JP2002275012A - Functional organic material for inhibiting plant disease injury - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a functional organic material by bearing antifungal matter- productive fungi on a carrier as a mixed compost prepared by compounding phytocompost and zoocompost together, in order to inhibit plant disease injury by biological control. SOLUTION: The functional organic material is a functional compost that is produced by preparing a mixed compost by compounding together the phytocompost with woody wastes as the main material and the zoocompost with fishery processing wastes as the main material and then bearing the above mixed compost as the carrier with at least one kind of antifungal matter- productive fungi belonging to the genus Burkholderia and having antifungal activity on plant pathogenic fungi, wherein the antifungal matter-productive fungi is e.g. Burkholderia cepacia. This functional organic material thus obtained can inhibit spinach stock rot.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a functional organic material which suppresses plant diseases by holding one or more antibacterial substance-producing bacteria having antibacterial activity against plant pathogenic bacteria.
More specifically, the present invention relates to a functional organic material that suppresses plant diseases holding Burkholderia cepacia, using a mixed compost prepared by blending and preparing plant compost and animal compost as a carrier.

[0002] Here, the Bar Cordelia cepacia (Burk)
holderia cepacia) is at least Rhizoctonia solanii (Rhizoctonia solan)
lani) and Fusarium, the causal agent of tomato root rot
It has been found to be an antibacterial substance-producing bacterium having antibacterial activity against oxysporum (Fusarium oxysporum). Hereinafter, antimicrobial activity (function), antagonism and antimicrobial activity are used interchangeably.

[0003]

2. Description of the Related Art Conventionally, it has been known that there is an environment-derived antibacterial substance-producing bacterium which does not show pathogenicity to plants but has antagonism (antibacterial activity) to plant pathogenic bacteria. The effectiveness (availability) as a means of biological control against plant diseases has been proposed.

[0004] For example, as proposed in Japanese Patent No. 2955642, a novel (unknown conventionally) antibacterial substance-producing bacterium having an antibacterial action against a specific plant pathogen is discovered (identified). Some of them have shown the reproducibility and completed the invention.

[0005] On the other hand, it is examined what kind of plant pathogenic bacteria the known antibacterial substance-producing bacteria have an antibacterial effect, and there are some which complete the invention.

In any case, it must be ensured that biological control is stably feasible and non-pathogenic upon human exposure.

[0007] Among these, Burkholderia cepacia [hereinafter referred to as cepacia. ]
Has attracted attention as a growth-promoting fungus for agricultural products that suppresses plant diseases. However, there is a current situation that cepacia bacteria (and their relatives) are listed as disease-derived bacteria [human pathogenic microorganisms] while they are environmentally-derived bacteria [environmental microorganisms]. Since there are many opportunities to be performed, the contact point (interaction) is regarded as a problem.

[0008] However, while verifying specific plant pathogens that exhibit antibacterial activity by cepacia bacteria isolated from the natural environment, and establishing culture conditions based on safety confirmation, a carrier that stably retains cepacia bacteria is developed. There is no doubt that developing is meaningful in industrial applicability. It goes without saying that development will proceed within the framework of microbial pesticide safety evaluation methods and biocontrol.
The background of the present invention is summarized below.

The present inventors have been conducting research and development of compost using the waste as a main raw material as part of effective utilization (reuse) of unused biological waste. The main purpose here was to develop soil and develop compost as fertilizer.

On the other hand, in terms of application of compost (fertilization), the pathogen of spinach rot is Rhizoctonia solani, and the pathogen of tomato root rot is Fusarium oxysporum.
rium oxysporum).

In the first place, compost has generally been required to have three functions. The first function is to improve the physical properties of the soil, such as drainage and drainage, and the soil is softened. The second is a function of improving chemical properties, such as enhancing the fertilizing ability and fertility and increasing the buffer capacity. The third is a biological improvement function, which is the diversification of microorganisms that necessarily results with application.

