CN108165498B - Penicillium griseus Pg-35 strain which antagonizes bacterial blight of rice and its fermentation filtrate and its application in plant disease control - Google Patents

Abstract

The invention belongs to the field of biotechnology microorganisms, and relates to 1 Penicillium griseofulvum Pg-35 strain for antagonizing rice bacterial blight and application thereof in plant disease prevention and treatment. Antagonizing rice bacterial blight (Xanthomonas oryzae pv.oryzae) Penicillium griseofulvum (A)Penicillium griseofulvum) Pg-35 strain, which is preserved in China center for type culture Collection with the preservation number: CCTCC M2017728, and the preservation date is 11 months and 27 days in 2017. Penicillium griseofulvum (I) screened by the inventionP.griseofulvum) The Pg-35 strain fermentation filtrate has obvious inhibition effect on rice bacterial blight. The penicillium Pg-35 strain can provide an excellent strain for the development of microbial pesticides, and has a good application prospect in biological control of rice bacterial leaf blight and the like.

Description

Penicillium griseus Pg-35 strain and its fermentation filtrate and its fermentation Application in Plant Disease Control

technical field

The invention belongs to the field of biotechnological microorganisms, and relates to a strain of Penicillium griseofulvum Pg-35 which antagonizes bacterial blight of rice and its application in plant disease prevention and smelting.

Background technique

Rice bacterial blight is also known as white leaf blast, thatch blast, and ground fire. It occurs in all rice regions in my country and is the main disease of rice. It is caused by Xanthomonas oryzae pv.oryza, Xoo. The cells are short rod-shaped, with a size of 1.0～2.7×0.5～1.0μm, solitary, single flagella, polar or subpolar, about 8.7μm long, 30nm in diameter, Gram stain negative, no spores and capsules, bacteria Surrounded in vitro by viscous exopolysaccharides. On artificial medium, the colony is honey-yellow, producing water-insoluble yellow pigment, aerobic, and respiratory-type metabolism. The optimum growth temperature of the bacteria is 25℃～30℃, and the optimum growth temperature is 6.5～7.0.

Penicillium is commonly found on citrus and other fruits, refrigerated cheeses and other foods contaminated with their spores, and its conidia are present everywhere in the soil, in the air and on decaying matter. Common Penicillium species are: Penicillium notatum, P. chrysogenum, P. oxalicum, P. citrinum, P. meleagrinum ), Penicillium griseofulvum (P.griseofulvum), etc. Penicillium is saprophytic, and its nutrient sources are extremely extensive, and it can grow on any substrate containing organic matter.

Penicillium is closely related to human life. In industry, it can be used to produce citric acid, fumaric acid, gluconic acid and other organic acids and enzyme preparations; the most famous antibiotic - penicillin is extracted from some strains of Penicillium, it is the earliest discovered, The first to be purified and the earliest antibiotic to be used clinically; another important antibiotic currently discovered, griseofulvin, is produced by Penicillium griseofulvum (P.griseofulvum).

SUMMARY OF THE INVENTION

In view of the increasingly exposed problems of chemical control and agricultural control of rice bacterial blight, the use of microorganisms and their secondary metabolites for biological control of rice bacterial blight has become a research direction.

The first object of the present invention is to provide a strain of Penicillium griseofulvum (P. griseofulvum) Pg-35 with high antagonism to bacterial blight and its application.

The second object of the present invention is to provide the above-mentioned Penicillium griseofulvum (P. griseofulvum) Pg-35 strain fermentation filtrate and its application.

The third object of the present invention is to provide a microbial-derived pesticide for the above-mentioned fermentation filtrate.

In order to realize the above-mentioned first purpose, the present invention adopts the following technical scheme:

A strain of Penicillium griseofulvum Pg-35 that antagonizes Xanthomonas oryzae pv.oryzae, the strain is preserved in the China Center for Type Culture Collection, the preservation number is: CCTCC M2017728, and the preservation date is 2017 November 27.

The application of the above-mentioned Penicillium griseus Pg-35 strain in the preparation of microbial pesticides.

Preferably, the microbial source pesticide is used for controlling bacterial blight of rice, Fusarium solani, Gibberella fujikuroi, Fusarium oxysporum f.sp. momordicae and early blight of tomato fungus (Alternaria solani).

In order to realize the above-mentioned second purpose, the present invention adopts the following technical scheme:

The fermentation filtrate of the above-mentioned Penicillium griseus Pg-35 strain.

The above-mentioned fermentation filtrate is used in the preparation of microbial pesticides.

Preferably, the microbial source pesticide is used for controlling bacterial blight of rice, Fusarium solani, Gibberella fujikuroi, Fusarium oxysporum f.sp. momordicae and early blight of tomato fungus (Alternaria solani).

