CN104974940B - A kind of Biocontrol Strain for preventing and treating dothiorella gregaria and its application - Google Patents
A kind of Biocontrol Strain for preventing and treating dothiorella gregaria and its application Download PDFInfo
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Abstract
The invention discloses a kind of Biocontrol Strain and its application process for preventing and treating dothiorella gregaria.Described bacterial strain is trichoderma aureoviride (Trichoderma aureoviride), strain number CGMCC8891.The bacterial strain fermentation liquor mixes with wheat bran and corn flour, and natural wind dry doubling, which pulverizes, after the 7d that ferments is made former powder；Microbial inoculum is made in addition 38wt% original powder, 38wt% kaolin, 13wt% sodium lignin sulfonates, 8.5wt%SDS, 2.5wt% humic acids；For preventing and treating dothiorella gregaria in a manner of spraying with pouring root after microbial inoculum is diluted.The bacterial strain has the advantages that varied to the mode of action of pathogen, reproduction speed is fast, action effect is stable, it is strong to dothiorella gregaria pathogen grape seat chamber bacterium (Botryosphaeria dothidia) inhibitory action, preventing and treating to dothiorella gregaria is significant, has good development prospect.
Description
Technical field：
The present invention relates to plant protection art, more particularly to a kind of Biocontrol Strain for preventing and treating dothiorella gregaria and its should
With relating in particular to a kind of efficiently preventing and treating dothiorella gregaria biocontrol bacterial strain trichoderma aureoviride CGMCC8891 and application.
Technical background：
Processed raw material as wood-based plate and the important of Fibre Wood, each kind willow and its Hybrid are worldwide
There is extensive distribution.In addition, willow, which is afforested and checked winds and fixed drifting sand to afforestation, ecological recovery, landscape, suffers from important work
With.Willow easily infects multiple diseases, and wherein dothiorella gregaria is one of China's Poplar Plantation important biological disaster, serious system
The about development of Poplar Plantation.This disease is mainly by fungies such as Botryosphaerias (Botryosphaeria spp.)
It is caused.Dothiorella gregaria can cause the death of poplar seedlings and hinder the normal growth of willow, have a strong impact on willow people
Work woods becomes a useful person, and restricts the stable development of China's forestry, industrial construction and ecological recovery.The disease China northeast, North China, East China and
The ground such as northwest generally occur, and cause nursery and the failure of afforestation, and economy and Ecological Loss are surprising, the morbidity of serious regional forest
Rate is up to 96.1%~100%, and disease-sensitive index is up to 6.7%~95.8%.
The preventing and treating to dothiorella gregaria is mainly realized by strengthening tending management and physics and chemical method at present.But
Pollution and destruction of the chemical prevention for environment turn into the problem of can not be ignored.People more and more pay close attention to the existence ring of oneself
Border, the preventing and treating to pest and disease damage propose higher and higher safety requirements.Anticipated with the development and people's environmental protection of social economy
The enhancing of knowledge, administering dothiorella gregaria with the method for biological control very likely turns into a kind of effective ways more received for people.
Separation screening obtains the biocontrol bacterial strain for having antagonism to dothiorella gregaria germ from Tissues of Poplar Clones, and by its
Develop and be referred to as active bacteria formulation, the preventing and treating to dothiorella gregaria has great importance.
The content of the invention：
It is an object of the invention to provide a kind of trichoderma aureoviride bacterial strain of efficiently preventing and treating dothiorella gregaria.The bacterial strain was in 2014
It was stored in the commonly micro- life of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 China Committee for Culture Collection of Microorganisms on March 4
Thing center, deposit number CGMCC8891.Trichoderma aureoviride CGMCC8891 is that a kind of separation screening from Tissues of Poplar Clones obtains
There is the biocontrol bacterial strain of antagonism to dothiorella gregaria germ, the preventing and treating to dothiorella gregaria has great importance.
The trichoderma aureoviride CGMCC8891 that another object of the present invention is to provide the efficiently preventing and treating dothiorella gregaria is being prevented and treated
A variety of applications in terms of dothiorella gregaria.