[0012] Today, under the circumstances in which the adverse effects of chemical pesticides are eliminated and environmentally conscious agriculture is promoted, attempts to discover and evaluate useful microbial genetic resources are accelerating. Regarding compost, it can be said that the above biological property improving function has become more important.

[0013] Therefore, a useful function of the group of microorganisms derived from the environment, that is, an antibacterial substance-producing bacterium having an antagonistic action (antibacterial activity function) against soil pathogens or plant pathogens, is extracted, and the added functionalities are obtained. Inspired by organic materials,
Several antibacterial substance-producing bacteria having antagonistic activity against plant pathogens were searched and isolated.

Then, Rhizocto sorani (Rhizocto)
nia solani), which was identified as a cepacia bacterium. Furthermore, after establishing the culture conditions for this strain, a cultivation test was performed by inoculating a prototype compost (carrier) to confirm the desired antibacterial activity. Thus, the invention of a functional organic material having an added antibacterial activity against plant pathogenic bacteria has been completed.

[0015]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and is intended to compost biological organic waste for reuse (resource recovery), and to use the compost as a carrier for antibacterial use. An object of the present invention is to provide a functional organic material which suppresses plant diseases which can be expected to contribute to the practical use of biological control means by adding an antibacterial activity to plant pathogenic bacteria by retaining a fungus that produces an active substance.

[0016]

Means for Solving the Problems In order to solve the problems, the present invention relates to an improvement of a functional organic material for suppressing plant diseases, which comprises a plant-based compost using woody waste as a main raw material and a marine processing waste. A mixed compost is prepared by blending an animal compost mainly composed of an antibacterial substance which belongs to the genus Burkholderia and has an antibacterial activity against plant pathogenic bacteria. It is characterized by being held.

Here, one antibacterial substance-producing bacterium is Burkholderia cepacia.

The plant pathogen is Rhizoctonia solani.

The plant pathogen is Fusarium oxysporum.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION In the embodiment of the present invention, one of the organic materials having the above constitution is a plant compost derived from food processing sludge or other organic waste containing woody and plant and animal residues, and a marine processing sludge. , Prepare animal compost derived from water-soluble protein of fish and other organic waste,
One or more antibacterial substance-producing bacteria having antibacterial activity against plant pathogenic bacteria are separated and extracted from the plant-based compost and subjected to liquid culture, and the antibacterial substance-producing bacteria after the cultivation are subjected to the plant-based compost and the animal-based compost. Is mixed into a primary mixing compost prepared and mixed and stirred to obtain an intermediate material having the following properties. (1) One antibacterial substance-producing bacterium is Burkholderia cepacia. (2) The plant pathogen is Rhizoctonia solani
a solani) or Fusarium oxysporum
oxysporum). (3) The antibacterial density [cfu / g] is at least 10 ¹⁰ orders. (4) Used as seed compost.

The organic material to be distributed as a product (final product) is used as a seed compost using the above-mentioned intermediate material and put into a dozen-fold amount of secondary mixed compost prepared in the same blend as the primary mixed compost. A product material obtained by diluting and mixing, and having the following properties. (1) One antibacterial substance-producing bacterium is Burkholderia cepacia. (2) The plant pathogen is Rhizoctonia solani
a solani) or Fusarium oxysporum
oxysporum). (3) The dilution ratio for seed compost is several tens of times, and the antibacterial density [cfu / g] is at least 10 ⁷ orders.

[0022]

DESCRIPTION OF THE PREFERRED EMBODIMENTS One embodiment of the present invention will be described in the following item order with reference to the accompanying drawings.

