JPH0235076A - Novel microorganism and plant blight controlling method using said microorganism - Google Patents

Abstract

NEW MATERIAL:A microbial strain belonging to Pseudomonas gladioli and capable of reducing nitrate and assimilating L-tartaric acid. EXAMPLE:Pseudomonas gladioli D-2251 (FERM P-9483). USE:Useful for the control of soil pathogens. It exhibits excellent effect especially for the control of rice blast caused by microbial strain belonging to genus Pyricularia. PREPARATION:Bulb or leaf of dendrobium, vanda or cymbidium is ground in 1% peptone water. The ground suspension is streaked on a bouillon-agar and cultured at 25 deg.C for 48hr. The formed colonies are fished.

Description

Detailed Description of the Invention [Objective of the Invention] (Field of Industrial Application) The present invention provides a novel microorganism, and uses the novel microorganism to treat Fusarium spp.
Control of soil pathogenic bacteria such as M genus bacteria, especially Pyricular
To develop a method for controlling a plant disease that is highly effective in controlling rice blast caused by a fungus of the genus Ia.

(Prior Art) Conventionally, soil diseases that cause harmful effects such as withering, damping, and vine cracking have been commonly treated by fumigating the soil using chlorpicrin and methyl bromide agents.
However, this method is economically expensive and extremely toxic, so the sprayer's HF! ! There is a risk of harming i.
Furthermore, even after spraying, the safety of surrounding residents cannot be ensured, which is a serious problem.

In addition, rice blast disease, which is the most widespread among the various diseases affecting paddy rice and causes serious damage, requires urgent control measures, and conventionally, anti-blast agents such as kasugamycin and plasticidin have been used to treat it. Methods have been adopted to control the disease, but the effects of these anti-rice blast agents tend to become unstable due to the appearance of anti-inflammatory bacteria, and there is also the risk of contaminating the natural environment due to drug damage.

(Problems to be Solved by the Present Invention) In view of the above circumstances, the present inventor discovered new microorganisms from panda, dendrobium, and cymbidium, which are types of orchids, and clarified their mycological properties. As a result of further experiments, the new microorganism, for example, Fusari
It has been confirmed that this product exhibits effective antibacterial activity against pathogenic bacteria such as U.S. It is intended to be

[Structure and Effects of the Invention] The microorganism of the present invention is a novel strain isolated from the bulbs or leaves of, for example, a type of orchid, (a) Dendrobium, (b) Panda, or (C) Cymbidium, and the isolation is Bulbs or leaves of Panda, Dendrobium, and Cymbidium were crushed in 1% peptone water, streaked onto bouillon agar, and cultured at 25° C. for about 48 hours, and the colonies formed were harvested. Pseudomonas gl has the mycological properties shown below, and for the reasons detailed later.
(a) The strain isolated from Dentrobium was identified as a new strain belonging to P. adioli.
The strain was named Pseudomonas gladioli D-2251 and deposited with the Institute of Microbiology, Agency of Industrial Science and Technology as "Feiko Kenbacterium No. 9483".
s gladioli V-2400 and deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as "Feikoken Bacterial Science No. 9484".
It was named w+onas gladioli C-0620 and has been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology as ``Keiken Bacterial Science No. 9963''.

Its mycological properties are as follows.

