CN106011032B - Pantoea for controlling bacterial wilt and its application - Google Patents

Abstract

The invention discloses a pantoea strain for preventing and treating bacterial wilt and its application. The name of this strain is Pantoea coffeiphila GZ‑33, the deposit number is CCTCC M 2016352, and it was deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China on June 27, 2016. The Pantoea strain grows well in LB medium, and the strain cells are rod-shaped. When cultured on LB agar plate, the surface of the colony is smooth and translucent, and the edges are neat. Bacterial wilt provides a way.

Description

Pantoea for controlling bacterial wilt and its application

technical field

The invention relates to the field of agricultural biotechnology, in particular to a Pantoea strain used for preventing and treating bacterial wilt and its application.

Background technique

Plant bacterial wilt is a devastating soil-borne bacterial disease of vascular bundles caused by Ralstonia solanacearum, which is a devastating soil-borne disease with widespread systemic infection in subtropical, tropical and temperate regions, commonly known as "Plant Blight". R. solanacearum has a wide range of hosts and can infect more than 450 species of plants from 54 families. At present, the prevention and control of bacterial wilt disease is a major problem recognized by the world. It is widely distributed in all parts of the world and occurs seriously in southern provinces and cities in my country, especially in Zhejiang. Mulberry bacterial wilt caused serious losses to sericulture production.

There are many races of R. solanacearum, which are divided into 5 physiological races according to the host range of R. solanacearum. Race 1 mainly harms most Solanaceae plants, including tomatoes, potatoes, eggplants and tobacco, etc., and also harms other plants; Species 2 can infect plants such as bananas, Heliconais and plantains; Race 3 mainly infects potatoes, but also weakly infects tobacco and tomato; Race 4 infects ginger and weakly infects other plants such as potatoes. Another researcher named the bacterial wilt strain isolated from mulberry trees in southern my country as race 5. R. solanacearum infects potatoes mainly race 1 and race 3.

At present, the control methods of bacterial wilt mainly include strengthening field management, cultivating disease-resistant varieties, using chemical agents and applying biocontrol agents, etc., but practice has proved that field management is difficult to effectively control the occurrence of bacterial wilt, and the breeding of resistant materials It takes a long time, and chemical control pollutes the environment and destroys the ecological balance, and the long-term use of chemical agents is easy to cause pathogenic bacteria to develop drug resistance. Therefore, the use of biocontrol strains and their active products to control bacterial wilt in ginger has attracted more and more attention. A large number of studies have shown that some bacteria and actinomycetes are ideal antagonists against bacterial wilt.

SUMMARY OF THE INVENTION

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide a pantoea strain for preventing and treating bacterial wilt.

Another object of the present invention is to provide the application of the described Pantoea for controlling bacterial wilt.

The object of the present invention is achieved through the following technical solutions: a strain of Pantoea coffeiphila for the prevention and treatment of bacterial wilt disease, named Pantoea coffeiphila GZ-33, with a deposit number of CCTCC M 2016352, deposited on June 27, 2016 at the Chinese Type Culture Collection at Wuhan University, Wuhan, China.

Application of the pantoea for controlling bacterial wilt disease in controlling bacterial wilt disease.

The application of the pantoea for controlling bacterial wilt disease in preventing and controlling bacterial wilt disease is to apply the pantoea bacterial solution in the soil for cultivating crops or spray it on the surface of the crops.

Described crops are crops that are easily infected by solanacearum; preferably Solanaceae crops; more preferably eggplants.

The crops can be crops that have been infected by R. solanacearum, or crops that have not been infected by R. solanacearum.

A bacterial wilt biological control fungicide contains the above pantoea for controlling bacterial wilt.

Compared with the prior art, the present invention has the following advantages and effects: the present invention provides a Pantoea strain with antagonistic effect on Ralstonia solanacearum, and provides another way for the prevention and treatment of bacterial wilt.

Description of drawings

Fig. 1 is the effect diagram of bacterial wilt antagonistic biocontrol test in the present invention.