1. Design and trial production 1-1) Search for antibacterial substance-producing bacteria and culture conditions 1-2) Identification and safety evaluation of antibacterial substance-producing bacteria 1-3) Propagation operation of antibacterial substance-producing bacteria 1-4) Seed compost (Intermediate material) design and trial production 1-5) Product compost (product material) design and trial production

2. Implementation and evaluation 2-1) Test plant and soil 2-2) Field test 1 and results 2-3) Field test 2 and results 2-4) Discussion 2-5) Functional compost (functional organic of the present invention) Materials)
2-6) Construction of manufacturing system and industrial applicability (outlook)

1. Design and Prototyping 1-1) Search for Antibacterial Substance Producing Bacteria and Culture Conditions One of the present inventors (Tamio Fujiwara) collected a sample of a plant-based compost (trade name: "Fujiro") involved in production. . This plant compost mainly consists of woody raw materials such as bark (bark), pruned foliage, forest deforestation waste, etc., and is used as a nitrogen source in the food industry sludge (milk drink, confectionery / bakery, noodle making, brewing, etc.) This is a fully-ripened compost that has been fermented and matured for 6 months or more by fusing animal and plant residues, waste milk, molasses, etc. [Hereinafter, plant test compost. ]

The target plant disease was spinach rot, and its pathogen, Rhizoctonia solani (Rhiz
octonia solani Kuhn) was obtained from Hiroshima Prefectural Agricultural Technology Center. [Hereinafter, target pathogens. ]

As a medium used in the confrontation test (detection of antibacterial activity), a PDA medium (potato dextrose agar medium) suitable for both pathogenic bacteria and antibacterial was used. PDA medium is PD
100 ml in which 3.9 g of A was dissolved was sterilized at 121 ° C. for 15 minutes, and then 20 ml was transferred to each dish and solidified.

Approximately 1 g of the plant test compost was collected in a 26 ml test tube, 10 ml of sterile physiological saline was added, and the mixture was stirred and mixed with a vortex mixer for about 5 minutes. After leaving to stand at room temperature for about 20 minutes, a circular filter paper (φ8 mm, thick) was immersed in the supernatant. This was placed in the center of a PDA medium, and the target pathogen was inoculated at equal intervals on all sides (four equal positions around the filter paper) and cultured in a constant temperature room at 25 ° C for 2 days.

If microorganisms that produce antibacterial substances are present in the suspension of the plant-based test compost soaked with the circular filter paper, the inoculated pathogen grows around the filter paper, so that the inhibition circle is reduced. It is formed. The streak smear culture was repeated for the antibacterial group collected from the inhibition circle, and the purified (isolated) antibacterial was transferred to the slant culture.

Here, from the bacteriological properties (Table 1) and 16S rRNA analysis described below, the antibacterial group
In addition to Burkholderia cepacia, its closely related Burkholderia glimae, Burkholderia vandii, Burkholderia plantari (Burkholderia plantari)
i), Burkholderia Cocovenance
cocovenenans), Bar Cordelia Gladioli (Burkh
It was estimated to be olderia gladioli, Burkholderia antimicrobica, Burkholderia glathei, Burkholderia caryophylli, and Burkholderia phenazinium.

Then, a new face-to-face test was carried out using the antibacterial cells cultured on the slope, and those which formed a clear inhibition circle were treated as antibacterial strains. [Hereinafter, the test antimicrobial strain. ]

1-2) Identification and Safety of Antibacterial Substance Producing Bacteria
Bacteriological properties of the evaluation test antibody strains shown in Table 1. Here, the test antimicrobial strain was found to belong to Burkholderia.

[0033]

[Table 1]

At the same time, 16S rRNA analysis of the test antimicrobial strain revealed that the test antimicrobial strain was cepacia (Burkho
lderia cepacia). [Data of gene base sequence is omitted]

Further, the safety of the test antibacterial strain (Bacillus cepacia) was evaluated by polymerase chain reaction (hereinafter, PCR). That is, it was determined whether or not the disease pathogen Burkholderia cepacia <genomovar III> was identified by genomovar identification. As a result of PCR using three pairs of primers, a pair of PC-SSF and PC-SSR (genomovar
I, III, IV), a pair of BC-GII and BC-R (gen
omovar II is not amplified by BC-GV and BC
It was amplified with the primer of the -R pair (recognizing genomovar V) and determined to be genomovar V. Therefore, the test antimicrobial strain was a cepacia bacterium derived from the natural environment, and even if it could be exposed to humans during its handling, it was evaluated that its pathogenicity could be denied for the time being.

