CN106399129A

CN106399129A - Trichoderma harzianum strain and application thereof

Info

Publication number: CN106399129A
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: 2017-02-15
Anticipated expiration: 2036-12-01
Also published as: CN106399129B

Abstract

The invention discloses a trichoderma harzianum strain and application thereof. The trichoderma strain is a trichoderma harzianum strain Hb13-3-2 CGMCC No. 13163. The strain and metabolites thereof have broad-spectrum activities of inhibiting pathogenic fungi. The contrast growth experiment proves that the trichoderma harzianum strain has the capacity of resisting red root pathogenic bacteria of rubber trees, colletotrichum gloeosporioides and other pathogenic bacteria, and further has the effects of controlling multiple other pollution microbes and promoting growth of rubber seedlings. Therefore, the trichoderma harzianum strain can be immediately applied to the cultivation process of the rubber trees at the seedling stage, healthy growth of the rubber seedlings is promoted, and occurrence of rubber tree root diseases can be shielded or inhibited.

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 application.

Background technology

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

Research shows, the pathogenic bacterium of Rubber Red Root Disease are Ganoderma pseudoferreum (Wakef.) Over.et Steinm, has that host is many, length incubation period, the features such as spread rapid in soil, is that the root of serious harm rubber tree infects Sexually transmitted disease (STD) does harm to.Red root disease pathogen belongs to mycota Hymenomyceteses, Basidiomycota, Ganoderma, endangers paragutta tree root cervical region, mainly Rely on root system contagion, also can pass through wind and rain and entomochory.Rubber Red Root Disease occurs generally in Ge Zhijiao area of China, Infect serious woods section sickness rate up to 4.0%, if not processing mortality rate in time can be 100%.The investigation of rubber tree root disease Need a large amount of manual labor, early stage follow-up investigation work is not in place, and generaI investigation efficiency is low.And work as rubber tree overground part and occur significantly During disease, it has been to infect serious occurrent time, cost accounting is high and preventive effect is poor.

Because rubber tree is perennial high megaphanerophyte, effectively prevent and treat the multiple diseases such as red root disease using biocontrol microorganisms, will be one Item extends rubber tree and adopts the glue time limit, the strong behave of environmental protection.

Although the resource microorganism that trichoderma is a class to have a extensive future, it is raw for carrying out Biological control using its Antagonism One importance of state agricultural sustainable development, is the focus that biocontrol agent is developed in recent years, but is intended to screen height Effect is prevented and treated the concrete pathogen of a certain plant and is purposefully used for Biological control, needs to do substantial amounts of element task.Such as Need to consider biological adaptation, its expanding propagation, preservation, production and the application cost of this trichoderma, the problems such as degenerating and make a variation.

Content of the invention

Trichoderma and plant cooperated evolution, unique and enrich, and also trichoderma has to Different Crop or even different cultivars Specificity, the experiment that inventor passes through for many years finds, trichoderma has very strong external antagonism energy to Rubber Red Root Disease pathogenic bacteria Power, it is contemplated that the Local Adaptation ability of trichoderma and the difference of antagonistic ability, therefore determines to screen local dominant strain.

It is an object of the invention to provide one plant has the Trichoderma harzianum (Trichoderma that wide spectrum suppresses pathogenic fungi Harzianum) Hb13-3-2 bacterial strain, it is to separate, from Hainan Tropical area, the local bacterial strain obtaining, and is not only able to suppress rubber Tree Arisaema balansae Engl. infection process rubber seedling, and rubber seedling can be promoted to grow, it is the probiotic strain with application value.

The bacterial strain of gained of the present invention has that growth and breeding is fast, low cost, antibacterial spectrum width and to leaf diseases and root disease Evil has the advantages that inhibitory action.It is an object of the invention to provide one plant can be suppressed Rubber Red Root Disease bacterium, rubber tree rod spore The trichoderma with wider antimicrobial spectrum of multiple pathogenic bacterias such as mould fallen leaves pathogenic bacteria, anthracnose of rubber trees bacterium, it grows fast, expanding propagation 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 passes through bolter from product glue rubber tree rhizosphere, Rubber plantation soils, the different tissues of rubber tree germplasm materials Choosing, purification obtains the Trichoderma harzianum biocontrol microorganisms Hb13-3-2 bacterial strain that In Vitro Bacteriostasis antagonistic effect reaches more than 80%；Using tissue culture material Material, inoculates trichoderma and Arisaema balansae Engl. pathogenic bacteria in vivo simultaneously, finds that trichoderma suppression infect efficiency on tissue cultured seedling for the Arisaema balansae Engl. pathogenic bacteria reaches To 100%, find after the biological characteristicses investigating this trichoderma strain and chlamydospore Formation and characteristics, this bacterial strain will be preventing and treating rubber The preferred strain of the bio-bacterial manure exploitation of gum red root disease, can apply to Biological control and promotes agricultural sustainable development application In.

