TWI510619B - New bacillus mycoides isolate pdml rs1013 effective in controlling cucurbitaceous vegetable seedling damping-off and cruciferous clubroot disease - Google Patents

Description

It has the control of melon vegetable seedlings and the cruciferous spores of cruciferous root nodule New strain of bacteria

The invention relates to collecting plant and soil samples from vegetable fields and paddy fields, and separating and screening tests one by one, and obtaining a new indigenous strain Bacillus mycoides having control of cucumber seedling disease and cruciferous vegetable root nodule disease. PDML RS1013 strain, and its use in plant protection agents.

Traditional agricultural farming systems use a large amount of agrochemicals to increase the production of crops, but cause the problem of resistance to pathogens and pests, and then the negative effects on the ecological environment and food safety. The use of beneficial microorganisms to control crop diseases can not only effectively reduce the impact of agrochemicals on human and animal health and the ecological environment, but also contribute to the sustainable operation of agricultural production. Looking at biopesticides, Bacillus is the most advantageous condition for bacteria, and it has been developed as a target for plant protection preparations (Ongena and Jacques, Trends Microbiol. 16:115-25, 2008).

Bacillus is a Gram-positive rod-shaped bacterium, which is ubiquitous in the surface soil and plant surface environment. The endospore formed by this bacterium is highly resistant to harsh living environment and can resist radiation, ultraviolet rays, high temperature and drought. Bad environmental factors such as osmotic pressure and chemical agents; if they encounter a suitable environment, they can be propagated again by endospores, so they are suitable for the development of long-shelf plant protection preparations. Bacillus spp. has been reported to have been used for the control of crop diseases, including Colletotrichum gloeosporioides (Lin, Master thesis, Department of Plant Pathology, National Chung-Hsing University, p63, 1987), and citrus green mold. Penicillium digitatum (Lin, 1987, with the above), carnation wilt pathogen Fusarium oxysporum f.sp. dianthi (Aldrich and Baker, Pl.Dis.Rep 54:. 446-448,1970) ., sugar beet damping-off pathogens Pythium sp (Dunleavy , Phytopathology 45 : 252-258, 1954), Verticillium sp. (Hall, Plant Dis. 70 :521-524, 1986), Monilinia fructicola (Pusey et al, Plant Dis. 72 : 622) -626,1988) and diseases caused by fungi and bacteria such as Xanthomonas campestris pv. mangiferaeindicae (Pruvost and Luisetti, Acta Hortic. 291 :324-337, 1991), some of which have been successfully developed into organisms. Pesticide related products. The disease prevention mechanisms of such biological pesticides include antibiosis, competition, induced resistance, and growth promotion (Whipps, J. Exp. Bot. 52:487- 511, 2001; Gardener and Driks, Phytopathology 94 : 1244, 2004). According to the literature, have heretofore been found to various Bacillus species having control effect, e.g. B.amyloliquefaciens, B.brevis Migula, B.megaterium de Bary , B.mycoides Flügge, B.penetrans (Thorne, 1940) Mankau, B. licheniformis, B.pumilis Meyer et Gottheil and B.subtilis (Ehrenberg, 1835) Cohn ( Bapat and Shan, Can J.Microbiol 46:. 125-132,2000 ) and the like.

In 1886, Flüggen first published a strain named Bacillus mycoides , whose taxonomic status was attributed to the Monera, Firmicutes, Bacilli. under (B.cereus group), Bacillus mesh (Bacillales), Bacillus Branch (Bacillaceae), Bacillus (Bacillus) of the B. cereus group. Bacillus licheniformis is found in a wide range of environments, such as soil and plants. When cultured on a plate medium, the growth colonies are special and appear to grow outwards in a rhizoidal colony manner, and the growth direction is clockwise and counterclockwise depending on the genetic traits between the strains ( Di Franco et al, Plasmid 44 : 280-284, 2002). At present, it has been reported that the application of biocontrol bacteria B. mycoides Bac J isolates in sugar beet crops can effectively promote the systemic disease resistance of plants, thereby achieving the effect of controlling beet leaf spot disease (Bargabus et al., Biol . Control 30 :342). -350, 2004). B. mycoides can induce plants to produce disease-resistant reactions by producing volatile or non-volatile substances, and also inhibit the growth of Botrytis cinerea (Pers. Ex Fr.), thereby achieving the effect of controlling gray mold (Guetsky et al. Human, Am . Phytopathological Society 91 :621-627, 2001). Czaban et al. (Polish J. Microbiol. 53 :101-110, 2004) pointed out that planting wheat seeds with B. mycoides after encapsulation can effectively increase wheat yield and can be reduced due to Gaeumannomyces graminis var. tritici and Fusarium culmorum . The incidence of disease.

