Method for producing seeds of plants resistant to seedling diseases, and method for preventing the onset of and controlling seedling diseases

Abstract

A method for producing a seed of a plant resistant to seedling diseases is provided which includes contacting a non-pathogenic microbe corresponding to a seed-borne pathogen with a flower part of a host plant around the flowering time and collecting a seed of the host plant colonized by the non-pathogenic microbe obtained thereafter, and a method for controlling seedling diseases using the seed.

Claims

1. A method for producing seeds of a plant resistant to seedling diseases, comprising the steps of: (a) contacting a separated non-pathogenic microbe with a flower part of a host plant around the flowering time, wherein the separated non-pathogenic microbe is applied in an amount sufficient for contact with and colonization in the host plant to control subsequent infection with a seed-borne pathogen, wherein the separated non-pathogenic microbe belongs to the same species or the same genus as the seed-borne pathogen; and (b) collecting seeds of the host plant colonized by the non-pathogenic microbe, obtained after step (a), wherein the separated non-pathogenic microbe is Fusarium oxysporum non-pathogenic strain W3 Accession Number NITE BP-01538 or Fusarium oxysporum non-pathogenic strain W5 Accession Number NITE BP-01539.

2. The production method according to claim 1, wherein the host plant is a gramineous plant.

3. The production method according to claim 2, wherein the gramineous plant is rice.

4. The production method according to claim 1, wherein the separated non-pathogenic microbe is in a liquid state comprising 110.sup.4 conidia, or more.

Description

EXAMPLES

(1) The Examples of the present invention will be described below with reference to specific examples. However, the present invention is not limited to the embodiments of the Examples.

Example 1

Effect of Controlling Seedling Disease in Plant Resistant to Seedling Disease (1)

(2) (Purpose)

(3) Microbial pesticides for controlling seedling diseases according to the present invention were prepared; seeds were prepared by the method for producing seeds of a plant resistant to seedling diseases using the microbial pesticides; and it was verified on a pot scale that seedling individuals were resistant to seedling diseases.

(4) (Material and Method)

(5) Non-pathogenic fungi, Fusarium oxysporum strains W3 (Accession Number: NITE BP-01538) and W5 (Accession Number: NITE BP-01539) were used as the biological control agent microbes.

(6) Each of the biological control agent microbes was subjected to shaking culture at 28 C. for 5 days in PSB (potato decoction) medium (200 g/L of potato decoction, 0.5% [w/v] saccharose). Conidia in the culture solution were collected by centrifugation (1,500g, 20 minutes), and a spore suspension diluted to 1.010.sup.5 to 1.010.sup.7/mL with sterile water was used as a microbial pesticide for controlling seedling diseases.

(7) The host plant used was rice (variety: Tangin-bozu). After placing 80 g of sterile culture soil as bed soil in a 200-mL plastic pot followed by watering, 2 seeds of rice were seeded as parent rice seeds, covered by 20 g of sterile culture soil, and again watered. After seeding, cultivation was performed in an artificial meteorological room set at 26 C. under conditions of natural light for 100 days.

(8) The method for contacting each of the microbial pesticides with the parent rice involved, 1 week after the first flowering of the parent rice, directly spraying the microbial pesticide on the flower part using a hand spray at a certain time during the morning (around 10 to 11 o'clock) when the rice flowers (contacting step). The amount of spray was about 5 mL per spica. The spray-treated parent rice was cultivated in an artificial meteorological room set at 26 C. under conditions of natural light until the seeds ripen. About the time when the terrestrial part of the parent rice turns yellow, it was threshed to collect seed paddies (collecting step). The seed paddies collected in the treatment are hereinafter referred to as the W3-treated seed paddies and W5-treated seed paddies (corresponding to seeds of a plant resistant to seedling diseases according to the present invention). On the other hand, control seed paddies collected by the same operation except for spraying sterile water in place of the microbial pesticide are hereinafter referred to as the untreated seed paddies. Each of the treated seed paddies and the untreated seed paddies were each dried before germination treatment.

(9) The respective seed paddies were soaked in a suspension of spores of pathogenic Fusarium fujikuroi as a causative fungus of rice bakanae disease (1.010.sup.3/mL, preparation method was the same as that for the suspension of spores of biological control agent microbes) at a bath ratio of 1:1 for soaking treatment at 15 C. for 4 days. Then, forced sprouting treatment was carried out at 30 C. for 1 day. After placing 80 g of sterile culture soil as bed soil in a 200-mL plastic pot followed by watering, the respective treated seed paddies and the untreated seed paddies were seeded in the pot, covered by 20 g of sterile culture soil, and again watered. The amount of seed paddies per pot was about 2 g (about 70 grains) by dry weight before soaking treatment. Pots in which the W3-treated seed paddies, the W5-treated seed paddies, and the untreated seed paddies were seeded were referred to as a W3 treatment zone, a W5 treatment zone, and an untreatment zone, respectively. After seeding, cultivation was performed in an artificial meteorological room set at 28 C. under conditions of natural light for 14 days, and the effect of the present invention was tested by comparing rice growth in the pots.

