CN106399129B

CN106399129B - One plant of trichoderma harzianum strain and its application

Info

Publication number: CN106399129B
Application number: CN201611087340.8A
Authority: CN
Inventors: 秦云霞; 阳江华; 唐朝荣; 肖小虎; 龙翔宇; 方永军; 戚继艳
Original assignee: Rubber Research Institute Chinese Academy Tropical Agricultural Sciences
Current assignee: Rubber Research Institute Chinese Academy Tropical Agricultural Sciences
Priority date: 2016-12-01
Filing date: 2016-12-01
Publication date: 2019-09-20
Anticipated expiration: 2036-12-01
Also published as: CN106399129A

Abstract

The invention discloses one plant of Trichoderma harzianum and its applications.The trichoderma strain is Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163.There is wide spectrum to inhibit disease fungus activity for the bacterial strain and its metabolite, face-off growth experiment shows, the trichoderma strain has antagonism Rubber Red Root Disease bacterium, and the ability of a variety of pathogens such as anthrax bacteria also has the function of the other a variety of pollution microbes of prevention and treatment and promotes rubber seedling growth.Therefore it can be applied to rubber tree seedling stage cultivating process immediately, promote the healthy growth of rubber seedling, shielding or the generation for inhibiting rubber tree root disease.

Description

One plant of trichoderma harzianum strain and its application

Technical field

The present invention relates to one plant of multi-functional trichoderma harzianum strain and its applications.

Background technique

Trichoderma Trichoderma Pers. (perfect stage was once known as " Hypocrea " Hypocrea Fr.) is under the jurisdiction of excrement Shell Gammaproteobacteria Sordariomycetes, Ascomycota Ascomycota, Hypocreaceae Hypocreaceae, meat seat mesh Hypocreales.The category fungal species are various, widely distributed, are a kind of important renewable natural resources, have higher economy Value and application prospect (Doi 1969,1972；Zhu Zhaoxiang and Zhuan Wenying, 2014).Trichoderma (Trichoderma spp.) is A kind of important multi-functional prebiotic fungi, is widely present in nature.They can not only control cause of disease by number of mechanisms The generation of bacterium infected with disease, and the ability for also having and promoting plant growth, improve crop antibiont and abiotic stress are A kind of generally acknowledged environmentally friendly fungi is applied to the fields such as agricultural, industry, environmental protection, and part of type is to phytopathy Fungal pathogens have stronger killing ability, and since generation nineteen fifty, fungus Trichoderma obtains people as the function of biocontrol microorganisms gradually Attention and application.

Studies have shown that the pathogenic bacteria of Rubber Red Root Disease are Ganoderma pseudoferreum (Wakef.) Over.et Steinm has the characteristics that host is more, incubation period is long, it is rapid to spread in the soil, is the root infection for seriously endangering rubber tree Venereal disease evil.Red root disease pathogen belongs to mycota Hymenomycetes, Basidiomycota, and Ganoderma endangers Para rubber tree rootstock, mainly By root system contagion, wind and rain and entomochory can also be passed through.Rubber Red Root Disease occurs generally in each area Zhi Jiao, China, Serious woods section disease incidence is infected up to 4.0%, if can be 100% without handling the death rate in time.The investigation of rubber tree root disease A large amount of manual labors are needed, early stage follow-up investigation work is not in place, generally investigates low efficiency.And when rubber tree overground part occurs significantly It has been to infect serious occurrent time when illness, control cost is high and preventive effect is poor.

Since rubber tree is perennial high megaphanerophyte, the multiple diseases such as red root disease are effectively prevented using biocontrol microorganisms, will be one Item extends rubber tree and adopts the glue time limit, protects the strong behave of environment.

Although Trichoderma is a kind of resource microorganism having a extensive future, it is raw for carrying out biological control using its Antagonism One importance of state agricultural sustainable development is the hot spot that biocontrol agent is developed in recent years, but to screen height Effect prevents and treats the specific pathogen of a certain plant and is purposefully used for biological control, needs to do a large amount of element task.Such as The problems such as needing to consider numerous biological adaptation, its expansion of the Trichoderma, preservation, production and application cost, degenerating and make a variation.

Summary of the invention

Trichoderma and plant cooperated evolution, it is unique and enrich, and also Trichoderma has Different Crop or even different cultivars Specificity, inventor have found that Trichoderma has very strong external antagonism energy to Rubber Red Root Disease germ by the experiment of many years Power, it is contemplated that the Local Adaptation ability of Trichoderma and the difference of antagonistic ability, therefore determine to screen local dominant strain.

The object of the present invention is to provide one plant of Trichoderma harzianum (Trichoderma with wide spectrum inhibition disease fungus Harzianum) Hb13-3-2 bacterial strain, it is the local bacterial strain isolated from Hainan Tropical area, is not only able to inhibit rubber Tree red root germ infects rubber seedling, and rubber seedling can be promoted to grow, and is the probiotic strain with application value.

The resulting bacterial strain of the present invention has fast, at low cost, the antibacterial spectrum width of growth and breeding and to leaf diseases and root disease Evil has the advantages that inhibiting effect.The object of the present invention is to provide one plant to be able to suppress Rubber Red Root Disease bacterium, rubber tree stick spore The Trichoderma with wider antimicrobial spectrum of a variety of germs such as mould fallen leaves germ, anthracnose of rubber trees bacterium, it grows fastly, expands numerous efficiency Height, and the growth of rubber seedling can be promoted, thus be expected to be applied to the bacterial strain that rubber seedling is commercially produced.

The present invention by from produce glue rubber tree rhizosphere, Rubber plantation soils, rubber tree germplasm materials different tissues in bolter Choosing, purifying obtain In Vitro Bacteriostasis antagonistic effect up to 80% or more Trichoderma harzianum biocontrol microorganisms Hb13-3-2 bacterial strain；Utilize tissue culture material Material, is inoculated with Trichoderma and red root germ simultaneously in vivo, and discovery Trichoderma inhibits infect efficiency of the red root germ on tissue-cultured seedling to reach It to 100%, is found after the biological characteristics and chlamydospore Formation and characteristics for investigating the trichoderma strain, which will be prevention and treatment rubber The preferred strain of the bio-bacterial manure exploitation of gum red root disease can be applied to biological control and promote agricultural sustainable development application In.

Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 of the present invention is from Hainan Province Danzhou City rubber It is isolated in the rubber tree rhizosphere soil of one plant of growing area infection red root disease, China is preserved on October 21st, 2016 Microbiological Culture Collection administration committee common micro-organisms collection (abbreviation CGMCC, address are as follows: city, BeiJing, China Haidian District Zhong Guan-cun north one No. 13), deposit number is CGMCC No.13163.

The basic culture morphological feature of Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 are as follows: in PDA On plate, the average growth rate of 28 DEG C of culture colony diameters is 4.9cm/ days.Mycelia have branch, have every, it is colourless, it is straight or curved Song is smooth；The symmetrical branch of conidiophore or once in a while verticillate branch, without every metulae is slightly thicker than stalk top, generally there is 2-3 times point Branch, sporogenic stigma doleiform estranged；Conidium monospore, ellipticity or spherical.

Also belong to using above-mentioned Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 as the bacteria agent of active constituent In protection scope of the present invention.It when needed, also may include common carrier and auxiliary material in microbial inoculum preparation in the microbial inoculum.

Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 is as pathogenic Application in bacterium Antagonistic Fungi also belongs to protection scope of the present invention.

Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 inhibits plant in preparation Application in the microbial inoculum of pathogen also belongs to protection scope of the present invention.Wherein, the phytopathogen is Rubber Red Root Disease Bacterium, the mould fallen leaves germ of rubber tree stick spore, anthracnose of rubber trees bacterium, banana blight bacteria and/or yampi anthrax bacteria.

A kind of phytopathogen drip irrigation is also claimed in the present invention, and active constituent is Trichoderma harzianum (Trichoderma harzianum)Hb13-3-2 CGMCC No.13163。

A kind of bio-bacterial manure, including Trichoderma harzianum (Trichoderma harzianum) Hb13- is also claimed in the present invention 3-2 CGMCC No.13163。

It further include molybdenum element, Zn-ef ficiency and/or copper in the bio-bacterial manure.The molybdenum element is added by ammonium molybdate Add, the mass percentage concentration of addition is 0.05%-0.3%；The Zn-ef ficiency by zinc sulfate add, the zinc ion of addition it is dense Degree is 1-4mM, and the copper is added by copper sulphate, and the mass percent concentration of addition is 0.0001-0.001%.