The technical scheme is that a kind of trichoderma aureoviride CGMCC8891 of efficiently preventing and treating dothiorella gregaria is provided, in PDA
On flat board, bacterium colony initial stage is white, and there are an obvious Chan Bao areas of a circle in the later stage among bacterium colony, and color is bottle green, light green or yellow
It is green.Mycelia has every conidiophore branch is more.Spore is smaller, spherical, is often attached to around mycelia.Pass through morphological feature
Initial analysis judges that CGMCC8891 bacterial strains are green mold category fungi.Performing PCR expansion is entered to bacterial strain CGMCC8891 ITS gene orders
Increase, be sequenced after product purification.The sequence length for measuring bacterial strain CGMCC8891 is 649bp.CGMCC8891 bacterium are found by analysis
It is one that the trichoderma aureoviride that strain is HQ596956 with numbering, which is gathered, and similarity 99%, affiliation is nearest, and homology is very high.It is comprehensive
The morphological feature and Molecular Identification result of bacterial strain are closed, it is trichoderma aureoviride (Trichoderma to determine CGMCC8891 bacterial strains
aureoviride).It is of the present invention efficiently preventing and treating dothiorella gregaria trichoderma aureoviride CGMCC8891 by flat board dual test,
Zymotic fluid secondary screening and in vitro tissue experiment sieving obtain.
Present invention also offers the trichoderma aureoviride CGMCC8891 of the efficiently preventing and treating dothiorella gregaria in preventing and treating willow ulcer
Application in disease, and can be used for the preparation for preparing controlling plant diseases.
The preparation process of the preparation of described preventing and treating dothiorella gregaria is as follows：
(1) first strain liquid is fermented：Fermentation condition is：Inoculum concentration is 3.5%, liquid amount 100mL/250mL, initial pH
Value 7.0,34 DEG C, rotating speed 190r/min, fermentation time 96h of temperature；Fermentation medium is：Potato soaks juice 200g/1000mL, fructose
1%, beef extract powder 1.4%, KCl0.1%；
(2) solid fermentation：Mixed from 50% corn flour with 50% wheat bran and be used as solid fermentation matrix, fermentation condition is
20% inoculum concentration, 30% water content, cultivation temperature are 30 DEG C.
(3) prepared by microbial inoculum：Former powder is made by being pulverized after solid fermentation thing natural air drying；Add 38wt% original powder, 38wt%
Microbial inoculum is made in kaolin, 13wt% sodium lignin sulfonates, 8.5wt%SDS, 2.5wt% humic acids.
After described microbial inoculum may also pass through dilution, directly it is used to prevent and treat dothiorella gregaria in a manner of spraying with pouring root.
Described microbial inoculum dilutes 100~1000 times.
The beneficial effects of the invention are as follows：
(1) trichoderma aureoviride CGMCC8891 has antagonism to dothiorella gregaria pathogen, and with to pathogen
The advantages that mode of action is varied, reproduction speed is fast, action effect is stable, played in dothiorella gregaria biological control
Act on extremely important；
(2) this kind of strain culturing condition is simple, easily preserves, is easy to industrialized production, before having good development and application
Scape.
Brief description of the drawings：
Fig. 1 is trichoderma aureoviride CGMCC8891 bacterial strain colonial morphology figures
Fig. 2 is trichoderma aureoviride CGMCC8891 bacterial strain hypha form figures
Fig. 3 is flat board face-off antagonism of the trichoderma aureoviride CGMCC8891 bacterial strains to dothiorella gregaria pathogen grape seat chamber bacterium
Action diagram
Embodiment：
The present invention provides trichoderma aureoviride CGMCC8891 new strains, and it is shown to dothiorella gregaria pathogen with stronger
Antagonistic activity.
The new strains of the present invention are isolated from willow cadre tissue.
For the trichoderma aureoviride CGMCC8891 of the present invention on PDA plate, bacterium colony initial stage is white, and the later stage has among bacterium colony
The one obvious Chan Bao areas of circle, color is bottle green, light green or yellowish green.Mycelia has every conidiophore branch is more.Spore is smaller,
It is spherical, often it is attached to around mycelia.Judge that CGMCC8891 bacterial strains are green mold category fungi by morphological feature initial analysis.
Performing PCR amplification is entered to bacterial strain CGMCC8891 ITS gene orders, is sequenced after product purification.Measure bacterial strain CGMCC8891 sequence
Row length is 649bp.It is one to find CGMCC8891 bacterial strains by analysis and number the trichoderma aureoviride for being HQ596956 to gather, phase
It is 99% like degree, affiliation is nearest, and homology is very high.The morphological feature and Molecular Identification result of comprehensive bacterial strain, it is determined that
CGMCC8891 bacterial strains are trichoderma aureoviride (Trichoderma aureoviride).The present invention is made into one with reference to embodiment
The detailed description of step, without limiting the present invention.
