CN101831393A - Theory for directed filtering of plant biological control strain and application thereof - Google Patents

Abstract

The invention relates to a novel theory method for separating a biological control strain and application thereof. The strain possibly having biological control potential is obtained by using traditional methods for filtering the plant biological control strain through a filtering way just as difficult as looking for a needle in a haystack. The filed effect of the strain has uncertainty to a larger extent. The invention provides a new method, i.e. a theory for directed filtering of the plant biological control strain, which has the key content comprising the steps of: selecting a special culture medium closely related to a target plant; filtering an anti-germ group which has an orientation relation and can hold a dominant ecological niche in a plant body or root; and proportioning a strain according with the biological control according to the occurring frequencies of various biological control strains for full-dimensionally inhibiting from invasion of harmful germs from a three-dimensional space. The theory overcomes the defects of the filtering of the traditional biological control strain, eliminates many biological control strains without practical application values, and can obtain the biological control strain with practical field effect prevention within shorter time. The biological control strain aiming at cotton Fusarium wilt and cotton Verticillium wilt, knojak soft rot, watermelon droop, tomato bacterial wilt, ginger Pseudomonas solanacearum and the like can be obtained by applying the invention, therefore, the biological control strain is applied to a pot culture and field experiment, results show that the biological control strain filtered by an orientated way has excellent effect prevention capable of reaching more than 80 percent; and the theory has huge application prospect.

Description

Theory for directed filtering of plant biological control strain and application

Technical field:

The invention belongs to the screening method of biocontrol microorganisms in the agriculture field, this method is the directed screening method, and the biocontrol microorganisms and the target plant of screening have close relation, has ensured the field validity of screening biocontrol microorganisms, overcome the blindness of biocontrol microorganisms screening in the past, got rid of many invalid biocontrol microorganisms.

Background technology:

The biocontrol microorganisms of bibliographical information screening now is of a great variety, comprise biocontrol fungi, biocontrol bacteria, biological and ecological methods to prevent plant disease, pests, and erosion actinomycetes etc., but the biocontrol fungicide that can really introduce to the market is considerably less.Trace it to its cause, mainly be that the microbial preparation of developing with the laboratory in the market has the following defective: 1) biocontrol microorganisms colonization ability under the natural condition of field is poor.Most of biocontrol microorganisms are Screening and Identification under laboratory condition, the substratum design has very big difference with the field natural condition, often the field colonization ability is not strong for Shai Xuan biocontrol microorganisms thus, the very fast decline of its population quantity also returns to itself ecologic niche, can not form enough ecological disease-resistant effects and do not reach due preventive effect, this becomes the commercial maximum restrictive factor 2 of biocontrol microorganisms) at different soils, the field prevention effect is stable inadequately under the weather condition, mainly be to the pH value because of this bacterium, soil nutrient, aeration conditions etc. require harsh, so in different areas, the effect difference of different fields piece; 3) present biological control is single microbial inoculum, does not unite the three-dimensional advantage of different biocontrol microorganisms, has reduced the prevention effect of biocontrol fungicide.

At above biocontrol microorganisms defective, how screening the biocontrol microorganisms that can bring into play the efficient disease-resistance effect in the field is the big difficulty that present biocontrol microorganisms faces.

Summary of the invention:

The present invention proposes " theory for directed filtering of plant biological control strain ", its core concept is utilized special culture medium directed screening and the closely-related biocontrol microorganisms of target plant exactly, and the biocontrol microorganisms of having guaranteed screening is the disease-resistant flora of advantage in rhizosphere or the plant materials; The biocontrol microorganisms frequency of occurrences is come its proportion in compound biocontrol fungicide of proportioning during simultaneously by screening, overcomes the deficiency of single bacterial strain.Use this theory and can fast, efficiently solve cancer in the class plant production such as vascular bundle diseases of plant, soil-borne disease, at present this class disease chemical pesticide substantially to no effect, disease-resistant variety also achieves no breakthrough.