(10) In disease symptom evaluation, a stock in which spindly growth of plant length and yellowing were observed was defined as a seedling with rice bakanae disease, and the rice bakanae disease infection rate of seedlings and the control effect were calculated using the following equations.
Infection Rate of Seedlings (%)=Number of Diseased Seedlings/Total Number of Seedlings100
Control effect=(Infection Rate of Seedlings in UntreatmentInfection Rate of Seedlings in Treatment)/Infection Rate of Seedlings in Untreatment100

(11) There was no significant difference in the germination rate between the treatment zones, in which the rates were roughly 90% or more.

(12) (Results)

(13) The results are shown in Table 1.

(14) TABLE-US-00001 TABLE 1 Infection Rate of Treatment Seedling (%) Control effect Untreatment 30.50 W3 Treatment 3.91 87.2 W5 Treatment 4.29 85.9

(15) As shown in Table 1, about 30.5% of rice seedling had the onset of rice bakanae disease in the untreatment zone as a negative control, while the infection rates of seedlings in rice seedling in the W3 and W5 treatment zones where the seeds of rice resistant to seedling diseases were seeded were each less than 5%. This demonstrated that the seeds obtained by the method for producing a seed of a plant resistant to seedling diseases using the microbial pesticide of the present invention acquired extremely high resistance to seedling diseases. Since the seed of rice resistant to seedling diseases obtained by treatment with a non-pathogenic fungus of Fusarium oxysporum, strain W3 or W5, showed resistance to rice bakanae disease caused by Fusarium fijikuroi belonging to the different species, it was shown that the seed of rice resistant to seedling diseases obtained by treatment with each biological control agent microbe and colonization by the microbe also provided high preventive and control activities against seedling disease caused by a pathogenic fungus belonging to species other than that of the biological control agent microbe.

Example 2

Effect of Controlling Seedling Disease in Plant Resistant to Seedling Disease (2)

(16) (Purpose)

(17) A microbial pesticide for controlling seedling diseases according to the present invention was prepared; seeds were prepared by the method for producing a seed of a plant resistant to seedling diseases using the microbial pesticide; and it was verified on a field scale that seedling individuals were resistant to seedling diseases.

(18) (Material and Method)

(19) Non-pathogenic fungus, Fusarium oxysporum strain W5 (Accession Number: NITE BP-01539) was used as the microbial pesticide for controlling seedling diseases, according to Example 1. The concentration of conidia in the culture solution was 1.010.sup.5/mL.

(20) Rice (variety: Tangin-bozu) was used as a host plant and cultivated by the following method for contact with the non-pathogenic fungus. The basic operation is according to Example 1. Seed paddies of the parent rice were first soaked in a disinfectant consisting of a 1:200 diluted solution of 250 mg/L of ipconazole/230 mg/L of wettable powder of copper hydroxide to disinfect the seeds. The resultant disinfection-treated parent rice seed paddies were subjected to raising seedling according to a known method (conventional method) in the art. No Rice bakanae disease control was carried out except treatment with the above disinfectant.

(21) Rice (variety: Koshihikari) naturally-infected with rice bakanae disease was used as a source of rice bakanae disease infection in the field. Seed paddies of the infection source were subjected to raising seedling without seed disinfection.

(22) Both seedlings were set in the major field at the 32nd day after seeding before onset.

(23) The non-pathogenic fungus of the present invention was contacted with the parent rice by directly spraying the non-pathogenic fungus on the flower part using a hand spray at a certain time during the morning (around 10 to 11 o'clock) when the parent rice flowers on two times, i.e., at heading stage at the 88th day after setting, at which the heading rate reaches 40 to 50% and at full heading stage at the 91st day after setting at which the heading rate reaches 80 to 90%. The amount of spray was about 140 mL/m.sup.2. The spray-treated parent rice was cultivated according to a conventional method. At the 155th day after setting the parent rice, rice was reaped and threshed to collect seed paddies.

