospora symptoms were carried out on grapevine leaf discs as follows. Ten leaf discs were first inoculated with prototypes for 20 min and then treated with a spore suspension (20000 sporangia/ml) of the fungus until zoospores were released from the sporangia. Afterwards, leaf disks were placed on water agar plates and incubated for 5 days at 23° C. (16 h light/8 h darkness). Consequently, disease severity was calculated by measurement of the percentage of diseased disk area. Control treatments were included by either using water or by application of the copper-based, commercially available plant protection product Cuprozin.

TABLE-US-00016 TABLE 16 Summary of variants tested in grapevine leaf disc assay. Prototypes Microbial Concentration Calcium Concentration Other Concentration G1 — Component A 2% G2 Component B 1% G3 Component B 1% Component A 2% PPP Cuprozine 0.30%

TABLE-US-00017 TABLE 17 Summary of the effect of different treatments on Peronospora in grapevine described by disease severity. Prototype Tissue Reduction of Symptoms used treated (Disease Severity %) Control Leaf disc — PPP Leaf disc 100***   G1 leaf 50.9** G2 leaf .sup. 7.3ns G3 leaf  68.3***

[0113] These data demonstrate that the composition of microorganisms as well as calcium ions are effective against a variety of plant diseases and pathogens and that this effect is stronger and more significant when applied in combination (see Table 17).

[0114] 3.2 Leaf Spot Diseases in Sugarbeet

[0115] Cercospora beticola and Ramularia betae are the causal fungi for leaf spot disease in sugar beet. Exemplary tests for control of leaf spot disease in sugar beet were carried out in the region of Lower Austria under real practical conditions. This means that the farmers applied the prototypes with their own equipment. The fields selected for the tests were normal fields used for farming.

[0116] Two adjacent rows were either treated with prototype W6 with the exemption, that the concentration of component A was lowered to 2 l/ha. A total of 4-5 treatments with W6 was carried out over the course of the season, starting around BBCH39 with the last application around BBCH 85. The second row was treated with a conventional plant protection plan using 3-5 treatments of commercial fungicides.

[0117] Natural infection occurred over the course of the field season, but especially in August and early September due to heavy rain falls.

[0118] Evaluation and rating of symptoms was based on the scaling procedure suggested by EPPO guideline PP1-4—foliar diseases of sugar beet. 20 plants per treatment that were located in vicinity to each other were evaluated. Only middle-aged leafs were considered for evaluation, between 7-15 leafs per plant were rated for symptoms. Disease incidence per treatment was calculated as the weighted mean of the over the scored plants in %.

TABLE-US-00018 TABLE 18 Summary of the effect of two different treatments on leaf spot disease in sugar beet Score Fungicide Prototype W6 0.1%  8 1  1% 7 5  2% 3 5  5% 2 6 10% 0 3 25% 0 0 35% 0 0 45% 0 0 60% 0 0 >60%  0 0 Average disease 1.19 3.75 incidence (%)

[0119] The fungicide treated sugarbeets showed a very low level of infection with leaf spot causing fungi of 1.19%. Treatment with prototype W6 also resulted in a low level of infection of 3.75%, even though weather conditions during the field season were very favorably for fungal growth (Table 18).