CN109652320B

CN109652320B - Biocontrol DSE strain LS1 and application thereof in banana wilt

Info

Publication number: CN109652320B
Application number: CN201811571220.4A
Authority: CN
Inventors: 张雯龙; 农倩; 谢玲; 苏琴; 陈艳露; 张艳
Original assignee: Guangxi Zhuang Nationality Autonomous Region Academy of Agricultural Sciences
Current assignee: Guangxi Zhuang Nationality Autonomous Region Academy of Agricultural Sciences
Priority date: 2018-12-21
Filing date: 2018-12-21
Publication date: 2021-11-16
Anticipated expiration: 2038-12-21
Also published as: CN109652320A

Abstract

The invention provides a biocontrol DSE strain LS1 and application thereof in banana wilt, wherein the preservation number of the strain is CGMCC NO.16498, and the preservation start time is 11 months and 8 days in 2018. The biocontrol DSE strain LS1 is screened through an indoor plate and pot experiment, the plate control effect is as high as 86.19%, and the pot control effect is 63.19%. The prevention performance of the strain in the plate and pot experiment is more stable.

Description

Biocontrol DSE strain LS1 and application thereof in banana wilt

Technical Field

The invention relates to the field of microorganisms, in particular to a biocontrol effect of a DSE strain Cladosporium chlorocephalum LS1 on banana vascular wilt.

Background

Bananas are important economic crops in subtropical regions in south China. The planting area of the Guangxi bananas in 2013 is about 9 million hectares, the yield is 275.2 million tons, and the yield is doubled compared with the yield in 2005. However, due to the large-scale continuous cropping of bananas, the soil-borne disease banana wilt (Fusarium oxysporum f.sp. cubense) is frequently damaged. The disease can cause withering of diseased plants and vascular bundle necrosis, the morbidity of a seriously ill field is up to 90 percent, and the development of Guangxi banana industry is seriously restricted. The economic and environment-friendly biological control technology is gradually the research focus of controlling the disease under the restriction of control means such as poor yield of disease-resistant varieties and unsatisfactory control of chemical agents.

Currently, Dark-color Septate endophytic fungi (DSE) are soil ascomycetes or fungi imperfecti which have Dark hyphae and can form microsclerotia, colonize in plant root tissue cells or in intercellular spaces and are not pathogenic to hosts. Many researches show that DSE can be symbiotic with a host in a reciprocal way, plays a positive ecological function in the aspects of stress resistance, growth promotion, disease prevention and the like, has wide colonization and nonspecific host, and leads the plant-DSE symbiont to be taken as an expression form of the symbiotic relationship between another plant and a microorganism after the leguminous plant-rhizobium symbiont and the mycorrhiza symbiont and to be gradually paid attention in the field of mycorrhiza research. In 2017, Zhang Xiao Rong and the like remarkably relieve the symptom that tomatoes are inhibited from growing by fusarium oxysporum by inoculating DSE fungi Phialophora musta K36 and Z48; a DSE strain L-14 (Schizothecium sp.) with strong biocontrol capability on banana wilt is obtained by screening of Naringin and the like. In 2018, Surono et al found that inoculation of DSE fungus Dialocepahala fortinii CKG.I.11 on asparagus can promote the growth of asparagus and has an inhibitory effect on wilt.

Disclosure of Invention

In previous researches, the applicant has found DSE strains with excellent biocontrol effect on banana vascular wilt, but the number of the DSE strains is still very limited, and the screening strength of the excellent strains is still required to be increased for the development of the compound microbial inoculum in the future. Therefore, the invention aims to measure the biocontrol effect of other DSE strains stored in the laboratory and classify and identify the strains. The invention aims to screen and obtain the high-efficiency and stable bio-control strain for the banana vascular wilt, and provides more abundant strain resources for the development of bio-control inoculants for the banana vascular wilt.

An endophytic fungus LS1 for preventing and treating banana wilt has a preservation number of CGMCC NO.16498 and a preservation time of 11 months and 8 days in 2018. The preservation unit: CGMCC-China general microbiological culture Collection center, classification and naming: cladosporium chlorocephalum; and (4) storage address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.