In order to realize the above-mentioned third purpose, the present invention adopts the following technical scheme:

A microbial source pesticide comprising the fermentation filtrate.

The Penicillium Pg-35 strain involved in the present invention is screened from paddy field soil. According to morphological characteristics and ITS rDNA gene sequence, it was identified as Penicillium griseofulvum.

Penicillium griseofulvum (P.griseofulvum) Pg-35 strain can be used to control rice bacterial blight. Cultivate the pure colony of 3d Pg-35 strain at 28°C, and inoculate the bacterial cake with a 0.5cm puncher into a 250mL conical flask containing 100mL PD liquid medium (shaker conditions 180r/min, 28°C) shaker After culturing for 7 days (180 r/min, 28° C.), a fermentation broth was obtained. Bacterial blight pathogen P6 race was inoculated into Xoo culture medium, activated and cultured in a shaker (180r/min, 28°C), when the bacterial density reached OD ₆₀₀ of 0.6, 100 μL of bacterial solution was uniformly spread on Xoo solid medium superior. 200 μL of fermentation broth was taken, and the inhibitory effect of the fermentation broth of Penicillium Pg-35 strain on P6 race was determined by Oxford cup method. Its fermentation broth inhibition zone reached 46mm (Figure 6).

The fermentation filtrate of Penicillium griseofulvum (P.griseofulvum) Pg-35 strain screened by the present invention has a significant inhibitory effect on bacterial blight of rice. When the initial pH of the fermentation medium is 6.0-6.5 and the culture temperature is 28°C, after 7 days of liquid fermentation, the diameter of the inhibition zone of the fermentation filtrate against B. oryzae can reach 46 mm (Fig. 6). The results of antibacterial spectrum test showed that the fermentation filtrate of Penicillium Pg-35 strain was resistant to Fusarium solani, Gibberella fujikuroi, Fusarium oxysporum f.sp. momordicae and tomato early blight. (Alternaria solani), the mycelial growth inhibition rates were 46.66%, 40.07%, 32.27% and 30.54%, respectively (Table 1, Figure 7). Penicillium Pg-35 strain can provide an excellent strain for the development of microbial pesticides, and has a good application prospect in biological control such as rice bacterial blight.

Description of drawings

Figure 1 The front (left) and back (right) of the colonies cultivated on PDA medium of Penicillium Pg-35 strain for 12 days.

Figure 2. Microscopic features of conidial heads of Penicillium Pg-35 strain (left, 100×; right, 400×).

Fig. 3 Microscopic characteristics of conidia of Penicillium Pg-35 strain (400×).

Fig. 4 ITS rDNA gene sequence (525bp) of Penicillium Pg-35 strain.

Fig. 5 Phylogenetic tree of Penicillium Pg-35 strain established based on ITS rDNA gene sequence.

Fig. 6 Inhibition circle of the fermentation broth of Penicillium Pg-35 strain against bacterial blight pathogen.

Figure 7 Inhibitory effect of the fermentation filtrate of Penicillium Pg-35 strain on four phytopathogenic fungi; Note: From left to right: Poplar Fusarium wilt, Wheat scab, Cucumber Fusarium wilt and Tomato Early Phytophthora; top row For treatment, the lower row is for control.

Detailed ways

The specific embodiments of the present invention will be described in detail below.

Example 1 Isolation, screening and identification of Penicillium griseofulvum (P.griseofulvum) Pg-35 strain

1 strain

(1) Penicillium strains: isolate, purify, identify and preserve Penicillium strains from different habitats (eg: paddy field soil, rotten fruit, bread, etc.).

(2) Rice bacterial blight (Xanthomonas oryzae pv.oryzae, Xoo) P6 race.

2 medium

(1) PDA medium: 200 g of potato, 20 g of glucose, 15-20 g of agar powder, 1 L of water, pH 5.5 to 6.0. For the isolation, purification and identification of Penicillium strains.

(2) PD culture medium: 200 g of potato, 20 g of glucose, 1 L of water, pH 5.5-6.0. For the fermentation of Penicillium strains.

(3) Bacterial blight solid medium (Xoo solid medium): potato 300 g, sucrose 15 g, Ca(NO ₃ ) ₂ 0.5 g, NaH ₂ PO ₄ ·12H ₂ O 2.0 g, tryptone 5.0 g, agar 15g, water 1000mL, pH 6.5. For solid culture of bacterial blight of rice.

(4) Bacterial blight culture solution (Xoo culture solution): 300g of potato, 5g of tryptone, 15g of sucrose, 0.5g of Ca( _NO3 ) _{· H2O} , _2g of _Na2HPO4 ·12H2O, 1L of water , pH 6.5. For bacterial blight pathogen P6 race fermentation culture.