It can also include a great number of elements and/or organic fertilizer in the bio-bacterial manure.

Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 is promoting rubber tree children Seedling or tissue-cultured seedling growth and/or the application in the bacteria agent or bio-bacterial manure that preparation promotes rubber seedling or tissue-cultured seedling growth Also belong to protection scope of the present invention.

Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC is also claimed in the present invention Application of the No.13163 in the preparation antibacterial gas of volatility；Wherein, escaping gas is to Rubber Red Root Disease bacterium, rubber bar spore Mould fallen leaves germ, anthracnose of rubber trees bacterium, banana blight bacteria and/or yampi anthrax bacteria have fungistatic effect.

The resulting bacterial strain of the present invention has fast, at low cost, the antibacterial spectrum width of growth and breeding and to leaf diseases and root disease Evil has the advantages that inhibiting effect.There is wide spectrum to inhibit disease fungus activity the bacterial strain and its metabolite, can generate and has Inhibit disease fungus active volatile substance, the bacterial strain can antagonism Rubber Red Root Disease bacterium, antagonism anthrax bacteria, to rubber Seedling is harmless, and after suitably increasing Trichoderma fast-propagation needed nutrient matter, it is numerous to can be widely applied for plant tissue culture Prevention and cure of pollution, the promotion growth educated, shorten the time of culture of rootage and strong sprout.

Using group training material, it is inoculated with Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 trichoderma simultaneously in vivo Bacterium and red root germ find that Trichoderma of the invention reaches 100% to the inhibiting rate of red root germ, in conjunction with plate face-off result and Rubber seedling co-cultures test result it is found that the bacterial strain will be the preferred strain preventing and treating the bio-bacterial manure of Rubber Red Root Disease and developing.

Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 of the invention and its generation Thank product with wide spectrum inhibit disease fungus activity, can generate with inhibit disease fungus active volatile substance.Face-off Growth experiment shows that the trichoderma strain has antagonism Rubber Red Root Disease bacterium, and the ability of a variety of pathogens such as anthrax bacteria also has There are the other a variety of living contaminants of prevention and treatment and promotes rubber seedling growth.During rubber seedling large-scale cultivation, have anti- The only contaminated effect of rubber seedling.Therefore it can be applied to rubber tree seedling stage cultivating process immediately, promote the health of rubber seedling raw It is long, shielding or the generation for inhibiting rubber tree root disease.The bacterial strain is harmless to rubber seedling, and fast suitably increasing Trichoderma After speed expands numerous needed nutrient matter, it can be widely applied for preventing and remedying pollution, promoting growth for plant tissue culture breeding, shorten training of taking root Support the time with strong sprout.

Heretofore described trichoderma strain Hb13-3-2 be can within be born in plant.The characteristic is by being inoculated with rubber seedling It, can isolated bacterial strain again after co-culturing a period of time.It is thinner by being carried out to its growth characteristics and possible function After the comparative analysis of cause, discovery such as microelement Cu²⁺、Mo²⁺With moderate-element Zn²⁺To Trichoderma conidium and chlamydospore It is formed with certain influence.Simultaneously to Trichoderma in the prevention and treatment of rubber tree tissue culture seedling pollution, root disease prevention and its to seedling growth Influence analyzed.The result shows that: it, can efficiently anti-decontamination under the conditions of the nutritional need of Reasonable Regulation And Control Trichoderma growth Dye, prevention and treatment root disease, and promote rubber seedling fast-growth, there are good development and application potentiality.In addition, the trichoderma strain It is also stronger to the patience of growth inhibitor potassium bichromate, it can survive under 1000 μ g/mL concentration, which can be applied to Detect its Field information situation.

Detailed description of the invention

Fig. 1 is growth and illumination situation of the Hb13-3-2 in PDA culture medium and in rose-bengal culture The growing state of 7d on base, in Fig. 1 A be Hb13-3-2 the case where being grown 4 days in PDA culture medium, B be that Hb13-3-2 adds in Meng Illumination situation after growing 10 days on red culture medium is drawn, the dyeing of cotton orchid shows its conidiophore and conidium shape State；C and D is the growing state of Hb13-3-2 7d on rose bengal medium in Fig. 1, shows that (C is special for front for its fold line Sign, D are back side growth characteristics).

Fig. 2 is the growth conditions of the Hb13-3-2 bacterial strain when cultivating 10 days on different culture medium plate；A is potato in Fig. 2 Culture medium, B are NA culture medium, and C is carrot culture medium, and D is CA culture medium, and E is maize powder medium, and F is Min culture medium.

Fig. 3 is growing state of the Hb13-3-2 on different carbon source culture medium, and A is mannitol, B is sorbierite, C is sweet Oil, D are starch, E is corn flour, F is galactolipin, G is glucose, H is fructose, I is sucrose, J is xylose.

Fig. 4 is growing state of the Hb13-3-2 under different carbon/nitrogen ratios, wherein A is carbon-nitrogen ratio 0:0 (only addition carbon source And nitrogen source is not added), B is carbon-nitrogen ratio 10:1, and C is carbon-nitrogen ratio 20:1, and D is carbon-nitrogen ratio 40:1, and E is carbon-nitrogen ratio 80:1.

Fig. 5 is growth-promoting functions of the trace element molybdenum to trichoderma strain Hb13-3-2.Mo0 is that molybdenum element is not added in figure, Mo1- Mo5 represents the mass percent concentration of molybdenum element as 0.05%, 0.1%, 0.15%, 0.2% and 0.3%.

Fig. 6 moderate-element Zn²⁺With microelement Cu²⁺To the growth-promoting functions of trichoderma strain Hb13-3-2, (in Fig. 6 I, A is 0PPMCuSO4, B 1PPMCuSO4, C 6PPMCuSO4, D 10PPMCuSO4, the II of Fig. 6, A 0mMZnSO4, B are 0.5mMZnSO4, C 1mMZnSO4, D 2mMZnSO4, E 4mMZnSO4, F 10mMZnSO4).

The face-off of Fig. 7 Trichoderma Hb13-3-2 and red root disease germ B05 grows 9 days effects, and wherein A is red root germ The case where the case where B05 is grown 13 days, B are to inoculate Hb13-3-2 after B05 is first inoculated with 4 days, and face-off co-cultures when growing 9 days.

Fig. 8 is the suppressed feelings of pathogen in Hb13-3-2 bacterial strain on PDA plate and plant pathogenic fungi opposite culture Condition；A and B is rubber tree anthrax bacterial strain HDHL in figure and Hz, C are the mould fallen leaves germ of rubber tree stick spore, E is banana blight bacteria FOC4, D and F respectively represent two plants of anthrax bacterias 12DP116 and 12DP06 of yampi.

Fig. 9 is that Hb13-3-2 and the mould fallen leaves germ YN49 of banana blight bacteria and rubber bar spore stand facing each other on PDA plate Cultivating 6 days test result I is stood facing each other 6 days with FOC4, and II is and YN49 opposite culture 6 days.

Figure 10 is the feelings that Hb13-3-2 inhibition yampi anthrax bacteria 12DP06 (I) and 12DP116 (II) infect cassava blade (A and C) is to be inoculated with pathogen and Trichoderma simultaneously above every leaf in condition figure, and lower section (B and D) be that only an inoculation pathogen makees For control.

Figure 11 is that Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 generates volatilization Property substance and the substance inhibiting effect of disease fungus is tested.A in figure, C, E, G are control, and B, D, F and H are produced by volatility Repressed situation after object influences.

Figure 12 is that Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 saves pollution The test A of rubber test tube seedling, (1-12) are that heat grinds 7-33-97 and gradually forms health from root clone embryo from sprouting respectively The young plant being contaminated during body；B is that pollution young plant shown in A is inoculated with to Hb13-3-2 nutrient solution immediately on the same day, 24 days Pollution young plant afterwards is rescued into appearance living, and (each test tube is inoculated with Hb13-3-2 conidium nutrient solution 1ml, and concentration is 10⁷A/ml).