The invention provides a biocontrol DSE strain LS1 and application thereof in banana vascular wilt, wherein the application method comprises the following steps:

step A, strain inoculation and influence on banana growth, wherein the strain to be tested is inoculated on an oat culture medium after being activated, banana tissue culture seedlings with consistent growth vigor are selected and transplanted to bacterial colonies, root systems are laid on the surfaces of the bacterial colonies, each bacterial colony is transplanted to one strain, a culture dish is placed into a tissue culture bottle, and the culture dish is placed into an incubator for culture; measuring the plant height, stem width, fresh weight and dry weight of the banana; each treated part of fresh banana root sample is used for the colonization observation and re-separation of DSE, the sample mass of the reserved part is recorded, the rest part is dried to constant weight by an oven after weighing the fresh weight, the dry weight is measured, the average water content is calculated, and the dry weight is calculated together after converting the previously reserved fresh root sample;

b, re-separating and colonizing the strain, namely taking a proper amount of fresh root samples, cutting into small sections, placing the small sections into a test tube, adding a KOH solution to completely immerse the root sections, dissociating the root sections in a constant-temperature water bath kettle to soften and transparent the root sections, dyeing the root sections with ink vinegar, rinsing the dyed root sections with clear water, soaking and decoloring the root sections, observing the colonizing condition of the DSE on the roots of the bananas, and calculating the colonizing rate (the colonizing rate is equal to (the number of colonized root sections/the total number of the root sections examined by the microscope) × 100%);

step C, measuring the control effect of the strain on the banana vascular wilt, namely transferring the DSE-banana symbiont (together with an oat culture medium) to a water agar culture medium with fusarium, and directly inoculating a control banana tissue culture seedling without inoculation to a water agar plate with fusarium; observing and recording the disease level, calculating the disease index and preventing and treating effects;

and D, carrying out classification and identification on the biocontrol strains, namely observing the colony characteristics of the strains on the PDA culture medium, enabling hyphae to grow on a cover glass by a cover glass inclined insertion method, and observing the hyphae form on the cover glass under an optical microscope.

Preferably, in the step a, the oat culture medium adopts: oat flour 10 g.L^-1Agar 18 g. L^-1，MgSO₄·7H₂O 1g·L^-1，KH₂PO₄ 1.5g·L^-1，NaNO₃ 1g·L^-13 clumps per dish were incubated at 28 ℃ for 10 days.

Preferably, in the step A, the tissue culture bottle is cultured at 28 ℃ and the light intensity is 180 mu mol.m^-2·s^-2And culturing for 40d in an incubator with a photoperiod of 16h to 8 h.

Preferably, in the step B, KOH solution with the mass concentration of 10% is added to completely immerse the root segment, the root segment is dissociated for 15-20min in a water bath kettle at the constant temperature of 90 ℃ to soften and transparent the root segment, the root segment is dyed by ink vinegar (volume ratio: ink 5: rice vinegar 95), and then the root segment is rinsed for 3 times by clear water and soaked for 12 hours for decolorization.

Preferably, in the step C, the disease grading standard is as follows: grade 0-no visible symptoms in appearance; grade 1-no obvious resistance to plant growth, and whole plant verticillium area less than or equal to 35%; 2 level-weak plant, the whole plant verticillium area is less than or equal to 80 percent more than 35 percent; grade 3-the whole plant verticillium area is more than 80%.

Preferably, in the step C, the banana seedlings with the size of 4 leaf stage are taken for the pot biological control test; preparation of 5X 10⁵cfu mL^-1The DSE bacterial liquid is treated by root irrigation. And (3) irrigating 50mL of bacterial liquid into roots of each banana seedling, and irrigating once every 15 days for three times. After the last root irrigation is finished for 10-15 days, the banana seedlings are subjected to root injury and fusarium spore liquid (1 multiplied by 10)⁶cfu mL^-1)。

Preferably, the Control (CK) is a treatment in which roots are drenched with clear water and inoculated with pathogenic bacteria. And (5) dissecting the banana corms 25 days after the inoculation of pathogenic bacteria, observing and recording the morbidity and calculating the control effect. Each treatment was repeated 3 times, 8 seedlings per repeat. Grading the disease condition standard: grade 0-healthy bulb without discoloration; level 1: the color change area of the corm is less than 20 percent; and 2, stage: the color change area of the bulb is more than or equal to 20 percent and less than 40 percent of the area of the bulb; and 3, level: the color change area of the bulb is more than or equal to 40 percent and less than 60 percent of the area of the bulb; 4, level: the color changing area of the bulb is more than or equal to 60 percent and less than 80 percent of the area of the bulb; and 5, stage: the color change area of the bulb accounts for more than or equal to 80 percent of the area of the bulb.