Figure 2 is a photograph of a single colony of strain GZ-33 in the present invention.

Detailed ways

The present invention will be described in further detail below with reference to the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

Example 1: Isolation and purification of bacteria:

1. Culture medium

LB agar medium (LB solid medium): peptone 10g, yeast extract 5g, NaCl 10g, agar 15g, make up to 1000ml with H ₂ O, pH 7.0-7.2. Sterilize at 121°C for 20 minutes.

2. Experimental steps

(1) Sample collection: Select a representative location (root soil of healthy plants in the field with bacterial wilt disease, in Guangdong Province), shovel off the topsoil, and use soil mining tools to collect the soil layer at a depth of 5 to 10 cm below the surface 5 samples from each site, bagged and recorded.

(2) Preparation of soil diluted solution: Weigh 10 g of soil, put it into a conical flask containing 90 mL of sterile water, and fully shake it to prepare a soil diluted solution with a concentration of 1:10. After the soil particles are settled, 1ml of the supernatant is drawn and transferred into a test tube containing 9ml of sterilized water to make a soil suspension with a concentration of 1:100, and so on to make a soil suspension of 1:1000 and 1:10000 , make 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 and 10 ^-6 times of soil solution for use.

(3) Bacterial isolation: choose 0.1 ml each of four concentrations of 10 ^-3 , 10 ^-4 , 10 ^-5 and 10 ^-6 , pour the diluted soil suspension into the LB medium petri dish, and place the coating rod on the The flame of the alcohol lamp is fully sterilized by burning, and the plate is coated after cooling. The inoculated petri dish was inverted and incubated at a constant temperature of 30 degrees Celsius, and three replicates were set.

(4) Results: 384 bacterial strains were isolated.

Example 2: Plate antagonism test of isolated bacteria against Ralstonia solanacearum

1. Culture medium

TTC solid medium: ①10g peptone, 1g Casein Acid Hydrolysate, 5g glucose, 15g agar, dilute to 1000ml with H ₂ O, pH 6.8-7.2; sterilize at 121°C for 20 minutes to obtain a solution I; ②TTC (2,3,5-triphenyltetrazolium chloride) was made into a solution of 1% by mass with distilled water, and sterilized by filtration with a 0.22 μm filter membrane to obtain solution II; ③Cool in solution I to about 45 At ℃, add 0.5 mL of solution II to every 100 mL of solution I, and then pour the plate.

TTC liquid medium: without adding agar and TTC.

2. Experimental steps

2.1 Plate antagonism test ---- primary screening

2.1.1 Activation of strains

Ralstonia solanacearum and bacteria are activated by strains: R. solanacearum GMI1000 (ATCC) and 384 strains of bacteria isolated in Example 1 were transferred to TTC solid medium plates and LB solid medium plates, respectively, and cultivated at 28 ° C for 2 days And incubate at 30°C for 1 day for use.

2.1.2 Preparation of R. solanacearum plates

The activated R. solanacearum was inserted into a TTC liquid medium for shake flask culture, and cultured at 28° C. and 200 rpm for 2 days to obtain a fermentation broth of R. solanacearum. The next day, pour the plate with TTC solid medium, and after the medium is solidified, add 0.1 ml (OD ₆₀₀ about 2.0) of the fermentation broth of R. solanacearum to each plate, spread evenly, and blow dry for use to obtain a plate of R. solanacearum.

2.1.3 Preliminary Screening of Plate Antagonism Test

Pick the activated single colonies of 384 strains of bacteria with sterilized toothpicks, and spot them on the R. solanacearum plates. Each plate is connected with 30 different antagonistic bacteria. The three replicates were cultured at 28°C and observed at 24h, 36h and 48h respectively. The experimental results mainly observed the existence of the inhibition zone.

2.2 Plate antagonism test-----rescreening

2.2.1 Strain activation

The same as 2.1.1, activated selection and primary screening of isolated bacteria with inhibition zone.