The importance and economic value of the melons and Cruciferae vegetable crops of the Cucurbitaceae in the agricultural market are highly valued. According to the 100-year agricultural statistics annual report, the vegetable acreage in Taiwan has been decreasing year by year (COA, 2012) from 91 to 100 years. Therefore, the yield and quality per unit of cultivated area are relatively increased to supply market demand. The important soil-borne diseases--the disease of vegetable seedlings and the root nodule of cruciferous vegetables are the research objects of biological control of vegetable crop diseases. Both pathogens belong to the original organism (Protista) in modern biological taxonomy, and the Pythium aphanidermatum belongs to the Chromista, Heterokontophyta, Oomycetes. The species under the plant; the plant parasitic Crusmodiophora brassicae belongs to the Rhizoria, Cercozoa, and Plasmodiophoromycota. Species. Each has a special structure of oospores and dormant spores, and it is easy to survive in the soil for a long time. When the environment conditions of the host crops are suitable, the two pathogens are easy to infect the base of the rhizome of the plant, causing the death of the seedlings in severe cases, or causing the disease to become rickets. The strain could not be harvested, which in turn caused serious economic losses.

The object of the present invention is to screen out the native strains of Bacillus licheniformis which have the effects of controlling the disease of the melon seedlings and the root nodule of cruciferous vegetables, as a key strain for the development of plant protection preparations. And further The selected beneficial microorganisms or their metabolites are used to develop low-toxicity and low-environmental plant protection preparations, in order to replace the application of chemical preparations in the crop disease management strategy, slow down the degree of crop rickets, and then achieve prevention and control. The effect of crop diseases.

One aspect of the present invention relates to a strain of Bacillus mycoides PDML RS1013 having the effect of preventing and treating melon seedling disease and cruciferous vegetable root nodule disease, and the colony morphology is rhizoidal colony. The Bacillus licheniformis PDML RS1013 strain was subjected to Gram staining and endophytic staining test and examined under a light microscope. The result showed that it was a Gram-positive bacillus producing endospores.

The Bacillus licheniformis PDML RS1013 strain of the present invention has an antibacterial ability to inhibit the production of zoospores for the melon seedlings. The Bacillus licheniformis PDML RS1013 strain of the present invention can also effectively reduce the chance of infection of cruciferous plants by Rhizobium.

In another aspect, the present invention relates to a method for controlling cucurbit vegetable seedling disease and cruciferous rhizobia, which comprises applying the Bacillus mycoides PDML RS1013 strain of the present invention to a seed or a plant. In a specific embodiment of the present invention, the method comprises treating the seed of the Bacillus licheniformis of the present invention in a soaking manner, and then cultivating the seed coated with the Bacillus licheniformis and then sown in the cultivation medium.

In still another aspect, the present invention provides a plant protection preparation comprising the Bacillus mycoides PDML RS1013 strain of the present invention. The plant protection preparation of the invention can be used for preparing a biological pesticide for preventing and treating melon vegetable seedling disease and cruciferous root tumor disease, and can also be used for preparing a growth promoter for enhancing the development of melon vegetables or cruciferous crops.

Figure 1 shows the inhibitory effect of the selected strains of Bacillus licheniformis PSO, PDML TS02, PDML RS1013, PDML RS1016 and PDML L0912 isolates on the sporulation ability of B. cucurbits (Kpa and Potapl). CK: The control group did not process.

Figure 2 shows the destruction of the zoospores of the sorghum seedlings by the Bacillus licheniformis (top left) and the gradual disintegration (sequentially from the top left to the bottom right).

In Fig. 3, the cut leaves of the cucurbits showed that the Bacillus licheniformis PDML RS1013 fermentation broth treatment group was compared with the control group (CK: no treatment), which effectively reduced the size of the browning tissue caused by the pathogenic bacteria on the melon leaves.

Figure 4 is a graph showing the effects of Bacillus licheniformis PDML RS1001, PDML RS1003, PDML RS1005, PDML RS1013, PDML RS1016 and PDML CS1001 isolates on the root hair infection rate of cabbage seedlings. CK: The control group did not process.