Calculating the formula: disease index ═ Σ (number of disease plants × representative value)/(total number of plants × highest disease-level representative value); the preventing and treating effect (%) is [ (control disease index-treating disease index) × 100 ]/control disease index

Preferably, in the step D, when inducing spore production, the culture medium is placed at 4 ℃ for 2-4 weeks.

Preferably, in the step D, the total DNA of the mycelium is extracted by using an OMEGA fungal DNA extraction kit, and the amplification primers are ITS 1: 5'-TCCGTAGGTGAACCTGCGG-3' and ITS 4: 5'-TCCTCCGCTTATTGATATGC-3' are provided. Amplification system 50. mu.l, containing: MasterMix (TIAngel) 25. mu.l, 20. mu. mol L-1 forward and reverse primers 1. mu.l each, DNA 1. mu.l, ddH2O to volume. And (3) amplification procedure: pre-denaturation at 98 ℃ for 2 min; denaturation at 94 ℃ for 40s, annealing at 50 ℃ for 1min, extension at 68 ℃ for 4min, 30 cycles; finally, extension is carried out for 10min at 72 ℃.

For a long time, the great difference of the control effects of the indoor bacteriostasis test and the field control test is a big problem of the effective application of the biological control technology. In view of the characteristic that DSE in the 'DSE-plant' symbiont is colonized in the interior of plant roots but not on the surface, the DSE is applied to the control of soil-borne disease banana vascular wilt so as to reduce the influence of other soil microorganisms on biocontrol strains and further reduce the instability of control effect.

The biocontrol DSE strain LS1 is screened through an indoor plate and pot experiment, the plate control effect is as high as 86.19%, and the pot control effect is 63.19%. The prevention effect of the strain in the plate and pot experiment is relatively stable, and further experiments prove how the field application prevention effect is needed. The other 4 DSE strains in the experiment can also show a certain biological control effect on the bananas, but the control effect of the pot experiment is not ideal.

Drawings

FIG. 1 is a morphometric identification of strain LS1 of the present invention. Wherein A is colony morphology on PDA medium; b is the produced sporophores and conidia; c is conidium.

FIG. 2 shows the hyphal and microsclerotia structure of DSE in banana root, wherein the arrow indicates microsclerotia.

FIG. 3 is a graph comparing the colonization rates of different DSEs of the present invention.

FIG. 4 biocontrol effects of different DSE strains on banana vascular wilt.

FIG. 5 shows an ITS fragment-based phylogenetic tree of the strain LS1 of the present invention and related strains. Detailed Description

Preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings:

example 1

Isolation of strain LS1 the materials and methods required were as follows:

1. material

Collecting soil samples around the roots of the suburb sugarcane field in the Nanning city, and storing the soil samples at 4 ℃ for later use.

Selecting eggplant seeds, wherein the method for disinfecting the surfaces of the seeds comprises the following steps: 20 seconds of 75 percent alcohol by volume concentration → 40 seconds of sodium hypochlorite by mass concentration of 1 percent for shaking and disinfection → three times of shaking and rinsing by sterile water → absorbing dry water by sterile filter paper → placing on a pure agar plate for culturing for 2 to 3 days, and sprouting and growing into sterile seedlings.

2. Isolation of Strain LS1

Transplanting the obtained aseptic eggplant seedlings into a mixed culture medium (the weight ratio of a soil sample to sterilized seedling culture soil is 2:1), planting for 50-60 days, washing the soil medium attached to eggplant roots with tap water, performing shake disinfection for 3 times by adopting Tween-20 with the mass concentration of 0.5% for 1 minute each time, performing shake washing for 3 times by using sterilized water, draining water, placing on a culture medium with 1/2 corn flour in weight concentration, culturing in an incubator at 28 ℃ and observing day by day, and timely transferring to a potato dextrose culture medium (PDA) after hyphae grow out from root sections to obtain pure culture strains.