Application example:

With the ginger bacterial wilt of ginger is example, serious novel bacterial wilt of ginger has taken place in area, Laiwu, Shandong, waits bitterly for chlorination and irritates root all to no effect, and we at first separate the morbidity sample, discovery is the Lao Ershi Ralstonia solanacearum of a variation, and virulence is far above coventional type Lao Ershi Ralstonia solanacearum.Have only from the biological control angle for this class disease and to go to handle, just can achieve good results.

1) screening culture medium preparation

The sandy soil of large beaker dress water content 7%, sterilising treatment.Get healthy ginger 500g, clean, with 75% alcohol surface sterilization, dry, imbed in the above-mentioned sandy soil, place 25 ℃ of incubators to take root, grow after 10 days by its germination, clean sandy soil with tap water, remove bud, take by weighing band root ginger 100g, in mortar, grind with the 100ml sterilized water, cross leaching filtrate, as substratum master composition; Add 0.1g peptone and 15g agar again, add water at last until 1000ml, the sterilization back is fallen dull and stereotyped, is screening biocontrol microorganisms flat board.The liquid screening substratum need not add agar.

2, screening process

Get healthy moistening ginger field soil sample, mixing is got the 5g soil sample in above-mentioned 100ml liquid screening substratum, carries out enrichment culture 3 days under 28 ℃.

Get above-mentioned nutrient solution 100 μ l and evenly coat on the screening flat board,, choose doubtful genus bacillus bacterium colony in 28 ℃ of following constant temperature culture 36 hours.

Making contains solid medium (1% peptone+0.5% yeast powder+1% glucose+1.5% agar of ginger plague germ, during sterilization postcooling to 50 ℃, the ginger plague bacteria culture fluid is added wherein in 0.2% ratio, make dull and stereotyped), to screen doubtful genus bacillus in a large number and choose in this flat board, and have the bacterium colony of transparent circle to carry out purifying around seeking with toothpick.Carry out postsearch screening then, have the bacterium colony of anti-microbial activity to focus on the screening flat board primary dcreening operation, the size of observing inhibition zone is selected active 5-10 the highest bacterium colony.

Further eliminate by pathogenic mensuration: after the ginger surface sterilization, be cut into the bulk of thickness 1cm, the biocontrol microorganisms of screening is inoculated into the ginger tangent plane one by one, observe this bacterium after 24 hours and whether ginger is had pathogenic effects, screen out bacterial strain with pathogenic effects.

Then remaining biocontrol microorganisms is done experiment under potted plant situation, observe the disease resisting effect of each bacterium, effect is measured its 16srDNA sequence preferably one by one, in conjunction with the kind status of biochemistry and morphology evaluation biocontrol microorganisms.

Bacterial strain to the different kind of above-mentioned evaluation cooperates, and observes its joint effect, after having the potted plant experiment of cooperation microbial inoculum process of stablizing good preventive effect, is generalized to the land for growing field crops, declares biological pesticide registration test at last, progressively introduces to the market.

This seminar adopts this separation method, therefrom screening has the biocontrol bacteria of high antibiosis ginger ginger plague germ, now be sieved to 3 strains have high anti-disease activity, can be at the quick rhizosphere dominant strain of breeding of cotton rhizosphere: subtilis (Bacillus subtilis) YUPP-8, bacillus cereus (Bacillus cereus) YUPP-9, series bacillus (Paenibacillus sp.) YUPP-10

Claims (4)

1. the present invention proposes a kind of " theory for directed filtering of plant biological control strain ", its core content is exactly to select the special culture medium relevant with suitable plant for use, screening and target plant have directional relation, can be in plant materials or rhizosphere occupy the disease-resistant flora of advantage ecological niche, and can come proportioning to meet biocontrol fungicide according to each biocontrol microorganisms frequency of occurrences, from the invasion of the harmful germ of the comprehensive prevention of solid space.。

2. the required special culture medium of claim 1 described " theory for directed filtering of plant biological control strain " preparation is characterized in that the substratum that the special culture medium main component is produced for the extract of the root exudates that is suitable for plant and foot end.

3. the biocontrol microorganisms of substratum screening according to claim 2.

4. the biocontrol microorganisms of substratum screening according to claim 3 is promoting plant-growth, is improving the purposes on plant resistance to environment stress and the raising plant biomass.