Separation, screening and the identification of raw biocontrol microorganisms in the dothiorella gregaria of embodiment 1
Take the blade, branch, cadre of willow to organize respectively and because of the callus formed after mechanical damage, load nothing
In bacterium valve bag, laboratory is taken back as being preserved in 4 DEG C of refrigerators, and separate endophyte as early as possible.It is special by cultural character and bacterium colony
Sign, judges to isolate 115 plants of microbial strains from Tissues of Poplar Clones, is stood facing each other and tested by flat board, isolated from 115 plants of bacterium
To 18 plants of 71 plants of bacterial strain for having fungistatic effect, wherein bacterium and actinomyces, 53 plants of fungi.Comprehensive " sandwich " method and improvement agar
Diffusion method carries out secondary screening to the zymotic fluid of primary dcreening operation bacterial strain, as a result filter out YZ1B3, YYSB1, YZ2F10, CGMCC8891 and
YYSF85 strains Antagonistic Fungi carries out in vitro tissue secondary screening.Final determination biocontrol bacterial strain CGMCC8891 antagonism is most strong.
The bacterial strain screened passes through morphological observation, bio-chemical characteristics and molecular biology identification, final to determine to be somebody's turn to do
Bacterium is trichoderma aureoviride.
Trichoderma aureoviride CGMCC8891 bacterial strains bacterium colony and hypha form figure of the present invention are shown in Fig. 1 and Fig. 2.
The trichoderma aureoviride CGMCC8891 strain fermentation process optimization and stabilities of embodiment 2
Through experiment of single factor, orthogonal design and response surface analysis, the final optimal fermentation medium for determining CGMCC8891
It is with condition：Potato soaks juice 200g/1000mL, fructose 1%, beef extract powder 1.4%, KCl0.1%, initial ph value 7.0, temperature
34.14 DEG C, rotating speed 188.80r/min, inoculum concentration 3.58%, liquid amount 100mL/250mL, predict that optimal bacteriostasis rate is
85.5667%.For the ease of the operation and control in actual production, CGMCC8891 fermentation condition is adjusted to originate pH
Value 7.0,34 DEG C, rotating speed 190r/min of temperature, inoculum concentration 3.5%, liquid amount 100mL/250mL.Bacterial strain CGMCC8891 after optimization
Bacteriostasis rate up to 84.98%, improve the bacteriostatic activity of biocontrol bacterial strain, be the subsequent development of bacterial strain using providing guarantee.
Trichoderma aureoviride CGMCC8891 has certain tolerance to soda acid processing, and under conditions of pH is 1, bacteriostasis rate is
63.12%, under the conditions of pH is 12,47.34% bacteriostasis rate can be still protected, is thus proved, trichoderma aureoviride CGMCC8891 is in acidity
Remain to keep high stability under environment, there is certain stability under strongly acidic conditions, and it is weaker to the stability of hot conditions.
It is demonstrated experimentally that the bacterial strain does not have stability to ultraviolet.After passage 10 times, the bacteriostatic activity of the bacterial strain still up to 73.46%,
Illustrate its inheritance stability.
The preparation of the trichoderma aureoviride CGMCC8891 biocontrol agent original powder of embodiment 3
Microbial inoculum original powder is prepared using solid fermentating mode.3 kinds of different solid fermentation matrix formulas are set：I. 400g is expected admittedly,
Wherein crack rice 50%, wheat bran 50%, and the ratio of solid material and water be 1: 1 (W/V), II. expects 400g admittedly, wherein crack rice 50%, jade
Ground rice 50%, and the ratio of solid material and water is 1: 1 (W/V), III. expects 400g, wherein wheat bran 50%, maize flour 50% admittedly, and consolidates
The ratio of material and water is 1: 1 (W/V).As a result show to mix with 50% wheat bran as solid fermentation matrix most from 50% corn flour
To be suitable, bacteriostasis rate 88.24%, spore count living is up to 1.51 × 1010cfu/g。
The screening of solid fermentation condition sets 9 groups of experiments altogether using the orthogonal design of Three factors-levels.It is final to obtain
The suitable solid fermentation condition of CGMCC8891 bacterial strains is 20% inoculum concentration, and 30% water content, cultivation temperature is 30 DEG C.At this
Under the conditions of kind, for bacteriostasis rate up to 90.36%, viable count is 8.08 × 109。
Former powder is made by being pulverized after solid fermentation thing natural air drying.
