Glomus iranicum var. tenuihypharum var. nov. strain and use thereof as bio-nematicide
09932647 ยท 2018-04-03
Assignee
Inventors
Cpc classification
A01N63/30
HUMAN NECESSITIES
A01H17/00
HUMAN NECESSITIES
International classification
A01N63/00
HUMAN NECESSITIES
A01H17/00
HUMAN NECESSITIES
Abstract
Glomus iranicum var. tenuihypharum var. nov. strain deposited under BCCM deposit number 54871, comprising the sequence identified by SEQ ID NO: 1; composition comprising said strain, 2:1 smectite clays, metal ions and chitin and use thereof as bio-nematicide. The invention also discloses a composition comprising said strain, fungicides, bio-fungicides, insecticides, bio-insecticides, nematicides and bio-nematicides.
Claims
1. A method of treating a plant nematode infection comprising applying a composition comprising a strain of Glomus iranicum var. tenuihypharum var. nov. deposited under BCCM deposit number 54871 comprising the sequence identified by SEQ ID NO: 1, 2:1 smectite clays, metal ions and chitin to the plant in need thereof, wherein said composition is applied to the plant by means of seed treatment, root treatment, roots embedded in an emulsion, addition to irrigation water, irrigation, application of powder to the root system or application of emulsion injected into the root system.
2. The method according to claim 1, wherein said 2:1 smectite clays are dioctahedral or trioctahedral.
3. The method according to claim 1, wherein said 2:1 smectite clays are selected from the group consisting of sepiolite, attapulgite, nontronite and saponite.
4. The method according to claim 1, wherein a concentration of said Glomus iranicum var. tenuihypharum var. nov. strain is between 0.05 and 4% by weight.
5. The method according to claim 4, wherein said concentration is between 0.1 and 3% by weight.
6. The method according to claim 1, wherein the chitin concentration is between 2 and 10% by weight.
7. The method according to claim 1, wherein said metal ions are selected from the group consisting of Fe, Ca and Mg.
8. The method according to claim 1, wherein the composition further comprises Fe, Ca and Mg.
9. The method according to claim 7, wherein the concentration of Fe is between 3 and 12% by weight, the concentration of Ca is between 0.5 and 5% by weight and the concentration of Mg is between 0.2 and 2% by weight.
10. The method according to claim 1, wherein the form of presentation of said composition is powder, emulsifiable concentrate or granules.
11. The method according to claim 1, wherein said composition is a liquid, a solid or a gel.
12. The method according to claim 1, wherein the composition further comprises at least one fungicide, at least one bio-fungicide, at least one insecticide, at least one bio-insecticide, at least one nematicide and/or at least one bio-nematicide.
13. The method according to claim 12, wherein said fungicide is selected from the group consisting of Maneb, Mancozeb, Metalaxyl-Ridomil, Myclobutanil, Olpisan, Propamocarb, Quintozene, Streptomycin, Sulfur, Thiophanate-methyl, Thiram, triforine, Vinclozolin, Zinc white, Zineb, Ziram, Banrot, Fixed copper, Chlorothalonil, Chlorothalonil, Captan, Chloroneb, Cyproconazole, Zinc ethelene, bisdithiocarbamate, Etridiazole, Fenaminosulf, Fenarimol, Flutolanil, Folpet, Fosetyl-AL and Iprodione.
14. The method according to claim 12, wherein said fungicide is selected from the group consisting of Trichodermas sp, Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus amyloliquefaciens, Streptomyces sp, Coniothyrium minitans and Pythium oligandrum.
15. The method according to claim 12, wherein said insecticide is selected from the group consisting of organophosphate, carbamate and neonicotinoid.
16. The method according to claim 12, wherein said bio-insecticide is selected from the group consisting of Bacillus sp. Chromobacterium sp., Beauveria sp. and Metarhizium sp.