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

The basic culture morphological characteristic of described Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 is：In PDA On flat board, the average growth rate of 28 DEG C of culture colony diameters is 4.9cm/ days.Mycelia have branch, have every, colourless, straight or curved Song is smooth；The symmetrical branch of conidiophore or once in a while verticillate branch, no every metulae is slightly thicker than stalk top, typically has 2-3 time point Branch, sporogenic stigma doleiform estranged；Conidium monospore, ellipticity or spherical.

Also belonged to above-mentioned Trichoderma harzianum (Trichoderma harzianum) bacteria agent as active component for the Hb13-3-2 In protection scope of the present invention.When needing, in this microbial inoculum, also can comprise conventional carrier and adjuvant in microbial inoculum preparation.

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

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

The present invention is also claimed a kind of phytopathogen drip irrigation, and its active component is Trichoderma harzianum (Trichoderma harzianum)Hb13-3-2 CGMCC No.13163.

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

Molybdenum element, zinc element and/or copper is also included in described bio-bacterial manure.Described molybdenum element is added by ammonium molybdate Plus, the mass percentage concentration of interpolation is 0.05%-0.3%；Described zinc element is added by zinc sulfate, the zinc ion of interpolation dense Degree is 1-4mM, and described copper is added by copper sulfate, and the mass percent concentration of interpolation is 0.0001-0.001%.

A great number of elements and/or fertilizer can also be included in described bio-bacterial manure.

Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 is promoting rubber tree children Application in Seedling or tissue cultured seedling growth and/or the bacteria agent in preparation promotion rubber seedling or tissue cultured seedling growth or bio-bacterial manure Fall within protection scope of the present invention.

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

The bacterial strain of gained of the present invention has that growth and breeding is fast, low cost, antibacterial spectrum width and to leaf diseases and root disease Evil has the advantages that inhibitory action.This bacterial strain and its metabolite have wide spectrum suppression pathogenic fungi activity, can produce and have Suppression pathogenic fungi active volatile material, this bacterial strain can antagonism Rubber Red Root Disease bacterium, antagonism anthrax bacteria, to rubber Seedling is harmless, and after suitable increase trichoderma fast-propagation desired nutritional material, can be widely applied for plant tissue culture numerous The prevention and cure of pollution educated, promotion grow, and shorten the time of root culture and strong sprout.

Using group training material, inoculate Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 Trichoderma spp. in vivo simultaneously Bacterium and Arisaema balansae Engl. pathogenic bacteria, find the present invention trichoderma 100% is reached to the suppression ratio of Arisaema balansae Engl. pathogenic bacteria, in conjunction with flat board face-off result and Rubber seedling co-cultures result of the test and understands, the preferred strain that the bio-bacterial manure being preventing and treating Rubber Red Root Disease is developed by this bacterial strain.

Trichoderma harzianum (Trichoderma harzianum) the Hb13-3-2 CGMCC No.13163 of the present invention and its generation Thank to product to there is wide spectrum suppression pathogenic fungi activity, the active volatile material with suppression pathogenic fungi can be produced.Face-off Growth experiment shows, this trichoderma strain has antagonism Rubber Red Root Disease bacterium, the ability of multiple pathogen such as anthrax bacteria, also has Play the role of to prevent and treat other multiple living contaminantses and promote rubber seedling growth.During rubber seedling large-scale cultivation, have anti- The only contaminated effect of rubber seedling.Therefore can immediately be applied to rubber tree seedling stage cultivating process, promote the health of rubber seedling raw Long, the generation of shielding or suppression rubber tree root disease.This bacterial strain is harmless to rubber seedling, and fast in suitable increase trichoderma After fast expanding propagation desired nutritional material, can be widely applied for that plant tissue culture breeds prevents and remedies pollution, promotes to grow, and shortens training of taking root Support the time with strong sprout.

Heretofore described trichoderma strain Hb13-3-2 be can within be born in plant.This characteristic is by inoculating rubber seedling After co-culturing a period of time, again can separately obtain this bacterial strain.Thinner by carrying out to its growth characteristics and possible function After the comparative analysiss causing, find such as trace element Cu²⁺、Mo²⁺With moderate-element Zn²⁺To trichoderma conidium and chlamydospore It is formed with certain impact.Trichoderma is taken precautions against and its to seedling growth in the preventing and treating of rubber tree tissue culture seedling pollution, root disease simultaneously Impact be analyzed.Result shows：Under the conditions of the nutritional need of Reasonable Regulation And Control Trichoderma spp. bacteria growing, can efficiently anti-decontamination Dye, preventing and treating root disease, and promote rubber seedling fast-growth, there are good development and application potentiality.In addition, this trichoderma strain Also stronger to the patience of growth inhibitor potassium dichromate, can survive under 1000 μ g/mL concentration, this characteristic can apply to Detect its Field information situation.