1-3) Proliferation operation of antibacterial substance-producing bacteria In an jar fermenter, NB medium (NUTRIENT BROTH; NB1
3g + AGAR15g / l) 3l, 150ml (5% of the medium volume) of the test antimicrobial strain collected by centrifugation after shaking culture was added,
Liquid culture was performed for 24 hours by a predetermined method. Estimate the peak time from the transition of the dissolved oxygen (DO), extract 10 ml of the culture solution every hour, measure the absorbance (OD660), and add the amount to be added to the growth apparatus described later in a low-temperature room at 4 ° C. Saved time. [Hereinafter, a culture sample. ]

A 1 ml culture sample was taken, diluted in 10 steps with 10 ml of sterile physiological saline (0.9% NaCl), inoculated on a PDA plate medium, and cultured at 30 ° C. for 2 days. The density was measured, and it was defined as the number of viable bacteria in the culture sample. A maximum antibacterial density of 10 ¹³ cfu / ml was obtained.

1-4) Design of seed compost and trial production The compost was set on a scale of 1 t, and 900 kg of test compost (plant) and 100 kg of animal compost were blended to prepare a mixed compost. Here, animal compost is formulated as a nutrient source, and is processed by fishery processing sludge, fish water-soluble protein, and other organic wastes, which are involved in the production by another of the present inventors (Tetsuo Hanakawa). Compost (product name "Hamayuki"). Nitrogen 4 ~
Contains 5%, phosphoric acid 9-11%, potassium 1-1.5%.

This mixed compost is put into a breeding apparatus,
In addition, 1000 ml of the culture sample was charged, and the mixture was rotated and stirred for 72 hours while maintaining the pH at 7 at 35 ° C., and the antibacterial density of the seed compost from which the sample was extracted was measured. The antibacterial density of 10 ¹⁰ cfu / g was secured until the 7th day of the material age. In some cases, seed compost is placed in circulation.

1-5) Design of product compost and effective antibacterial density of compost used for trial production and application are 10 ⁷ cfu /
Since g is used as an index, if 1% of seed compost is added to the product compost capacity to dilute it uniformly,
Antimicrobial density of 10 ⁸ orders is inferred shall be guaranteed. So we set the product compost to 100t scale,
First, a mixed compost 99t prepared by mixing test compost (plant-based) 98t and animal compost 1t was prepared. Here, the reason for declining by adding seed compost instead of directly adding a predetermined amount of culture sample to the set volume of product compost is an advantage in manufacturing control that the volume of product compost can be set freely. Because.

Then, the mixed compost (99 t) and the seed compost (1 t) were put into a mixing device (mixer), and the antibacterial density of the product compost from which the sample was extracted was measured.
Until the 18th material age, an antibacterial density of 10 ⁸ cfu / g was secured. In the case where the product compost to provide the market, since it is there is of the order of one month expiration date, effective antibacterial density 10 ⁷ cfu / g at the time of application if the ship products compost having antimicrobial density of 10 ⁸ order enough Think it is guaranteed.

2. Implementation and Evaluation 2-1) Spinach [Cultivar: Okame (Spinacia)
oleracea L.)], and cultivated soil in a planter. As the cultivated soil (raw soil or control soil), a mixture of 10 kg of hard acid soil (masa soil) and 3.5 L of pearlite per planter was used. Fertilizer is ammonium sulfate (300
kgN / ha equivalent), superphosphate (344kg P ₂ O ₅ / ha equivalent)
, Potassium sulfate (equivalent to 120 kgK ₂ O / ha) and formic lime (equivalent to 4000 kg / ha) were mixed into the soil.