Figure 5 is a test of the effect of the selected Bacillus licheniformis PDML RS1001, PDML RS1003, PDML RS1005, PDML RS1013, PDML RS1016 and PDML CS1001 isolates against cabbage nodule disease (reducing plant rickets). CK: The control group did not process.

Figure 6 shows the incidence of Bacillus licheniformis PDML RS1013 strain in the greenhouse test. CK: The control group did not process.

Figure 7 is a test of the effect of Bacillus licheniformis PDML RS1013 and PDML RS1016 strains on the degree of rickets after application to cabbage in different ways. Culture medium containing the Agrobacterium pathogens of cruciferous ⁽¹⁰⁷ dormant spores / gram dry weight of the medium), CK: control group not treated, C: PDML RS1013 antagonistic bacteria and PDML RS1016 bacteria (10 ⁴ cfu / ml) seed-coat; D: The roots of cabbage seedlings were irrigated with antagonistic PDML RS1013 and PDML RS1016 (10 ⁸ cfu/ml); M: mixed with the antagonists PDML RS1013 and PDML RS1016 (10 ⁸ cfu/ml).

Figure 8 shows the growth of cabbage in Bacillus licheniformis PDML13 and PDML RS1016 strains treated with different application methods after inoculation of Brassicaceae. The cultivation medium contained pathogens of 10 ⁷ (dormancy spores/gram dry weight), CK: no control in the control group, C: powdered seeds with antagonistic PDML RS1013 and PDML RS1016 (10 ⁴ cfu/ml); D: The roots of the cabbage seedlings were irrigated with antagonistic PDML RS1013 and PDML RS1016 (10 ⁸ cfu/ml); M: mixed with the media with antagonistic PDML RS1013 and PDML RS1016 (10 ⁸ cfu/ml).

The other features and advantages of the present invention are further exemplified and illustrated in the following examples, which are intended to be illustrative only and not to limit the scope of the invention.

Isolation and culture of Bacillus mycoides strain

First, we went to all parts of Taiwan to collect vegetables and paddy crops and their soils. Then we treated each plant and soil sample at a temperature above 45 °C, and then evenly spread each sample to the nutrient medium (Nutrient Agar; NA, by dilution plate method). DIFCO ^TM ) was placed in a thermostat at room temperature and 30 ° C. A single colony of Bacillus licheniformis appeared, and after colonization, it was cultured purely for testing. From the results of the culture, more than 100 bacterial strains were isolated from the roots and roots of the plant. After preliminary identification, most of them were Bacillus spp., and more than 20 bacterial colonies were rooted. The shape is outwardly grown, and the appearance appears as a colony of Bacillus licheniformis reported in academic journals.

The Bacillus licheniformis strain obtained by screening is streaked in a nutrient medium or Tryptic Soy Agar (DIFCO ^TM , USA) medium, and then placed at 30 ° C for 12-24 hours, and then transplanted with a colonization ring. Colonies of each strain were taken, and sterile water was added one by one to prepare a bacterial suspension, and the concentration was adjusted to 1×108 cfu/ml or more for use in the control of plant diseases.

Screening of Bacillus licheniformis strains that can prevent melon mites Isolation and inoculation source preparation of melon

1. Separation of Pythium aphanidermatum : Cut the rickets from the rickets of the squash seedlings and rinse them three times with sterile water, place on water Agar plate medium, then at room temperature. After 1 day of culture, the mycelium was isolated and cultured in a medium of V8 (V8 ^® vegetable juice agar) and potato dextrose agar (PDA, Potato Dextrose Agar, DIFCO ^TM ) to obtain a seedling strain of the seedlings for testing.

2. Preparation of melon sputum inoculation source: The seedling blast fungus strain (Kpa and Potapl) was cultured in V8 plate medium (diameter 9 cm culture dish) for 4 days, and the sterilized No. 3 puncher (diameter 0.7) was used. Centimeter) or scalpel cut (1 cm square) Take the whole dish of the hyphae (disk), then place all the hyphae in an empty culture dish of 15 cm diameter, add 100 ml of sterile water, soak at room temperature After 8-12 hours of mycelial masses, a zoospore suspension can be produced for inoculation testing.