(a) Morphological properties Same as left Cell shape Bacillus, 1.01.2 and size Presence of spores with 1 to 2 monopolar hairs No Durham staining Negative acid-fastness Negative accumulation of PBH granules Positive (b) Growth status in each medium D-2251V-2400 Same as left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Left -left gravy culture culture growth growth well -colored colony Colorony Colorony Coloni Same left, left, left same left, left colony transparency of transparent left colony. No growth on liquid surface Liquefied alkaline meat juice Gelatin Puncture Lidomus milk (c) Physiological properties a) - Boat fishing Physiological properties Positive Negative Negative Negative Reduction denitrification reaction of nitrate MR test VP test Formation of indole Same left circular convex circular shape Same left circular convex circular, left left, left same left, left same left same left same left same left same left same left same left same left same left same left d -2251V -2400 hydrogen sulfide hydrogen sin sulfide positive hydrolyzed podnidic hydrolysis positive use of positive left quenched. Utilization of source Positive Generation of the same fluorescent dye as left Negative Formation of the same water-soluble dye Positive Same urease Negative Same oxidase East neighborliness
Same as left Catalase Positive Same as left Growth range 8-41”C Same as left Attitude towards oxygen Aerobic Same as left 0-F Test
1) -2251V-21100C-06201)-22
511 (51 North Positive Same as left Same left Negative D
-751 Knee North Positive Same as left Same as left Negative 0-
4 Solo Solo -positive, left negative, left negative, left negative same left same left, left left same left left left gas left left gas left gas left left gas left gas left gas left gas left left gas left d -2251V -24200C -06200-22510
-ri-11] -λ Positive Same left Same left Negative Qi
Nose positive Same left Same left Negative D-75 Masu
Positive Same left Same left Negative 0-h-rough 1-s
Positive Same left Same left Negative maltose Negative Same left Same left Negative Sucrose Negative Positive Negative Negative Lactose Positive Same left Same left Negative Trehalose Positive Same left Same left Negative D-So IH: -
) Positive Same as left Same as left Negative 0-7 positive Same as left Negative Innosoft positive Same as left
Same as left Negative Ku II t 'J' positive Same as left Same as left Negative Teishibushi Negative Same as left Same as left Negative? Do2)-1I Positive Same left Same left Negative C) Part II! ! Physiological properties of D-2251V-2400 Use of alcohol Positive Same left Hydrolysis of Aesculin Positive Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left D-2251V-2400 Degradation of hippuric acid Positive Utilization of malonic acid as on the left Positive Hydrolysis of arginine as on the left Negative
Decarboxylation reaction of lysine (same as left) Negative (same as left 5% chloride) Growth of +7um (same as left) Lipase (same as left)
Positive Same as left Lecithinase Positive Same as left 2) Use #l: Test D-2251V-2400C-0620 Satsuh'D acid
Positive Same as left Same as left Firin M
Positive Negative Positive me9kosyic acid
positive rtrue positive acetic acid
Negative Positive Positive phosphoric acid Positive Same as left Same as left Kim Positive Same as left
Same as left Humano+acid Positive Same as left Linchoic acid Same as left Same as left Oxalic acid
Negative Same as left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Brohyne Acid Succinic acid Lactic acid - Tartaric acid - Tartaric acid Benzoic acid Coolconic acid? R: / Acid L Pintothenic Acid L Pyroxylic Acid II Tamic Acid Hydromeric Acid - Hydro ~ Cybenzoic Acid α ? [Mi81 Min-2] Malonic acid hippuric acid Negative Positive Positive Positive Positive Positive Positive Positive Negative Positive Positive Negative Negative Positive Positive Positive Same left Same left Same left Same left Negative Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Negative left positive left positive, positive positive positive left bird] ゛ ネ 酸 ソ ソ ソ ソ ソ ソ ソ ソ ソ ソ ソ ソ ソ ソ ソ ソ ソ ソ ソ ソ ソ ソ ソ ソ ソ 酸 ソ 酸 酸 酸Ntran-11-acid iso-anthoranic acid n-caffeinated acid tecanic acid coulter j1-acid polycylinyl-citrulline beta -75 nin positive positive negative negative negative negative positive positive negative positive positive negative positive positive positive positive positive negative east neighbor same left same left same left same left Same left East East Night Neighborhood Same Same Sono left negative left negative left negative left negative positive positive positive positive East East East East East Neighborhood, left positive left positive left positive positive positive positive positive positive positive positive v -2400C -0620 p -7 mino chicken frostolic acid Shi-ornithinerohefu0tan Acid butyric acid Ichirokufuron Acid sulfurin Acid fluoresceon Iso0 Inositutanthiol? Sericic acid? So 0 phosphoric acid Itacosic acid J Tracon acid 04 Mine 2- Nurcaf 6 Toetano-II Negative Positive Negative Positive Positive Positive Positive Positive Negative Positive Negative Positive Negative Positive Negative Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left Same left left, left positive left positive left positive left positive left positive left -positive left -positive left, left positive left -positive left, left left, left, left left, left left, left, left, left, left, left, left, left, left, left, left, left, left, left, left, left, left positive left positive left positive left positive left positive left positive left positive, left positive, left positive left positive left, left positive left positive, left positive left positive left.
A search using Systematicbacteriology (Volume 1) shows that it is a Durham-negative bacillus, grows aerobically, has positive oxidase activity, does not form spores, and has a good growth status on bouillon agar. Book D-225 in terms of good quality, etc.
1V-2400 and C-0620 are clearly Pseud
It belongs to the genus Omonas. And as a related species, Ps
eudomonas caryophy and Pseud
omonas cepacia exists.