Brief description

Fig. 1 is growth in PDA culture medium for the Hb13-3-2 and illumination situation and cultivates in rose-bengal The growing state of 7d on base, in Fig. 1 A for Hb13-3-2, the situation of 4 days is grown on PDA culture medium, B be Hb13-3-2 in Meng plus Illumination situation after growing 10 days is drawn on red culture medium, the blue dyeing of cotton shows its conidiophore and conidium shape State；In Fig. 1, C and D is the growing state of Hb13-3-2 7d on rose bengal medium, shows that (C is that front is special to its fold stricture of vagina Levy, D is back side growth characteristics).

Fig. 2 is the growth conditions of Hb13-3-2 bacterial strain when cultivating 10 days on different culture media flat board；In Fig. 2, A is Rhizoma Solani tuber osi Culture medium, B is NA culture medium, and C is Radix Dauci Sativae 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 in different carbon source culture medium for the Hb13-3-2, and A is Mannitol, B is Sorbitol, C is sweet Oil, D are starch, E is Semen Maydis powder, F is galactose, G is glucose, H is Fructose, I is sucrose, J is xylose.

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

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

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

The face-off growth effect of 9 days of Fig. 7 trichoderma Hb13-3-2 and red root disease pathogenic bacteria B05, wherein A is Arisaema balansae Engl. pathogenic bacteria B05 grows the situation of 13 days, after B first inoculates 4 days for B05, inoculates Hb13-3-2, face-off co-cultures situation when growing 9 days.

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

Fig. 9 is Hb13-3-2 and banana blight bacteria and rubber bar spore mould fallen leaves pathogenic bacteria YN49 face-off on PDA plate 6 days result of the tests I of culture are and FOC4 stands facing each other 6 days, and II is and YN49 opposite culture 6 days.

Figure 10 suppresses yampi anthrax bacteria 12DP06 (I) and 12DP116 (II) to infect the feelings of Maninot esculenta crantz. blade for Hb13-3-2 Above every leaf of condition in figure (A and C) be inoculation pathogen and trichoderma simultaneously, lower section (B and D) is that only an inoculation pathogen makees For comparison.

Figure 11 is that Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 produces volatilization Property material and this material the inhibitory action of pathogenic fungi is tested.In figure A, C, E, G are comparison, and B, D, F and H are to be produced by volatility Repressed situation after thing impact.

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) is that heat is ground 7-33-97 and gradually formed a health from root clone embryo from sprouting respectively The young plant being contaminated during body；B, is that the pollution young plant shown in A inoculates Hb13-3-2 nutritional solution on the same day immediately, 24 days Pollution young plant afterwards is rescued into appearance alive, and (Hb13-3-2 conidium nutritional solution 1ml inoculated by each test tube, and concentration is 10⁷Individual/ml).

Figure 13 is that Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 promotes rubber Test I and II of test tube seedling growth is independent repeated trials twice, and after display is processed with Hb13-3-2, rubber seedling grows than comparison Faster, more strong result, in II, arrow indication is that Newborn Leaves are fluffy, and comparison does not have the fluffy appearance of Newborn Leaves.

Figure 14 is can to 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, main root or lateral root section in plant different tissues for the Hb13-3-2；B, stem section；C, inferior leads Section piece；D, top tender leaf is cut into slices.

Specific embodiment

Method in following embodiments, if no special instructions, is conventional method.

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

1st, 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 Danzhou City rubber planting area In soil, separation obtains, the soil sample of collection rubber tree rhizosphere 5-10cm soil layer, by locality order and separation sequencing volume Number.Using dilution-plate method, 10 are pressed to each soil sample^-3、10^-4Variable concentrations dilution, be inoculated in (PDA on rose bengal medium + chloromycetin 0.015g/L+ streptomycin 0.015g/L+ rose bengal 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 purification, obtain trichoderma Strain, then numbering identification.

The trichoderma strain isolating and purifying is carried out with the screening of pathogen Antagonistic Fungi, finally obtains one plant to multiple pathogen spy It is not the trichoderma that Pathogenic Fungi of Rubber Red Root Disease has excellent antagonism performance, be named as Hb13-3-2.

