CN109294961B

CN109294961B - Biocontrol strain PNC25 for preventing and treating litchi downy blight and application thereof

Info

Publication number: CN109294961B
Application number: CN201811457701.2A
Authority: CN
Inventors: 郑丽; 吴健雄; 蒋仁娇
Original assignee: CHINESE ACADEMY OF TROPICAL AGRICULTURAL SCIENCE GUANGZHOU EXPERIMENTAL STATION
Current assignee: CHINESE ACADEMY OF TROPICAL AGRICULTURAL SCIENCE GUANGZHOU EXPERIMENTAL STATION
Priority date: 2018-11-30
Filing date: 2018-11-30
Publication date: 2021-07-27
Anticipated expiration: 2038-11-30
Also published as: CN109294961A

Abstract

The invention discloses a biocontrol strain PNC25 for preventing and treating litchi phytophthora blight and application thereof. A biocontrol strain PNC25 for preventing and treating litchi phytophthora blight is Microbacterium acetylicum (Exiguobacterium acetylicum) which is preserved in Guangdong province microorganism culture collection center in 2016, 11 and 15 days, and the strain preservation number is GDMCC No. 60108. The acetobacter xylinum has an inhibiting effect on peronophythora litchi, has an excellent biocontrol effect on peronophythora litchi under multiple experimental conditions, and can be applied to control of peronophythora litchi. Meanwhile, the acetobacter xylinum has a good fresh-keeping effect on litchi, and can be used for preparing litchi fresh-keeping preparations.

Description

Biocontrol strain PNC25 for preventing and treating litchi downy blight and application thereof

Technical Field

The invention belongs to the technical field of plant protection, and relates to a biocontrol strain PNC25 for preventing and treating litchi phytophthora blight and application thereof.

Background

Litchi (lichi chinensis Sonn) is an important subtropical economic crop in southern areas, is called as the precious fruit of China, has high economic value and is one of the most competitive fruits in the international market of China. However, in the main litchi producing area of China, downy mildew caused by Peronophythora litchii Chen ex Ko et al is one of the most important diseases causing the yield reduction of litchi. The disease has the characteristic of indirect outbreak, is greatly influenced by climate, and can cause more than 80 percent of loss of litchi in suitable years. At present, effective measures for controlling litchi frost blight mainly adopt chemical prevention and control, but because environmental pollution problems and drug resistance problems of pathogens caused by the use of a large amount of chemical pesticides are obvious day by day, research and development of low-toxicity and residue-free biological source pesticides for litchi disease prevention and control have very important significance for controlling pesticide residue, ensuring edible safety and improving international competitiveness of products.

Bacteria of the genus microbacterium are a common type of bacteria, and current methods are used to control pollution. 201110195307.8 discloses a strain MG2 of the genus Microbacterium, which has high efficiency of decolorization and can be used for degrading triphenylmethane dye malachite green. 201610012010.6 discloses a strain of Microbacterium mexicana 2a, 2a with maximum degradation rate of 72.8% and 57.9% to colloid and asphaltene in crude oil, which can reduce crude oil viscosity by 11.1%; the solubility of the solid paraffin in normal hexane can be obviously improved, the amount of the soluble paraffin accounts for 62.0 percent of the total amount of the paraffin, the amount of the crystalline paraffin is reduced by 64.7 percent compared with a control group, and the paraffin removal rate and the paraffin prevention rate respectively reach 77.1 percent and 96.9 percent; the cells are highly hydrophobic and can be firmly attached to the surface of the metal to form a biological film and prevent wax crystallization and deposition. 200810063812.5 discloses a biological demanganization functional bacterium MB4(Exiguobacterium sp.) belonging to the genus Microbacterium and having the function of removing Mn in water²⁺Ability of (C), Mn²⁺The removal rate is higher than 91%. At present, no report that the acetobacter xylinum has the function of preventing and treating the litchi downy blight is found.

Disclosure of Invention

The invention aims to provide a biocontrol strain PNC25 for preventing and treating litchi phytophthora blight, aiming at the defects in the prior art.

Another object of the present invention is to provide the use of the strain.

The invention also aims to provide a microbial inoculum containing the strain and a preparation method thereof.