Figure 13 is that Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 promotes rubber The test I and II of test tube seedling growth are independent repeated trials twice, and rubber seedling is than control growth after display is handled with Hb13-3-2 Faster, more strong as a result, in II arrow meaning be Newborn Leaves it is fluffy, compare the fluffy appearance of no Newborn Leaves.

Figure 14 is that can open examination in advance using the protection of Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 The test of the lid nurturing staff of Guan Miao.

Figure 15 is colonisation A of the Hb13-3-2 in plant different tissues, main root or lateral root section；B, stem section；C, inferior leads It is sliced piece；D, top tender leaf slice.

Specific embodiment

Method in following embodiments is unless otherwise instructed conventional method.

The screening and identification of embodiment 1, Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 bacterial strain

1, the screening of trichoderma strain

Hb13-3-2 bacterial strain is us from the rubber tree rhizosphere of the one plant of infection red root disease in Hainan Province, rubber planting area, Danzhou City It is isolated in soil, the soil sample of rubber tree rhizosphere 5-10cm soil layer is acquired, is compiled by locality sequence and separation sequencing Number.Using dilution-plate method, 10 are pressed to each soil sample^-3、10^-4Various concentration dilution, be inoculated on rose bengal medium (PDA + chloramphenicol 0.015g/L+ streptomysin 0.015g/L+ Bengal rose red 0.05g/L), 28 DEG C of culture 3d；Picking Trichoderma bacterium colony It is inoculated on rose bengal medium (not added with antibiotic), 28 DEG C of culture 3d, further single spore separation purifying obtains Trichoderma Strain, then number identification.

The screening of pathogen Antagonistic Fungi is carried out to the trichoderma strain isolated and purified, it is final to obtain one plant to a variety of pathogens spies It is not the Trichoderma that Pathogenic Fungi of Rubber Red Root Disease has excellent antagonism performance, is named as Hb13-3-2.

2, the morphological feature of Hb13-3-2 bacterial strain

Hb13-3-2 strain inoculated is cultivated on PDA culture medium plate, the morphological feature of the bacterial strain are as follows: in PDA plate On, 28 DEG C of colony diameters after culture 1 day are 4.9cm, there is a substrate mycelium and aerial hyphae, and mycelia has branch, have every, it is colourless, directly Or bending, it is smooth；The symmetrical branch of conidiophore or once in a while verticillate branch, without every metulae is slightly thicker than stalk top, generally there is 2-3 Secondary branch, sporogenic stigma doleiform estranged；Conidium monospore, ellipticity or spherical.Basic growth characteristics and classification are such as Shown in table 1 and Fig. 1.

The basic growth characteristics and classification of 1 Hb13-3-2 of table

Hb13-3-2 is on PDA plate, and mycelia growth is vigorous, and bacterium colony is smooth, and edge is relatively regular；In rose bengal medium The upper flat plate back side generates obvious and relatively regular fold line (see Fig. 1-D rose bengal medium plate back side fold line).

3, the present invention in trichoderma strain Hb13-3-2 Molecular Identification

ITS1-5.8S-ITS2 sections of sequences are expanded using universal primer ITS4, ITS5, are sent to Shanghai biotechnology clothes The sequencing of business Co., Ltd, gained sequence information carry out sequence alignment by TrichoKEY software, identify bacterial strain type.

A. the extraction of reesei gene group DNA

Referring to the method and slightly modified of (2008) such as Cao Wenbo, the specific steps are as follows: collection, which is separately cultured, is grown in PDA The mycelia of Hb13-3-2 bacterial strain on culture medium weighs 0.1g liquid nitrogen grinding, and the CTAB extracting solution having had been warmed up is added (100mmol/L Tris-HCl, pH8.0,1.4mol/L NaCl, 20mmol/L EDTA, pH8.0,2%CTAB), and add few Perhaps the quartz sand to have sterilized is moved into centrifuge tube after being fully ground spore in mortar, and is incubated for 15 minutes at 65 DEG C, is added Enter isometric phenol/chloroform/isoamyl alcohol (25:24:1v/v/v) solution, firmly shakes mixing of turning upside down, 12000r/min It is centrifuged 10min, supernatant is moved into another new centrifuge tube, isometric chloroform/isoamyl alcohol (24:1v/v/v) is added Solution, firmly shakes mixing of turning upside down, and 12000r/min is centrifuged 10min；Supernatant is taken to be added into another new centrifuge tube The 3mol/LNaAc solution (pH5.2) of isometric isopropanol and 1/10 volume, one 20 DEG C of placements 20min, 12000r/min from Heart 5min, abandoning supernatant, inversion flow to end tube wall liquid, and adding the rinsing of 70% dehydrated alcohol, DNA is precipitated twice, centrifugation 1min, in abandoning Clearly, it is air-dried at room temperature volatilization ethyl alcohol, aqua sterilisa dissolution is added.Place 4 DEG C of refrigerator overnight dissolutions；One 20 DEG C of preservations.

Mono- area ITS sequencing analysis of b.rDNA

The primer sequence: ITS4:5 '-TCCTCCGCTTATTGATATGC-3 '；ITSS:5'- GGAAGTAAAAGTCGTAACAAGG-3'；PCR reaction system (25 μ L): 0.20 2.5 μ L of μ L, 10xbuffer of Taq enzyme, dNTP Each 21.1 μ L of 0.5 μ L, ddH2O of primer of 0.2 μ L, 10 μ L.DNA profiling 25ng PCR amplification program: 94 DEG C of initial denaturations 4min, 94 DEG C are denaturalized 30 seconds, 57 DEG C of annealing 1min, 72 DEG C of extension 1min, and totally 30 recycle；Last 72 DEG C of extensions 10min.

As a result rDNA-ITS sequence ITS1 as shown in sequence 1 in sequence table of the bacterial strain, the region ITS2 partial sequence are sent out It is 98~100% that existing its, which belongs to the rDNA-ITS region sequence similitude of bacterial strain from different Trichoderma,.

Tef1 identifies that sequence as shown in sequence 2 in sequence table, selects the α-genetic fragment of extension factor 1 (tef1) for reflecting It is fixed, it is to be capable of in the kind of discrimination, kind because theoretically introne is longer containing the 4th long introne in the segment Between sequence difference sequence it is abundanter, can be preferably applied to identification difference.

Hb13-3-2 bacterial strain is accredited as Trichoderma harzianum (Trichoderma by above-mentioned phenotypic characteristic and characterization of molecules Harzianum), China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on October 21st, 2016 (abbreviation CGMCC, address are as follows: city, BeiJing, China, north, Haidian District Zhong Guan-cun one No. 13), deposit number is CGMCC No.13163.

4, Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 difference store method Research

Using PDA culture medium as activation medium.Four kinds of methods are mainly used, are 4 DEG C of plate preservation methods respectively, often Fire stons wax oil preservation method, -80 DEG C of ultra-low temperature preservation methods, inclined-plane preservation method.4 DEG C of plate preservation methods: PDA plate is inoculated with Hb13-3-2 After fungus block, 28 DEG C are stored in 4 DEG C after dark culture 1 week；- 80 DEG C of ultra-low temperature preservation methods: after PDA plate is inoculated with Hb13-3-2 fungus block, 28 DEG C after dark culture 2 weeks, are washed conidium get off, be uniformly mixed with sterile glycerol with nutrient solution, are saved in -80 DEG C；Inclined-plane Preservation method: slant activation is cultured, there are a large amount of mature conidiums to protect 4 DEG C of tube preservations.Room temperature paraffin oil preservation method: It is cultured to slant activation, there are a large amount of mature conidiums to protect in tube the paraffin oil (paraffin oil being added after stringent sterilization Amount sufficiently to cover culture face) sealed membrane closing, room temperature preservation.

As a result, it has been found that room temperature paraffin oil preservation method can keep the activity of bacterium, but mycelia relative growth is slow when resurrection；4 DEG C plate holding time more long contaminated possibility is big.To reduce pollution, in conditional situation, -80 DEG C can be used Preservation method makees long-term preservation.