2nd, the morphological characteristic of Hb13-3-2 bacterial strain

Hb13-3-2 inoculation is cultivated on PDA culture medium flat board, the morphological characteristic of this bacterial strain is：In PDA plate On, after 28 DEG C of cultures 1 day, colony diameter is 4.9cm, has substrate mycelium and an aerial hyphae, mycelia has branch, has every colourless, directly Or bending, smooth；The symmetrical branch of conidiophore or once in a while verticillate branch, no every metulae is slightly thicker than stalk top, typically has 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 of table 1 Hb13-3-2 and classification

, on PDA plate, mycelial growth is vigorous for Hb13-3-2, and bacterium colony is smooth, and edge is relatively regular；In rose bengal medium The upper flat plate back side produces obvious and relatively regular fold stricture of vagina (see Fig. 1-D rose bengal medium flat board back side fold stricture of vagina).

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

Expand ITS1-5.8S-ITS2 section sequence using universal primer ITS4, ITS5, deliver to Shanghai biotechnology clothes The sequencing of business company limited, gained sequence information carries out sequence alignment by TrichoKEY software, identifies bacterial strain species.

A. the extraction of reesei gene group DNA

With reference to the method for (2008) slightly modified such as Cao Wenbo, comprise the following steps that：Collect separation and Culture and be grown in PDA The mycelia of the Hb13-3-2 bacterial strain in culture medium, weighs 0.1g liquid nitrogen grinding, adds the extract with CTAB liquid having been warmed up (100mmol/L Tris-HCl, pH8.0,1.4mol/L NaCl, 20mmol/L EDTA, pH8.0,2%CTAB), and add few Permitted the quartz sand having sterilized, moved in centrifuge tube after spore being fully ground in mortar, and be incubated 15 minutes at 65 DEG C, plus Enter isopyknic phenol/chloroform/isoamyl alcohol (25:24:1v/v/v) solution, firmly shakes mixing of turning upside down, 12000r/min Centrifugation 10min, supernatant is moved in another new centrifuge tube, adds isopyknic chloroform/isoamyl alcohol (24:1v/v/v) Solution, firmly shakes mixing of turning upside down, and 12000r/min is centrifuged 10min；Supernatant is taken to add in another new centrifuge tube The 3mol/LNaAc solution (pH5.2) of isopyknic isopropanol and 1/10 volume, one 20 DEG C placement 20min, 12000r/min from Heart 5min, abandons supernatant, inversion makes tube wall liquid flow to end, plus 70% dehydrated alcohol rinsing DNA precipitation twice, is centrifuged 1min, abandons Clearly, room temperature air-dries volatilization ethanol, adds sterilizing water dissolution.Place 4 DEG C of refrigerator overnight dissolvings；One 20 DEG C of preservations.

B.rDNA mono- ITS area sequencing analysis

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

ITS1 as shown in sequence 1 in sequence table for the rDNA-ITS sequence of this bacterial strain, ITS2 region partial sequence, result is sent out The existing rDNA-ITS region sequence similarity that it belongs to bacterial strain from different Trichoderma is 98～100%.

As shown in sequence 2 in sequence table, the α-genetic fragment from EF-1 (tef1) is used for reflecting tef1 identification sequence Fixed, it is because containing the 4th long intron in this fragment, intron is longer in theory, be capable of in the kind of discrimination, plant Between sequence difference sequence abundanter, can be preferably applied to identify difference.

By above-mentioned phenotypic characteristic and characterization of molecules, Hb13-3-2 identification of strains is Trichoderma harzianum (Trichoderma Harzianum), it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 21st, 2016 (abbreviation CGMCC, address is：City of BeiJing, China Haidian District Zhong Guan-cun north one No. 13), preserving number is CGMCC No.13163.

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

By the use of PDA culture medium as activation medium.Mainly employ four kinds of methods, be 4 DEG C of flat board preservation methods respectively, often Fire stons wax oil preservation method, -80 DEG C of ultra-low temperature preservation methods, inclined-plane preservation methods.4 DEG C of flat board preservation methods：PDA plate inoculates Hb13-3-2 After truffle, 28 DEG C of light culture were stored in 4 DEG C after 1 week；- 80 DEG C of ultra-low temperature preservation methods：After PDA plate inoculation Hb13-3-2 truffle, 28 DEG C of light culture, after 2 weeks, are washed conidium get off with nutritional solution, are mixed homogeneously with sterile glycerol, preserve in -80 DEG C；Inclined-plane Preservation method：Slant activation is cultured, there is ripe conidium in a large number and protect 4 DEG C of preservations of tube.Room temperature paraffin oil preservation method： Cultured to slant activation, there is ripe conidium in a large number and protect the paraffin oil (paraffin oil after adding stringent sterilization in tube Amount to fully cover culture face) sealing membrane closure, room temperature preservation.