The purpose of the invention can be realized by the following technical scheme:

a biocontrol strain PNC25 for preventing and treating litchi phytophthora blight is Microbacterium acetylicum (Exiguobacterium acetylicum) which is preserved in Guangdong province microorganism culture collection center in 2016, 11 and 15 days, and the strain preservation number is GDMCC No. 60108.

The invention relates to an application of a biocontrol strain PNC25 with a deposition number of GDMCC No.60108 in preparation of a medicament for preventing and treating litchi frost blight.

A biocontrol microbial inoculum for preventing and treating litchi phytophthora blight takes the tiny acetyl bacillus PNC25 with the preservation number of GDMCC No.60108 as an effective component, wherein the concentration of viable bacteria of the tiny acetyl bacillus PNC25 is 1 multiplied by 10⁸～10⁹CFU/mL。

The preparation method of the biocontrol microbial inoculum is prepared by carrying out liquid fermentation on tiny acetyl bacillus PNC25, and specifically comprises the following steps: and (3) inoculating the acetyl micro-bacterium PNC25 into an LB culture solution for culture to obtain the biocontrol microbial inoculum.

Wherein, the pH value of the LB culture solution is preferably 7.0-7.2.

The preferable culture is 28-30 ℃, and the shaking rate of a shaking table is 180-200 rpm for 24 hours.

The invention relates to application of a biocontrol strain PNC25 with a deposition number of GDMCC No.60108 in preventing and treating litchi phytophthora blight.

The biocontrol microbial inoculum is applied to preventing and treating litchi frost blight.

The biocontrol microbial inoculum can be sprayed for use when 65-75% of field litchis are mature or sprayed for use in the process of room-temperature storage after picking when preventing and treating the litchi frost blight.

The invention relates to application of a microbacterium acetobacter PNC25 with a deposition number of GDMCC No.60108 in litchi preservation.

The biocontrol microbial inoculum is applied to litchi preservation.

The invention has the beneficial effects that:

the obtained acetobacter xylinum is derived from litchi endophytic bacteria and can be well colonized on the surfaces of litchi fruits; in addition, the endophyte screened from the litchi is used for preventing and treating litchi diseases, the endophyte is expressed by the increase or reduction of the number of certain microorganisms among specific populations, the category of the microorganism population on the litchi is not changed, so that the stability of the populations is influenced, in addition, as the endophyte coexists with the litchi for a long time and is mutually beneficial and symbiotic, a series of survival mechanisms similar to the endophyte are formed, and the endophyte is sprayed on the surfaces of litchi fruits for preventing and treating the diseases, so that the edible litchi fruits and processed products thereof are safer and more reliable.

The acetobacter xylinum has an inhibiting effect on peronophythora litchi, has an excellent biocontrol effect on the peronophythora litchi under a plurality of experimental conditions, and is a biocontrol preparation developed specially for the peronophythora litchi. Because the litchi chinensis benth is a biological preparation, a series of problems caused by the use of chemical pesticides are completely avoided, so that the litchi chinensis benth is beneficial to pollution-free production of litchi, farmers can not use or reduce the use amount of other chemical pesticides, the cost of the farmers can be saved, and the litchi chinensis benth is beneficial to fruit export. Meanwhile, the acetobacter xylinum has a good fresh-keeping effect on litchi, and can be used for preparing litchi fresh-keeping preparations.

The obtained acetobacter xylinum has low requirement on culture conditions and good development and application prospects.

Biological sample preservation information

The biocontrol strain PNC25 is classified as Exiguobacterium acetylicum, is preserved in Guangdong province microbial strain preservation center in 2016, 11 and 15 days, is preserved in Guangzhou, Michelia Tokyo No. 100, No. 59, and is preserved in Guangdong province microbial research institute, and the strain preservation number is GDMCC No. 60108.

Detailed Description

Example 1

The strain source is as follows: PNC25 was obtained by separating the "Yuannuo" pulp from Whilae certain litchi orchard, uncovering Yang, Guangdong province.