The biological characteristics of embodiment 2, Hb13-3-2 bacterial strain

One, the carbon source of Hb13-3-2 bacterial strain, nitrogen source demand characteristics

1, Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 the most suitable growth culture The determination of base

Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 consolidates in as described below The comparison of growing state on body culture medium (pH6) plate:

Min (Minimal agar medium) culture medium: KNO₃10g, KH₂PO₄5g, MgSO₄·7H₂O 0.5g, FeCl₃0.02g, glucose 10g, agar 20g, constant volume to 1000mL；

PDA (potato glucose) culture medium: peeled potatoes 200g, glucose 20g, agar 14g, peeled potatoes boil Filtrate is settled to 1000mL with distilled water after boiling 30min；

CMV (corn flour) culture medium: filtrate is settled to after boiling 30min with distilled water by corn flour 30g, agar 14g 1000mL；

OA (oat) culture medium: filtrate is settled to after boiling 30min with distilled water by oatmeal 30g, agar 14g 1000mL；

CA (carrot) culture medium: carrot 200g, glucose 20g, agar 14g will be filtered after boiling 30min with distilled water Liquid is settled to 1000mL；

NA culture medium: beef extract 3g, peptone 5g, agar 14g are settled to 1000mL；

Above-mentioned culture medium is at 121 DEG C, and sterilize 20min.

The above-mentioned six kinds of solid mediums for pouring into 20mL thawing in the culture dish of diameter 9cm respectively, up to each after solidification The equal thickness solid plate of kind culture medium, in the Trichoderma harzianum (Trichoderma of plate center inoculation diameter about 4mm size Harzianum) Hb13-3-2 CGMCC No.13163 fungus block, every kind of culture medium is set to be repeated three times, 28 DEG C of dark cultures, is surveyed daily Bacterium colony size is measured, colony growth rate is calculated, takes pictures after 10 days.

The results are shown in Table 2, and the speed of growth is most fast on CA plate, is secondly PDA plate, Min plate, OA plate, NA Plate and CMV plate, the speed of growth on CMV plate are most slow.As shown in Fig. 2, Hb13-3-2 bacterial strain is in 6 kinds of different culture mediums On mycelial growth rate difference it is little, but there are notable differences for illumination amount, from it in minimum nutrient medium From the point of view of the growing state in corn culture medium, it can be seen that the strain growth and nutritional need be not high, belongs to and is easy to expand numerous class Type.In CMV, NA, bacterium colony quality is loose on PDA and CA culture medium, and bacterium colony quality is closely on Min and OA culture medium.Point The speed sequence of sporogenic formation is Min > CMV > OA > CA > NA > PDA plate, and bacterium colony becomes green, bottle green from light green.

The speed of growth (colony diameter/incubation time) of the table 2.Hb13-3-2 in different culture medium

2, the carbon source of Hb13-3-2 bacterial strain, nitrogen source demand characteristics

Different carbon/nitrogen sources are raw to Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 Long influence

It prepares the culture medium of different carbon source: using Czapek culture medium as minimal medium, using mannitol, glycerol, jade respectively Rice flour, D-Glucose, galactolipin, starch, D-glucitol, D-Fructose, D- xylose replaces the sucrose in Czapek culture medium, other Components unchanged, preparation is using these substances as the culture medium (agar is added and prepares solid medium) of sole carbon source.

Prepare different nitrogen sources culture medium: using Czapek culture medium as minimal medium, respectively use potassium nitrate, ammonium nitrate, Ammonium sulfate, yeast extract, beef extract, yeast extract, peptone replace the sodium nitrate in Czapek culture medium, other ingredients Constant, preparation is using these substances as the culture medium (agar is added and prepares solid medium) of only nitrogen source.

The formula of above-mentioned Czapek culture medium are as follows: MgSO47H₂O 0.5g, K₂HPO₄1g, KCl 0.5g, FeSO₄ 7H₂O 0.01g, NaNO₃2g, sucrose 20g, are settled to 1000mL, and pH7.0 (is added agar and prepares solid medium).Through 121 DEG C, 20min sterilizing.

Its bacterium colony average diameter calculated growth speed is surveyed using solid plate method: that above-mentioned different carbon originals and nitrogen are former respectively After solid medium sterilizing, poured into the culture dish of diameter 9cm respectively, 20mL/ ware, the above-mentioned difference of equal thickness after solidification to obtain the final product Solid medium tablets.Plate center inoculation diameter about 4mm size fungus block, if repeating three times, 28 DEG C dark culture 3 days, use Crossing method measures colony diameter.The results are shown in Table 3, the results showed that, on the plate for being sucrose containing sole carbon source, life Length it is fastest, and containing sole carbon source be D-glucitol plate on grow it is worst, with D- xylose (J in Fig. 3) and gala Sugared (F in Fig. 3) is that its speed of growth is not so good as in glycerol, starch, corn flour, D-Fructose, PEARLITOL 25C on the plate of sole carbon source With (Fig. 3) on D-Glucose plate.

Speed of growth difference of Hb13-3-2 bacterial strain under the conditions of 10 kinds of carbon sources is unobvious, but different carbon source is to mitogenetic spore Sub- forming quantity and speed influence are very big, such as on glycerol, starch and maize powder medium, can quickly form a large amount of point Raw spore.The factors such as the price of the product of consideration, so when expanding numerous culture Hb13-3-2 later, Ying Xuanyong corn flour, glycerol, shallow lake Powder is as carbon source.

As shown in table 3, it on plate containing only nitrogen source, is grown most on the plate using yeast extract as only nitrogen source It fastly, is secondly respectively yeast extract, sodium nitrate, potassium nitrate, ammonium sulfate, soy peptone, and it is flat in beef extract and ammonium nitrate It is grown on plate most slow.Though the speed of growth of the Hb13-3-2 bacterial strain under 8 kinds of nitrogen conditions is variant, it is contemplated that product price Etc. factors, bacterium cultivate when we select ammonium sulfate as nitrogen source.

Hb13-3-2 strain growth diameter (mm/ days) on the different unique carbon nitrogen sources of table 3., carbon source plate

Two, the carbon/nitrogen ratio demand characteristics of Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 bacterial strain

Since Hb13-3-2 can take into account ready availability, the present invention of cost and material well using corn flour as carbon source It is that (0 refers to 0:0 using corn flour and ammonium sulfate as carbon source (30g/L) and nitrogen source (molar ratio that ratio is respectively carbon source and nitrogen source) Be plus carbon source, nitrogen source is not added), 10:1,20:1,40:1,80:1), add K₂HPO₄1g/L, KCl 0.5g/L, FeSO₄.7H₂O 0.01g/L, MgSO₄.5H₂O 0.5g/L, Agar 14g/L, investigates its carbon, nitrogen ratio demand characteristics.

As a result as shown in table 4 and figure 4, in identical incubation time Hb13-3-2 bacterial strain on the culture medium of 5 kinds of ratios There is illumination, it is more with the quantity that the ratio (ammonium sulfate is not added and only adds carbon source) of 0:0 is formed, illustrate bacterium growth to carbon Source demand is more, seldom to nitrogen source demand.

The growing state of table 4, Hb13-3-2 bacterial strain on different carbon/nitrogen ratio culture medium

Three, Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 strain growth temperature Range research

The preparation of PDA plate: pouring into the PDA solid medium that 20mL dissolves in the culture dish of diameter 9cm, after solidification i.e. The PDA solid plate for obtaining equal thickness, the Hb13- for being 3 days in the cell age for the diameter about 4mm size that the inoculation punching of plate center obtains 3-2 fungus block is respectively placed in (4 DEG C (processing 3 days), 16 DEG C (processing 3 days), 20 DEG C (processing 3 days), 28 DEG C, 37 DEG C of different temperature With 45 DEG C (processing 1h), 50 DEG C (processing 1h), 55 DEG C (processing 1h)) after be placed in dark culture at 28 DEG C, each test temperature again If repeating three times, bacterium colony growing state is observed.In used 8 temperature, 28 DEG C grow optimum temperature, 55 DEG C of processing for it 1h will not also be dead although mycelia growth is reduced to 1cm/ days, illustrates that the bacterium all has certain fit to high temperature and low temperature It should be able to power.

