Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Bacteria or actinomycetales
Reexamination Certificate
2000-03-09
2003-05-20
Marx, Irene (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Bacteria or actinomycetales
C424S093460, C424S093470, C424S093480, C435S252100, C435S253300, C435S252900, C435S252500
Reexamination Certificate
active
06565846
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for screening microbes antagonistic to pathogenic bacteria that emerge during raising of seedlings of gramineous plants by selection from a plant body, soil, a seed or seed soaking water utilizing tropolone resistance and tropolone non-producing characteristics as indices. Also, the present invention relates to a microbial pesticide containing as an active ingredients a bacterium and spores thereof characterized by having a resistance to tropolone and not producing tropolone and also having a antagonistic activity against pathogenic bacteria that emerge during raising of seedlings of gramineous plants.
BACKGROUND ART
Diseases that emerge during raising of seedlings of gramineous plants include rice “Bakanae” disease, Helminthosporium leaf spot, and blast typically caused by fungi, rice bacterial gram rot, bacterial seedling blight, bacterial brown stripe, etc. caused by bacteria. To prevent these diseases, seed disinfectants, soil drenching agents, soil drench agents, foliage application agents after greening are said to be effective and these are used systematically by using them singly or incombination. For the disease damages by fungi, it has recently become possible to prevent them at high degree with EBI agents having high efficacy. Also, the disease damages caused by bacteria are prevented by use of chemical pesticides. However, chemical pesticides are generally low in their effect when bacterial diseases outburst and so that the problem remains that no sufficient prevention can be obtained. In case bacterial diseases outburst, at present the affected seedlings must be abolished. Therefore, development of a pesticide that can prevent or control the outburst of bacterial diseases has been desired.
Under the circumstances, the present inventors have made intensive investigation with view to making a research on bio-controlling material that is superior in controlling effect to chemical pesticides on the bacterial diseases of gramineous plants. Focusing on tropolone, which is a pathogenic toxin of pathogenic bacteria on rice bacterial seedling blight disease, they have searched a bacterium that is resistant to tropolone but does not produce tropolone from rice plants and established a method for screening a antagonistic bacteria to the pathogenic bacteria. Using this screening method, they have made an extensive search for microbial materials that can be used for controlling bacterial diseases. As a result, they have been successful in isolating antagonistic bacteria from rice seedlings that are antagonistic to the pathogenic bacteria.
DISCLOSURE OF THE INVENTION
The present invention relates to a method for screening a microbe antagonistic to pathogenic bacteria that emerge during raising of seedlings of gramineous plants by selecting a microbe from plant body, soil, seeds or seed soaking water utilizing tropolone resistance and tropolone non-producing property as indices. Also the present invention relates to a microbial pesticide containing as an active ingredient, the bacterium that is characterized by tropolone resistance and tropolone non-producing property selected by the screening method or spores thereof. Specifically, the present invention relates to a microbial pesticide comprising as a bacterium having a antagonistic action against pathogenic bacteria on rice plants, especially those that emerge during raising rice seedlings, a bacterium selected from the group consisting of bacteria belonging to the genera Pseudomonas, Bacillus, and Enterobacter. Also the present invention relates to a microbial pesticide comprising a mixture of two or more out of these. Specifically, the present invention relates to amicrobial pesticide for controlling bacterial diseases of gramineous plants, characterized in that one of bacteria belonging to either one of the genera Pseudomonas, Bacillus, and Enterobacter that have a antagonistic activity to pathogenic bacteria for bacterial diseases emerging in rice plants or a mixture of two or more of these is used for seed treatment or caused to be contained by soil for raising seedlings, or the bacterial cells are added to a seed soaking water, or the bacterium-containing solution is sprayed onto a rice plant body in the field.
BEST MODE FOR CARRYING OUT THE INVENTION
The screening method of the present invention is carried out by placing or spreading a fragment of a plant such as rice plant, homogenate of plant, soil, a seed, and a seed soaking water, on a suitable solid medium containing tropolone transplanting the resulting colonies of a tropolone resistant bacterium on a suitable solid medium containing iron ion, and selecting a colony that shows no red color on the iron-containing medium, cultivating the bacterium on a suitable medium to grow it, collecting the bacterial cells, converting the cells to a formulation or directly treating the infected seeds with a live bacterium suspension, and then raising seedlings and examining the protective activity to select an effective strain. In the screening of the present invention, tropolone derivatives may also be used instead of tropolone.
The bacterium that can be selected by the screening of the present invention and has a antagonistic activity to the pathogenic bacteria emerging during raising seedlings of gramineous plants, or spores of the bacterium can be utilized as an active ingredient of a microbial pesticide. The bio-controlling material that are used in the present invention are obtained by the above screening method and include, for example, bacteria belonging to either one of the genera Pseudomonas, Bacillus, and Enterobacter or two or more of them may be used in admixture. The bacterium belonging to the genus Pseudomonas includes, for example,
Pseudomonas aureofaciens,
and more specifically
Pseudomonas aureofaciens
B5. The bacterium belonging to the genus Bacillus includes, for example,
Bacillus polymyxa
and more specifically Bacillus is
Bacillus polymyxa
B36. The bacterium belonging to the genus Enterobacter includes, for example,
Enterobacter cloacae
and more specifically
Enterobacter cloacae
B51. The bacteriological characteristics of
Pseudomonas aureofaciens
B5,
Bacillus polymyxa
B36 and
Enterobacter cloacae
B51 will be indicated below.
TABLE 1
Characteristics of
Pseudomonas aureofaciens
B5
Test Item
Results
Morphology
Bacillus
Gram stain
−
Spore
−
Mobility
+
Flagella
Extreme polytrichate
Oxygen requirement
Aerobic
Oxidase
+
Catalase
+
O/F
O
Color of colony
Not producing
Characteristic pigment
Fluorescent pigment generation
+
Water-soluble pigment generation
+ (Orange)
PHP accumulation
−
Growth at 41° C.
−
Levan generation
−
Arginine dihydrolase
+
Denitrification
−
Reduction of nitrate
−
Liquefaction of gelatin
+
Hydrolysis of starch
−
Decomposition of Tween 80
+
Carbon source assimilation
Glucose
+
Trehalose
+
2-Ketogluconic acid
+
Inositol
+
Geraniol
−
L-Valine
+
&bgr;-Alanine
+
DL-Arginine
+
Adonitol
−
Sorbitol
−
Quinone system
Q-9
GC Content in intracellular DNA (mol %)
63
TABLE 2
Characteristics of
Bacillus polymyxa
B36
Test Item
Results
Morphology
Bacillus
Gram stain
+
Spore
+
Shape
Ellipsodial
Position
Terminal
Sporangium
evaginating
Mobility
+
Oxygen requirement
Facultative aerobic
Catalase
+
Growth under anaerobic conditions
+
VP reaction
+
pH of VP broth
4.7
Acid formation from glucose
+
Gas formation from glucose
+
Liquefaction of gelatin
+
Hydrolysis of starch
+
Utilization of citrate
−
Utilization of propionate
−
Yolk reaction
+
Reduction of nitrate
−
Generation of dihydroxyacetone
+
Growth at pH 6.8 (nutrient broth)
+
Growth at pH 5.7
+
Growth in the presence of 5% NaCl
−
Growth in the presence of 7% NaCl
−
Growth at 5° C.
−
Growth at 10° C.
+
Growth at 40° C.
+
Growth at 50° C.
−
GC Content in intracellular DNA (mol %)
Hollander Law Firm, P.L.C.
Kureha Chemical Industry Co. Ltd.
Marx Irene
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