The collected samples were quickly filled into numbered plastic bags and taken back to the laboratory for separation. Shearing pulp 3g, soaking in 1mL of 1% sodium hypochlorite for 5min, soaking in 70% ethanol for 2min, and washing with sterile water for 3 times. (0.1 mL of the last wash solution was taken and R2A was applied, or 0.1mL of the last wash solution was taken and R was added₂And (3) performing shaking culture on the culture solution A at 30 ℃, and considering that the surface is disinfected if the culture solution grows aseptically after 48 hours). Respectively grinding the surface sterilized pulp with sterilized grinding bowel, adding 3mL sterile water, filtering with sterile cotton cloth, diluting with sterile water 10 times, sequentially diluting with 0.1mL, respectively taking 10^-4、10^-5、10^-6Three gradients 0.1mL R₂Plates A, cultured at 28 ℃ for 48h, plates with colony counts between 50-300 were selected, counted and all colonies picked, purified, stored at-70 ℃ in 40% glycerol (Berg et al, 2000).

Plate antagonism activity assay employs antagonism culture method, and Phytophthora bloom preserved at 20 deg.C is inoculated onto Carrot (CA) plate for activation, and after the plate is overgrown with fungus, the fungus is uniformly beaten into round fungus block with diameter of 5mm from the outer edge of colony by sterilized puncher. The hypha blocks were inoculated in the center of a CA-PDA medium mixed at a volume of 1:1, and a strain of bacteria was inoculated at a distance of about 27mm from the center at the periphery thereof, in four replicates. Culturing at 25 deg.C for 5 days, and recording the size of antibacterial band after the control hypha grows over the plate.

Determination of the Activity of strains hydrolases and metabolites protease (Yang et al, 2008), chitinase (Roberts and Selitrennikoff, 1988), beta-1, 3-glucanase (Yang 2011), cellulase (Ghose T.K 1987), siderophin activity (Shin et al, 2001), Indoleacetic activity (Sawar and Kremer 1992) were carried out according to the methods of the above-mentioned references. The results show that the strain produces protease and beta-1, 3-glucanase and siderophore, and has anti-fudge-phytophthora-robusta-microplate antagonist activity (Table 1).

TABLE 1 analysis of basic characteristics of the strains

The strain PNC25 is cultured on LB solid medium 28du for 24-48h, and the colony is round, medium in size, flat, opaque, yellow, viscous, irregular in edge and smooth. The gram stain shows bluish purple, the shape is short rod-shaped, the thallus is approximately spherical after being cultured for 48 hours, and the cells have periphytic flagella; the spore is red in color, is a vegetative somatic cell, and no obvious colored green spore is formed. Catalase is positive, and can decompose starch and liquefy gelatin.

Sequencing of the 16S r DNA gene fragment identified this strain as Exiguobacterium acetylicum (Table 2).

TABLE 2 sequencing alignment results

Example 2 preparation of microbial inoculum

(1) Culturing seed bacterial liquid: inoculating PNC25(GDMCC No.60108) into LB culture solution, carrying out shaking culture at 28 ℃ and 200rpm for 24h, sampling and measuring OD values at 600nm positions every 3h in an ultra-clean workbench after 16h, and zeroing with a culture solution without inoculation in the measuring process until the OD values of seed bacterial solutions are all between 0.5 and 0.8, thus obtaining the seed bacterial solution;

(2) preparing wet bacteria: mixing seed bacterial liquid in a ratio of 1: adding 500 volume ratio into LB culture solution, shaking culturing at 28 deg.C and 200rpm for 24 hr to obtain biological control bacteria with viable bacteria concentration of 1 × 10⁸～10⁹CFU/mL。

Example 3 evaluation of indoor (LT) disease prevention effect of biocontrol bacteria PNC25 on litchi fruits/leaves

The method comprises the following steps of (1) carrying out the following treatment on healthy litchi picked in the field: A. diluting the biocontrol bacteria stock solution to a final concentration of 5 x 10⁷CFU/mL, spray fruit/dilute to final concentration 1 x 10⁷CFU/mL spray blade; B. LB solution with the same dilution factor. Every treatment is repeated for 3 times, every fruit is repeated for 30 fruits/5 branches, and the fruits/branches are placed in a preservation box (the box bottom is paved with sterilized filter paper, the diameter is 18cm, the sterilization box is wetted by sterilized water, the specification of the preservation box is 323 x 220 x 100mm, and the manufacturer is Tan plastics factory in Changchong city of Buddha mountain city). The inoculum and control groups were sprayed for a total of 300mL per treatment (3 replicates). 24hpt (waters post treatment) spray inoculation of pathogen P. litchii (5 x 10)⁴sporangia/mL). And (5) investigating disease indexes and calculating the biocontrol effect.