17. The method according to claim 12, wherein said nematicide is organophosphate or carbamate.
18. The method according to claim 12, wherein said bio-nematicide is Pasteuria sp.
19. Method for obtaining a composition according to claim 1, comprising: (a) coating inoculation of a seed of a host plant with the Glomus iranicum var. tenuihypharum var. nov. strain deposited under BCCM deposit number 54871, (b) cultivating said plant in watering cycles of between 7 to 10 days on a reproduction substrate comprising smectite clays in a percentage above 52% of the total weight of said substrate, (c) discontinuing said watering for a period equal to or greater than 20 days, (d) removing the aerial part of the plant and removing the substrate and (e) milling said substrate below 80 microns at a temperature between 25 and 30 C. to obtain said composition.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1)
(2)
PREFERRED EMBODIMENTS
Example 1
(3) Genetic Identification of the Strain of the Invention
(4) DNA Extraction
(5) Isolated hyphae and spores were transferred to 1.5 ml Eppendorf tubes with 0.2 g of glass beads (2 mm diameter) and 100 L of CTAB buffer (2% CTAB=Cetyl trimethyl ammonium bromide, 1.4 M NaCl, 0.1 M Tris-HCl pH 7.5, 0.2 M Na-EDTA).
(6) This mixture was homogenized using a ball mill type Retsch MM301 at full speed for 30 seconds. Additional 400 L of CTAB buffer were added and the mixture was incubated at 65 C. for one hour. Subsequently 400 L of Chloroformo-isoamyl alcohol (24:1) were added and mixed by inverting the reaction tubes and then it was centrifuged for 5 min at 10,000g, and the upper layer was recovered in a clean Eppendorf tube. This step was repeated twice. 200 L of 5 M ammonium acetate were added to this suspension, the mixture was incubated at 4 C. for 30 minutes, followed by 20 minutes of spinning at 4 C. and 13,000 rpm. The DNA was precipitated with 700 L of isopropanol at 20 C. overnight. The DNA pellet obtained was precipitated with isopropanol and washed with 70% ice-cold ethanol, air dried and re-dissolved in 50 L of Tris ethylene diamine buffer (10 mM Tris, 10 mM EDTA, pH 8)+4.5 U RNase/ml.
(7) PCR Conditions
(8) The primers used for PCR amplification and for sequencing the internal transcribed spacers region of the 18S rDNA gene were Glom1310 and ITS4i (Redecker, 2000). Amplifications were performed in 0.2 mM dNTP-mix, 1 mM of each primer, 10% of PCR reaction buffer and double distilled sterile water. GoTaq DNA polymerase (Promega, Mannheim, Germany) was added to 3.75 u/100 L of reaction mixture; 2 L of genomic DNA template was used in each 20 L/reaction. Amplifications were carried out in a advanced thermal cycler Primus 96(peqLab Biotechnology) in 200 reaction tubes mu 1 (94 C., 120 s initial denaturation, followed by 30 cycles of 94 C. 15 s, 52 C., 30 s, 72 C., 45 s, and a final slope at 72 C. for 120 s).
(9) Data Analysis
(10) The alignment was carried out initially using the computer program BioEdit 7.0. Phylogenetic analyses by maximum likelihood (ML) were carried out with the PHYML program. The nucleotide substitution model GTR was used with ML estimates of the base frequencies. The proportion of invariable sites were estimated and optimized. Four categories of the substitution rate were developed and the gamma distribution parameter was also estimated and optimized. Bootstrap analysis was used with 100 replicates to test the statistical support of the branches.
(11) Sequencing
(12) Excess primers and dNTPs were removed by column chromatography (Microspin S-300 HR, Amersham Biosciences). For partial sequencing of the 18S-ITS1-5.8S region primers Glom1310 and ITS4i were used in a concentration of 1.6 mM. Sequencing was carried out with PRISM BigDye Terminator Cycle Sequencing Kit from ABI (Applied Biosystems) according to the manufacturer's recommendations. The parameters for sequencing were delay 18 seconds at 96 C., followed by 25 cycles with 18 sec at 96 C., 5 seconds at 50 C. and 4 min at 60 C. Sequence analysis was performed using an automated sequence analyzer (ABI PRISM 3130, Applied Biosystems) in conjunction with the ABI Prism Auto Assembler software (version 140, Applied Biosystems).
(13) Ribosomal RNA 18S sequence (partial sequence), ITS1 (complete sequence) and ribosomal RNA 5.8S gene (partial sequence) were obtained from the strain of the invention, which sequence is identified by SEQ ID NO: 1.