However, regarding D-2251, it is appropriate to consider it as a new bacterial species belonging to Pseudomonas gladioli for the following reasons. Namely, a) Hydrolysis of arginine, liquefaction of gelatin, adonitol, saccharic acid, levulinic acid, D-tartaric acid, benzoic acid, L-citrulline, L-ornithine, n-hebutanoic acid, speric acid, pimelic acid, adipic acid. , Pscudom, which is one of the closely related bacteria in terms of utilization of citraconic acid.

Unlike Pseudomonas caryophylli, the size of Pseudomonas cepacia is 0.8 to 1. OX1.6-3.2 microns, while the main side is 1.0-1.2X1.6-2.2 microns, production of metallic purple pigment, reduction of nitrates, butylamine, tryptamine, α - Pseu in terms of utilization of amylamine, m-hydroxybenzoic acid, butanediol, butrescine, spermine, D-tartaric acid, mesaconic acid
It is different from Pseudomonas cepacia, and in the end, it is most reasonable to judge that this species belongs to Pseudomonas gladioli. However, while this method utilizes pimelic acid, sheric acid, and levulinic acid, Pseudomonas gladio
Pseudomonas gla
The conventional bacteria of Dioli differ in that they do not reduce nitrate. Therefore, the main point is Pseudomo.
We believe that it is impossible to assign it to the conventional strain of nas gladioli, and it is appropriate to treat it as a new strain.
seudomonas gladioli D-2251
It was named.

Also, regarding V-2400, Ps
It is appropriate to consider it as a new bacterial species belonging to eudomonas 51adioli. Namely, a) hydrolysis of arginine, nighting of gelatin, adonitol, saccharic acid,
Pseudomonas caryo is a closely related bacterium that utilizes L-ornithine, n-hebutanoic acid, sperinic acid, azelaic acid, adipic acid, and citraconic acid.
Unlike phyl l i, the size of the bacteria is Ps
eudom.

nas cepacia is 0.8 to 1. OX1.6~3
．． 2 microns, whereas the main surface is 1.0 to 1.2 x 1
．． 6-2.2 microns, production of metallic purple pigment, reduction of nitrates, utilization of butylamine, tobutamine, pimelic acid, alpha-amylamine, m-hydroxybenzoic acid, butrescin, spermine, levulinic acid. Unlike Pseudomonas cepaca, magnolia is actually Pseudomonas gladi.

It is considered most appropriate to judge that it belongs to .

However, this method utilizes superric acid and butanediol and does not utilize D-tartaric acid and mesaconic acid to reduce nitrate, whereas Pseudomonas 31ad
The conventional strain No. 01 differs in that it does not utilize speric acid or butanediol and does not reduce nitrate. Therefore, the magnolia as it is is Pseudomonas gladi.
It is considered that it is impossible to assign it to the conventional bacteria of Pseudomonas, and it is appropriate to treat it as a new strain.
sgladioli V-2400.

Furthermore, regarding C-0620, P
It is appropriate to consider it as a new bacterial species belonging to seudomonas gladioli. Namely, a) Hydrolysis of arginine, liquefaction of gelatin, citrulline, anthranilic acid,
heptanoic acid, caprylic acid, adipic acid, azelaic acid,
Pseudomonas c. is one of the most closely related bacteria in terms of utilization of citraconic acid, adonitol, and ornithine.
Unlike Pseudomonas cepacia, the size of Pseudomonas cepacia is 0.8-1.
．． OX is 1.6-3.2 microns, whereas magnolia is 1
．． 0-1.2 Xl, 6-2°2 microns,
Pseudomonas c. for its production of metallic purple pigments and utilization of tryptamine, metahydroxybenzoic acid, bicudiol, butrescin, spermine, and mesaconic acid.
Unlike epacia, in the end magnolia is pseudomo
It is considered most appropriate to judge that it belongs to nas gladio'li. However, whereas magnolia uses butylamine, pimelic acid, α-amylamine, speric acid, realic acid, and L-tartaric acid to reduce nitrate, Pseudom.

The conventional strain of nas gladioli differs in that it does not utilize butylamine, pimelic acid, α-amylamine, superric acid, realic acid, and tartaric acid, and does not reduce nitrate. Therefore, magnolia is just Pseudo.
Kota said that it is impossible to assign Pseudomonas 31adioli to the conventional strain, but it is appropriate to classify it as a new strain, and it is classified as Pseudomonas gladioli C-0.
It was named 620.