Screening for Bacillus licheniformis

In order to evaluate the direct effect of the fermentation broth of Bacillus licheniformis on the pathogen, the seedlings of K. pylori (Kpa and Potapl) were incubated at 30 ° C for 12 hours, and after 3-5 days of culture, 3 tablets were taken directly. The hyphae disc was placed in a petri dish (3 cm in diameter) containing 3 ml of sterile water, and a yield of nearly 10 ⁵ viable spores per ml was obtained in 5-8 hours.

The selected strains of Bacillus licheniformis PSO, PDML TS02, PDML RS1013, PDML RS1016, PDML L0912, etc. were respectively fermented in a nutrient medium at 200 rpm, 30 ° C for more than 5 days, and the spore-like spores were detected according to the above-mentioned production of the spore platform. The effect of bacilli on the migration of spores by Kappa and Potapl. The results showed that the fermentation broths of PDML TS02, PDML RS1013 and PDML RS1016 strains had the best inhibition of sporulation (Fig. 1). After observing the fermentation broth to treat the spore-forming hyphae disc, the viable spores were found. The phenomenon of rupture (Figure 2).

Using the first true leaf of the courgette seedlings to inoculate the pathogen (Kpa), the spores were used as a platform to evaluate the control effect; according to the above-mentioned pathogen inoculation source preparation method, the seedlings were cultured and made into hyphae (round) After the dish), use sterile water to promote the pathogen to produce the spores, then take 5μl of spore suspension (containing about 200-400 spores), drip on the surface of the 2 week old courgette leaves, and moisturize for 12 hours. In a 30 ° C constant temperature chamber, the browning area on the leaves was observed after 24 hr. In this way, a strain of Bacillus licheniformis capable of controlling cucurbits of cucurbits is screened by the step of 1:1 (v/v) ratio of the obtained fermentation broth of each strain of Bacillus licheniformis to the aforementioned tourospores The mixture was suspended and mixed, and then the mixture was inoculated to the first true leaf of the 2-week-old courgette, and after 24 hours, the effect of each strain on inhibiting the pathogen-infected leaves was observed.

The above-mentioned sporulation platform can be used to analyze and screen out the strains that can best inhibit the migration of Kpa from the seedlings, and the strains of Bacillus licheniformis PDML TS02, PDML RS1013 and PDML RS1016 are made into cell suspension (OD620 value). About 0.8, the amount of bacteria reached 1x108 cfu/ml or more, and the germination was carried out by treating each of the B. faecalis seeds with a soaking method, and then the treated seeds were sown in the cultivation medium, and after the seedlings were cured for one week, Each of the squash seedlings was inoculated with 1 ml of a spore suspension of the seedling Kappa Kp (1 x 105 migrating spores/ml), and each treatment was repeated 3 times, and the incidence of the plants was observed and counted 7 days after the inoculation.

The results showed that after treatment with the fermentation broth of Bacillus licheniformis PDML RS1013, the number of spores produced by the pathogenic bacteria decreased, which effectively reduced the size of the browning tissue caused by the pathogenic bacteria on the melon leaves, even without disease (Fig. 3).

Screening of Bacillus sp. strains for controlling cruciferous vegetable root nodule disease Preparation of cruciferous vegetable root nodule bacteria inoculation source

The nodule tissue stored at -20 °C was washed with water and surface-sterilized with 0.5% (w/v) sodium hypochlorite (NaClO), then fully ground with a high-speed stirrer, and then filtered with 8 layers of gauze to obtain the pathogen. The resting spores suspension was counted and the spore concentration was calculated using a Hemocytometer. The prepared spore suspension was uniformly mixed in a peat soil (BVB No. 4, Netherlands) cultivation medium, and the carrier medium having a concentration of 10 ⁸ dormant spores/gram dry weight of the medium was adjusted for inoculation test.

Disease investigation method

Refer to the method of grading and calculating the degree of rickets used by Dixon and Robinson in 1986: the disease classification has 0 grades for healthy roots without any nodulation; grade 1 is for slight root nodules; The volume of nodulation with roots accounted for less than 50% of the total root system; the volume of nodulation of the main roots and roots of the third grade accounted for more than 50% of the total root system. The calculation formula for the conversion 罹 disease is as follows:

Rapid assessment technique for prevention and treatment of cruciferous vegetable root nodule disease

1. the cabbage seedlings infected with clubroot of cruciferous Platform Evaluation: The cabbage seeds were sown in a concentration of ¹⁰⁷ containing pathogens dormant spores / g dry weight medium medium, at 24 deg.] C growth chamber moisturizing plant 8, and then every machine 10 strains of cabbage were selected and stained with cotton blue dye (cotton blue: phenol 200 g, cotton blue 0.5 g, glycerol 400 ml, lactic acid 200 ml, ddH ₂ O 200 ml) after dyeing for 5 minutes, under optical microscope. Microscopic examination of root hair infection; observe the main root part of the cabbage seedlings within 2-4 cm, if there is a spore sac in the root hair cells, it is the infected cabbage seedlings.