Disease investigation method (cai chu qing, 2010): level 0: no disease symptoms; level 1: the inoculation spot has slight lesion spots, and the necrotic area is less than 5%; and 3, level: the necrotic area is 5% -10%; and 5, stage: the necrotic area is 10% -25%; and 7, stage: the necrotic area is 25% -50%; and 9, stage: necrotic areas exceed 50% or total white mold (fruit) or browning (leaf). The statistical calculation method of the disease index and the biocontrol effect comprises the following steps: disease index (%) [ Σ (number of disease stages × number of fruits of the disease stage)/(highest number of fruits of the disease stage × total number) ] × 100; biocontrol effect (%) [ (control disease index-treatment group disease index)/control disease index ] × 100.

The content of the experimental control effect analysis and determination comprises the following steps: LT2016-1, the material is from healthy 'Huai branch' fruits in a certain orchard of Guangzhou Huadu, the maturity is about 85%, and the control effect is observed by pathogen spray inoculation after the fruits are picked to a laboratory and are subjected to PNC25 spray treatment for 24 hours; LT2016-2, the material is from diseased 'Huai Branch' fruits in a certain Guangzhou Huadu orchard, the maturity is about 85%, and the control effect is observed by pathogenic bacteria spray inoculation after the fruits are picked to a laboratory and are subjected to PNC25 spray treatment for 24 hours; LT2016-3A/3B, material from the "sweet osmanthus flavor" and "Huai branch" of healthy leaves in certain Guangzhou Huadu orchard, adopt the laboratory to carry on PNC25 spray treatment 24h, the pathogenic bacterium sprays and inoculates and observes the control effect; LT2017-1, the material is from healthy Feizixiao fruits in a certain orchard in Hainan delirium, the maturity is about 80%, and the control effect is observed by pathogenic bacteria spray inoculation after the fruits are picked into a laboratory and are subjected to PNC25 spray treatment for 24 hours; LT2017-2, the material is from healthy Feizixiao fruits in Guangzhou Huadu orchards, the maturity is about 85%, and the control effect is observed by pathogenic bacteria spray inoculation after the fruits are picked to a laboratory and are subjected to PNC25 spray treatment for 24 hours.

The experimental data show that PNC25 shows better control effect on litchi fruits in two-year experimental tests, the average control effect in LT2016-1 and LT2017-1/2 is respectively 29.05%, 44.56% and 43.54% (table 3), and compared with a control group, the time points of the disease index and the time points of the control group reaching significant difference are respectively 1, 4 and 5 (table 3).

TABLE 3 analysis of control effect of biocontrol bacteria PNC25 on indoor fruit of frostbite of Litchi (LDB) (LT 2016-1, LT2017-1, LT2017-2)

^yLt (laboratory tertiary), laboratory experiments; flt (field plus lab Trial), field-laboratory experiments; FT, (field trial), field trial; hpi (hours post inoculation), hours after inoculation with a pathogen.

^x1Biocontrol bacteria fermentation diluent (5X 10)⁷CFU/mL) spray pretreatment of litchi fruits Huaizhi' (85% maturity, harvested in the field to a laboratory preservation box), room temperature culture (25 ℃),24 hpt (waters post treatment) spray inoculation of pathogenic bacteria P. litchii (5 × 10)⁴sporangium/mL)。

^x2Biocontrol bacteria fermentation diluent (5X 10)⁷CFU/mL) sprayPretreatment of litchi fruits ` Feizixiao ` (80% maturity, delirium, Hainan) as Fruit Sprays (FS) at room temperature (25 ℃),24 hpt (waters post treatments) spray inoculation of pathogen P⁴sporangium/mL)。

^x3Biocontrol bacteria fermentation diluent (5X 10)⁷CFU/mL) spray pretreated litchi fruits (85% maturity, Guangdong) as Fruit Sprays (FS) were incubated at room temperature (25 ℃),24 hpt (waters post treatment) spray inoculated with pathogen P. litchii (5X 10)⁴sporangium/mL)。

^ZAE, average efficacy, indicates average control efficacy. Data are the average of three replicates, and the english letters after the data indicate significant variability at the 95% level between treatments (Duncan' test).