The development of the trichoderma aureoviride CGMCC8891 biocontrol agents of embodiment 4
38wt% original powder is added, 38wt% kaolin, 13wt% sodium lignin sulfonates, 8.5wt%SDS, 2.5wt% are rotten to plant
Acid is microbial inoculum optimization formula.Wetability of this lower microbial inoculum of formula, dispersiveness, suspension are all up optimum level, to ultraviolet steady
It is qualitative also most strong.Viable count is 6.25 × 10 in microbial inoculum finished product10Cfu/g, bacteriostasis rate is up to 89.12%.After measured, the shelf of microbial inoculum
Storage life is longer, and bacteriostasis rate is still up to 75.38% after preserving 6 months.The trichoderma aureoviride CGMCC8891 biocontrol agents of embodiment 5
Woodland application method and effect
(1) optimal application method
The microbial inoculum configured is diluted 500 times with clear water, is applied in after well mixed on willow cuttage seedling.Set 5 kinds
Different administration method：A：Only spray CGMCC8891 microbial inoculums (YP)：Microbial inoculum dilution is uniformly sprayed at cuttage with miniaturised nebuliser
Seedling tissue surface, it is advisable with soaking body surface, but being dripped without obvious drop；B：Only use CGMCC8891 microbial inoculum pouring roots：Using pouring root device
Dilution bacterium solution is filled in cuttage seeding root, 50~100mL/ strains, be advisable with to soak rhizosphere soil (YG) C：Spray simultaneously
CGMCC8891 microbial inoculums and pouring root CGMCC8891 microbial inoculums (YP+YG)；D：To spray with pouring root clear water as negative control simultaneously；E：With
The carbendazim of sprinkling simultaneously and the dilution times of pouring root 500 is positive control.It is identical each to handle 10 plants of growing ways of each selection, it is healthy healthy and strong
Willow cuttage seeding be experiment material.
Beaten with a diameter of 4mm card punch and take bark disk, be placed in the triangular flask for filling distilled water, moist heat sterilization is cold
But after, bark disk is placed on the culture medium flat plate for covering with dothiorella gregaria bacterium, after 25 DEG C of culture 15d, on bark disk
Spore device is grown, using this bark disk as inoculum.
Preventive effect and therapeutic effect of the biocontrol agent to dothiorella gregaria under measure different administration mode respectively：
(1) measure of preventive effect：After biocontrol agent 72h, canker inoculum is inoculated into cuttage seeding, use is wet
Absorbent cotton moisturizing, periphery are tightened with preservative film.
(2) measure of therapeutic effect：First canker inoculum is inoculated into cuttage seeding, with wet absorbent cotton moisturizing, periphery
Tightened with preservative film.Biocontrol agent is applied after 72h.
The incidence of disease and disease index of observation statistics each group seedling canker after microbial inoculum 20d is applied.Such as following table, by inoculation
Scab is divided into 5 grades by point Spot expansion diameter.
The potted plant inoculation scab grade scale of table 1
Disease index=and ∑ (sick level strain number × represent numerical value)/total strain number × morbidity most heavy duty typical value × 100
Prevention effect=(control disease index-processing disease index)/control disease index × 100%
Influence of the table 2CGMCC8891 microbial inoculum different administration modes to dothiorella gregaria prevention effect
The potted plant experiment result of CGMCC8891 biocontrol agents shows that microbial inoculum has preferable prevention effect to dothiorella gregaria.
When method of application is combined for sprinkling and pouring root, its preventive effect and optimal is reached to the control effect after morbidity,
And the disease index of dothiorella gregaria is greatly reduced.CGMCC8891 biocontrol agents are excellent to the therapeutic effect of dothiorella gregaria
In preventive effect, preventive effect up to 73.27% and therapeutic effect be 51.04%.The prevention effect of 500 times of dilutions of microbial inoculum is equal
Better than chemical pesticide carbendazim.
(2) optimal application concentration
Application concentration is set in the following manner：
Test process A (blank control)：Spray clear water, clear water root irrigation；
Test process B (control effect testing)：The biocontrol agent that 100 times of above-mentioned Antagonistic Fungi CGMCC8891 is diluted with clear water sprays
Spill, root irrigation.
Test process C (control effect testing)：The biocontrol agent that 500 times of above-mentioned Antagonistic Fungi CGMCC8891 is diluted with clear water sprays
Spill, root irrigation.