3 Experimental methods

3.1 Isolation, purification and preservation of Penicillium strains

(1) Isolation and purification method of surface Penicillium strains such as rotten fruits: Pick the surface mold mycelium of the fruit with tweezers and inoculate it in PDA solid medium, cultivate at 28°C, and pick the apical fungus after the white fluffy mycelium grows. The silk was purified and cultured 3 times to obtain pure strains.

(2) Separation and purification method of Penicillium strains in soil: Take 1 g of soil sample and put it in 99 mL of sterile water, shake it on a shaking table at 120 r/min for 30 min. Take the supernatant for gradient dilution (10 ^-2 to 10 ^-8 ), choose an appropriate concentration (10 ^-6 ), and spread 100 μL on the PDA medium evenly, incubate at 28°C for 36 hours, until different filamentous forms appear. Fungal colony. Pick a single colony, transfer it to PDA medium, and purify it for 3 passages to obtain a pure strain.

(3) Preliminary identification and numbering preservation of Penicillium strains: The above pure strains were cultured on PDA medium at 28°C for 3 days, and the strains were preliminarily identified according to the colony morphology and microscopic morphology, and the strains with typical characteristics of Penicillium were numbered , and stored in a 4°C refrigerator.

3.2 Screening of Penicillium strains against rice bacterial blight

3.2.1 Preliminary screening by co-culture method

Bacterial blight P6 race was inoculated into Xoo culture medium, and the culture was shaken (180 r/min, 28° C.). When the bacterial density reaches OD ₆₀₀ of 0.6, take 100 μL of bacterial liquid and spread it evenly on the Xoo solid medium, inoculate the activated Penicillium strain cake (diameter 6 mm) in the middle of the Xoo solid medium, and cultivate at 28°C for 48 hours. Observe the zone of inhibition. Penicillium strains with antagonistic effects were initially screened according to the presence and size of the inhibition zone, and were preserved in 25% glycerol.

3.2.2 Oxford Cup re-screening

(1) Fermentation culture of Penicillium strains: Inoculate the antagonistic Penicillium strains into a fresh PDA medium plate, cultivate in a 28°C incubator for 3 days, and use a 6mm puncher to take 1 bacterial cake and inoculate it into a 100 mL After 7 days of shaking culture (180 r/min, 28° C.) in a 250 mL conical flask of PD medium, a fermentation filtrate was obtained.

(2) Cultivation of the P6 race of B. oryzae: inoculate the P6 race in the Xoo medium, and activate the culture on a shaking table (180 r/min, 28°C). When the bacterial density reaches OD ₆₀₀ of 0.6, take 100 μL The bacterial liquid was evenly spread on the Xoo solid medium. The fermentation filtrate of 200 μL of Penicillium strains was taken, and the inhibitory effect of each Penicillium strain fermentation broth on P6 race was determined by Oxford cup method. The Penicillium strains with stronger antagonistic effect were selected according to the size of the inhibition zone.

3.3 Identification of the target strain of Penicillium antagonism against rice bacterial blight

Pick a small amount of Penicillium target strain mycelium with tweezers, transfer it to a PDA medium plate, and activate it for 3 days; take 1 bacterial cake (6 mm in diameter) from the plate and inoculate it on a new PDA medium plate, and cultivate it at 28°C for 3 days. The morphological characteristics of the hyphae, conidial head, conidiophore, conidia, etc. of the target strain were observed under the microscope, and photographs were taken.

Extract the genomic DNA of the target strain, amplify the ITS rDNA gene sequence, and send it to Shanghai Bioengineering Company for sequencing. The sequenced ITS rDNA sequences were submitted to GenBank and analyzed by BLAST to preliminarily determine the species they belonged to.

4 Experimental results

4.1 Obtaining pure strains of Penicillium

A total of 60 pure Penicillium strains were isolated and purified from natural fermentation samples (soil, fruit, organic matter, etc.) collected from different habitats.

4.2 Screening of Penicillium strains against rice bacterial blight

The co-culture method and Oxford cup method were used to screen 60 pure strains of Penicillium oryzae for their inhibitory effects on B. oryzae. The results showed that the inhibitory effects of different Penicillium strains on B. oryzae were quite different. After screening, a Penicillium strain with strong antagonistic effect was obtained, which was numbered as Pg-35 strain. The diameter of the inhibition zone of the fermentation broth was 46 mm. Penicillium Pg-35 was isolated from the paddy field soil.

4.3 Identification results of Penicillium Pg-35 strain

Morphological characteristics: cultured on PDA at 28°C for 12 days, the colony diameter is 30-40mm; a small amount of water droplets are produced on the surface; a small amount of white mycelium is formed on the edge, and a yellow-brown pigment is produced on the back (Fig. 1). The conidiophore occurs in the substrate or in the sporophore bundles and hyphae, and the wall is smooth; the broom-like branches are more complex, observed under a 400-fold microscope as two whorls, forked, and the branch point is usually one and scattered. 5 to 7 phialides per round, 5.0 to 7.5 × 2.0 to 2.5 μm (Fig. 2); the conidia are nearly spherical, with smooth walls, and the conidia chains are cylindrical or nearly cylindrical with relatively close and forked chains. (Figure 3).