It was found that room temperature paraffin oil preservation method can keep the activity of bacterium, but when bringing back to life, mycelia relative growth is slow；4 DEG C flat board holding time more long contaminated probability is big.For reducing pollution, in the case of conditional, -80 DEG C can be adopted Preservation method makees long-term preservation.

Embodiment 2, the biological characteristicses of Hb13-3-2 bacterial strain

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

1st, 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) flat board：

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 With distilled water, filtrate is settled to 1000mL after boiling 30min；

CMV (Semen Maydis powder) culture medium：Filtrate is settled to after boiling 30min by Semen Maydis powder 30g, agar 14g with distilled water 1000mL；

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

CA (Radix Dauci Sativae) culture medium：Radix Dauci Sativae 200g, glucose 20g, agar 14g, boil filtrate after 30min with distilled water It is settled to 1000mL；

NA culture medium：Carnis Bovis seu Bubali cream 3g, peptone 5g, agar 14g, are settled to 1000mL；

All at 121 DEG C, sterilize above-mentioned culture medium 20min.

Pour above-mentioned six kinds of solid mediums of 20mL thawing respectively in the culture dish of diameter 9cm, obtain final product each after solidification Plant the equal thickness solid plate of culture medium, in the Trichoderma harzianum (Trichoderma of flat board central authorities inoculation diameter about 4mm size Harzianum) Hb13-3-2 CGMCC No.13163 truffle, every kind of culture medium sets three repetitions, 28 DEG C of light culture, daily survey Amount bacterium colony size, calculates colony growth rate, takes pictures after 10 days.

As shown in table 2, on CA flat board, the speed of growth is the fastest for result, is secondly PDA plate, Min flat board, OA flat board, NA Flat board and CMV flat board, the speed of growth on CMV flat board is the slowest.As shown in Fig. 2 Hb13-3-2 bacterial strain is in 6 kinds of different culture medias On mycelial growth rate difference less, but there is notable difference in illumination amount, from it in minimum Nutrient medium It can be seen that this strain growth and nutritional need be not high, belong to easy expanding propagation class with from the point of view of the growing state in corn culture medium Type.On CMV, NA, PDA and CA culture medium, bacterium colony quality is loose, and in Min and OA culture medium, bacterium colony quality is closely.Point The speed order of sporogenic formation is Min>CMV>OA>CA>NA>PDA plate, bacterium colony is changed into green, bottle green from light green.

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

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

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

Prepare the culture medium of different carbon source：With Czapek culture medium as minimal medium, use Mannitol, glycerol, jade respectively Rice flour, D-Glucose, galactose, starch, D-glucitol, D-Fructose, the sucrose in Czapek culture medium replaced by D- xylose, other Components unchanged, the culture medium (adding agar to prepare solid medium) with these materials as sole carbon source for the preparation.

Prepare the culture medium of different nitrogen sources：With Czapek culture medium as minimal medium, respectively with potassium nitrate, ammonium nitrate, Ammonium sulfate, yeast extract, beef extract, yeast extract, peptone replaces the sodium nitrate in Czapek culture medium, other compositions Constant, the culture medium (adding agar to prepare solid medium) with these materials as only nitrogen source for the preparation.

The formula of above-mentioned Czapek culture medium is：MgSO4·7H₂O 0.5g, K₂HPO₄1g, KCl 0.5g, FeSO₄7H₂O 0.01g, NaNO₃2g, sucrose 20g, are settled to 1000mL, and pH7.0 (adds agar to prepare solid medium).All through 121 DEG C, 20min sterilizes.

Its bacterium colony average diameter calculated growth speed is surveyed using solid plate method：Respectively will be former former with nitrogen for above-mentioned difference carbon After solid medium sterilizing, pour in the culture dish of diameter 9cm respectively, 20mL/ ware, the above-mentioned difference of the equal thickness obtaining final product after solidification Solid medium flat board.In the truffle of flat board central authorities inoculation diameter about 4mm size, if three repetitions, 28 DEG C of light culture 3 days, use Crossing method measures colony diameter.As shown in table 3, result shows result, containing sole carbon source on the flat board of sucrose, its life Length is fastest, and grows worst on containing sole carbon source for the flat board of D-glucitol, with D- xylose (J in Fig. 3) and gala Sugared (F in Fig. 3) is that on the flat board of sole carbon source, its speed of growth is not so good as in glycerol, starch, Semen Maydis powder, D-Fructose, PEARLITOL 25C With (Fig. 3) on D-Glucose flat board.