2. The effect of the inoculation concentration of cruciferous rhizobia on the rickets of plants: the cabbage seedlings cultivated for 3 weeks were transplanted, and the concentration of pathogens were ⁰ , 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ^6, are inoculated with a growing medium in the dry weight of ¹⁰⁷ and ¹⁰⁸ resting spores / gram of medium. After 8 weeks of cultivation in the pathogen medium, the cabbage plants were sampled and observed, and the degree of rickets was recorded, and each treatment was repeated 3 times.

3. The effect of root infection rate of cabbage seedlings on the disease degree: After collecting the above-mentioned inoculation concentration test records, the data of root hair infection rate and adult disease degree of cabbage seedlings were collected, and the results were determined by SAS ^® (Statistics Analysis System) biostatistical software. , analyze each other's relevance.

Effect of Bacillus licheniformis strain on the infection rate of seedlings

After containing the Bacillus mycoides strains of the bacterial suspension (10 ⁸ cfu / ml) and cruciferous nodule bacteria ⁽¹⁰⁷ dormant spores / gram dry weight of the medium) of cabbage cultivation medium for 3 weeks, the cabbage seedlings hairs infection investigations, The results showed that the strains of Bacillus licheniformis PDML RS1001, PDML RS1003, PDML RS1005, PDML RS1013, PDML RS1016 and PDML CS1001 could reduce the infection rate of root hairs of cabbage seedlings by pathogens from 80.6% (control group) to 16.6-31.8. %, among which PDML RS1013 strain was the best to inhibit root hair infection rate (Fig. 4).

Effect of Bacillus licheniformis strain on plant rickets

The above test results were screened, and the strains of Bacillus licheniformis PDML RS1001, PDML RS1003, PDML RS1005, PDML RS1013, PDML RS1016 and PDML CS1001 which can reduce the root hair infection rate to 30% or less were cultured, and the bacterial strains were taken. The suspension was separately added to the cultivation medium and uniformly mixed to a concentration of 10 ⁸ (cfu/ml). Seedlings of cabbage seeds were incubated for 3 weeks, and then transplanted to a medium with a pathogen concentration of 10 ⁷ dormant spores/gram dry weight of the medium. The results showed that the above 6 strains of Bacillus licheniformis could effectively treat the plant rickets from 100.0%. Reduced to 55.56% (Figure 5).

Identification of Bacillus mycoides PDML RS1013 strain Physiological and biochemical test

The above test was used to screen the strain PDML RS1013, which has the effect of controlling the two diseases and promoting the growth of the plant, according to the physiological and biochemical characteristics of the Bacillus properties described in the Bergey's Manual, respectively, Gram staining and endospore staining. ), anaerobic growth test, motility test, catalase test, casesin hydrolysis test, starch hydrolysis test ), hydrolysis of gelatin test, citrate utilization test (alkali on citrate-salts agar) and salt tolerance test (7% NaCl tolerance test). The colony morphology of the PDML RS1013 strain resembled rhizoidal colony. After Gram staining and endospore staining tests, microscopic examination was performed under an optical microscope, and the results showed that they were Gram-positive bacilli producing endospores. Physiological and biochemical test strains are aerobic, non-motility, containing hydrogen peroxide enzymes, with starch, casein and gelatin hydrolyzing enzymes, can not use citrate, can not survive in 7% sodium chloride NB (Nutrient The properties of Broth) culture fluid (see Table 1 below) are consistent with the characteristics of Bacillus mycoides reported by Parry et.al (1983) and Sneath (1986).