In the LT2016-2 experiment, the data show that PNC25 still shows control effect on litchi fruits, the average control effect is 23.32%, (table 4), and compared with a control group, the time points of the disease index and the control group reaching significant difference are respectively 2 (table 4).

Table 4 fruit control analysis of natural infection p.litchii by biocontrol bacterium PNC25 (LT2016-2)

^xLitchii infests litchi fruits under natural conditions, and white mildew layers are found on 12-24hpt fruit surfaces after the litchi fruits are picked up in a laboratory; fruits 'Huaizhi' (85% maturity) were pretreated by spraying biocontrol bacteria liquid diluent (5X 107 CFU/mL). Data are the average of three replicates, and the english letters after the data indicate significant variability at the 95% level between treatments (Duncan' test).

^ZAE, mean control.

In the LT2016-3A/3B experiment, the average control effects of PNC25 on the osmanthus flavor and the Huai branch of litchi leaves are respectively 20.81 and 24.17 percent (table 5), and compared with a control group, the time points of the disease indexes and the time points of the control group reaching the significant differences are respectively 2 and 1 (table 5).

TABLE 5 analysis of LDB controlling effect of litchi leaves by strain PNC25 (LT2016-3)

^xBiocontrol bacteria liquid diluent (1X 10)⁷CFU/mL) spraying the litchi leaves in the preservation box in the pretreatment chamber; 24hpt spray inoculation of pathogen P. litchii (5X 10)⁴sporangium/mL.) data are averages of three replicates, with english letters after the data indicating significant variability at the 95% level between treatments (Duncan' test).

^ZAE, mean control.

Example 4 determination of the Effect of biocontrol bacteria on litchi fruit control under field-indoor (FLT) conditions

The experimental design is as follows: in Guangzhou of 2016, fruits of glutinous rice cake with 75% maturity in the field are picked up and placed in a fresh-keeping box in a laboratory after being pretreated by biocontrol bacteria spray for 3 days, and are inoculated with pathogenic bacteria P. In 2017, fruits of glutinous rice cakes with the maturity of 65% in the field are picked up in a laboratory after being pretreated by biocontrol bacteria spray for 7 days and placed in a preservation box, and the fruits are inoculated with pathogenic bacteria P.litchii for 24 hours, wherein the treatment is repeated for 3 times and 30 fruits are repeated for each time. 36hpi began to observe disease severity.

The experimental contents of the experiment are as follows: FLT2016, prepared by spray pretreatment of healthy glutinous rice fruits (ripeness of about 75%) from Guangzhou Huadu orchard, harvested 3 days later (ripeness of about 85%) and inoculated with pathogenic bacteria spray in laboratory for observation of control; FLT2017-1, the material is from healthy Feizixiao fruits (maturity of about 65%) in certain orchard in Hainan delirium, the fruits are pretreated by spraying, and after 7 days, the fruits are picked (maturity of about 85%) and are subjected to pathogen spraying inoculation in a laboratory to observe the prevention effect.

In the FLT2016and FLT2017-1 experiments, the average control effect of PNC25 on litchi fruits is 55.67% and 66.32% respectively (Table 6), and compared with a control group, the time points of the disease index and the control group reaching significant difference are 2 and 2 respectively (Table 6).

TABLE 6 analysis of LDB control of litchi fruits by strains (FLT2016and FLT-2017-1)

^x1Biocontrol bacteria liquid diluent (5X 10)⁷CFU/mL) spray pretreatment of field litchi fruits "Nuomici" (75% maturity, guangdong); picking litchi fruits at 3dpt, placing the litchi fruits in a laboratory, and placing the litchi fruits in a preservation box (with the maturity of about 85 percent); spraying after 24h to inoculate pathogen P. litchii (5X 10)⁴sporangium/mL).