Speed of growth difference under the conditions of 10 kinds of carbon sources for the Hb13-3-2 bacterial strain is inconspicuous, but different carbon source is to mitogenetic spore Sub- quantity of formation and speed impact are very big, such as on glycerol, starch and maize powder medium, can quickly form substantial amounts of point Raw spore.The factors such as the price of product considering, so during later expanding propagation culture Hb13-3-2, should select Semen Maydis powder, glycerol, shallow lake Powder is as carbon source.

As shown in table 3, on flat board containing only nitrogen source, the flat board with yeast extract as only nitrogen source grows Hurry up, be secondly respectively yeast extract, sodium nitrate, potassium nitrate, ammonium sulfate, soy peptone, and put down in beef extract and ammonium nitrate Grow the slowest on plate.Though the speed of growth under 8 kinds of nitrogen conditions for the Hb13-3-2 bacterial strain is variant, it is contemplated that product price Etc. factor, during bacterium culture, we select ammonium sulfate as nitrogen source.

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

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

Because Hb13-3-2 can take into account ready availability, the present invention of cost and material well by the use of Semen Maydis powder as carbon source Using Semen Maydis powder and ammonium sulfate as carbon source (30g/L) and nitrogen source (ratio is the mol ratio of carbon source and nitrogen source respectively) for 0:0 (0 finger Be plus carbon source, be not added with nitrogen source), 10:1,20:Isosorbide-5-Nitrae 0:1,80:1), 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 shown in table 4 and Fig. 4, in identical incubation time, Hb13-3-2 bacterial strain is in the culture medium of 5 kinds of ratios for result There is illumination, with 0:The quantity that 0 ratio (be not added with ammonium sulfate and only add carbon source) is formed is many, illustrates this bacteria growing to carbon Source demand is many, little to nitrogen source demand.

Table 4, the Hb13-3-2 bacterial strain growing state in different carbon/nitrogen ratio culture medium

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

The preparation of PDA plate：Pour the PDA solid medium that 20mL dissolves in the culture dish of diameter 9cm, after solidification i.e. Obtain the PDA solid plate of equal thickness, the cell age of the diameter about 4mm size obtaining in the inoculation punching of flat board central authorities is the Hb13- of 3 days 3-2 truffle, be respectively placed in different temperature (4 DEG C (processing 3 days), 16 DEG C (processing 3 days), 20 DEG C (processing 3 days), 28 DEG C, 37 DEG C With 45 DEG C (process 1h), 50 DEG C (processing 1h), 55 DEG C (processing 1h)) after be positioned over light culture at 28 DEG C again, each test temperature If three repetitions, observe colony growth situation.In 8 temperature being adopted, 28 DEG C grow optimum temperature, 55 DEG C of process for it 1h although mycelial growth is reduced to 1cm/ days, but also will not be dead, illustrate that this bacterium all has to high temperature and low temperature certain suitable Should be able to power.

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

First, the Salt And Alkali Tolerance characteristic of Hb13-3-2 bacterial strain

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

Prepare the flat board of above-mentioned culture medium, and inoculate Hb13-3-2 bacterial strain truffle, three repetitions of each medium treatment.28 DEG C light culture 7 days, measures colony diameter with crossing method.

As shown in table 5, Hb13-3-2 bacterial strain is at least resistant to the NaCl that mass percent concentration is 7% to result, at least can Enough tolerate 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 grow 7 days on salt containing variable concentrations, the PDA plate of alkali after bacterium colony average diameter (cm)

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

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

The PDA plate of the different pH value of preparation：Respectively with 1M HCl and 1M NaOH regulation liquid PDA culture medium pH value to 4, 5,6,7,8,9,10,11,12, after regulating pH, add agar to be configured to solid medium, 121 DEG C, 20min sterilizing obtains PH Value is respectively 4,5,6,7,8,9,10,11,12 PDA plate.

The PDA plate central authorities being respectively 4,5,6,7,8,9,10,11 or 12 in the pH value of above-mentioned preparation inoculate one respectively Beaten with sterilization punchers and take the cell age of diameter about 4mm size to be that PDA solid plate cultivates the truffle of 4 days, put 28 DEG C respectively and dark train Support, cultivate 3 days, measure colony diameter with crossing method respectively, three repetitions, ask for average and standard deviation.Result such as table 6 Shown, result shows, this bacterial strain can grow in the range of for examination pH4～8, and less, the most suitable growth pH is speed of growth difference 5 although also can grow, speed significantly slows down on alkaline medium, it is also contemplated that during pH11 and pH12, acid-base value is not Accuracy, but at least can illustrate that this bacterium can survive under slight alkali environment.