(24) The rice bakanae disease infection rate was verified for the obtained seed paddies. The basic operation was according to Example 1. However, in this Example, since natural infection from bakanae disease-infected Koshihikari in the field was used as a pollution source, it was not necessary in principle to inoculate seed paddies with the pathogenic fungus. Thus, only the soaking treatment of seed paddies was performed using sterile water in place of the rice bakanae disease fungus spore suspension and the effect of suppressing disease onset was tested according to Example 1. In disease symptom evaluation, a stock in which spindly growth of plant length and yellowing were observed was defined as a diseased seedling, and the disease infection rate of seedlings and the control effect were calculated using the equations described in Example 1.

(25) (Results)

(26) The results are shown in Table 2.

(27) TABLE-US-00002 TABLE 2 Infection Rate of Treatment Seedling (%) Control effect Untreatment 89.76 W3 Treatment 29.38 67.3 W5 Treatment 6.59 92.7

(28) As shown in Table 2, the rice bakanae disease infection rate of seedlings in the rice seedling in the non-pathogenic fungus W3 treatment zone or W5 treatment zone where seeds of rice resistant to seedling diseases were seeded remained at about 30% with the control effect being more than 65% for the W3 treatment zone, and was only about 6.6% with the control effect being more than 90% for the W5 treatment zone. This experiment demonstrated the effect of the present invention even on a field scale.

Example 3

Effect of Controlling Seedling Disease in Plant Resistant to Seedling Disease (3)

(29) (Purpose)

(30) It was verified that seedling individuals of plants resistant to seedling diseases according to the present invention was resistant to seedling diseases by inoculating a pathogenic fungus under more severe conditions than those for Example 2.

(31) (Material and Method)

(32) The materials and the basic operation were according to Examples 1 and 2. However, in this Example, treatment with a non-pathogenic fungus was carried out in a field and seed paddies were prepared using natural infection from bakanae disease-infected Koshihikari as a pollution source as in Example 2, but in forced sprouting treatment, seed paddies were additionally inoculated with the pathogenic fungus by soaking not in sterile water but in a suspension of spores of a rice bakanae disease fungus, Fusarium fujikuroi under the same conditions as those in Example 1. This is an control test carried out supposing that some seeds polluted with the bakanae disease fungus remain and that bakanae disease spread to adjacent stocks in raising seedling.

(33) (Results)

(34) The results are shown in Table 3.

(35) TABLE-US-00003 TABLE 3 Infection Rate of Treatment Seedling (%) Control effect Untreatment 52.33 W3 Treatment 19.88 62.0 W5 Treatment 14.29 72.7

(36) In spite of the fact that treatment under more severe onset conditions was performed by inoculation with spores of the rice bakanae disease fungus in the form of a suspension in addition to the same natural infection as that in Example 2, the rice bakanae disease infection rate of seedlings was about 20% with the control effect being 62% in the non-pathogenic fungus W3 treatment zone, and the rice bakanae disease infection rate of seedlings was about 15% with the control effect being more than 70% in the non-pathogenic fungus W5 treatment zone. Thus, the effect of the present invention was demonstrated regardless of the difference in severity of infection conditions of seedling disease fungus.

Example 4

Effect of Controlling Seedling Disease in Plant Resistant to Seedling Disease (4)

(37) (Purpose)

(38) It was verified on a pot scale using a fungal strain of the genus Trichoderma that seedling individuals of plants resistant to seedling diseases according to the present invention became resistant to seedling diseases even when a fungal strain other than a fungus of the genus Fusarium was used.

(39) (Material and Method)

(40) The materials and the basic operation were according to Example 1. However, in this Example, Trichoderma atrovidide strain SKT-1 commercially available as a microbial pesticide (product name: Ecohope, Kumiai Chemical Industry Co., Ltd.) was used together with a non-pathogenic fungus, Fusarium oxysporum strain W5. Conidia were used by scraping the ones formed not in liquid culture but in plate culture. The concentration of conidia was 1.010.sup.5/mL, and spray on the flower part was carried out 4 times in total every 2 days from the 87th day after setting.

(41) (Results)

(42) The results are shown in Table 4.

(43) TABLE-US-00004 TABLE 4 Infection Rate of Treatment Seedling (%) Control effect Untreatment 13.0 W5 Treatment 1.8 86.5 Trichoderma Treatment 6.0 53.6

(44) As shown in Table 4, in the Trichoderma fungus treatment zone, the disease infection rate of seedlings remained at about 6.0% with the control effect being more than 50%. This experiment demonstrated the effect of the present invention not only when a non-pathogenic fungus Fusarium oxysporum, strain W3 or W5, was used but also when a fungus of the genus Trichoderma was used. Thus, it is considered that the present invention can be practiced using a wide range of fungi including fungi of the genus Fusarium and the genus Trichoderma.

(45) All publications, patents, and patent applications cited in this application are incorporated herein by reference in their entirety.