Biolog Identification System

The PDML RS10013 strain was renewed and cultured on a BUG ^TM agar (Biolog, Inc., USA) plate supplemented with 0.25% maltose and 0.9% thioglycolate according to the proposed method and procedure of the Biolog Bacterial Identification System, and cultured at 30 ° C for 16 hours. The bacterial suspension was configured with an aqueous solution of GN/GP-IF (Biolog, Inc., USA) and adjusted to a concentration of 28% T ± 3% with a Biolog spectrophotometer, and then the bacterial suspension droplets were injected into the Biolog GP2 MicroPlate 96 In a well, the 96-well plate was placed at 30 ° C for 4 to 24 hours, and each reaction value was interpreted by a Biolog microplate reader. The Biolog system was used to identify the results of the GP2 MicroPlate reaction of PDML RS1013 strain. The results are shown in Table 2 below. The PDML RS1013 strain is Bacillus mycoides strain.

Molecular biotechnology identification

The DNA of the PDML RS1013 strain was first extracted using a GeneMark Tissue & cell genomic DNA purification kit. The strain was inoculated into NB medium, cultured at 28 ° C for 24 hours, centrifuged at 12000 rpm for 10 minutes, and after removing the supernatant, 180 μl of lysozyme buffer (20 mM Tris-HCL pH 8.0, 2 mM EDTA, was added. 1.2% Triton X-100 contains 40 mg of lysozyme) to resuspend the lower layer of the precipitate. After extracting and purifying the bacterial suspension according to the method provided by Kit, the DNA was converted to OD ₂₆₀ by spectrophotometer. Concentration, and the ratio of OD ₂₆₀ / OD _{280 were} determined to determine DNA purity. The DNA purified material was placed at -20 ° C until use.

Next, the DNA extract of the PDML RS10013 strain was amplified as a template required for the PCR reaction. The primer pair required for the PCR reaction is ITS-U1F (5'CTYAAAKRAATTGRCGGRRRSSCS 3') and ITS-U1R (5'CGGGCGGTGT GTRCAARRSSC 3') published by Rivas et al. (2004). The PCR reaction mixture contained 2 μl (100 ng) of template DNA, 0.5 μl of Taq DNA polymerase (MD Bio Inc., Taiwan), 5 μl of 10× PCR reaction buffer (MD Bio Inc., Taiwan), and 2 μl of mM dNTPs (GeneMark Technology Co., Ltd., Taiwan), 1 μl primer (GeneMark Technology Co., Ltd., Taiwan) and 33.5 μl dH2O, the total reaction volume was 50 μl. The replication of the DNA fragments was then carried out in an automatic temperature cycle control reactor (Programmable Thermal Controller, PTC-200, MJ Research, Inc., MA, U.S.A.). The operating conditions were denaturation at 95 ° C for 2 minutes, followed by a cycle of denaturation at 95 ° C for 30 seconds, 55 ° C for 30 minutes, and 72 ° C for 30 seconds, and finally at 72 ° C. The PCR reaction was completed by further extension for 5 minutes.

The DNA reaction solution which was replicated by the aforementioned PCR was 1.5% (w/v) acacia, 0.5 times TAE buffer [(20 mM Tris-acetate, 0.5 mM EDTA-Na2 (pH 8.5), 10 mM). Glacial acetic acid) was electrophoresed at a voltage of 100 volts. The electrophoretically separated film was stained with ethidium bromide (EtBr) for 10 minutes, and then the excess dye was degraded with deionized water. The dyed film was recorded on a UV camera system to record the DNA fragments on the colloidal gel, and then sent to the source of International Biotechnology Co., Ltd. for sequencing. The sequence of the ITS segmentation sequence of the obtained PDML RS1013 strain is utilized by the US Biotechnology Information The sequence registered in the database of the Center (National Center for Biotechnology Imformation, NCBI, http://www.ncbi.nlm.nih.gov/), is analyzed by the nucleic acid gene library sequence group, and according to The DNA distance matrix deduced the parental relationship, showing that it is closer to Bacillus mycoides strain 273 (accession no.NR036880.1).

Control effect of Bacillus mycoides PDML RS1013 strain on melon seedling disease and cruciferous vegetable root nodulation Prevention of melon vegetable diseases

The isolated Bacillus licheniformis strains were prepared into a cell suspension (OD ₆₂₀ value of about 0.8, the amount of bacteria reached 1×10 ⁸ cfu/ml or more), and the courgette seeds were immersed and coated with each Bacillus licheniformis strain. After the suspension treatment, the germination is carried out, and then the seed treated seed is incubated in the cultivation medium for 1 week, and each of the cucurbit seedlings is inoculated with 1 ml of a spore suspension of the seedling Kappa Kp (1×10 ⁵ tour spores/ml), each One treatment was repeated 3 times, and then the onset of the cucumber seedlings was observed and recorded every seven days.

