Ustilago trichophora mycoherbicidal compositions and methods of use

A herbicide useful for controlling weeds belonging to genus Echinochloa such as barnyardgrass, a method for controlling such weeds and novel microorganisms which can be used as an effective ingredient of the herbicide are disclosed. The present invention provides a herbicide comprising as an effective ingredient a microorganism belonging to genus Ustilago. The present invention also provides a method for controlling weeds by utilizing a microorganism belonging to genus Ustilago. The present invention also provides Ustilago trichophora B-171 (FERM BP-3968). The present invention further provides Ustilago trichophora B-174 (FERM BP-3969). The present invention further provides Ustilago trichophora B-185 (FERM BP-3970).

This application is filed under 35 USC 37 from PCT/JP92/01184. 
TECHNICAL FIELD 
This invention relates to a herbicide, a method for controlling weeds and 
novel microorganisms belonging to genus Ustilago which can be used 
therefor. 
PRIOR ART 
Barnyardgrass (belonging to genus Echinochloa) which is an important weed 
in paddy and plowed fields is conventionally controlled mainly by chemical 
herbicides. In view of the environmental pollution caused by using 
agricultural chemicals in large amounts, it is desired to develop a 
herbicide and use thereof, which does not employ an agricultural chemical. 
A method for controlling weeds, which employs a pathogenic fungus which 
specifically attacks barnyardgrass, is known (Japanese Laid-open Patent 
Application (Kokai) No. 2-13367). However, the effectiveness of this 
method is not satisfactory, so that the herbicide and weeds-controlling 
method utilizing this pathogenic fungus are not widely used. Further, the 
conventional methods for controlling weeds utilizing chemical herbicides 
and the above-mentioned microorganism mainly aim at killing barnyardgrass. 
DISCLOSURE OF THE INVENTION 
An object of the present invention is to provide a novel microorganism 
which has an ability to control weeds such as barnyardgrass. Another 
object of the present invention is to provide a herbicide by which weeds 
such as barnyardgrass can be effectively and selectively controlled. Still 
another object of the present invention is to provide a method for 
effectively and selectively controlling weeds such as barnyardgrass. 
The present inventors intensively studied to discover novel microorganisms 
pathogenic to barnyardgrass and that weeds such as barnyardgrass can be 
controlled by using the novel microorganisms, thereby completing the 
present invention. 
The present invention provides a herbicide comprising as an effective 
ingredient a microorganism belonging to genus Ustilago. The present 
invention also provides a method for controlling weeds by utilizing a 
microorganism belonging to genus Ustilago. The present invention also 
provides Ustilago trichophora B-171 (FERM BP-3968). The present invention 
further provides Ustilago trichophora B-174 (FERM BP-3969). The present 
invention further provides Ustilago trichophora B-185 (FERM BP-3970). 
These microorganisms are deposited at the Fermentation Research Institute 
of Industrial Science and Technology, 1-3, Higashi 1-chome Tsukubashi 
Ibaraki-keen, 305, Japan (FERM BP-3970). 
The microorganisms according to the present invention exhibit high 
virulences and selective pathogenicities to weeds such as barnyardgrass 
and do not adversely affect the growth of crops such as rice. Therefore, 
by using the microorganisms of the present invention, weeds can be 
effectively and selectively controlled. Further, unlike agricultural 
chemicals, the herbicide according to the present invention does not 
pollute or destroy environment. 
BEST MODE FOR CARRYING OUT THE INVENTION 
As mentioned above, the present invention provides Ustilago trichophora 
B-171 (FERM BP-3968), Ustilago trichophora B-174 (FERM BP-3969) and 
Ustilago trichophora B-185 (FERM BP-3970). 
Synonyms of Ustilago trichophora include Ustilago crus-galli and Ustilago 
sphaerogena. 
The novel microorganisms according to the present invention were first 
provided by collecting diseased barnyardgrass plants from various places 
in Japan, separating fungal strains pathogenic to barnyardgrass therefrom, 
pure culturing the separated strains, and by screening the pure cultured 
strains which are pathogenic to barnyardgrass but not pathogenic to other 
crops, particularly rice. The detailed method for first providing the 
microorganisms are described in the examples hereinbelow described. 
The major mycological properties of the novel microorganisms are as 
follows: 
They are aerobic. They grow well at a pH between 5 and 9. They grow at a 
temperature between 15.degree.-35.degree. C. and optimum growth 
temperature is 30.degree.-35.degree. C. The morphology and color in potato 
dextrose agar medium (hereinafter referred to as "PDA medium") are as 
follows: 
The morphology of the colonies on PDA medium at the initial stage is white 
or pink, and smooth like colonies of yeasts. In the late stage, white 
mycelia grow from the surfaces of the colonies. Sexual spores are called 
teliospores or smut spores and are formed in the body of barnyardgrass 
plant which is the host. The sexual spores are black to dark brown and are 
spherical or oval with a diameter of about 9 .mu.m. They have no septa and 
a number of spinelike protuberances are observed on the surfaces thereof. 