And (3) classification and identification of biocontrol strains:

1. morphological Observation of Strain LS1

The strain is activated on a potato dextrose medium (PDA), and the colony morphology characteristics are observed after 14 days of culture. Inoculating the purified strain to oat culture medium by inserting method, culturing at 28 deg.C for 2 weeks, inducing to generate conidium in refrigerator at 4 deg.C for 4-6 weeks when mycelium is obviously observed on the cover glass, taking out the glass slide, and observing under optical microscope.

2. Molecular characterization of Strain LS1

Culturing endophytic fungus strain LS1 in potato glucose liquid culture medium (PDB), shake culturing in shaking table at 28 deg.C and rotation speed of 120r/min for 10-14d, filtering, and collecting mycelium. The total DNA of the mycelium is extracted by adopting an OMEGA fungus DNA extraction kit, and amplification primers are ITS 1: 5'-TCCGTAGGTGAACCTGCGG-3' and ITS 4: 5'-TCCTCCGCTTATTGATATGC-3' are provided. Amplification system 50. mu.l, containing: MasterMix (TIAngel) 25. mu.l, 20. mu. mol L-1 forward and reverse primers 1. mu.l each, DNA 1. mu.l, ddH2O to volume. And (3) amplification procedure: pre-denaturation at 98 ℃ for 2 min; denaturation at 94 ℃ for 40s, annealing at 50 ℃ for 1min, extension at 68 ℃ for 4min, 30 cycles; finally, extension is carried out for 10min at 72 ℃. Sequencing of the product was performed by Shanghai Bioengineering technology, Inc.

The sequence was analyzed using ClustalX 1.81 and BioEdit v7.0 software, the experimentally obtained ITS1/ITS4 sequence and ITS most similar sequence downloaded in GenBank, the Neighbor-Joining phylogenetic tree was constructed using MEGA6.06 software based on the Kimura two-parameter model, and the reliability of the phylogenetic tree was checked by bootstrap1000 cycles.

3. Morphological characterization of Strain LS1

Morphological identification: the growth rate of the strain LS1 on the PDA culture medium is moderate, after 2 weeks of culture at 28 ℃, the diameter of a bacterial colony is about 70mm, the bacterial colony is round, brown to brownish green, felted, flat in edge, provided with shallow radial grooves and dark brown in the back (figure 1A). Microscopic observation shows that LS1 has hypha branch with septa, yellow brown to brown, smooth, thick wall, and diameter of 1.4-4.7 μm. Conidiophores are vertically grown on the top or lateral branches of hyphae, and have no branching, brown color, smoothness, and septum, and size of 22.4-386.7 × 2.7-5.2 μm (FIG. 1B). The top and middle parts of the conidiophores expand to form spore-forming cells, and the expanded parts are 3.7-6.3 mu m. The branched conidia are mostly cylindrical and partially elliptical, most of the branched conidia are slightly contracted in the middle and have 0-2 partitions, on which conidia are grown, cladospores are grown or regenerated, and the size of the regenerated cladospores is 6.4-28.5 multiplied by 2.0-5.6 mu m. Conidiophore is round, elliptical, lemon or spindle-shaped, yellowish brown, without separation, with umbilicus at two ends or one end of partial conidiophore of 3.4-8.4 × 2.2-4.7 μm; the diameter of the near-circular spore is 2.4-7.6 μm (FIG. 1C). The above characteristics are substantially in accordance with the morphological characteristics of Cladosporium chlorocephalum described in the prior art.

4. Sequence analysis and phylogenetic analysis

An ITS fragment of the LS1 strain is amplified, purified and sequenced to obtain a target fragment of about 550bp, GenBank is submitted to obtain accession number MH376857, the sequence is subjected to Blastn analysis in NCBI, and the sequence similarity of LS1 and each isolate of Cladosporium is found to be more than 99 percent; a phylogenetic tree (FIG. 5) based on ITS sequences was constructed using sequences in Bensch K (students in Mycology,2015,82(82):23-74) and related sequences downloaded in GenBank, and LS1 was found to have one strand with C.chlorocephalum and C.tenuissimum, with a support rate of 89%, and with a strain LS1 having 100% similarity to the ITS sequences of C.chlorocephalum. And comprehensively analyzing the result, and identifying the strain LS1 as Cladosporium chlorocephalum.