And the above D-2251, V-2400 and C-06
20 is the conventional Pseudomonas gladio
Bergey's manual o
fsystematic bacteriolo8y
(according to Volume 1), both reduce nitrate and L-
Since it has the property of assimilating tartaric acid, it is inappropriate to include it in the conventional Pseudomonas gladioli, and it is considered that it constitutes a new and different phylogenetic group.

Now, this Pseudomonas gladioli
As a result of further investigation of the antibacterial effects of new microorganisms (hereinafter referred to as the new microorganisms) constituting a new phylogenetic group represented by D-2251 + V-2400 and C-0620, we found that Fusarium spp. and Rhizoctonia spp. , Cortesium, Viricularia, Alternaria, Verticillium, Bicillum, Sclerotium, etc.

The antibacterial activity was measured using a petri dish with a diameter of 90 mm.
and PPG^ medium (5 g of peptone,
xtrose broth 24g,'iKten+sg
) was cultured against pathogenic bacteria and magnolia at 25°C for -weeks.

The results are shown in the table below.

Plant Name Disease Name Pathogen Name Activity〕UkaOkyuritai〕Ntamane4'' Rough Bird〕 ?Suha0 Lakase blast disease Vine split disease Wilt disease Vine crack disease Vine split disease Yellowing disease Dry rot disease Dry rot disease Damping off disease D- 2251V-2400C-0620Pyricul
aria +++ +++ +
++ryzae F, oxysporum ++ +++
++f, sp, lagenar+ae F, oxysporum +++ +++
+++f, sp, 1ycopersici F, oxysporum ++ +
++ ++f, Sp, cucumerinu
m F, oxysporum ++ ++++
+f, sp, niveum F, oxysporum +++ +++
+f, sp, raphan F, oxysporuIll ++
10+ +f, sp, cepae F, oxysporull ++ +
+f, sp, al F, oxysporum ++ ++
++f, sp, asparag New cyclamen garlic] Restock corn cyclamen garlic F, oxysporum ++f, sp,
5p1nac+ae F, oxysporum ++f, sp, f
ragar1ae F, oxysporum +f, sp, cy
clamin+s F, oxysporum ÷+f, 5p4a
rl +c F, oxysporum +++f, sp, 1
illii F, oxysporum +f, sp, c
onglut+nans F, oxysporum ++f, sp, ar
ct F, oxysporua + + (Cymbi
dium) F, oxysporum +++ (Chine
se chive) F, oxysporu @ ++++ + +++ + +++ +++ + ++ +++ ++ ++ So] Gypsophila cyclamen (Zygocacutus) Bakanae disease F, moni l i form half-body withering Verticilliu m +++ malignancy d
ahl +ae Macrophomina phase.

Botrytis disease cinerea black spot Alternaria alternata damping off Rh1zoctonia oan black rot Sclerotium disease cep+voruWl anthracnose CCo11etotrichu ++ +c
yclamenae Corticium olfs Root rot Pythium sp.

10++ Tomato Corynebacterium
+++ disease michiganense Note) +: Active ++: Strong activity +++: Very strong activity In other words, from the above antibacterial activity test table, the new microorganisms are Fusarium spp., Verticillium spp., Botrytis spp. It is clear that it exhibits strong antibacterial activity against bacteria of the genus Alternaria, genus Piricularia, bacteria of the genus Sclerotium, bacteria of the genus Colletotrichum, bacteria of the genus Corchicilium, and bacteria of the genus Bicillum.

Therefore, we focused on this antibacterial effect and investigated ways to control the above-mentioned diseases. First, when we tried inoculating various plants with this new microorganism, we found that, for example, Cymbidium, Dendrobium,
Panda, Cadrea, Boinola, Pilstechera, Mildonia, Phalaenopsis, Paphiobedilum, Gladiolus, Iris, Onion, Leek, Leek, Corn,
It was confirmed that this new microorganism can be grown in the leaves, stems, roots, and bulbs of rice, Hytranger, persimmon, masaki, tomato, eggplant, and lettuce. The leaves, stems, roots, and bulbs of the above plants were inoculated to try to propagate them. As a result, as shown in the above-mentioned antibacterial activity test table, rice blast, sagebrush vine crack disease, strawberry chlorosis, tomato wilt, and eggplant half-wilt, which frequently occur in plants such as rice, sagebrush, tomato, cucumber, and watermelon, were detected. It has been shown that this method can control strawberry half wilt, damping-off, white silk disease, tomato canker disease, etc. Among these, it is particularly noteworthy that it is highly effective against rice blast disease, which is considered difficult to eradicate. In addition, it was confirmed that this new microorganism does not suffer from the adverse effects of side effects caused by conventional agricultural chemicals.