The teliospores germinate on the medium and form promycelium (basidium) 
having septa. On the promycelium, sporidia which are basidiospores are 
formed. The sporidia asexually grow by means of budding. The sporidia are 
hyaline, have cylindrical shape with a length of about 9 .mu.m and a width 
of about 3 .mu.m, and have no septa. 
From these mycological properties, these microorganisms were identified as 
Ustilago trichophora. The strains B-171, B-174 and B-185 belonging to this 
species were deposited with FERMENTATION RESEARCH INSTITUTE OF JAPAN on 
Sep. 4, 1991, Feb. 5, 1992 and Feb. 5, 1992, respectively. All of these 
depositions were converted to international depositions under THE BUDAPEST 
TREATY on Aug. 18, 1992. The accession numbers of the international 
depositions are FERM BP-3968, FERM BP-3969 and FERM BP-3970, 
respectively. All of these strains have the above-described mycological 
properties. However, since their pathogenic powers are different as shown 
in the examples below, different strain names were given. 
The novel microorganisms according to the present invention are pathogenic 
to the weeds belonging to genus Echinochloa such as barnyardgrass in paddy 
and plowed fields while not pathogenic to plants belonging to family 
Gramineae such as rice, wheat, barley, corn and Japanese barnyard millet; 
plants belonging to family Leguminosae such as soybean; plants belonging 
to family Solanaceae such as eggplant; and to plants belonging to family 
Cruciferae such as cabbage. As shown in the examples hereinbelow 
described, these microorganisms can be cultured in large scale and a large 
amount of spores can be obtained. 
The herbicide according to the present invention is characterized by 
containing a microorganism belonging to genus Ustilago as an effective 
ingredient. Although the microorganism may be in the form of fungal 
mycelium, it is preferred that the microorganism be in the form of spores 
because spores have higher durability. The spores may be fresh spores 
immediately after cultivation or may be those restored from storage by 
using an aqueous medium. The storage can be carried out by the storage at 
a super low temperature (-80.degree. C.) or by lyophilization, which are 
well-known as the storing methods of microorganisms. 
Although the herbicide according to the present invention may contain the 
above-described microorganism alone, it preferably contains one or more 
agricultural carriers widely used. Any agricultural carrier can be used as 
long as it does not adversely affect the microorganism, and water is best 
preferred. The population density of the spores suspended in water is not 
restricted but preferably not less than 10.sup.6 spores/ml, more 
preferably 10.sup.7 -10.sup.9 spores/ml. In suspending the spores in 
water, one or more adjuvants such as surfactants, and spreader and sticker 
may be added. 
The herbicide according to the present invention may be applied at any 
growth stage of the weeds and before the germination of the weeds. Thus, 
the herbicide according to the present invention can be directly applied 
to the weeds or to the ground of paddy or plowed fields in which 
prevention of emergence of the weeds is desired. The amount of the 
herbicide to be applied can be appropriately selected depending on the 
growth stage, number and size of the weeds. When the herbicide is spread 
on the paddy or plowed field, the amount of the herbicide is preferably 
10.sup.9 to 10.sup.11 in terms of the number of microorganisms per 1 
m.sup.2. 
The novel microorganisms according to the present invention are pathogenic 
to the weeds belonging to genus Echinochloa such as barnyardgrass, which 
are weeds in paddy and plowed fields, and form gall or swelling in leaf 
sheaths so that the infected barnyardgrass plants are killed or their 
growth is extremely inhibited. Killing the weeds is not the only method 
for controlling the weeds in paddy fields. If the growth of the weeds is 
inhibited by the above-described microorganism, the growth of rice 
prevails the growth of the weeds, so that most of the sun light is 
received by the rice and the growth of the weeds in the shade of rice is 
further inhibited. As a result, it is thought that the damage by the weeds 
can be substantially reduced. Further, the barnyardgrass plants which are 
diseased and whose growth is inhibited cannot form ears, or even if they 
can form ears, they are sterile. When ears are infected with the 
microorganisms, the seeds are filled with the teliospores and are sterile, 
so that the emergence of the weeds in the next year is prevented. On the 
other hand, cultivated plants such as rice, wheat, Japanese barnyard 
millet, soybean, eggplant and cabbage are not infected with the 
microorganisms. Therefore, the target weeds can be selectively controlled.

(EXAMPLES) 
1) Process for First Obtaining Novel Microorganisms of the Present 
Invention 
Barnyardgrass plants grown on paddy and plowed field, which exhibited 
diseased symptoms were collected. The diseased portions were cut out and 
the mass of teliospores therein were taken out. The mass were immersed in 
70% ethanol for 30 to 60 seconds and suspended in 0.5% sodium hypochlorite 
solution to which equivalent volume of 0.1% TWEEN 80 had been added, for 
1-3 minutes to sterilize the surfaces thereof. 
After washing the spores once with 0.1% TWEEN 80 solution and twice with 
sterilized water, they were streaked on an antibiotic-containing potato 
dextrose agar medium (hereinafter referred to as "APDA medium") and were 
germinated in an incubator at 25.degree. C. Each of single colonies was 
transferred to a fresh PDA medium to carry out pure isolation of the 
microorganism and cultured in the incubator. 