Example 2

1. Materials and methods

1.1 Experimental materials

Test strains: obtained by being separated from the soil around the roots of sugarcane in 2016, and the serial numbers of the soil are LS1, LC8, B2, MS38 and TD1 respectively.

Testing banana tissue culture seedlings: gui Banana No. 6, supplied by Guangxi plant tissue culture seedling Co., Ltd.

Pathogenic bacteria to be tested: fusarium # 4 physiological race (FOC 4).

1.2 Experimental methods

1.2.1 Strain inoculation and its Effect on Banana growth

The DSE strain to be tested is activated and inoculated on an oat culture medium (10 g.L oat flour)^-1Agar 18 g. L^-1，MgSO₄·7H₂O 1g·L^-1，KH₂PO₄ 1.5g·L^-1，NaNO₃ 1g·L^-1) 3 pieces of bacteria are cultured in each dish at 28 ℃ for 10-14 days. Selecting banana tissue culture seedlings with consistent growth vigor, transplanting the banana tissue culture seedlings onto bacterial colonies, paving root systems on the surfaces of the bacterial colonies, transplanting one bacterial colony for each bacterial colony, placing a culture dish into a tissue culture bottle, and culturing at the temperature of 28-30 ℃ under the light intensity of 160-^-2·s^-2And culturing for about 40 days in an incubator with a photoperiod of 16h:8h, and taking the treatment without inoculating the DSE strain as a control, wherein each treatment is repeated for 3 times and each time is repeated for 10 dishes. The plant height, stem width, fresh weight and dry weight of bananas were determined. And (3) each treated part of fresh banana root samples are used for the colonization observation and re-separation of DSE, the sample mass of the reserved part is recorded, the rest part is weighed, dried in an oven at 70 ℃ for 70-75h to constant weight, the dry weight is measured, the average water content is calculated, and the dry weight is calculated together with the reserved fresh root samples after conversion.

1.2.2 Re-isolation and colonization detection of strains

Taking a proper amount of fresh root sample, washing the fresh root sample with tap water, cutting the fresh root sample into small sections of about 1cm, placing the small sections into a clean 50mL test tube, adding 10% KOH solution to completely immerse the root sections, dissociating the root sections in a constant-temperature water bath kettle at 90 ℃ for 15-20min to soften and transparent the root sections, dyeing the root sections with ink vinegar (the volume ratio of the ink to the ink is 5: rice vinegar 95), rinsing the root sections with clear water for 3 times, soaking the root sections for 10-14h for decolorization, observing the colonization condition of DSE on the roots of the bananas under an optical microscope, and calculating the colonization rate (the colonization rate is: (the number of the colonized root sections/the total number of the microscopic root sections). times.100%). And (5) re-separating the DSE.

1.2.3 determination of prevention and treatment effects of bacterial strains on banana vascular wilt

Plate and pot biocontrol experiments: the DSE-banana symbiont (together with oat culture medium) is transferred to a water agar medium with fusarium, and the control banana tissue culture seedlings without inoculation are directly inoculated to a water agar plate with fusarium. Each treatment was repeated for 10 dishes. Observing and recording the disease level, calculating the disease index and preventing and treating effect. Grading the disease condition standard: grade 0-no visible symptoms in appearance; grade 1-no obvious resistance to plant growth, and whole plant verticillium area less than or equal to 35%; 2 level-weak plant, the whole plant verticillium area is less than or equal to 80 percent more than 35 percent; grade 3-the whole plant verticillium area is more than 80%.

The pot biological control test is carried out by taking banana seedlings with the size of 4 leaves. Preparation of 5X 10⁵cfu mL^-1The DSE bacterial liquid is treated by root irrigation. And (3) irrigating 50mL of bacterial liquid into roots of each banana seedling, and irrigating once every 15 days for three times. After the last root irrigation is finished for 15 days, the banana seedlings are subjected to root injury and fusarium spore liquid (1 multiplied by 10)⁶cfu mL^-1). The roots were drenched with clear water and inoculated with pathogenic bacteria as Control (CK). And (5) dissecting the banana corms 25 days after the inoculation of pathogenic bacteria, observing and recording the morbidity and calculating the control effect. Each treatment was repeated 3 times, 8 seedlings per repeat. Grading the disease condition standard: grade 0-healthy bulb without discoloration; level 1: the color change area of the corm is less than 20 percent; and 2, stage: the color change area of the bulb is more than or equal to 20 percent and less than 40 percent of the area of the bulb; and 3, level: the color change area of the bulb is more than or equal to 40 percent and less than 60 percent of the area of the bulb; 4, level: the color changing area of the bulb is more than or equal to 60 percent and less than 80 percent of the area of the bulb; and 5, stage: the color change area of the bulb accounts for more than or equal to 80 percent of the area of the bulb.