The speed of growth in different pH culture medium for the table 6 Hb13-3-2 CGMCC No.13163

Embodiment 4, trichoderma (Trichoderma) Hb13-3-2 bacterial strain CGMCC No.13163 are to variable concentrations dichromic acid The tolerance research of potassium

Potassium dichromate is a kind of heavy metal salt, can suppress the growth of funguses, when separating soil actinomycete, culture medium The potassium dichromate of middle addition 50 μ g/mL preferable can must play the effect of suppression fungal contamination.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 The PDA culture medium of potassium dichromate flat board, inoculate trichoderma (Trichoderma) Hb13-3-2 truffle, the weight of each concentration Neutral potassium chromate processes and carries out three repetitions, and flat board is placed in 28 DEG C of light culture, measures colony diameter daily.

Result as shown in table 7, with the rising of potassium dichromate concentration, successively decrease by the speed of growth of bacterial strain Hb13-3-2.Hb13- On the PDA plate containing 1000 μ g/mL potassium dichromates, after 1 day, colony diameter is 1.3cm to 3-2 bacterial strain, and after 5 days, colony diameter can Reach 7.1 ± 0.5cm.Because this bacterial strain has stronger patience to potassium dichromate, investigate its field planting in plant root afterwards In qualification process, can be very good to suppress living contaminantses using this characteristic, such that it is able to by itself or related bacterium from host plant Separate.

Table 7. trichoderma (Trichoderma) Hb13-3-2 puts down in the bacterium colony of the PDA plate containing variable concentrations potassium dichromate All diameters (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

First, the growth-promoting functions to trichoderma strain Hb13-3-2 for the trace element molybdenum

Molydbenum fertilizer has 1) purity is high, is harmful to magazine composition 2 without to crop) solubility property is good：Effective ingredient is completely water-soluble, 3) absorption efficiency is high：Soda acid appropriateness, nutrient form is suitable for Crop；4) fertilizer efficiency is lasting：There is good blade adhesive ability, Resistance of rainwater washing against, fertilizer efficiency is lasting；5) the features such as raise productivity and improve the quality.In order to explore molydbenum fertilizer whether can promote trichoderma growth this ask Topic, it is minimal medium that the present invention utilizes PDA, 5 mass percent concentrations of interpolation (0,0.05%, 0.1%, 0.15%, 0.2%, 0.3%) ammonium molybdate, inoculation flat board is placed on 28 DEG C of light culture 10 days, with crossing method measurement colony diameter simultaneously Observe illumination amount.Result shows, in the range of 0.05%-0.3%, adds molydbenum fertilizer and trichoderma mycelia can be promoted quick Growth, thicken and illumination, best with the molydbenum fertilizer effect of adding 0.2%.Specifically as shown in figure 5, in Fig. 5, A is to be not added with Molybdenum element；B is the molybdenum element of interpolation 0.05%；C is the molybdenum element of interpolation 0.1%；D is the molybdenum element of interpolation 0.15%；E is to add The molybdenum element plus 0.2%；F is the molybdenum element of interpolation 0.3%.

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

Be minimal medium using PDA, add the zinc sulfate of 6 concentration (0,0.5,1,2,4,10mM) or concentration be 0, 1PPM, 6PPM, 10PPM (it is 1/1000000th that 1PPM is equivalent to mass percentage concentration) copper sulfate is configured to corresponding flat board, with It is placed on 28 DEG C of light culture 10 days after inoculation in a day, measure colony diameter with crossing method and observe illumination amount.

Result shows, in 1-10PPM Cu²⁺With 1-4mM Zn in ion range²⁺Concentration range in, add zinc element 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 multiple The test of pathogen antagonism

First, Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 and pathogen is right Stand erect test

Detection Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 and pathogenic fungi Antagonistic activity, including Rubber Red Root Disease bacterium, rubber tree rod spore mould fallen leaves pathogenic bacteria, anthracnose of rubber trees bacterium, banana blight Bacterium, yampi anthrax bacteria etc..