Example 2
(14) Effectiveness of the Composition of the Invention on Tomato Crop
(15) The assay was divided into two parts, preventive treatment and curative treatment, divided into 5 treatments: T.sub.1: Control Treatment T.sub.2: Treatment with Glomus iranicum var. tenuihypharum var. nov mycorrhizal inoculum. T.sub.3: Treatment with product to be tested. (Composition of the invention, Glomus iranicum var. tenuihypharum var. nov. with metal ions and chitin) T.sub.5: Comparative treatment with commercial product (VYDATE, oxamyl)
(16) The amount present of the fungus Glomus iranicum var. tenuihypharum var. nov. in the treatments in which the fungus was inoculated has been estimated. Between 1 and 5 grams of mycelium-spores of this species was produced in 1 kg of soil, which is a range between 0.1 and 0.5%. On the other hand, the root system in which it is produced, reached 10% of the gross weight (100 g). Fine rootlets, which are those mycorrhized, were 40 g per 1 kilogram of soil, i.e. 4% of the total weight. From these fine rootlets, only 75% has mycorrhizal propagules, therefore, the percentage of mycorrhizae in one kilogram of soil was equivalent to 3%.
(17) The composition of the invention used in Treatment T.sub.3 contains Glomus iranicum var. tenuihypharum var. nov.; the concentrations of chitin and metal ions in said composition were: chitin 5% by weight, 5% by weight Fe, Ca 4% by weight and Mg 1% by weight.
(18) 4 repetitions of each treatment were carried out for preventive treatment and similarly for curative treatment. Each experimental unit consists of 2 growing containers of 30 L volume, containing 2 plants each. Plants were selected randomly in each treatment, or plants that had suffered viruses, mishap or abnormality were selected. The rows and containers corresponding to the entire periphery of the assay were not part of it to avoid the edge effect.
(19) Irrigation and fertilization was common throughout the test plot. Discharge was established at 4 l/h per growing container, divided into two discharge points for all treatments.
(20) The irrigation water used for carrying out the assay came from the Tajo-Segura transfer system. The irrigation water was alkaline and slightly saline, with somewhat elevated levels of sodium and chloride ion, but in no way limiting for the crop object of the assay.
(21) The inoculum used was taken from a batch of peat with tomato roots infested with phyto-parasitic nematodes from greenhouses from Mazarrn, province of Murcia. The inoculum was homogenized and multiplied to ensure adequate distribution of the nematode population and ensure sufficient population for the second inoculation.
(22) In the previous analysis for the identification of phyto-parasitic nematodes, a high population of Meloidogyne was identified, followed by Heterodera and hardly any Globodera individual.
(23) The first inoculation was made to coincide with the date of planting of the curative part of the assay. The second inoculation was made only in the preventive part of the assay, two days before the first application of the products. The growing cycle was 9 months.
(24) Nematode inoculation of the curative assay was performed coinciding with the transplantation of the tomato. One month later, the nematode inoculation of the preventive assay was performed. Two days later it was performed the first application (inoculations of the composition of the invention and application of Vydate) and the second application (inoculations of the composition of the invention and application of Vydate) was performed 2 months later.
(25) The balance of the elements N, P, K, Ca and Mg used during the growing cycle was: N, 12.8 mM; P, 1.4 mM; K, 5.5 mM Ca, 4.8 mM; Mg, 2.4 mM. Fertilizer concentrations provided to achieve this balance were: 72% phosphoric acid 0.117 cm.sup.3/l, calcium nitrate 0.379 g/l, potassium nitrate 0.547 g/l, ammonium nitrate 0.120 g/l, micronutrient mixture 0.020 g/l. The following parameters were used in the irrigation controller: final pH 5.5, in order to obtain pH 6 in the dropper, estimated consumption 59% nitric acid 0.07 l/m.sup.3, final electrical conductivity (EC) 2.1 dS/m, EC increase 0.8 dS/m.
(26) The following table shows the phytosanitary treatments performed.