2-2) Field Test 1 and Result Processing The target pathogenic bacteria (hereinafter simply referred to as pathogenic bacteria) are placed in an Erlenmeyer flask (100 m
After growing to vermiculite in l), one Erlenmeyer flask was inoculated per planter. Product compost (hereinafter referred to as functional compost) was used as a test compost, and 200 g per planter was added and mixed when the fertilizer was charged. In the treatment group to which the functional compost was administered, half of the ammonium sulfate was administered. The treatment plots were a control plot (only in the original soil), a pathogen inoculation plot (in the original plot only), a functional compost administration plot, a pathogen inoculation plot and a functional compost administration plot. [See Table 2 below]

Seeding and Cultivation Seeding was carried out three times at different times. At the first time, 15 strains were sown at a rate of about 3 cm between the strains, two to three grains per strain. Second
And at the third time, one seed was sown for each of 15 strains. After sowing, each in the growth cabinet [(day
28 ° C / 25 ° C at night), natural light, and a relative humidity of 70%].

Results The results of surveys on the time of sowing, the number of germinated individuals (total number of strains), the number of affected individuals, etc. are shown in Table 2, and a graph of the morbidity based on the data without regard to the sowing time is shown in FIG. Table 3 shows the results of the re-aggregation of the morbidity by seeding time, and the graph is shown in FIG.

[0046]

[Table 2]

[0047]

[Table 3]

As can be seen from the total morbidity without regard to the sowing time (FIG. 1) and the morbidity at each sowing time (FIG. 2), the functional compost-administered group not inoculated with the pathogenic bacteria (the sign on the horizontal axis) In 3), the morbidity was 0%, which was completely suppressed, and it was confirmed that the disease was significantly suppressed in the functional compost administration group (2 + 3 on the horizontal axis) inoculated with pathogenic bacteria. Moreover, since there is no significant difference in the morbidity according to the sowing time in the functional compost-administered group inoculated with the pathogen (2 + 3 on the horizontal axis), it is recognized that the compost effect is not impaired.

2-3) Field test 2 and results Separately, the effect on the germination (number of individuals) of the functional compost was carried out as field test 2, but the treatment, sowing, and cultivation were almost the same as those in field test 1 above. Details are omitted, and FIG.
Only the results are described. In FIG. 3, sterile soil (control)
Is a test soil in which the test soil is sterilized, compost A is a test plot in which functional compost prepared in the laboratory is administered to non-sterile soil, and compost B is a test plot in which functional compost experimentally prepared in a non-sterile soil is administered using a growth apparatus. , And compost C are test plots in which mixed compost (without adding antimicrobial) was administered to non-sterile soil, and the sign (+-) indicates the presence or absence of pathogen inoculation.

The administration of the functional compost significantly increased the number of germinated individuals irrespective of the presence or absence of the inoculation of pathogenic bacteria, and significantly suppressed the decrease in the number of germinated individuals due to inoculation of the pathogenic bacteria. In addition, the morbidity was significantly reduced, and poor growth (inhibition) of germinated individuals was hardly observed.

2-4) Discussion It was recognized that the application of the functional compost of the present invention was effective in suppressing spinach rot. In addition, it was inferred that healthy germinated individuals had vigorous growth, and in particular, had remarkable growth of leaves and area, and exhibited a composting effect.

2-5) Evaluation of Functional Compost The functional compost of the present invention is a compound which holds a carrier producing an antibacterial substance in a suitable environment and exhibits a desired antibacterial function, and is used as a biological control means. Evaluated as valid.

Moreover, the mixed compost as a carrier is:
As a support for soil microorganisms (an energy supply source such as the supply of soil minerals), it is evaluated that it has not only impaired the original composting effect but also supplemented it.

[0054] Therefore, it is evaluated that it contributes to avoiding soil contamination and crop contamination due to pesticides, and to restore the material circulation including the cultivated land ecosystem to its original shape.