Embodiment 3, Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 bacterial strain to specific culture condition Patience

One, the saline-alkali tolerant characteristic of Hb13-3-2 bacterial strain

It prepares and contains salt NaCl (mass percentage concentration is respectively 1%, 3%, 5%, 7%, 10% or 15%) or KCl The PDA culture medium of (mass percentage concentration is respectively 1%, 3%, 5%, 7%, 10% or 15%) contains alkali part KOH (quality hundred Point concentration be respectively 1%, 3%, 5%, 7% or PDA culture medium 9%), containing NaOH (mass percentage concentration is respectively 1%, 3%, 5%, 7% or PDA culture medium 9%) or contain Na₂CO₃(mass percentage concentration be respectively 1%, 3%, 5%, 7% or 9%) PDA culture medium, for detecting the tolerance saline and alkaline to various concentration of Hb13-3-2 bacterial strain.

The plate of above-mentioned culture medium is prepared, and is inoculated with Hb13-3-2 bacterial strain fungus block, each medium treatment repeats three times.28 DEG C dark culture 7 days, colony diameter was measured with crossing method.

The results are shown in Table 5, and Hb13-3-2 bacterial strain is at least resistant to the NaCl that mass percent concentration is 7%, at least can Enough it is resistant to 11%KCl.But it is very poor to the tolerance of alkali, in three kinds of alkali KOH, NaOH or Na₂CO₃Mass percent concentration is 1% Concentration on can not grow.

Table 5.Hb13-3-2 bacterial strain salt containing various concentration, alkali PDA plate on grow 7 days after bacterium colony average diameter (cm)

Three, the pH characteristic of Hb13-3-2 bacterial strain

Trichoderma (Trichoderma) Hb13-3-2 CGMCC No.13163 grows pH value range detection:

It prepares the PDA plate of different pH value: adjusting liquid PDA culture medium pH value to 4 with 1M HCl and 1M NaOH respectively, 5,6,7,8,9,10,11,12, after regulating pH, agar is added and is configured to solid medium, 121 DEG C, 20min sterilizes to obtain PH Value is respectively 4,5,6,7,8,9,10,11,12 PDA plate.

It is inoculated with one respectively in the PDA plate center that the pH value of above-mentioned preparation is respectively 4,5,6,7,8,9,10,11 or 12 Beaten with sterilization punchers take diameter about 4mm size cell age be the culture 4 days of PDA solid plate fungus block, set 28 DEG C of dark trainings respectively It supports, cultivates 3 days, measure colony diameter with crossing method respectively, repeat three times, seek average and standard deviation.As a result such as table 6 It is shown, the results showed that, which can grow in for the examination range of pH4~8, and speed of growth difference is little, and the most suitable growth pH is 5, although can also grow on alkaline medium, speed significantly slows down, it is also contemplated that pH value is not when pH11 and pH12 Accuracy, but can at least illustrate that the bacterium can survive under slight alkali environment.

The speed of growth of the 6 Hb13-3-2 CGMCC No.13163 of table on different pH culture mediums

Embodiment 4, Trichoderma (Trichoderma) Hb13-3-2 bacterial strain CGMCC No.13163 are to various concentration dichromic acid The tolerance of potassium is studied

Potassium bichromate is a kind of heavy metal salt, is able to suppress the growth of fungi, when separating soil actinomycete, culture medium It is middle be added 50 μ g/mL potassium bichromate can be preferable play inhibit fungal contamination effect.This experiment 10 μ g/mL, 50 μ g/ ML, 100 μ g/mL, 150 μ g/mL, 200 μ g/mL, 300 μ g/mL, 400 μ g/mL, 500 μ g/mL, 600 μ g/mL or 1000 μ g/mL Potassium bichromate PDA culture medium plate, be inoculated with Trichoderma (Trichoderma) Hb13-3-2 fungus block, the weight of each concentration Potassium chromate processing is repeated three times, and plate is placed in 28 DEG C of dark cultures, measures colony diameter daily.

The results are shown in Table 7, and with the raising of potassium bichromate concentration, the speed of growth of bacterial strain Hb13-3-2 is successively decreased.Hb13- 3-2 bacterial strain is on the PDA plate containing 1000 μ g/mL potassium bichromates, and colony diameter is 1.3cm after 1 day, and colony diameter can after 5 days Up to 7.1 ± 0.5cm.Since the bacterial strain has stronger patience to potassium bichromate, it is investigated afterwards in the field planting of plant root In qualification process, can be very good to inhibit living contaminants using the characteristic, so as to by itself or related bacterium from host plant It separates.

7. Trichoderma of table (Trichoderma) Hb13-3-2 is flat in the bacterium colony of the PDA plate of the potassium bichromate containing various concentration Equal diameter (cm/ days)

Concentration (μ g/mL)	10	50	100	150	200
						Average diameter (cm)	2.72±0.12	2.51±0.11	2.17±0.15	2.12±0.08	1.84±0.09
Concentration (μ g/mL)	300	400	500	600	1000
						Average diameter (cm)	1.57±0.09	1.50±0.07	1.47±0.14	1.33±0.08	1.30±0.1

Embodiment 5, trace element molybdenum, copper and moderate-element zinc are to Trichoderma harzianum (Trichoderma harzianum) The growth-promoting functions of Hb13-3-2

One, growth-promoting functions of the trace element molybdenum to trichoderma strain Hb13-3-2

Molydbenum fertilizer have it is 1) with high purity, without to crop nocuousness magazine ingredient 2) solubility property is good: effective component is completely water-soluble, 3) absorption efficiency is high: soda acid appropriateness, nutrient form are suitble to crop to absorb；4) fertilizer efficiency is lasting: there is good blade adhesive ability, Resistance of rainwater washing against, fertilizer efficiency are lasting；5) the features such as raising productivity and improving the quality.In order to explore molydbenum fertilizer whether can promote Trichoderma growth this ask Topic, the present invention using PDA be minimal medium, add 5 mass percent concentrations (0,0.05%, 0.1%, 0.15%, 0.2%, 0.3%) ammonium molybdate, inoculation plate are placed on 28 DEG C of dark cultures 10 days, simultaneously with crossing method measurement colony diameter Observe illumination amount.The result shows that addition molydbenum fertilizer can promote Trichoderma mycelia quick within the scope of 0.05%-0.3% It grows, thicken and illumination, it is best with the molydbenum fertilizer effect for adding 0.2%.Specifically as shown in figure 5, in Fig. 5, A is to be not added Molybdenum element；B is the molybdenum element for adding 0.05%；C is the molybdenum element for adding 0.1%；D is the molybdenum element for adding 0.15%；E is to add Add 0.2% molybdenum element；F is the molybdenum element for adding 0.3%.

Two, the growth-promoting functions of moderate-element zinc and copper to Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2

Using PDA be minimal medium, add 6 concentration (0,0.5,1,2,4,10mM) zinc sulfate or concentration be 0, 1PPM, 6PPM, 10PPM (it is hundred a ten thousandths that 1PPM, which is equivalent to mass percentage concentration) copper sulphate is configured to corresponding plate, together 28 DEG C of dark cultures are placed on 10 days after inoculation in one day, measure colony diameter with crossing method and observe illumination amount.

The result shows that in 1-10PPM Cu²⁺In ion range and 1-4mM Zn²⁺Concentration range in, addition Zn-ef ficiency and Copper can promote Trichoderma mycelia fast-growth, thicken or illumination (Fig. 6).

Embodiment 6, Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 are to a variety of The test of pathogen antagonism

One, pair of Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 and pathogen It stands erect test

Detect Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 and disease fungus Antagonistic activity, including Rubber Red Root Disease bacterium, the mould fallen leaves germ of rubber tree stick spore, anthracnose of rubber trees bacterium, banana blight Bacterium, yampi anthrax bacteria etc..