(27) TABLE-US-00002 TABLE 8 Phytosanitary treatments Date Active matter Product Dose Mode of use Cause 06/10/11 Pymetrozine 50% WG Plenum 50 g/ 16L Whitefly Hl Backpack 11/10/11 Cyprodinil 37.5% + Switch 100 g/ 16L Botrytis fludioxinil 25% hl Backpack 11/10/11 Bacillus thuringiensis Turex 20 g/ 16L Worm 2.5% WG Hl Backpack 16/11/11 Pymetrozine 50% WG Plenum 50 g/ 16L Whitefly Hl Backpack 24/11/11 Pymetrozine 50% WG Plenum 50 g/ 16L Whitefly Hl Backpack 09/11/11 Pymetrozine 50% WG Plenum 50 g/ 16L Whitefly Hl Backpack 09/11/11 Bacillus thuringiensis Turex 20 g/ 16L Worm 2.5% WG Hl Backpack 03/02/12 Imidacloprid 20% SL Confidor 75 cc/ 16L Fly, Aphid Hl Backpack 15/02/12 Bacillus thuringiensis Turex 20 g/ 16L Worm 2.5% WG Hl Backpack 07/03/12 Methiocarb 50% WP Mesurol 100 g/ 100 l Cask Trip Hl 07/03/12 Bacillus thuringiensis Turex 20 g/ 100 l Cask Worm 2.5% WG Hl 21/03/12 Pyriproxyfen 10% EC Brai 75 cc/ 100 l Cask Fly, Aphid Hl 12/04/12 Imidacloprid 20% SL Confidor 75 cc/ 100 l Cask Fly, Aphid Hl 12/04/12 Bacillus thuringiensis Turex 20 g/ 100 l Cask Worm 2.5% WG Hl 02/05/12 Flubendiamide 24% WG Fenos 25 g/ 100 l Cask Tuta, Moth Hl 09/05/12 Flubendiamide 24% WG Fenos 25 g/ 100 l Cask Tuta, Moth Hl 17/05/12 Spinosad 48% SC Spintor 25 cc/ 100 l Cask Tuta, Trip, Hl Fly 01/06/12 Spinosad 48% SC Spintor 25 cc/ 100 l Cask Tuta, Trip, Hl Fly 01/06/12 Cyprodinil 37.5% + Switch 100 g/ 100 l Cask Botrytis fludioxinil 25% hl 20/06/12 Spinosad 48% SC Spintor 25 cc/ 100 l Cask Tuta, Trip, Hl Fly 20/06/12 Bacillus thuringiensis Turex 20 g/ 100 l Cask Worm 2.5% WG Hl
Evaluation of Galling
(28) For each treatment 2 extractions were performed at the beginning under curative to assess the infestation level and under preventive at the end of cultivation for the same purpose.
(29) The first extraction was performed at 45 days after inoculation of nematodes and 7 days after application of the product to be tested. High resolution scanning was performed, enlarging live roots submerged in osmosis water, observing a high percentage of galls in all treatments, indicating a high population of nematodes.
(30) The second extraction was performed at 120 days after inoculation and 83 days after application of the product to be tested. At this point, a significant difference between treatments was already clearly appreciated, the treatments with the composition of the invention having a smaller number of galls and a greater number of active rootlets.
(31) A third extraction was carried out at the end of cultivation of plants belonging to the preventive portion to estimate the degree of infestation of nematodes in all treatments.
(32) The same difference was observed in root architecture and development than at 120 days, the product to be tested showing better results, as it was shown in the scanning of living roots submerged in osmosis water.
(33) Root Analysis
(34) 9 soil and 2 root extractions were carried out for each treatment to assess root thickness, root colonization, external mycelium, total microflora and nematology.
(35) The following tables set out the total values of mycorrhizal colonization, extramatrical mycelium and nematology corresponding to data taken in the nine extractions made throughout the growing cycle.
(36) Mycorrhizal Colonization
(37) TABLE-US-00003 TABLE 9 Mycorrhizal colonization in curative treatment Glomus iranicum var. tenuihypharum var. nov. Composition Days Control (%) (%) of the invention (%) Vydate (%) 0 d 11 4 2 10 30 d 15 19 10 2 60 d 18 38 40 5 90 d 16 56 57 4 120 d 9 63 59 8 150 d 15 65 63 9 180 d 33 67 71 18 210 d 20 69 82 18 240 d 18 74 89 22
(38) TABLE-US-00004 TABLE 10 Mycorrhizal colonization in preventive treatment Glomus iranicum var. tenuihypharum var. nov. Composition Days Control (%) (%) of the invention (%) Vydate (%) 0 d 13 2 5 12 30 d 4 32 27 2 60 d 10 36 39 8 90 d 2 25 70 12 120 d 22 66 70 22 150 d 28 75 82 24 180 d 22 78 82.3 16 210 d 28 85 87 22 240 d 24 71 89 24
(39) Rootlets samples were taken, washed with deionized water and clarified with a 10% solution of KOH for 10 minutes at 90 C. Subsequently they were passed through 2N HCl for 10 minutes and finally they were stained with a 0.05 lacto glycerinTrypan Blue solution.