Test process D (control effect testing)：The biocontrol agent that 1000 times of above-mentioned Antagonistic Fungi CGMCC8891 is diluted with clear water sprays
Spill, root irrigation.
Test process E (chemical pesticide control effect testing)：Sprayed with 75%500 times of carbendazim, root irrigation.
The potted plant cuttage seeding of 9 plants of growing way identicals of each processing selection is carried out.Apply microbial inoculum once within every 15 days, add up to and co-administer
3 times.Pathogen vaccination ways are the same.10d is once investigated after last time is handled.Disease index is recorded, it is final to calculate
Prevention effect.Influence of the biocontrol agent to plant strain growth is determined simultaneously in investigation, mainly determines the height of each strain willow cuttage seeding
Degree, stem are thick etc..
Influence of the table 3CGMCC8891 microbial inoculum different administration concentration to dothiorella gregaria prevention effect
It can be seen from the results that by microbial inoculum dilute 100 times, 500 times, 1000 times of preventive effect can reach 70% with
On.Compared with compareing carbendazim, clear superiority is then presented to the prevention effect of dothiorella gregaria in CGMCC8891 microbial inoculums.By pair
The growing state investigation of each experimental group seedling finds that facilitation is played in growth of the CGMCC8891 microbial inoculums to willow cuttage seeding.Through
After crossing nearly 60d growth, using the seedling of 100 times and 500 times dilution microbial inoculum groups in average stem high growth amount, the thick incrementss of stem etc.
Aspect is superior to control group, and incrementss are 50% or so.
(3) the optimal application time
The occurrence regularity of dothiorella gregaria is：Start April to fall ill, first onset peak of formation in late May to June, 7~
Patient's condition slows down when August temperature increases, and second onset peak, point that this germ is formed from spring scab then occurs in September
Raw spore, stops after October.Experiment selection is carried out in Langfang in Hebei Province proving ground.Respectively in March, May, July, September, November
Using biocontrol agent, monthly applied twice within 15 days by interval.Young growth area experiment uses RANDOMIZED BLOCK DESIGN, sets 5 cells altogether, often
15 plants of cell, every plant of spacing is 2.5m × 3m, and minizone sets protection row.Respectively April 10, June 10, August 10 days,
October 10, the incidence of disease, Lesion number and the disease occurrence degree of investigation on December 10 sample tree.
Disease occurrence degree is described using the susceptible area ratio of trunk (Infected Area Ratio of Trunk), is used for
The index that the order of severity occurs for individual trees disease is described, with the total face of scab in the range of 0.8~1.8m of isolated tree wood trunk height
The percentage that product accounts for trunk total surface area represents.
The scab gross area/this section of trunk total surface area × 100% on susceptible area ratio (%)=0.8~1.8m trunks of trunk
Influence of the table 4CGMCC8891 microbial inoculum different administration times to dothiorella gregaria prevention effect
It can be seen that by 3 incidence of disease, scab number, disease occurrence degree indexs and apply microbial inoculum to canker in March
Prevention effect it is optimal, next to that July apply effect it is also preferable.Although the incidence of disease in dothiorella gregaria in November,
Scab number, disease occurrence degree are minimum, but this natural conditions such as low to cold weather at that time, air humidity is related, so
Natural conditions be not suitable for growth of pathogenic bacteria, it is impossible to completely embody biocontrol agent effect.
Synthesis result, the optimum application process of CGMCC8891 microbial inoculums are：CGMCC8891 biocontrol agent clear water is dilute
500 times are released, the microbial inoculum suspension after dilution is applied to willow root and cadre by the way of pouring root and sprinkling are combined.Using
Dosage is every plant of 200mL or so, is advisable with soaking rhizosphere 20cm scopes soil and being dripped without obvious drop.Time of application is annual
3m, 7m, applied once per 15d in these months.
Claims (3)
1. a kind of Biocontrol Strain for preventing and treating dothiorella gregaria, it is characterised in that described bacterial strain is trichoderma aureoviride
(Trichoderma aureoviride), bacterial strain deposit number are CGMCC No.8891.
2. application of the Biocontrol Strain in dothiorella gregaria is prevented and treated described in claim 1.
3. application according to claim 2, it is characterised in that described Biocontrol Strain is used to prepare preventing and treating willow ulcer
The preparation of disease.
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| 适宜黄绿木霉T1010生长培养料筛选;王未名等;《中国生物防治》;20090715;第25卷(第S1期);第59页1.2-1.3、第60页2.2和表2 * |
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