^X2Biocontrol bacteria liquid diluent (5X 10)⁷CFU/mL) spray pretreatment of field litchi fruits "Feizixiao" (65% maturity, hainan delirium); 7dpt, picking the litchi fruits, placing the litchi fruits in a fresh-keeping box (about 85 percent maturity) in a laboratory, and spraying and inoculating pathogenic bacteria P after 24 hours⁴sporangium/mL).

Example 5 evaluation of Field (FT) control Effect of biocontrol bacteria on litchi fruits

The 2016-year field control variety is 'Huai Zhi' (about 75% mature lichee) of a certain farmer in Guangzhou Huadu area, the biocontrol bacteria is pretreated by field spraying for 3d (about 85% maturity), then the pathogenic bacteria are inoculated by spraying, and after the fruit becomes white mold, the disease index is observed once a day.

In the FT2016 experiment, the average control effect of PNC25 on litchi fruits was 46.73% (table 7), and the time points for significant differences between disease index and control group compared to control group were 4 (table 7). The strain has better control effect under field conditions.

TABLE 7 analysis of LDB field control of litchi fruits by strains (FT2016)

^XBiocontrol bacteria liquid diluent (5X 10)⁷CFU/mL) spray pretreatment fieldLitchi fruit Huaizhi "(75% maturity, guangdong, guangzhou); picking fruits at 3dpt, placing the fruits in a laboratory in a preservation box (about 85% maturity), and spraying and inoculating pathogenic bacteria P after 24h (5 multiplied by 10)⁴sporangium/mL.) data are averages of three replicates, with english letters after the data indicating significant variability at the 95% level between treatments (Duncan' test).

Example 6 evaluation of the fresh-keeping Effect of biocontrol bacteria PNC25 on litchi fruits

Healthy fruits picked in the field are treated as follows: A. diluting the biocontrol bacteria stock solution to a final concentration of 5 x 10⁷Spraying the fruits in a CFU/mL mode; B. LB solution with the same dilution factor. Every treatment is repeated for 3 times, every treatment is repeated for 30 fruits, and the fruits are placed in a preservation box (the box bottom is paved with sterilized filter paper, the diameter is 18cm, the sterilized water is used for wetting, the specification of the preservation box is 323 x 220 x 100mm, and the manufacturer is a Hualong plastic plant in Changchan city, Buddha mountain city). The inoculum and control groups were sprayed at 300mL per treatment (3 replicates).

The fruit picking batches were the same as those of example 3(LT2017-3/4) and example 4(FLT2017-2), and were picked into laboratories for corresponding treatment without inoculation of pathogenic bacteria. And observing the browning condition of the fruits. The determination of the browning index refers to a Jiangxing method and the like (2000), and the determination of the browning grade is as follows: and (3) fruit grade I: all red, grade 3 fruit: slight browning of the pericarp, grade 5 fruit: the browning area of the peel is more than or equal to 5% and less than 25%, and the browning area of the 7-grade fruit is as follows: the browning area of the peel is more than or equal to 25% and less than 50%, and the browning area of the 9-grade fruit is as follows: the browning area of the peel is more than 50 percent. Browning index ═ Σ (number of browning stages × number of fruits)/(number of highest stage × total fruits); the fresh-keeping effect (%) [ (control browning index-treatment group browning index)/control browning index ] × 100.

The present embodiment determines the following three: LT2017-3, which is prepared from healthy Feizixiao fruits (the maturity is about 80%) in an orchard of Hainan delirium, and is picked up in a laboratory for fresh-keeping effect observation; LT2017-4, the material is from healthy Feizixiao fruits (the maturity is about 85%) in Guangzhou Huadu orchard, and the fruits are picked to a laboratory for fresh-keeping effect observation; FLT2017-2, the material is from healthy 'Feizixiao' fruits (maturity is about 65%) in certain orchard in Hainan delirium, the fruits are pretreated by spraying, and the fruits are picked after 7 days (maturity is about 85%) to a laboratory for fresh-keeping observation.

The experimental data show that PNC25 has a good fresh-keeping effect on litchi fruits in two-year experimental tests, the average control effects in LT2017-3, LT2017-4 and FLT2017-2 are respectively 21.95%, 44.60% and 68.98% (Table 8), and compared with a control group, the time points when the disease indexes and the control group reach significant differences are respectively 2, 1 and 1 (Table 8).