(40) As shown by the data, the treatment with the composition of the invention has been the one that has obtained greater mycorrhizal colonization reaching 89% both in the curative part and in the preventive part of the assay. Mycorrhizal values achieved in the treatment with Glomus iranicum var. tenuihypharum var. nov. were lower in the presence of metal ions and chitin. Both curative and preventive conditions affected the colonization of the native AMF, becoming more negative in the presence of Vydate, which undoubtedly affected native mycorrhization.
(41) Extramatrical Mycelium
(42) TABLE-US-00005 TABLE 11 Extramatrical mycelium in curative treatment Glomus iranicum var. Composition tenuihypharum of the Vydate Control (mg/kg var. nov. invention (mg/kg Days soil) (mg/kg soil) (mg/kg soil) soil) 30 d 223.5 260.1 248.3 173.8 60 d 173.8 360.1 260.8 223.5 90 d 273.2 434.6 422.2 161.4 120 d 223.5 571.2 471.8 173.8 150 d 347.7 347.7 682.9 173.8 180 d 347.7 533.9 720.7 186.3 210 d 173.8 260.8 397.3 235.0 240 d 484.3 645.7 608.4 183.6
(43) TABLE-US-00006 TABLE 12 Extramatrical mycelium in preventive treatment Glomus iranicum var. Composition tenuihypharum of the Vydate Control (mg/kg var. nov. invention (mg/kg Days soil) (mg/kg soil) (mg/kg soil) soil) 30 d 223.5 347.7 285.6 161.4 60 d 260.8 360.1 335.3 186.3 90 d 298.0 298.0 434.6 161.4 120 d 322.8 397.3 409.8 161.4 150 d 248.3 682.9 620.8 149.0 180 d 447.0 397.3 645.7 223.5 210 d 235.9 260.8 447.0 149.0 240 d 422.7 471.8 831.9 322.8
(44) Following centrifugation and decanting of a 50 g soil aliquot, all the material collected in the 40 micron sieve was taken, it was passed through a mixer at 900 rpm, it was vacuum filtered and the collected material was taken in 5 40 mg replicates, and it was evaluated under dissecting microscope. Two imaginary lines, horizontally and vertically, were counted and the average of mycelia counted was multiplied by the correction factor, 0.000745 and it was extrapolated to mg of mycelium per gram of sample.
(45) The extramatrical mycelium evolution was similar to mycorrhizal colonization, as well as in the percentage of colonization the higher values were reported for the treatment with the composition of the invention. The mycelium values were higher in the presence of metal ions and chitin, however, plants treated only with Glomus iranicum var. tenuihypharum var. nov. also achieved high concentration of this parameter, in comparison with the control and treatment with Vydate, which further decreased the expression of the native fungi extramatrical mycelium. Like the colonization percentage, values were very similar for the two conditions of application, the curative treatment producing the greatest stimulation.
(46) Nematode Counts in the Curative Treatment
(47) TABLE-US-00007 TABLE 13 Live juvenile nematodes count Glomus iranicum var. tenuihypharum Composition var. nov. of the Days Control (n) (n) invention (n) Vydate (n) 0 52.5 27 51.5 55 30 46.5 48 36.5 15.5 60 61 44 23 16.5 90 63 57.5 42 29.5 120 44.5 32.5 30.5 78.5 150 155.5 104.5 22.5 33 180 137 67 31 42.5 210 59 62.5 14 23 240 182 16.5 19.5 73.5
(48) TABLE-US-00008 TABLE 14 Dead juvenile nematodes count Glomus iranicum var. tenuihypharum Composition var. nov. of the Days Control (n) (n) invention (n) Vydate (n) 0 68.5 26.5 54 40 30 31.5 71.5 88.5 30.5 60 102 153 114 86.5 90 65 106 185 67.5 120 106.5 86.5 113 118 150 66 251 127 49.5 180 82 143.5 158.5 33 210 20 27 41 5.5 240 34 6 54 38.5
(49) TABLE-US-00009 TABLE 15 Mortality rate Glomus iranicum var. Composition tenuihypharum of the var. nov. invention Vydate Days Control (% m) (% m) (% m) (% m) 0 56.8% 63.0% 51.4% 42.1% 30 40.4% 59.9% 70.7% 66.5% 60 62.6% 77.7% 83.2% 84.2% 90 50.8% 64.9% 81.8% 69.2% 120 70.5% 72.4% 79.8% 53.9% 150 29.8% 72.0% 84.8% 60.0% 180 39.6% 68.2% 83.7% 43.8% 210 25.3% 32.1% 73.9% 19.1% 240 16.0% 27.0% 73.4% 34.7%
(50) The analysis of the evolution of the nematode population in soil was carried out from the juveniles count (J2) and the mortality rates of their populations. It was found that both Vydate treatment and the treatment with the composition of the invention, kept very similar J2 levels throughout the assay, showing the control by these treatments, but not for the case of the application of Glomus iranicum var. tenuihypharum var. nov. and absolute control.