2-6) Construction of Manufacturing System and Industrial Use
Utilization (outlook) Unused woody waste, which is the main raw material for plant-based compost, is pruned foliage and forest deforestation waste, and has a C / N ratio of 40.
Since it is about 70 to about 70, which is much smaller than the bark of sawn wood bark 200 to 300, it can be said that there is almost no technical problem (difficulty) in composting. This kind of raw material resources is easy to obtain in large quantities, and product compost can be produced and supplied sequentially at least on a scale of at least 100 tons, so that it has both supply and economics that contribute to biological control of vast arable soil. ing.

The immediate problems related to the implementation are expected to be the place where the raw materials are generated (including collection) and the logistics cost of the composting factory. In recent years, as part of a resource recycling society system promoted by local governments, etc. If it can be incorporated into the local recycling circle (processed items), it can be expected that it will greatly advance.

[Brief description of the drawings]

FIG. 1 is a graph showing the disease-suppressing effect based on Table 2 obtained by Field Test 1.

FIG. 2 is a graph showing the disease-suppressing effect based on Table 3 in the results of field test 1.

FIG. 3 is a graph showing the effect on the number of germinations in field test 2.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) C05F 11/00 C05F 11/00 C05G 3/02 C05G 3/02 C12N 1/20 C12N 1/20 ED / / (C12N 1/20 (C12N 1/20 C12R 1:01) C12R 1:01) F term (reference) 2B022 BA11 BA18 BB01 4B065 AA01X AC14 BA22 BB23 BB26 BD05 CA48 CA55 4H011 AA01 AA03 BA01 BB21 DD04 DG06 4H061 AA01 CC32 CC60 DD07 EE66 FF06 GG48 HH08 LL24

Claims (7)

[Claims] 1. To improve a functional organic material for controlling plant diseases, a mixed compost is prepared by blending a plant compost composed mainly of woody waste and an animal compost composed mainly of marine processing waste. And a plant disease-suppressing function characterized by comprising one or more antibacterial substance-producing bacteria belonging to the genus Burkholderia and having antibacterial activity against plant pathogens. Organic materials. 2. For 100 parts by weight of the mixed compost,
The functional organic material for suppressing plant diseases according to claim 1, which has a compounding ratio of 90 parts by weight or more of plant compost and 10 parts by weight or less of animal compost. 3. The functional organic material for controlling plant diseases according to claim 1, wherein the one antibacterial substance-producing bacterium is Burkholderia cepacia. 4. The plant pathogen is Rhizoctonia solani (Rh
The functional organic material for controlling plant diseases according to claim 1, which is izoctonia solani). 5. The functional organic material for suppressing plant diseases according to claim 1, wherein the plant pathogenic fungus is Fusarium oxysporum. 6. A plant compost derived from food processing sludge or other organic waste containing woody or animal and plant residues, and a water-soluble protein of marine processing sludge and fish, for improving a functional organic material for suppressing plant diseases. Animal compost derived from other organic waste is prepared, and one or more antibacterial substance-producing bacteria having antibacterial activity against plant pathogenic bacteria are separated and extracted from the plant compost, and liquid culture is performed. An intermediate material obtained by adding the subsequent antibacterial substance-producing bacterium to the primary mixed compost in which the plant-based compost and the animal-based compost are blended and prepared, and agitating, and having the following properties. Functional organic materials that control plant diseases. (1) One antibacterial substance-producing bacterium is Burkholderia cepacia. (2) The plant pathogen is Rhizoctonia solani
a solani) or Fusarium oxysporum
oxysporum). (3) The antibacterial density [cfu / g] is at least 10 ¹⁰ orders. (4) Used as seed compost. 7. In the improvement of a functional organic material for suppressing plant diseases, the material according to claim 6 is used as a seed compost, and several tens times the amount of a secondary mixed compost prepared in the same composition as the primary mixed compost. A functional organic material which suppresses plant diseases, characterized by having the following properties: (1) One antibacterial substance-producing bacterium is Burkholderia cepacia. (2) The plant pathogen is Rhizoctonia solani
a solani) or Fusarium oxysporum
oxysporum). (3) The dilution ratio for seed compost is several tens of times, and the antibacterial density [cfu / g] is at least 10 ⁷ orders.