Each of the thus isolated strain was checked again for the pathogenicity to 
barnyardgrass, and its pathogenicities to rice, wheat, barley, corn, 
Japanese barnyard millet, soybean, eggplant and cabbage were examined. The 
strains B-171, B-174, B-185 according to the present invention which 
exhibit excellent herbicidal activities to barnyardgrass and which do not 
exhibit pathogenicities to rice, wheat, barley, corn, Japanese barnyard 
millet, soybean, eggplant and cabbage were isolated. 
2) Identification of First Provided Microorganisms 
The identification of the microorganisms according to the present invention 
was carried out by observing the morphologies of the teliospores and 
morphologies of germs on the medium. As a result, as described above, the 
microorganisms according to the present invention were identified as 
Ustilago trichophora. 
3) Method for Culturing in Large Scale 
To the microorganisms grown on PDA medium, sterilized water is added and 
stirred to prepare a suspension containing spores at a high concentration. 
About 100 .mu.l of the suspension is dropped on a fresh medium and the 
drop is spread by using a sterilized L-shaped glass rod. By this method, 
inoculation of the microorganism to a large number of petri dishes 
(diameter: 9 cm) can be accomplished at one time and the time required for 
growing the microorganisms was largely reduced to 1-2 days. By this 
method, 5.times.10.sup.10 spores per one petri dish were formed. 
The microorganisms grown on PDA medium were inoculated to modified YPD 
liquid medium (containing 10 g of trypton, 5 g of yeast extract and 10 g 
of dextrose per one liter of water) by using a platinum loop and cultured 
under shaking for 2 days. As a result, 5.times.10.sup.8 spores per 1 ml of 
culture medium were formed. 
Thus, spores of the microorganisms according to the present invention can 
be easily obtained in a large amount by using the plate medium or liquid 
medium. 
4) Test for Attacking Barnyardgrass (Virulence) 
Barnyardgrass plants were grown in commercially available pots and the 
plants in 1-5 leaf stage were used as test materials. 
The spores of the microorganisms according to the present invention, which 
were obtained by culturing the microorganisms on PDA medium or in modified 
YPD liquid medium were suspended in 0.1% TWEEN 80 solution and each of the 
suspensions was applied to barnyardgrass plants with an air spray. The 
inoculated plants were incubated at 22.degree. C. for 24 hours in a moist 
chamber and then incubated for 5 weeks at 25.degree. C. in a green house. 
Thereafter, the rates of diseased barnyardgrass plants were determined, 
which are shown in Table 1. By any of the strains, not less than 60% of 
the barnyardgrass plants were diseased. B-171 which exhibited the highest 
rate of diseased plants was checked for the relationship between the 
concentration of the inoculum and the rate of diseased plants. As shown in 
Table 2, it was proved that there is a correlation between the 
concentration and the rate of diseased plants. The heights of the 
barnyardgrass plants diseased by B-171 and those of healthy barnyardgrass 
plants were compared, which is shown in Table 3. 
TABLE 1 
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Strain and Rate of Diseased Plants 
Strain Rate of Diseased Plants (%) 
______________________________________ 
B-171 100 
B-174 70 
B-185 60 
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TABLE 2 
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Concentration of Inoculum and Rate of Diseased Plants 
Concentration of 
Inoculum (spores/ml) 
Rate of Diseased Plants (%) 
______________________________________ 
0 0 
10.sup.7 69 
10.sup.8 78 
10.sup.9 100 
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TABLE 3 
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Comparison of Heights of Diseased Barnyardgrass Plants 
and Healthy Barnyardgrass Plants 
Height of Plant (cm) 
______________________________________ 
Healthy Barnyardgrass 
90 
Diseased Barnyardgrass 
22 
______________________________________ 
As is apparent from Tables 1, 2 and 3, the microorganisms according to the 
present invention exhibited excellent controlling effects against 
barnyardgrass. 
5) Influence on Cultivated Plants 
Pathogenicity tests against cultivated plants were carried out for B-171, 
B-174 and B-185 in the same manner as in the test of virulence against 
barnyardgrass. The concentration of the spores in the inoculum was 
10.sup.9 spores/ml. 
Rice, wheat, barley, corn, Japanese barnyard millet, soybean, eggplant and 
cabbage were used as the test plants. The results of the tests are shown 
in Table 4. 
TABLE 4 
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Pathogenicities Against Cultivated Plants 
Plant Pathogenicity 
______________________________________ 
Rice - 
Wheat - 
Barley - 
Corn - 
Japanese Barnyard Millet 
- 
Soybean - 
Eggplant - 
Cabbage - 
Barnyardgrass + 
______________________________________ 
As is apparent from Table 4, the microorganisms according to the present 
invention did not exhibit pathogenicities to rice, wheat, barley, corn, 
Japanese barnyard millet, soybean, eggplant and cabbage.