(51) However, it was found that the application of the composition of the invention promoted the highest mortality rates, also these were constant from day 60 until the end of the assay, which demonstrates its power as bio-nematicide, due to the effect promoted by the combined action on one part of the mycorrhizal fungi, and on the other part of metal ions and chitin which stimulated nematode destruction in the rhizosphere. The chemical control treatment reached the highest rates at 60 days, but its effectiveness was closely related to the punctual moments of its application and not sustained as with the previous case.
(52) The Glomus iranicum var. tenuihypharum var. nov. strain alone had mortality values above 60%, and after 6 months, its activity declined significantly. Absolute control had the worst mortality rates in soil.
(53) Nematode Counts in the Preventive Treatment
(54) TABLE-US-00010 TABLE 16 Live juvenile nematodes count Glomus iranicum var. tenuihypharum Composition var. nov. of the Days Control (n) (n) invention (n) Vydate (n) 0 70.5 130 53 53 30 91.5 43 45.5 14 60 41.5 15.5 4.5 3.5 90 143.5 58 38 49.5 120 52.5 33 32.5 7 150 45 168 62 41 180 162.5 173 108.5 37 210 63 63.5 14.5 30.5 240 82 71.5 14 65
(55) TABLE-US-00011 TABLE 17 Dead juvenile nematodes count Glomus iranicum var. tenuihypharum Composition var. nov. of the Days Control (n) (n) invention (n) Vydate (n) 0 119 221.5 99.5 116 30 21.5 56 99.5 30 60 184.5 103 45 29.5 90 95 130 212 105 120 109 146 125.5 47.5 150 108 201.5 177.5 140 180 107 197 179.5 50 210 17 24 44 43.5 240 17 18.5 63.5 54
(56) TABLE-US-00012 TABLE 18 Mortality rate (%) Glomus iranicum var. tenuihypharum Composition var. nov. of the Days Control (n) (n) invention (n) Vydate (n) 0 41.0% 49.7% 46.6% 53.1% 30 19.1% 56.7% 68.6% 67.8% 60 81.6% 86.6% 90.9% 89.7% 90 41.7% 68.9% 84.8% 70.3% 120 67.7% 83.4% 81.9% 87.0% 150 71.1% 59.7% 72.3% 77.8% 180 39.5% 53.7% 62.0% 57.3% 210 21.4% 28.6% 75.8% 58.4% 240 17.2% 20.7% 81.0% 45.3%
(57) In the preventive case the Vydate treatment kept J2 levels lower than the treatment with the composition of the invention, however, in both cases it was possible to control the nematode population compared with the rest of the treatments that maintained very similar levels throughout the assay.
(58) The analysis of the mortality rate in these conditions yielded results that were very similar to the curative case, so the application of the composition of the invention promoted the highest mortality rates and these were also constant from day 60 until the end of the assay. The chemical control treatment reached the highest rates at 60 and 120 days, but then progressively decreased. Also here its effectiveness was closely related to the moments of its application and it was not sustained.
(59) Both in curative and preventive conditions, the composition of the invention has shown to have a high mortality rate of nematodes, which remains at around 70-80% throughout the growing cycle. Vydate treatment, however, lost effectiveness over days.
(60) Rhizosphere Activity in the Curative Treatment
(61) Serial dilutions of rhizosphere soil samples were prepared. Subsequently, the concentrations of rhizosphere bacteria and fungi present in the different treatments were quantified by determining the most probable number of microorganisms. For bacterial growth nutrient agar medium was used and for fungi growth, Rose Bengal and potato dextrose agar.