Table 8: analysis of litchi fruit fresh-keeping effect of the Strain (LT2017-3, LT2017-4, and FLT2017-2)

^y LT,laboratory trial；hpt,hours post treatment.

^x1Biocontrol bacteria liquid diluent (5X 10)⁷CFU/mL) indoor spray pretreatment of litchi fruit 'Feizixiao' (approximately 80% maturity, delirium in Hainan); but not inoculated with the pathogen p.

^x2Biocontrol bacteria liquid diluent (5X 10)⁷CFU/mL) indoor spray pretreatment of litchi fruits Feizixiao' (85% maturity, guangdong); but not inoculated with the pathogen p.

^X3Biocontrol bacteria liquid diluent (5X 10)⁷CFU/mL) field pretreatment of litchi fruit 'Feizixiao' (65% maturity, hainan delirium); 7dpt, fruits (approx. 85% maturity) were picked to the laboratory and placed in crisps without inoculation of pathogen P.

^ZData are averages of three replicates, and the english letters after the data indicate significant variability at the 95% level between treatments (Duncan' test).

Example 7 analysis of the Effect of biocontrol bacteria PNC25 on fruit quality

PNC25 spray pretreatment field litchi fruit (Hainan Feizixiao and Guangzhou Huai)Branches ", about 65% maturity), litchi fruits (about 90% maturity) were picked at 18d to the laboratory and tested for: soluble protein Pr (μ g/g), soluble sugar content (μ g/g), free amino acids (mg/g), vitamin C (μ g/g). Randomly sampling litchi fruits in each treatment group, wherein each group is repeated for three times, and each group is repeated for 5 fruits; according to

And Hanyasan (1992).

The analysis data shows that the quality of the fruit with delirium "Feizixiao" treated by PNC25 is respectively that the content of free amino acid (7.02mg/g) is higher than that of the control (5.46mg/g), the content of soluble protein (25.43 mu g/g) is higher than that of the control (5.98 mu g/g), and the content of vitamin c (18.02 mu g/g) is higher than that of the control (16.35 mu g/g) (Table 9). The quality of Guangzhou 'Huai branch' fruits shows similar rules, and the content of soluble protein (15.83 mu g/g) of the fruits treated by PNC25 is higher than that of a control (28.01 mu g/g); the vitamin c content (9.99. mu.g/g) was higher than the control (8.58. mu.g/g) (Table 9). The PNC25 treated litchi fruits 18d have no influence on the fruit quality, and the PNC25 sprayed pretreated fruits are safe and reliable.

Table 9: quality determination of litchi fruits by biocontrol bacteria PNC25

Claims

1. A biocontrol strain PNC25 for preventing and treating downy mildew of litchi is Exiguobacterium acetylicum (Exiguobacterium acetylicum) which is preserved in Guangdong province microorganism strain collection center in 2016, 11 and 15 days, and the strain preservation number is GDMCC number 60108.

2. The biocontrol strain PNC25 as claimed in claim 1, for use in the preparation of a medicament for the prevention and treatment of downy mildew of litchi.

3. A biocontrol microbial inoculum for preventing and treating litchi downy mildew is characterized in that: microbacterium acetobacter with deposition number GDMCC number 60108PNC25 is effective component with viable bacteria concentration of 1 × 10⁸～10⁹CFU/ml。

4. The method for preparing the biocontrol microbial inoculum of claim 3, which is characterized by comprising the following steps: prepared by carrying out liquid fermentation on Acetobacter xylinum PNC25, and specifically comprises the following steps: and (3) inoculating the acetyl micro-bacterium PNC25 into an LB culture solution for culture to obtain the biocontrol microbial inoculum.

5. The method of claim 4, wherein: the pH value of the LB culture solution is 7.0-7.2.

6. The method of claim 4, wherein: the culture refers to culturing for 24 hours at the temperature of 28-30 ℃ and the shaking speed of a shaking table of 180-200 rpm.

7. The biocontrol strain PNC25 as claimed in claim 1, for use in controlling downy mildew of litchi.

8. The application of the biocontrol microbial inoculum of claim 3 in preventing and treating peronophythora litchi.