In addition to the method described in the upper column of the antibacterial activity test table, the antibacterial activity may be determined by a counterculture method inoculating plants of the genus Allium, a pot test method, or a test method in the field. That is, for example, according to the counterculture method in which plants of the genus Allium are inoculated, D-2251, V-2400, and C-0620 are adjusted to 10 cells/ml in chive stem disks punched out aseptically with corkpolar particles having a diameter of 6+ am. Inoculate the solution with
The antibacterial activity can be measured by implanting this on a PPGA medium and culturing it with Pythium genus bacteria (strawberry root rot fungus) at 25°C for 1 week.

As a result, the effects shown in the table below were obtained.

Treatment method Inhibition circle size Confrontation culture of chives only 1 mm or less V-24
00 only confronting culture 16.7mm V-2400 was inoculated 19.0mff1 chive and confronting culture D-2251 only confronting culture II 3.3mm D-2251 was inoculated 7.0mm chive and confronting tea C-0620 only confronting culture! I 2.5mmC-
Confrontation culture with 4.5m+* chives inoculated with 0620. These results indicate that inoculation of Allium genus plants in Japan increases its antibacterial activity.

As described above, this new microorganism has great industrial significance in that it can control soil diseases by biological means as an anti-filamentous fungus, and in particular, its ability to control rice blast without side effects is attracting much attention. be.

(Example 1) To explain an example of the method of culturing this new microorganism, 40.5 g of 2HPO was used as the medium.
, MgSO4,7H200,2g NaC15g, yeast extract 0.8g, 7bistoll 2g, pure water 10
Put the composition of 100O and pH 6.8 into a flask,
D-2 of Pseudomonas gladioli
Inoculated with 251, V-2400 and C-0620, 25
When incubated for 96 hours at a temperature of 10C, each bacterium
Grow to FU/ml.

(Example 2) Next, to explain a disease control method using this new microorganism, Bacterium D-2251 was treated with PPGA (Peptone 53.Potato dextrose broth).
24g of agar and 15g of agar) at a temperature of 25°C.
Cultured for 6 hours. Then, this was dissolved in sterilized water or water, and the number of bacteria was further adjusted to 10 cells/ml. Then, on the night of the adjustment, rice seeds or leaves were dipped or sprayed.

V-2400 and C-0620 were also tested under the same conditions, and the results shown in the table below were obtained.

Treatment method: No treatment 100% Seed inoculation 0% 100% 0% Disease rate 100% 0% Note) D-2251, V-2400 and C-0620 were re-isolated from rice.

(Re-separation conditions) NH4)12PO40.5g K2HPO40.5g MgSO4,7H200,2g NaC15g Agar 15g ``11 throat-11'' t0ne"0shi Pure water 25pprt1 000m , 22-3'-1 Procedural amendment (spontaneous) Supplementary Positive of Inside capacity April 13, 1989 On page 4, line 4 of the specification, Insert the following sentence.

1.Display of the incident 2. Name of the invention 1999 Patent Application No. 6965 New microorganisms and their use Plant disease control methods 3. Person who makes corrections Relationship with the incident address full name

Claims (1)

[Claims] 1) belonging to Pseudomonas gladioli;
A new microorganism that reduces nitrate and assimilates L-tartaric acid. 2) Institute of Microbial Technology, Agency of Industrial Science and Technology
The novel microorganism according to claim 1, which is identified as "No. 1". 3) Institute of Microbial Technology, Agency of Industrial Science and Technology
The novel microorganism according to claim 1, which is identified as "No. 1". 4) Institute of Microbial Technology, Agency of Industrial Science and Technology
The novel microorganism according to claim 1, which is identified as "No. 1". 5) Belongs to Pseudomonas gladioli,
A method for controlling plant diseases using a novel microorganism that reduces nitrate and assimilates L-tartaric acid, and has antimicrobial activity against any of Deuteromycetes, Ascomycetes, Basidiomycetes, Flagellate fungi, and bacteria.