Calculating the formula: disease index ═ Σ (number of disease plants × representative value)/(total number of plants × highest disease-level representative value); the preventing and treating effect (%) is [ (control disease index-treatment disease index) × 100 ]/control disease index.

1.2.4 Classification and identification of biocontrol strains

The morphological observation of DSE was performed by referring to the method of echinacea purpurea and the like, and the colony characteristics of the strain on PDA medium were observed, and hyphae were allowed to grow on a cover glass by the cover glass oblique insertion method, and the hyphae morphology on the cover glass was observed under an optical microscope. When inducing spore production, the culture medium is placed at 4 ℃ for 2-4 weeks.

The total DNA of the mycelium is extracted by adopting an OMEGA fungus DNA extraction kit, and amplification primers are ITS 1: 5'-TCCGTAGGTGAACCTGCGG-3' and ITS 4: 5'-TCCTCCGCTTATTGATATGC-3' are provided. Amplification system 50. mu.l, containing: MasterMix (TIAngel) 25. mu.l, 20. mu. mol L-1 forward and reverse primers 1. mu.l each, DNA 1. mu.l, ddH2O to volume. And (3) amplification procedure: pre-denaturation at 98 ℃ for 2 min; denaturation at 94 ℃ for 40s, annealing at 50 ℃ for 1min, extension at 68 ℃ for 4min, 30 cycles; finally, extension is carried out for 10min at 72 ℃. Sequencing of the product was performed by Shanghai Bioengineering technology, Inc. The obtained sequences were analyzed by alignment by NCBI Blastn and phylogenetic tree construction was performed using the MEGA6.0 program.

2. Results and analysis

2.1 Effect of the inoculum strains on Banana growth

After inoculation of the DSE strain for 40 days, it was observed that each DSE-inoculated banana plant grew better than the non-inoculated DSE. As can be seen from Table 1, the plant height, stem width, fresh weight and dry weight of banana plants can be effectively improved by inoculating DSE, the growth rates are respectively 4.49% -21.91%, 3.66% -30.37%, 18.42% -47.36% and 35.9% -42.40%, wherein the plant height, stem width, fresh weight and dry weight of the banana plants after being inoculated with the strain LS1 reach significant difference levels compared with a control, and the fresh weight and the dry weight are respectively increased by 47.36% and 42.40% compared with the control.

TABLE 1 Effect of different DSE strains on growth of tissue culture seedlings of bananas

2.2 colonization of DSE in Banana root systems

The banana tissue culture seedling root sample inoculated with the DSE strain for 40 days under the aseptic condition is taken as a material, and endophytic fungi with culture properties and morphological structures consistent with those of the inoculated DSE are separated from the root system, which shows that a DSE-banana symbiotic system is established after 40 days of inoculation. After the banana root sample is dissociated and dyed, hyphae with dark colors and transverse septa are found through microscopic observation, and DSE typical structures such as 'microsclerotia' and the like can be observed in part of samples (figure 2). The colonization rate of 5 DSE strains is 30% -45%, the colonization rate of LS1, TD1 and B2 is high and has no significant difference, and the colonization rate of LC8 is the lowest (FIG. 3).

2.3 biocontrol effects of DSE on Banana wilt

As shown in figure 4, after 5 DSE strains are inoculated, each treated banana plant generates disease resistance with different intensity to banana vascular wilt, the plate control effect is between 57.76% and 86.19%, and the pot control effect is between 12.69% and 63.19%. The banana plants treated by the strain LS1 have the best prevention and control effect and reach a remarkable difference level with other strains. The visible strain LS1 is an endophytic fungus with biocontrol potential, and is further classified and identified.