Have for examination disease fungus: No. 4 microspecies of banana blight bacteria (Fusarium oxysporium f.sp.cubense) (FOC4) (Sun Yong, Zeng Huicai, Peng Ming, Wang Xuchu, Yi little Ping * banana blight pathogenic molecular mechanism and study on prevention are in progress [J] tropical crops journal 2012,33 (4): 759-766), rubber anthrax bacteria (Colletotrichum Gloeosporioides is bought in Chinese agriculture microbial strains preservation administrative center, deposit number ACCC No.30012), Following numbers according to it in antagonistic experiment are named as HDHL, the mould fallen leaves germ (Corynespora of rubber tree stick spore Cassicola is bought from China General Microbiological culture presevation administrative center, deposit number CGMCC No.3.10072), following It is named as HbYN58 according to its number in antagonistic experiment, in addition voluntarily acquires and identifies that one plant of rubber tree stick spore is mould and fall Leaf disease bacterium is named as YN49 according to its number in antagonistic experiment；Two plants of yampi anthrax bacterias are conventionally from Hainan Yampi field acquires and identifies, is named as 12DP06 and 12DP116 according to its number in antagonistic experiment, rubber tree is red Root disease pathogen (G.pseudoferreum) bacterial strain YN1 (quick etc., the RAPD analysis of Rubber Red Root Disease bacterium genetic polymorphism is applied, 2012, Chinese Plants pathology can Annual Conference collection of thesis in 2012), B05 and Rubber Red Root Disease bacterium (Hainan separation strains) DF, Different rubber planting areas are picked up from by doctor Tu Min of germ plasm resource seminar, our unit, conventionally from variable rate technology red root Sample, purifying, culture, identification traits etc. are acquired on the apparent rubber tree of disease symptoms.This experiment further molecule before application Identification confirmation, is stored in 4 DEG C of refrigerators with PDA plate, periodically recovery culture.

The Multiplying culture method of Rubber Red Root Disease germ: pass through (A, 1/2MS+CMA；B,CMA；C,PDA；D, PDA+ Hu Radish E, 1/2MS+PDA) 5 kinds of culture mediums test, find red root germ in 1/2MS+CMA (maize powder medium) and 1/2MS+ The amplification cultivation speed of growth is fast in PDA culture medium.

Opposite culture and bacteriostasis rate: on identical 1/2MS+PDA culture medium, compared with the Trichoderma speed of growth, red root The growth of germ B05 or more relatively slow (7.8 ± 0.45mm/ days) uses for the antagonistic effect of more acurrate identification Trichoderma First the cultured Rubber Red Root Disease bacterium of rejuvenation is inoculated on culture plate, is cultivated in 28 DEG C of constant incubators, waits germs Diameter it is long to about 3-4cm when, inoculate Hb13-3-2 CGMCC No.13163 Trichoderma, 2~3cm of the two interval, repeated inoculation 3 times.It culture and routine observation, records and takes pictures in 28 DEG C of constant incubators.The growth diameter of periodic measurement germ, is surveyed simultaneously Amount is not inoculated with the growing state (control) of the germ of Trichoderma, calculates inhibiting rate.And fallen leaves germ mould for rubber bar spore HbYN58 (speed of growth is 6.3mm ± 0.09/ day), (speed of growth of HDHL is 6.0mm ± 0.12/ to anthracnose of rubber trees bacterium It；The speed of growth of Hz is 4.0mm ± 0.15/ day), banana blight bacteria (speed of growth of FOC4 is 3.9mm ± 0.3/ day), (speed of growth of the 12DP116 bacterial strain on culture medium is 10mm ± 0.08/ day to yampi anthrax bacteria, and 12DP06 bacterial strain is 13mm ± 0.05/ day) it is also relatively slow, therefore also as red root germ, it is first inoculated with pathogen, then it is inoculated with Hb13-3-2 wood Mould, the purpose for the arrangement is that in order to more determine whether Trichoderma really is able to kill or inhibit the growth of pathogen, because Culture pathogen, pathogen occupy advantage on plate in advance, if Trichoderma can be last in face-off in the case where being in a disadvantageous position Pathogen is killed or inhibited, could preferably prove its function.Then we have also been made pathogen and Hb13-3-2 on the same day Stand facing each other the test being inoculated on culture plate, and face-off in 6 days is the result is that 100% inhibits (Fig. 9) of pathogen；Red root germ is also The same, while being inoculated with pathogen and Hb13-3-2, Hb13-3-2 is to the inhibiting rate of red root germ up to 100%.Bacteriostasis rate (%) The colony radius of=(colony radius of control group germ-processing group germ colony radius) × 100/ control group germ.Data Processing is carried out using statistical analysis software DPS (7.5 editions).

8. 9 days opposite cultures of table, inhibiting rate of the Hb13-3-2 to Different Kinds of Pathogens fungi

Trichoderma Hb13-3-2 CGMCC No.13163 is to opposite culture the experimental results showed that (Fig. 7), leading in B05 After inoculation 4 days, the diameter of B05 inoculates Hb13-3-2, after opposite culture 9 days, Hb13-3-2 is to red root in 2.7-4.0cm Germ has significant ground antagonistic effect, can quickly inhibit, occupy, even killing red root disease germ mycelium.B05 is complete in figure It is surrounded by Hb13-3-2, non-renewable length.B05 is surrounded by Hb13-3-2 completely in Fig. 7, non-renewable length.In addition, to YN1 and The antagonistic effect of BT also reaches DF similar horizontal (not showing in chart).Trichoderma Hb13-3-2 CGMCC No.13163 couple The mould fallen leaves germ of rubber tree stick spore (C in Fig. 8), anthrax bacteria (A and B in Fig. 8), banana blight bacteria (E in Fig. 8), yampi charcoal The antagonistic activity qualification result of subcutaneous ulcer germ (D and F in Fig. 8) is as shown in Figure 8: Trichoderma Hb13-3-2 can significantly inhibit rubber tree The growth of pathogen HDHL, Hz and HbYN58 also have apparent inhibition to other crop disease FOC4,12DP06 and 12DP116 Effect.

Table 8 is 9 days data results of antagonism: Hb13-3-2 Trichoderma has significant antagonistic effect to 4 kinds of pathogens；Wherein 86.7%-90% is reached to the inhibiting rate of rubber tree rubber bar spore mould and anthrax-bacilus.To banana blight bacteria and yampi anthrax The inhibiting rate of germ is also 50% or more.So Hb13-3-2 is multi-functional Trichoderma.

The test result that pathogen and trichoderma strain Hb13-3-2 are inoculated with simultaneously demonstrates again that Hb13-3-2 is withered to banana The significant antagonism of germ FOC4 and the mould fallen leaves germ YN49 of stick spore.Fig. 9 is Hb13-3-2 bacterial strain and banana on PDA plate Wilt FOC4 and rubber bar spore 6 days test result photos of mould fallen leaves germ YN49 opposite culture.I A is banana in Fig. 9 The case where wilt FOC4 individually cultivates 6 days；B is that opposite culture shows the repressed situation of FOC4.A is rubber in Fig. 9 II The case where tree mould fallen leaves germ YN49 of stick spore individually cultivates 6 days；B is that opposite culture shows the repressed situation of YN49.

Two, Hb13-3-2 is to the in vitro of the inhibition situation of yampi anthrax bacteria 12DP06 (I) and 12DP116 (II) infecting potential Leaf assay

Using in vitro yampi blade, using mechanical damage and the method that is inoculated with fungus block, observation is having feelings existing for Antagonistic Fungi Under condition, anthrax-bacilus is to the suppressed situation of the infection ability of blade.The results are shown in Figure 10, and A and C is Hb13-3-2 in Figure 10 (I) It is inoculated with simultaneously with anthrax pathogen 12DP06；B is only to be inoculated with anthrax-bacilus 12DP06.A is Hb13-3-2 and anthrax in Figure 10 (II) Pathogen 12DP116 is inoculated with simultaneously；B is only to be inoculated with anthrax-bacilus 12DP116.Dark culturing 4 days, take pictures under 28 degree.As a result table It is bright in the presence of Antagonistic Fungi Hb13-3-2, compared with the control, have the withered and yellow area of the damaged blade of Antagonistic Fungi smaller, explanation Pathogenic significant decrease of the anthrax pathogen 12DP06 and 12DP116 to yampi blade, it was demonstrated that Trichoderma Hb13-3-2 inhibits charcoal The infecting potential of subcutaneous ulcer bacterium is able to suppress pathogen infecting on blade.Test is repeated 3 times, as a result close.

Embodiment 7, Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 generation are waved Hair property gaseous compound and the gas test the inhibiting effect of disease fungus

1, PDA plate is prepared with the culture dish that diameter is 9cm, and one piece of diameter of center inoculation on each plate is about The Hb13-3-2 of 4mm size cultivates 2 days fungus blocks, and 28 DEG C are cultivated 4 days, after it covers with plate substantially, i.e., for activity volatilization Property gaseous compound test.