2.1.2 Preparation of R. solanacearum plates

The activated R. solanacearum was inserted into a TTC liquid medium for shake flask culture, and fermented at 28° C. and 200 rpm for 2 days to obtain a fermentation broth of R. solanacearum. The next day, pour the plate with TTC solid medium, and after the medium solidifies, add 0.1ml (OD ₆₀₀ about 2.0) bacterial wilt fermentation broth to each plate, spread evenly, and then blow dry with a punch (Φ5mm) to punch holes. Punch a hole for each plate.

2.2.3 Isolation of bacterial liquid fermentation

On the second day of R. solanacearum liquid shake-flask culture, the activated isolated bacteria were transferred to LB liquid medium for shake-flask culture at 30° C., 200 rpm for 24 hours (OD ₆₀₀ about 2.0).

2.2.4 Rescreening of plate antagonism test

Pipette 10 μl of the isolated bacterial fermentation broth into the wells of the R. solanacearum plate with a pipette, blow dry, repeat 3 times, and cultivate at 28°C. The experimental results were observed at 24h, 36h and 48h respectively, mainly to observe the existence and size of the inhibition zone.

3. Results

The 53 bacterial strains with antagonistic effect were obtained through the primary screening test, and the plate antagonism test was punched for re-screening. The more obvious the inhibition zone was, the better the antagonistic effect was. After rescreening, 6 strains of bacteria with better antagonistic effect were obtained, and the strain GZ-33 was one of them, and its inhibition zone against R. solanacearum was obvious.

Example 3: Biological control effect test of strain GZ-33

In order to test the biocontrol effect of the strain GZ-33, the biocontrol effect of bacterial wilt was tested on the fermentation broth of the strain GZ-33 in pots.

Experimental steps:

1.1 Buy eggplant seedlings

Buy eggplant seedlings grown in greenhouses.

1.2 Transplanting seedlings

Fill the flowerpot (specification 170mm*160mm) with the base soil, press it slightly, and then move 1 eggplant seedling into each pot. After watering, the two fingers are slightly pressed at the root, and cultivated in the greenhouse. Cultivated in pots for about 4 weeks.

1.3 R. solanacearum and antagonistic bacteria activation and fermentation broth preparation

Ralstonia solanacearum GMI1000 and antagonist GZ-33 were activated respectively, and then single colonies were respectively inserted into TTC liquid medium and LB liquid medium for shake flask culture, and were incubated at 28°C and 30°C, respectively, at 200 rpm for about two days (to OD). ₆₀₀ are about 2.5).

1.4 Ralstonia solanacearum and antagonistic inoculation

Two control groups were set up in this experiment, namely CK1 and CK2, CK1: no bacteria was added, only 5ml TTC liquid medium was added; CK2: only 5ml bacterial wilt fermentation broth was added, no antagonistic bacteria fermentation broth was added. Strain GZ-33 treatment: according to the volume ratio of R. solanacearum: antagonistic bacteria are 1:1, 1:2.5 and 1:5, do 3 treatments, each treatment has 5 pots, and the roots of each strain are first poured 5ml of R. solanacearum fermentation broth , and then pour 5ml, 12.5ml, 25ml of antagonistic antibacterial fermentation broth.

1.5 Records

The number of diseased plants and the severity of disease were recorded every other day.

2. Results

As shown in Figure 1, after artificial inoculation, the control group CK1 that only received TTC liquid medium did not develop disease, and the mortality rate was 0. The control group CK2, which was directly inoculated with the fermentation broth of R. solanacearum GMI1000, all developed disease, and the mortality rate was 100%. Inoculated with R. solanacearum The mortality rates of 1:1, 1:2.5, and 1:5 treatment groups of GMI1000 fermentation broth and antagonistic GZ-33 fermentation broth were 20%, 0, and 0, respectively. It can be seen that the treatment of antagonistic GZ-33 fermentation broth can make The occurrence of bacterial wilt in eggplant plants was effectively controlled.