According to the results of the control group up to 14 days, it showed that the seedlings were treated with PDML RS1013 strain, the incidence of seedlings was about 30%, and the disease prevention effect was the best, while the PDML RS1016 strain was the second, but the PDML RS1016 and PDML L0912 strains were reduced. The unearthed rate of the jujube seedlings relatively affects the disease prevention effect. However, although the treatment with PDML TS02 strain has no seedling mortality, most plants have gangrene symptoms at the base of the stem. In comparison, the seedling mortality rate of the PDML RS1013-treated group was 0%, and the incidence of the disease was low and the development was optimal (Fig. 6).

Control cruciferous vegetable root nodule disease

The strains of Bacillus licheniformis with the effect of controlling the root nodule disease of cruciferous vegetables were selected by the application methods such as seed coating, mixing medium and drenching plants to further evaluate the cross-flower control of each strain. The effect of vegetable root nodule disease. Seed coating treatment: The selected strains were cultured in increments of plates to prepare a concentration of 10 ⁴ cfu/ml bacterial suspension, and then the cabbage seeds were soaked for 24 hours, and then the germinated seeds were sown in the cultivation medium. Mixing medium treatment: The culture medium is uniformly mixed with the bacterial suspension, and the concentration is adjusted to 10 ⁸ cfu/ml, and then the germinated cabbage seeds are seeded. Watering plant treatment: After germination of the germinated cabbage seeds in the cultivation medium, a bacterial suspension of 10 ⁸ cfu/ml is prepared, and 10 ml of the bacterial liquid is poured around the root ring of the cabbage plant in each hole (7 x 7.2 cm). Watering once a week (3 times in total). After each treatment the cultivation of cabbage plug 3 weeks were transplanted in resting spores having a concentration of ¹⁰⁷ / g dry culture medium with a weight of cruciferous vegetables nodule bacteria, the cultivation to collect and record 8 weeks cabbage plant The disease and fresh weight, each treatment contains 3 repetitions.

The results showed that the treatment of powdered seeds with PDML RS1013 strain had the most significant disease prevention effect (Fig. 7), which could reduce the rickets of 51.85%. In the part of cabbage plant growth, the treatment with PDML RS1013 powder seed could be observed. In the case of promoting root growth, the fresh weight of the shoots and the fresh weight of the lower part of the ground were increased by 5.4 times compared with the control group (Fig. 8).

In summary, the present invention has been isolated and screened from vegetable fields or paddy fields, and has a native strain of Bacillus licheniformis which is resistant to vegetable crop diseases, and it has been found that PDML RS1013 strain inhibits the production of zoospores against seedling disease. The ability of bacteria can effectively achieve the effect of preventing and curing the disease of courgette seedlings; and adding the strain to the cultivation medium containing the rhizobium of cruciferous vegetables can also greatly reduce the infection rate of pathogenic bacteria to cabbage seedlings, thereby reducing the strain of cabbage The degree of rickets, therefore, in addition to the prevention and treatment of melon seedlings, the Bacillus licheniformis PDML RS1013 strain has the ability to control cruciferous nodulopathy. It is shown that the native Bacillus licheniformis PDML RS1013 strain can be used as a source of bacteria for the development of microbial source plant protection preparations.

Claims (6)

A Bacillus mycoides PDML RS1013 strain having the ability to inhibit the migration of Pythium aphanidermatum and the infection of Plasmodiophora brassicae , the accession number is BCRC910584, The colony morphology resembled rhizoidal colony and was shown to be Gram-positive bacilli producing endospores by Gram staining and endosperm staining tests. A method for jointly preventing and treating melon vegetable seedling disease and cruciferous root tumor disease, comprising applying a strain of Bacillus mycoides PDML RS1013 according to the scope of claim 1 to a seed or a plant, wherein the PDML RS1013 strain The registration number is BCRC910584. The method of claim 2, wherein the Bacillus licheniformis application method comprises treating the seed by soaking and coating, and then cultivating the seed coated with Bacillus licheniformis and then sown in the cultivation medium. A plant protection preparation for controlling cucurbit vegetable seedling disease and cruciferous root tumor disease, comprising Bacillus mycoides PDML RS1013 strain according to item 1 of the patent application. A plant protection preparation according to item 4 of the patent application for the preparation of a biological pesticide. A plant protection preparation according to item 4 of the patent application for the preparation of a crop growth promoter.