2.4 taxonomic identification of biocontrol Strain LS1

Morphological identification: the growth rate of the strain LS1 on the PDA culture medium is moderate, after 2 weeks of culture at 28 ℃, the diameter of a bacterial colony is about 70mm, the bacterial colony is round, brown to brownish green, felted, flat in edge, provided with shallow radial grooves and dark brown in the back (figure 1A). Microscopic observation shows that LS1 has hypha branch with septa, yellow brown to brown, smooth, thick wall, and diameter of 1.4-4.7 μm. Conidiophores are vertically grown on the top or lateral branches of hyphae, and have no branching, brown color, smoothness, and septum, and size of 22.4-386.7 × 2.7-5.2 μm (FIG. 1B). The top and middle parts of the conidiophores expand to form spore-forming cells, and the expanded parts are 3.7-6.3 mu m. The branched conidia are mostly cylindrical and partially elliptical, most of the branched conidia are slightly contracted in the middle and have 0-2 partitions, on which conidia are grown, cladospores are grown or regenerated, and the size of the regenerated cladospores is 6.4-28.5 multiplied by 2.0-5.6 mu m. Conidiophore is round, elliptical, lemon or spindle-shaped, yellowish brown, without separation, with umbilicus at two ends or one end of partial conidiophore of 3.4-8.4 × 2.2-4.7 μm; the diameter of the near-circular spore is 2.4-7.6 μm. (FIG. 1C). The above characteristics are substantially in accordance with the morphological characteristics reported in the literature for Cladosporium chlorocephalum.

And (3) molecular identification: an ITS primer is utilized to carry out ITS amplification on total DNA of an LS1 strain to obtain a target PCR product band of about 550bp, the PCR product is purified and then sequenced, a GenBank is submitted to obtain a login number MH376857, the sequence is subjected to Blastn analysis in NCBI, and the sequence similarity of LS1 and each isolate of Cladosporium is found to be more than 99 percent; a phylogenetic tree (FIG. 5) based on ITS sequences was constructed using sequences in Bensch K (students in Mycology,2015,82(82):23-74) and related sequences downloaded in GenBank, and LS1 was found to have one strand with C.chlorocephalum and C.tenuissimum, with a support rate of 89%, and with a strain LS1 having 100% similarity to the ITS sequences of C.chlorocephalum.

And comprehensively analyzing the result, and identifying the strain LS1 as Cladosporium chlorocephalum.

The classification status of DSEs is one of the problems to be solved urgently in the current DSE research. Since most DSEs are present in vegetative form already and few sexual states occur under culturable conditions, this presents difficulties in morphological classification. Currently, with the help of molecular identification technology, DSEs have been identified that comprise more than 10 genera, about 30 species of fungi, and exhibit multiple origins. The strain LS1 is identified as the Cladosporium chlorocephalum by a means of combining morphology and molecular identification.

By inoculating Cladosporium chlorophyllium LS1 separated from sugarcane root-surrounding soil on bananas, the plant height, stem width, fresh weight and dry weight of bananas are effectively improved. After inoculation is finished, the plate control effect of the LS 1-banana symbiont on banana wilt disease reaches 86.19%, and the pot control effect reaches 63.19%. Therefore, the strain is a beneficial biocontrol strain with great research and utilization values.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Sequence listing

<110> Guangxi Zhuang nationality college of autonomous region agro-sciences

<120> biocontrol DSE strain LS1 and application thereof in banana vascular wilt

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> DNA

<213> ITS1

<400> 1

tccgtaggtg aacctgcgg 19

<210> 2

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<212> DNA

<213> ITS4

<400> 2

tcctccgctt attgatatgc 20

Claims

1. A biocontrol DSE strain LS1 is characterized in that the preservation number is CGMCC NO.16498, and the preservation time is 11 months and 8 days in 2018.

