CN100411520C

CN100411520C - Biological bactericide for resisting grey mould one its preparation method

Info

Publication number: CN100411520C
Application number: CNB200510039128XA
Authority: CN
Inventors: 蒋立科; 岳永德; 罗曼; 花日茂; 汤锋; 操海群
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2005-04-25
Filing date: 2005-04-25
Publication date: 2008-08-20
Anticipated expiration: 2025-04-25
Also published as: CN1685834A

Abstract

The present invention relates to biological bactericide for resisting gray mold disease, which is prepared by that trichoderma spp is directly fermented to obtain the biological bactericide. The trichoderma spp is fermented in a liquid culture medium which comprises wheat bran or rice chaff or potato as base material, chitin as an inducer and other basic nutrient components under the condition of a temperature range of 25 to 40DEG C for 5 to 10 days. The acute oral toxicity (LD50) of the biological bactericide is more than 4640 mg/kg, the prepared biological bactericide is sprayed on leaf surfaces after being blended with water, the resisting efficiency reaches 80% after 3 days, and the resisting efficiency reaches 99% after 5 days. The present invention has simple technology, easy use, no pollution to the environment and no remnant after fermentation, and the present invention is genuine green bactericide without environmental pollution.

Description

Biological bactericide of a kind of botrytis resistant and preparation method thereof

One, technical field

The present invention relates to a kind of agricultural biological bactericide and processing method thereof, exactly is biological bactericide of a kind of botrytis resistant and preparation method thereof.

Two, background technology

By the gray mold (Botrytis cinerea Pers) due to the Botrytis cinerea (Botrytis cinerea PersexFr), still China strawberry does not endanger than one of severe diseases, and bring disaster to other horticultural crops, also be global important horticultural crop disease.Occur at present monoclonal antibody, two anti-even many anti-ash arrhizus bacteria bacterial strains again, prevent the task arduousness of such disease.Sick the removing of chemical control strawberry brings environmental pollution and pathogen to produce the antibiosis, and importantly strawberry is as a kind of fresh fruit product, because of to the residue problem of agricultural chemicals sensitivity especially, brings psychological impact to people's health.The production and consumption upsurge of current non-polluted farm product is risen, along with the restriction that chemical substance is used in agricultural production is more and more, for the agricultural chemicals that reduces the important production-increasing function of the sick performance of plant pest in the agricultural production, requirements at the higher level have been proposed undoubtedly, the various countries researcher is seeking the natural biological source pesticide, by screening with utilize the beneficial microbe of anti-botrytis cinerea and the biological control method of metabolite thereof, become the important and valid approach of of gray mold control.

Trichoderma (Trichoderma spp) belongs to the clump spore Gammaproteobacteria of Fungi Imperfecti, and the Moniliales Moniliaceae exists extensively that soil, rhizosphere, leaf enclose, in the ecotopes such as seed and bulb.Because extensive adaptability, broad spectrum activity and the many mechanisms of Trichoderma, it is the key object of disease flocking biocontrol scholar research always, at present the focus of research be by biological technique method on purpose transform from the natural world separation screening to Trichoderma, to obtain novel strain, for example merge with ultraviolet radiation mutagenesis, protoplast and gene transformation technology etc. developed novel trichoderma strain.

Three, summary of the invention

This biological bactericide is according to the mould good biological control characteristic of wood, and adopting wood mould is starting strain, and by the biological bactericide that the fermented and cultured acquisition has anti-botrytis cinerea activity, problem to be solved is the screening of medium and the control of fermentation process.

This biological bactericide carries out fermented and cultured on the liquid nutrient medium (culture fluid) after separation obtains (being zymotic fluid) with mould being seeded in of wood.

Described culture fluid with wheat bran or/and rice chaff or/and potato is base-material (carbon source), chitin is an inducer, other auxilliary basic nutrient components and water preparation and.Wheat bran separates or/and the rice chaff drying adds the suitable quantity of water decoction after pulverizing, and get its juice preparation culture fluid, and potato is to pulverize after the bright product peeling, gets bright powder preparation culture fluid, and wheat bran, rice chaff, potato are hereinafter to be referred as the carbon source powder.Each component has following percentage by weight:

Carbon source powder 1～5%

Chitin 0.5～3.5%

FeSO ₄ 0.01～0.03％

KH ₂PO ₄ 0.02～0.2％

K ₂HPO ₄ 0.05～0.25％

MgSO ₄ 0.05～0.35％

Water surplus.

Preferably

Carbon source powder 2～4%

Chitin 1～3%

FeSO ₄ 0.015～0.025％

KH ₂PO ₄ 0.05～0.15％

K ₂HPO ₄ 0.1～0.2％

MgSO ₄ 0.1～0.3％

Water surplus.

Best

Carbon source powder 3%

Chitin 2%

FeSO ₄ 0.02％

KH ₂PO ₄ 0.1％

K ₂HPO ₄ 0.15％

MgSO ₄ 0.2％

Water surplus.