Example 4: Identification of the isolated bacteria

Tested by the Guangdong Provincial Microbiological Analysis and Testing Center, the test is based on the "Common Bacterial System Identification Manual" (edited by Dong Xiuzhu, Science Press) "Berger Bacterial Identification Manual" ninth edition and "Molecular Cloning Experiment Guide" The second edition translated by Huang Peitang 2002 Science Press), the identification result is: the 16S rDNA sequence of the strain GZ-33 has a homology of 98.06% with Pantoea coffeiphila (Pantoea), and its morphological and physiological and biochemical characteristics are most similar to Pantoea coffeiphila (Pantoea) . The specific test results are as follows:

(1) The features such as morphology, culture, physiology and biochemistry of the preserved strain are as follows:

Pantoea coffeiphila GZ-33: belongs to Pantoea coffeiphila; it grows well in LB medium, the strain cells are rod-shaped, and the colony surface is smooth and translucent when cultured on LB agar plate, with neat edges (as shown in Figure 2).

(2) The results of physical and chemical experiments of strain GZ-33 are shown in Table 1.

Table 1 Physicochemical experimental results of strain GZ-33

Note: "+" means positive, "-" means negative.

(3) The results of the 16S rRNA gene sequence determination of strain GZ-33 are as follows:

AAGTGGTAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCCGATCCGGACTACGACGCACTTTATGAGATCCGCTTGCCCTCGCGAGGTCGCTTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCCCCTGAGTTCCCGACCGAATCGCTGGCAACAGAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCACAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTGTGGATGTCAAGGCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCGTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTCGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACGAGACTCAAGCCTGCCAGTTTCAAATGCAGTTCCCGGGTTGAGCCCGGGGATTTCACATCTGACTTAACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGGGTAACGTCAATCGACGC GGTTATTAACCGCATCGCCTTCCTCCCCGCTGAAAGTACTTTACAACCCGAAGGCCTTCTTCATACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGAGCCATTACCCCACCTACTAGCCTAATTCCCATCTGGGCACATCCGATGGTGTGAGGCCCGAAGGTCCCCCACTTTGGTCCGAAGACGTTATGCGGTATTAGCTACCGTTTCCAGTAGTTATCCCCCTCCATCGGGCAGTTTCCCAGACATTACTCACCCGTCCGCCACTCGTCACCCAAGAAGCAGGCTTCT。

The 16S rRNA gene sequence was analyzed by Blast, and the similarity with Pantoea coffeiphila was 98.06%, and its morphological and physiological and biochemical characteristics were most similar to Pantoea coffeiphila. , this strain can be classified as Pantoeacoffeiphila.

GZ-33 was named as Pantoea coffeiphila GZ-33, and the deposit number was CCTCC M2016352, which was deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China on June 27, 2016.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

Claims (5)

1. one plant for preventing and treating the general bacterium of bacterial wilt, it is characterised in that: entitled general bacterium (Pantoea coffeiphila) GZ- 33, deposit number is CCTCC M 2016352, is preserved in the China positioned at Wuhan, China Wuhan University on June 27th, 2016 Type Tissue Collection.

2. for preventing and treating application of the general bacterium of bacterial wilt in the bacterial wilt of prevention and treatment solanaceous crops infection described in claim 1.

3. according to claim 2 for preventing and treating general bacterium the answering in the bacterial wilt of prevention and treatment solanaceous crops infection of bacterial wilt With, it is characterised in that: it is the surface that general bacterium bacterium solution is applied to in the soil of culture crops or is sprayed at crops.

4. according to claim 3 for preventing and treating general bacterium the answering in the bacterial wilt of prevention and treatment solanaceous crops infection of bacterial wilt With, it is characterised in that: the crops are the crops for being easy to be infected by Ralstonia solanacearum.

5. a kind of bacterial wilt biological control microbial inoculum, it is characterised in that: containing described in claim 1 for preventing and treating the general of bacterial wilt Bacterium.