2, using the method for being similar to opposite culture, pathogen fungus block (rubber tree red root is first inoculated on culture medium flat plate Germ B01, B05, anthracnose of rubber trees bacterium HDHL, yampi anthrax bacteria 12DP06), when to wait its size be 3cm or so, according still further to Following methods co-culture.

3, the Hb13-3-2 plate for taking step 1 to cultivate 4 days, gently the ware bottom and 4 by lower section with Hb13-3-2 bacterium colony PDA plate ware bottom (center is with inoculated disease fungus fungus block) back-off that kind connects disease fungus together, is used Parafilm " M " sealing co-cultures；And to tip upside down on ware bottom and the PDA plate ware of disease fungus of not cultivating Hb13-3-2 Bottom buckle together, is co-cultured with parafilm " M " sealing and is compared, repeated three times, and 28 DEG C are observed after dark culture 10 days, according to reality The method for applying example 6 calculates bacteriostasis rate.

The suppressed growing state of pathogen is as shown in figure 11, and right figure is respectively to be produced by Hb13-3-2 in A-D in Figure 11 Liveliness proof escaping gas substance inhibit influence Rubber Red Root Disease bacterium B05 (A and B in Figure 11), B01 (C and D in Figure 11), Anthracnose of rubber trees bacterium HDHL (E and F in Figure 11) and yampi anthrax bacteria 12DP06 (G and H in Figure 11) bacterium colony upgrowth situation, Left figure is the growth shape for not covering the control at the ware bottom (covering sterile ware bottom) for cultivating Hb13-3-2 in A-D in Figure 11 Condition.It can be seen that all growths for trying disease fungus are all suppressed, wherein to Rubber Red Root Disease bacterium (protecting pavilion separation strains B01) disease Bacteria strain inhibiting rate highest shows that bacterial strain Hb13-3-2 can generate the life that active volatile gaseous matter inhibits disease fungus It is long.

Embodiment 8, Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 save dirty Contaminate the growth test of rubber test tube seedling

During the cultivation process, the later period will appear pollution rate and constantly increases, is growth retardation, developmental deformity, old rubber tissue-cultured seedling Phenomena such as seedling, the survival rate transplanted after not only reducing, commodity selling rate and price, and cost is increased (see Figure 12 Middle A).The development to cotyledon, stem, root is just formed from plumule completely to during the young plant of a healthy and strong suitable transplanting, The pollution for the different bacterium that this new individual can all be met with and it is dead.With probiotics Hb13-3-2 deprotect this new individual at It is long, the pollution of other miscellaneous bacterias can be effectively prevented, and promote its healthy Fast Growth, thus will be in the industry development of rubber seedling Play a significant role.

Experiment is to prove Trichoderma harzianum (Trichoderma harzianum) Hb13-3- to the present invention for the first time Whether 2CGMCC No.13163 can save pollution rubber test tube seedling.September is inoculated into 29 totally 24 days from September 5 in 2016, benefit 7-33-97 is ground with heat to gradually form the young plant being contaminated during a healthy individuals from root clone embryo from sprouting and such as scheme A (1-12) shown in, followed by scheme A in pollution young plant on the same day immediately inoculation Hb13-3-2 nutrient solution (connect in each test tube Kind Hb13-3-2 conidium nutrient solution 1ml, concentration 10⁷A/ml), take pictures after 24 days it is handled after contaminated materials whether It is rescued into work.It in each test tube inoculation Hb13-3-2 conidium nutrient solution is configured with PDB+1/2MS inorganic salt solution The conidium newly cultivated, and constant volume is 10⁷A/ml.As a result as shown in figure 12, in the test material in different stages of growth (1-12 of figure A), Hb13-3-2 processing can kill contaminated bacteria.Test result of the invention shows that Hb13-3-2 can be effective It prevents and remedies pollution rubber seedling, makes it from death, reduce economic loss.

In order to investigate whether Hb13-3-2 can promote the growth of rubber seedling, from June 21 in 2016 or 27 days to August 12nd Total 40-50 days.The heat that we select growth potential relatively uniform grinds 7-33-97 rubber tissue-cultured seedling and has carried out second and third time two Secondary inoculation test, the interior inoculation Hb13-3-2 conidium quantity of each test tube of processing group is 10⁷The nutrient solution 1ml of a/ml (is used PDB+1/2MS inorganic salt solution is prepared), it is conidial that the interior addition equivalent of each test tube of control group does not add Hb13-3-2 PDB+1/2MS inorganic salt solution.

As a result as shown in figure 13, Figure 13 is the B in the Test Drawing I of Hb13-3-2 promotion rubber test tube seedling growth, C, D and figure A in II, B, C, D are processing groups；Scheme the E in the A and figure II in I, F, G are the growing state for compareing strain test tube seedling.In Figure 13 I B, C, D plant height and new fluffy leaf obviously increase, and Hb13-3-2 processing averagely increases by 1.78 ± 1.51cm than control plant height；In Figure 13 II New fluffy leaf is expedited the emergence of earlier than control, and Hb13-3-2 processing averagely increases by 0.76 ± 0.83cm than control plant height.Test of the invention The result shows that Hb13-3-2 there are apparent growth-promoting functions to rubber seedling, increase the vigor of rubber seedling, therefore can be applied to rubber The strong seedling culture of seedling.Note: the growth conditions of the test tube seedling of different batches are different, but the case where the growth-promoting functions of response Trichoderma It is the same.The growth characteristics of rubber tree seedling stem and leaf are that one fluffy (a fluffy leaf is an elongation unit) is extracted out to a fluffy leaf, It is fluffy with it is fluffy between differ 20-30 days.Therefore the morning that a new fluffy leaf occurs also means that ahead of time at least 6-7 days time.

In order to preferably utilize Trichoderma Hb13-3-2, we are devised according to production practices demand in Trichoderma Hb13- Under the protection of 3-2, the lid for removing test tube seedling in advance practices the test of seedling.In conventional manufacturing procedures, tissue culture seedling direct transplantation is husky Bed practices this stage of seedling without the lid for first opening culture test tube, is because opening lid 3-4 days, rubber seedling just will receive It pollutes and reduces survival rate and growth potential.It is that lid is opened from September 2 in 2016 to totally 10 days (each of September 12 days in the present invention Test tube is inoculated with Hb13-3-2 conidium nutrient solution 1ml, concentration 10⁷A/ml (is prepared) with PDB+1/2MS inorganic salt solution, Contrast test tube seedling is to open simultaneously, and is only inoculated with equivalent and does not add the conidial PDB+1/2MS inorganic salt solution of Hb13-3-2). Test result be under the protection of Trichoderma Hb13-3-2, open lid 10 days will not by the pollution of other miscellaneous bacterias, and Hb13-3-2 handle plant than compare plant strain growth it is more vibrant, and grow fresh and tender fluffy leaf (in such as Figure 14 1,2,3 and 4) and 4 Strain control strain (5,6,7 and 8 in such as Figure 14) has all been contaminated.Should the result shows that: under the protection of Trichoderma Hb13-3-2, Test tube seedling can exempt the invasion of other miscellaneous bacterias, can obtain the exercise of better light, temperature, water, gas in advance, reach transplanting in advance Requirement.And contrast test tube seedling a dozen uncap son then influenced its growth and development by the invasion of miscellaneous bacterias various in environment.In Figure 14 The case where 5,6,7 and 8 pollution.Therefore the measure can transplant first 20 days addition Trichoderma liquors in sand bed, beat using in production It uncaps son, practice seedling and strong seedling culture and do not have to worry again actually be also the young plant pollution problem that can occur.