The culture fluid for preparing is transferred pH5.7～6.5, inoculate behind the autoclaving, cultivated 5～10 days in 25～40 ℃ of condition bottom fermentations.Isolated by filtration after the fermentation ends if ferment pH＞4 when stopping, is then transferred pH≤4, and sedimentation is filtered, and filtrate is transferred pH5.8～6.2, promptly is the biological bactericide with botrytis resistant activity.

Preferred culture fluid pH6.0～6.2 are inoculated behind the autoclaving, cultivate 6～8 days in 26～28 ℃ of condition bottom fermentations.Last handling process is the same.

Described chitin is the chitin that the shell by crustaceans such as crab, shrimp, shellfish, insects obtains through acidolysis, washing, drying, and main component is linear polymeric polysaccharide and a spot of protein.Watery hydrochloric acid is generally used in acidolysis, also can use technical hydrochloric acid.

This zymotic fluid carries out toxicity test (test number 20020571), rat acute per os LD through Jiangsu Prov. Disease Preventing and Controlling Center ₅₀＞4640mg/Kg belongs to low toxicity level.

This zymotic fluid uses as bactericide need convert the water dilution more than 50 times, 50 times of dilution mu consumption 20～30Kg, and foliage-spray, preventive effect is that preventive effect reaches 99% after 80%, five day after three days, meanwhile plant growth is had significant spread effect.

Raw material of the present invention is easy to get, cheap, technology is simple, and is easy to use, environmentally safe, no fermentation product is residual, is genuine green non-pollution bactericide.But the product heat endurance is relatively poor, and different temperatures is preserved half a year, and 10 ℃ of following preventive effects are more than 95%, and 15 ℃ of following preventive effects are more than 80%, and 25 ℃ of following preventive effects should 10 ℃ of following low temperature storages more than 65%.

Four, embodiment

1, the preparation of carbon source powder

Get wheat bran or rice chaff crushed after being dried, perhaps pulverize fresh potato peeling back, standby.

2, chitinous preparation

Get crab shell, dried small shrimp or shell and reject remaining (remaining) slough, clean the back oven dry, pulverize, drop in the technical hydrochloric acid and soaked 24 hours, and constantly stir, be washed to neutrality, redistilled water flush away chlorion, crushed after being dried, standby.

3, culture fluid preparation and fermented and cultured

Get wheat bran or rice husk meal 25～30g, add 200～350ml, boiled 0.5～1 hour, filter, treat that filtrate cooling back adds 0.2g FeSO ₄, 1g KH ₂PO ₄, 1.5g K ₂HPO ₄, 2g MgSO ₄With the 20g chitin, perhaps get fresh potato powder 25～30g, add in 200～350ml water, add 0.2g FeSO then ₄, 1g KH ₂PO ₄, 1.5g K ₂HPO ₄, 2g MgSO ₄With the 20g chitin, be settled to 1000ml with distilled water diluting, transfer pH to 6.0～6.2 with sodium citrate solution at last.

To inoculate behind the culture fluid autoclaving, adding does not have the mould spore suspension 1ml of assorted bacterium wood, contains spore 1 * 10 ⁶Individual/ml.In 26～28 ℃ of condition bottom fermentations about 1 week, measure terminal point pH, isolated by filtration when pH≤5.3, and transfer pH≤4 with oxalic acid, the sedimentation isolated by filtration is transferred pH5.8～6.2 with sodium tripolyphosphate solution, promptly is biological bactericide.10 ℃ of following low temperature storages.

Claims

1. the preparation method of the biological bactericide of a botrytis resistant, comprise preparation, sterilization, inoculation, the fermented and cultured of liquid nutrient medium and separate, it is characterized in that: on the liquid nutrient medium of the mould pH5.7 of being seeded in of wood～6.5 in 25～40 ℃ of condition bottom fermentations 5～10 days; Described liquid nutrient medium carbonaceous sources powder 1～5%, chitin 0.5～3.5%, FeSO ₄0.01 KH～0.03%, ₂PO ₄0.02 K～0.2%, ₂HPO ₄0.05 MgSO～0.25%, ₄0.05 water surplus～0.35%; Zymotic fluid after the separation is transferred pH5.8～6.2; Described carbon source powder is selected from the bright powder of wheat bran juice or rice chaff juice or potato.

2. preparation method according to claim 1 is characterized in that: on the liquid nutrient medium of the wood mould pH6.0 of being seeded in～6.2 in 26～28 ℃ of condition bottom fermentations 6～8 days; Described liquid nutrient medium carbonaceous sources powder 2～4%, chitin 1～3%, FeSO ₄0.015 KH～0.025%, ₂PO ₄0.05 K～0.15%, ₂HPO ₄0.1 MgSO～0.2%, ₄0.1 water surplus～0.3%.

3. preparation method according to claim 1 and 2 is characterized in that: liquid nutrient medium carbonaceous sources powder juice 3%, chitin 2%, FeSO ₄0.02%, KH ₂PO ₄0.1%, K ₂HPO ₄0.15%, MgSO ₄0.2%, water surplus.