ITS rDNA gene sequence: The analysis results showed that the length of the ITS rDNA gene sequence of Penicillium Pg-35 strain was 525 bp (Fig. 4). Penicillium (P. griseofulvum) has the closest evolutionary distance (Fig. 5).

According to the morphological characteristics and ITS rDNA gene sequence of Penicillium Pg-35 strain, combined with the Penicillium (Penicillium) classification key table, Pg-35 strain was identified as Penicillium griseofulvum.

Embodiment 2 Penicillium griseofulvum (Penicillium griseofulvum) Pg-35 strain fermentation broth antibacterial spectrum determination

1 strain

(1) Penicillium griseofulvum Pg-35 strain.

(2) 4 kinds of plant pathogenic fungi: Gibberella fujikuroi, Alternaria solani, Fusarium solani, Fusarium oxysporum f.sp. momordicae. Fungal Antimicrobial Spectrum Test.

2 medium

(1) PDA medium: 200 g of potato, 20 g of glucose, 20 g of agar powder, 1 L of water, pH 6.0-6.5. Used for activation culture of Pg-35 strain and culture of 4 antibacterial fungi.

(2) PD culture medium: 200 g of potato, 20 g of glucose, 1 L of water, pH 6.0-6.5. For fermented seed culture of Penicillium Pg-35 strain.

(3) Fermentation culture medium: 200 g of potato, 20 g of glucose, 5 g of peptone, 1 L of water, pH 6.0-6.5. For Pg-35 strain fermentation culture.

3 Experimental methods

3.1 Preparation of Penicillium Pg-35 strain fermentation filtrate

A small amount of spores from the PDA solid medium of Penicillium Pg-35 strain were scraped and inoculated into PD medium, with a liquid volume of 50/250 mL, an initial pH of 6.5, 180 r/min, and a constant temperature shaker at 28 °C for 48 h as the seed solution. The seed liquid was inoculated into the fermentation medium according to the inoculation amount of 4%, and the fermentation culture was carried out on a shaking table for 7 days. The fermentation broth was placed in a 50 mL centrifuge tube and centrifuged at 12,000 r/min for 10 min, and the supernatant was filtered with a 0.5 μm organic filter to remove residual spores to obtain a fermentation filtrate.

3.2 Activation and cultivation of four plant pathogenic bacteria

Four kinds of pathogenic bacteria stored at 4°C were inoculated into PDA medium respectively, and activated and cultured at 28°C for 2 days. Then it was transferred to a new PDA medium and cultured at 28°C for 4 days for the determination of antibacterial spectrum.

3.3 Determination of antibacterial spectrum of Penicillium Pg-35 strain

The fermentation filtrate and the PDA solid medium in a molten state at 55°C were evenly mixed at a volume ratio of 1:9, and poured into a petri dish to prepare a plate containing the fermentation filtrate. A fungus cake with a diameter of 6 mm with four phytopathogenic fungi to be tested was removed and placed in the center of the above plate with a hole punch. Incubate at 28°C for 72h. The colony growth diameters of the four phytopathogenic fungi were measured by the crisscross method, and three groups of parallel replicates were set up to take the average value. Calculate the inhibition rate of mycelial growth according to the following inhibition rate calculation formula.

Inhibition rate%=control colony diameter-treated colony diameter)/(control colony diameter-6)×100

4 Experimental results

The determination results showed that the fermentation broth of Penicillium Pg-35 strain had good inhibitory effect on the four phytopathogenic fungi. The mycelial growth inhibition rates of Fusarium solani, Gibberella fujikuroi, Fusarium oxysporum f.sp. momordicae and Alternaria solani were 46.66%, respectively. , 40.07%, 32.27% and 30.54% (Table 1, Figure 7).

Table 1 Inhibitory effect of Penicillium Pg-35 strain fermentation filtrate on four plant pathogens

Note: Mycelial growth inhibition rate (%)=(control colony diameter-treated colony diameter)/(control colony diameter-6)×100

Claims (2)

1. Antagonizing rice bacterial blight (Xanthomonas oryzae pv. oryzae) Penicillium griseofulvum (A)Penicillium griseofulvum) Pg-35 strain, which is preserved in China center for type culture Collection with the preservation number: CCTCC NO: m2017728, preservation date of 2017, 11 months and 27 days.

2. The use of the penicillium griseofulvum Pg-35 strain of claim 1 for preparing a microbial pesticide for controlling rice bacterial blight and poplar blightFusarium solaniWheat scabGibberella fujikuroiAnd early blight of tomatoAlternaria solani。

Images