2. The use of the biocontrol DSE strain LS1 as claimed in claim 1 for controlling banana vascular wilt comprising the steps of:

step A, strain inoculation and influence on banana growth, wherein the strain to be tested is inoculated on an oat culture medium after being activated, banana tissue culture seedlings with consistent growth vigor are selected and transplanted to bacterial colonies, root systems are laid on the surfaces of the bacterial colonies, each bacterial colony is transplanted to one strain, a culture dish is placed into a tissue culture bottle, and the culture dish is placed into an incubator for culture; measuring the plant height, stem width, fresh weight and dry weight of the banana; each treated part of fresh banana root sample is used for colonization observation and re-separation of DSE strains, the sample mass of the reserved part is recorded, the rest part is dried to constant weight in an oven after weighing fresh weight, the dry weight is measured, the average water content is calculated, and the dry weight is calculated together after converting the previously reserved fresh root sample;

and step B, re-separation and colonization detection of the strains, namely taking a proper amount of fresh root samples, cutting the fresh root samples into small sections, placing the small sections into a test tube, adding KOH solution to completely immerse the root sections, dissociating the root sections in a constant-temperature water bath kettle to soften and transparent the root sections, dyeing the root sections with ink vinegar, rinsing the root sections with clear water, soaking and decoloring the root sections, observing the colonization condition of the DSE strains at the roots of the bananas, and calculating the colonization rate: colonization rate% = (number of colonized root segments/total number of roots microscopic examined) × 100%;

step C, measuring the prevention and treatment effect of the strain on banana vascular wilt: transferring the DSE strain-banana symbiont and the oat culture medium to a water agar medium with fusarium, and directly inoculating the control banana tissue culture seedlings without inoculation to a water agar plate with fusarium;

3. The use of claim 2, wherein in step a, the oat culture medium is: oat flour 10 g.L^-1Agar 18 g. L^-1，MgSO₄·7H₂O 1g·L^-1，KH₂PO₄1.5g·L^-1，NaNO₃1g·L^-13 clumps per dish were incubated at 28 ℃ for 10 days.

4. The use of claim 2, wherein in step A, the tissue culture bottle is at 28 deg.C and light intensity of 180 μmol-m^-2·s^-2And culturing for 30-40 days in an incubator with a photoperiod of 16h to 8 h.

5. The use of claim 2, wherein in step B, KOH solution with the mass concentration of 10% is added to completely immerse the root segment, the root segment is dissociated in a water bath kettle at the constant temperature of 90 ℃ for 15-20min to soften and transparent the root segment, the root segment is dyed by ink vinegar, then rinsed for 3 times by clean water, and soaked for 12h to decolor.

6. The use of claim 2, wherein in step C, the disease staging criteria are: grade 0-no visible symptoms in appearance; grade 1-no obvious resistance to plant growth, and whole plant verticillium area less than or equal to 35%; 2 level-weak plant, the whole plant verticillium area is less than or equal to 80 percent more than 35 percent; grade 3-the whole plant verticillium area is more than 80%.

7. The use of claim 2, wherein in step C, the potted plant biocontrol test is performed on 4-leaf stage sized banana seedlings; preparation of 5X 10⁵cfu mL^-1Performing root irrigation on the DSE bacterial liquid; irrigating root of each banana seedling with 50mL of bacterial liquid once every 15 days for three times, and after the last root irrigation for 15 days, root-damaging and inoculating fusarium spore liquid at 1X 10 times⁶cfu mL^-1。

8. The use according to claim 7, wherein the control CK is the treatment of irrigating roots with clear water and inoculating pathogens, the banana corms are dissected 25 days after the inoculation of the pathogens, the morbidity is observed and recorded, and the control effect is calculated, wherein each treatment is repeated for 3 times, and each repetition is 8 seedlings.

9. The use according to claim 2, wherein in step D, when inducing sporulation, the cells are incubated at 4 ℃ for 2 to 4 weeks.

10. The use of claim 2, wherein in step D, the total DNA of the mycelium is extracted using an OMEGA fungal DNA extraction kit, and the amplification primers are ITS 1: 5'-TCCGTAGGTGAACCTGCGG-3' and ITS 4: 5'-TCCTCCGCTTATTGATATGC-3', amplification system 50. mu.l, containing: mastermix TIAngel 25. mu.l, 20. mu. mol L-1 forward and reverse primers 1. mu.l each, DNA 1. mu.l, ddH₂O, fixing the volume; and (3) amplification procedure: pre-denaturation at 98 ℃ for 2 min; denaturation at 94 ℃ for 40s, annealing at 50 ℃ for 1min, and extension at 68 ℃4min, 30 cycles; finally, extension is carried out for 10min at 72 ℃.