(62) The graphs of the microbial evolution of the total microflora (bacteria and fungi) throughout the assay in the curative treatments (
(63) Rhizosphere Activity in the Preventive Treatment
(64) The results are shown in
(65) It is good to note the low microbial activity found in the control treatment and in the Vydate treatment. This low activity is due to severe effects in control plants by nematode attack and a disinfectant effect of Vydate that caused a very significant decrease of microbial populations.
(66) Production and Quality
(67) Each treatment was harvested under curative and preventive conditions and the fruits were counted and weighed. The fruits affected with some defect or disease were discarded.
(68) Only the fruits having some defect or disease were considered as rejections, and they are discarded. Small fruits were not discarded. Once the collection started, it was harvested about once a week until the end of the crop.
(69) The total data of each of the varieties in the preventive and curative treatment are shown below:
(70) TABLE-US-00013 TABLE 19 Daniela Tomato (curative treatment) Number Weight Weight Number Average of Fruits/ (g/ (g) of fruits weight plants plant plant) Control 27113.7 315 101.7 6 52.5 4519.0 Glomus 45453.4 388 115.2 5 77.6 9090.7 iranicum var. tenuihypharum var. nov. Composition 49444.2 390 124.1 4 97.5 12361.1 of the invention Vydate 46672.3 385 130.5 7 55.0 6667.5
(71) TABLE-US-00014 TABLE 20 Daniela Tomato (preventive treatment) Number Weight Weight Number Average of Fruits/ (g/ (g) of fruits weight plants plant plant) Control 40625.8 380 118.5 6 63.3 6771.0 Glomus 55495 375 147.8 6 62.5 9249.2 iranicum var. tenuihypharum var. nov. Composition 64839.1 414 149.0 5 82.8 12967.8 of the invention Vydate 57619.9 452 134.4 6 75.3 9603.3
(72) TABLE-US-00015 TABLE 21 Raf Tomato (curative treatment) Number Weight Weight Number Average of Fruits/ (g/ (g) of fruits weight plants plant plant) Control 23161.1 229 105.8 7 32.7 3308.7 Glomus 28997.4 241 115.6 6 40.2 4832.9 iranicum var. tenuihypharum var. nov. Composition 22626.6 235 96.7 5 47.0 4925.3 of the invention Vydate 23876 244 105.6 6 40.7 3979.3
(73) TABLE-US-00016 TABLE 22 Raf Tomato (preventive treatment) Number Weight Weight Number Average of Fruits/ (g/ (g) of fruits weight plants plant plant) Control 27512.8 275 94.5 6 45.8 4585.5 Glomus 28334.3 234 127.5 6 39.0 4722.4 iranicum var. tenuihypharum var. nov. Composition 29132.4 243 114.2 6 40.5 4855.4 of the invention Vydate 24129.1 253 97.9 7 36.1 3447.0
(74) Data of Brix degrees and hardness of the fruit were taken in alternate collections, five times in total throughout the production period in order to assess whether the quality of the fruit was affected in some way.
(75) Total average data of each of the varieties in the preventive and curative treatment are shown below:
(76) TABLE-US-00017 TABLE 23 Raf Tomato (hardness) RAF Hardness T1 T2 T3 T4 Cur 5.5 5 5 4.7 Prev 4.5 4.5 5.5 5.1
(77) TABLE-US-00018 TABLE 24 Raf Tomato ( Brix) RAF Brix T1 T2 T3 T4 Cur 7.7 7.7 8.1 7.8 Prev 7.4 7.4 8.1 7.5
(78) TABLE-US-00019 TABLE 25 Daniela Tomato (hardness) Daniela Hardness T1 T2 T3 T4 Cur 4.8 5.6 5 5.6 Prev 4.8 5.3 5.1 4.9
(79) TABLE-US-00020 TABLE 26 Daniela Tomato ( Brix) Daniela Brix T1 T2 T3 T4 Cur 6.2 6.1 6.4 6 Prev 6.1 5.8 6.1 5.9
(80) No drastic changes were appreciated in Brix degrees caused by the nematode population or by the applications of the products. The values were normal for both varieties reaching maximum Brix degrees midway through the cycle (8.1 Brix measured in the Raf variety and 6.4 Brix in the Daniela variety, both data belong to treatment T3).
(81) The hardness of the fruit was not significantly affected since the penetrometer values are normal for each variety. In Raf the values are between 4.5 and 5.5 for all treatments and in Daniela 4.8 to 5.6. Therefore the fruits showed normal consistency both in the presence of the pest and after the application of the tested products.