In order to preferably verify Trichoderma of the present invention to the growth-promoting of rubber seedling and the effect prevented and remedied pollution, the present invention is devised Following experiment: selecting 5 groups of rubber tree heat to grind 7-33-97 test tube seedling, according to its growth potential difference, is divided into five groups of A, B, C and D, E, Every group of 40 plants of test tube seedlings, E group are control.Wherein A group test tube seedling root, stem, leaf are various, but leaf color shades, and growth retardation has 4- 5 leaves；B group test tube seedling growing way is poor, and leaf jaundice, plant is small and weak, there is 3-4 piece leaf；The long potential difference of C group test tube seedling, plant It is small and weak, there is 1-2 piece leaf, and turn to be yellow；D group test tube seedling growing way is very poor, and plant is small and weak, and some is one, light stem.Compare E group It is that 10 plants are respectively taken from A-D, totally 40 plants；Add 1ml PDB+1/2MS solution in contrast test tube seedling, handles in test tube seedling and 1ml is respectively added Hb13-3-2 conidium liquid, spore count 10 are prepared with PDB+1/2MS solution⁷A/ml.The test tube seedling respectively handled is placed on greenhouse It is grown under interior similarity condition.Handling the time is totally 24 days 27 days-October 20 of September in 2016.Test result is counted such as 9 institute of table Show:

Table 9, Trichoderma are to the growth-promoting of rubber seedling and the effect prevented and remedied pollution

* expression has significant difference compared with the control

Should the result shows that, with Trichoderma Hb13-3-2 processing rubber tree 7-33-97 strain tissue-cultured seedling can substantially reduce dirt Dye rate (from 20% to zero) and the death rate significantly promote rubber tree 7-33-97 strain test tube seedling from 7% to 1-5% Growth.

The interior life of embodiment 8, Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 Property identification

With Hb13-3-2 conidium nutrient solution (concentration 10⁷A/ml) it is inoculated with the tissue culture seedling of pollution pathogen, it connects altogether 100 plants of kind, survival rate 95.2%.Co-culturing the period is respectively to randomly select 3 plants of processes respectively after 10 days, 15 days, 30 days The processed tissue-cultured seedling of Hb13-3-2, first flowing water sufficiently rinse 30 minutes, then through 70% (volumn concentration) alcohol disinfecting 3-5 Minute, then the liquor natrii hypochloritis disinfection 20min for being 20% with mass percentage concentration, sterile water wash 5 times, aseptic filter paper blots Afterwards, different tissues dissection or piece are placed on 1/2MS plate, using the same coated plate of last sterile water as control, with investigate whether Disinfection is complete.Plate is put in 28 DEG C dark culture 2-3 days, observes daily and counts band hyphostroma, and records data, the results showed that The survival rate of the seedling root of Hb13-3-2 after treatment is 99 ± 0.21%, and stem is 40 ± 0.11%, and spire and old leaf are 14.37 ± 0.62%.Therefore after handling rubber seedling with Hb13-3-2, rubber seedling available lasting protection and growth-promoting functions. Hb13-3-2 is shown in Figure 15 in the colonisation of plant different tissues.

Sequence table

<110>Rubber Institute, Chinese Academy of Agricultural Science

<120>one plants of trichoderma harzianum strains and its applications

<130> WHOI160078

<160> 2

<210> 1

<211> 644

<212> DNA

<213>Trichoderma harzianum (Trichoderma harzianum)

<400> 1

ttcctccgct tattgatatg cttaagttca gcgggtattc ctacctgatc cgaggtcaac 60

atttcagaag ttgggtgttt aacggctgtg gacgcgccgc gctcccgatg cgagtgtgca 120

aactactgcg caggagaggc tgcggcgaga ccgccactgt atttcggaga cggccaccgc 180

caagaggcag ggccgatccc caacgccgac cccccggagg ggttcgaggg ttgaaatgac 240

gctcggacag gcatgcccgc cagaatactg gcgggcgcaa tgtgcgttca aagattcgat 300

gattcactga attctgcaat tcacattact tatcgcattt cgctgcgttc ttcatcgatg 360

ccagaaccaa gagatccgtt gttgaaagtt ttgattcatt ttcgaaacgc ctacgagagg 420

cgccgagaag gctcagatta taaaaaaccc gcgagggggt atacaaaaag agttttggtt 480

ggtcctccgg cgggcgcctt ggtccggggc tgcgacgcac ccggggcaga gatcccgccg 540

aggcaacagt ttggtaacgt tcacattggg tttgggagtt gtaaactcgg taatgatccc 600

tccgctggtt caccaacgga gaccttgtta cgacttttac ttcc 644

<210> 2

<211> 1320

<212> DNA

<213>Trichoderma harzianum (Trichoderma harzianum)

<400> 2

aacttgcagg caatgtgggc agtgtggcag tcaagaacgg gggcgtagcc ggcaccgacc 60

tggccagggt ggttcatgac gatgacctga gcggtgaacg aagcggcacc catggggggg 120

tcgttcttgg agtcaccggc aacgttacca cggcgaattt ccttaacgga aacgttcttg 180

acgttgaaac caacgttgtc accgggaaca ccctcgacga gctgctcgtg gtgcatctcg 240

acggacttga cttcagtggt gacgttggag ggagcgaagg tgacgaccat accgggcttg 300

aggataccag tctcgatacg gccgacggga actgttccaa taccaccgat cttgtagaca 360

tcctggaggg gaagacggag gggcttgtcc gtgggacgct tggggggctc gatggagtcg 420

atggcctcaa ggagggtctt gccggtgaac ttgccagcct tggtctcctt ctcccagccc 480

ttgtaccagg ggcagttggt ggagggctgg agcatgttgt caccgttgaa accggagatg 540

gggacgaaag caacagcctt ggggttgaag ccgaccttct tgatgaagtt ggaggtctcc 600

ttgatgattt cctggtaacg agcctcggcc cagttggcag tgtccatctt gttgatggca 660

acgatgagct gcttgacacc cagggtgtag gcgagcagag cgtgctcacg ggtctggcca 720

tccttggaga taccagcctc gaactcacca gtaccggcgg caatgatgag gatagcgcaa 780

tcggcctggg aagtaccagt gatcatgttc ttgatgaaat cacggtggcc gggagcgtct 840

gtgaattgcc tgttagcact ggattgcaat tgcagcatga agttgatgaa gtagacatac 900

caatgacggt gacatagtac ttgggagtct cgaacttcca cagagcaatg tcgatggtga 960

taccacgctc acgctcggcc ttgagcttgt caagaaccca agcgtacttg aaggaaccct 1020

tgccgagttc ggcggcttcc tattgatgga aaagtggtta gcatcgttga ataaccaaag 1080

acacagagca cgttgaatga tgactgggaa gtgaatgaag cacaaaaaaa gcagtgaggt 1140

agtggggttg cacagagaac cccactaaaa atcaaacggc agcaaaaaaa atttgcgtcg 1200

ctgcagaggg gtaatggaaa gcggggtgac gaaaaattgt cgacccaaaa agtctctgag 1260

gaattgtcgg gcacaattga atatgaaaga agaggaatcg aggcgaaaat cagttgacgc 1320

Claims

Trichoderma harzianum 1. (Trichoderma harzianum) Hb13-3-2, in Chinese microorganism strain preservation conservator The deposit number of meeting common micro-organisms center is CGMCC No.13163.
2. Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 is in controlling plant diseases Application.
3. Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 prevents and treats phytopathy in preparation Application in harmful microbial inoculum.
4. application according to claim 2 or 3, it is characterised in that: the plant disease is Rubber Red Root Disease bacterium, rubber Set the mould fallen leaves germ of stick spore, anthracnose of rubber trees bacterium, the disease of rubber tree caused by banana blight bacteria and/or yampi anthrax bacteria Evil.
5. a kind of phytopathogen drip irrigation, active constituent is Trichoderma harzianum (Trichoderma harzianum) Hb13- 3-2 CGMCC No.13163。
6. a kind of bio-bacterial manure, including Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163。
7. bio-bacterial manure according to claim 6, it is characterised in that: further include molybdenum element, zinc member in the bio-bacterial manure Element and/or copper.
8. bio-bacterial manure according to claim 7, it is characterised in that: further include in the bio-bacterial manure a great number of elements and/ Or organic fertilizer.
9. Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 promote rubber tree seedling, Application in tissue-cultured seedling growth or prevention and treatment rubber tree disease.
10. Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 is antibacterial in preparation volatility Application in gas；Wherein, escaping gas is to Rubber Red Root Disease bacterium, the mould fallen leaves germ of rubber tree stick spore, rubber tree anthrax Germ, banana blight bacteria and/or yampi anthrax bacteria have fungistatic effect.