Have for examination pathogenic fungi：No. 4 microspecies of banana blight bacteria (Fusarium oxysporium f.sp.cubense) (FOC4) (Sun Yong, Zeng Huicai, Peng Ming, Wang Xuchu, easily little flat * banana blight pathogenic molecular mechanism is in progress with study on prevention [J]. tropical crops journal 2012,33 (4)：759-766), rubber anthrax bacteria (Colletotrichum Gloeosporioides, buys in Chinese agriculture microbial strains preservation administrative center, deposit number ACCC No.30012), Following numberings according to it in antagonistic experiment are named as HDHL, rubber tree rod spore mould fallen leaves pathogenic bacteria (Corynespora Cassicola, buys from China General Microbiological culture presevation administrative center, deposit number CGMCC No.3.10072), following HbYN58 is named as according to its numbering in antagonistic experiment, in addition voluntarily gather and identify one plant of rubber tree rod spore mould fall Leaf disease bacterium, is named as YN49 according to its numbering in antagonistic experiment；Two plants of yampi anthrax bacterias are conventionally from Hainan Yampi field gathers and identifies, is named as 12DP06 and 12DP116 according to its numbering in antagonistic experiment, rubber tree is red Root disease pathogen (G.pseudoferreum) bacterial strain YN1 (Tu Min etc., the RAPD analysis of Rubber Red Root Disease bacterium genetic polymorphism, 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 of our unit, conventionally from variable rate technology Arisaema balansae Engl. Sample, purification, culture, identification traitses etc. are gathered on the obvious rubber tree of disease symptoms.This experiment further molecule before application Identification confirms, is stored in 4 DEG C of refrigerators with PDA plate, periodically recovery culture.

The enrichment culture method of Rubber Red Root Disease pathogenic bacteria：Through (A, 1/2MS+CMA；B、CMA；C、PDA；D, PDA+ are recklessly Radix Raphani E, 1/2MS+PDA) 5 kinds of culture medium test, find Arisaema balansae Engl. pathogenic bacteria in 1/2MS+CMA (maize powder medium) and 1/2MS+ Amplification cultivation fast growth in PDA culture medium.

Opposite culture and bacteriostasis rate：In identical 1/2MS+PDA culture medium, compared with the trichoderma speed of growth, Arisaema balansae Engl. The growth of pathogenic bacteria B05 or more relatively slow (7.8 ± 0.45mm/ days), for the antagonistic effect of more precise Identification trichoderma, adopt First cultured for rejuvenation Rubber Red Root Disease bacterium is inoculated on culture plate, cultivates in 28 DEG C of constant incubators, wait pathogenic bacteria When diameter length is to about 3-4cm, inoculate Hb13-3-2 CGMCC No.13163 trichoderma, the two is spaced 2～3cm, repeated inoculation 3 times.Culture routine observation, record and take pictures in 28 DEG C of constant incubators.The growth diameter of periodic measurement pathogenic bacteria, surveys simultaneously Amount the growing state (comparison) of the pathogenic bacteria not inoculating trichoderma, calculates suppression ratio.And for rubber bar spore mould fallen leaves pathogenic bacteria 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 My god；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 in culture medium for the 12DP116 bacterial strain 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 the same with Arisaema balansae Engl. pathogenic bacteria, first inoculate pathogen, then inoculation Hb13-3-2 wood Mycete, the purpose of do so is to more determine whether trichoderma really is able to kill or suppress the growth of pathogen, because Culture pathogen in advance, pathogen occupies advantage on flat board, if trichoderma can be last in face-off under being in a disadvantageous position Kill or suppress pathogen, could preferably prove its function.Then we have also been made by pathogen and Hb13-3-2 on the same day Face-off is inoculated in the test on culture plate, and the face-off result of 6 days is 100% suppression pathogen (Fig. 9)；Arisaema balansae Engl. pathogenic bacteria is also The same, inoculation pathogen and Hb13-3-2 simultaneously, Hb13-3-2 reach 100% to the suppression ratio of Arisaema balansae Engl. pathogenic bacteria.Bacteriostasis rate (%) The colony radius of=(colony radius of the colony radius of matched group pathogenic bacteria-treatment group pathogenic bacteria) × 100/ matched group pathogenic bacteria.Data Process and carried out using statistical analysis software DPS (7.5 editions).

8. 9 days opposite culture of table, the suppression ratio to Different Kinds of Pathogens funguses for the Hb13-3-2

Trichoderma Hb13-3-2 CGMCC No.13163 to opposite culture test result indicate that (Fig. 7), leading in B05 After inoculation 4 days, the diameter of B05, in 2.7-4.0cm, inoculates Hb13-3-2, and, after 9 days, Hb13-3-2 is to Arisaema balansae Engl. for opposite culture Pathogenic bacteria all has significantly antagonistic effect, can quickly suppress, occupy, even killing red root disease pathogenic bacteria mycelium.In figure B05 is complete Surrounded by Hb13-3-2, non-renewable length.In Fig. 7, B05 is surrounded by Hb13-3-2 completely, non-renewable length.In addition, to YN1 and The antagonistic effect of BT also reaches the similar level of DF (not showing in chart).Trichoderma Hb13-3-2 CGMCC No.13163 couple Rubber tree rod spore mould fallen leaves pathogenic bacteria (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 cellulitis pathogenic bacteria (D and F in Fig. 8) is as shown in Figure 8：Trichoderma Hb13-3-2 can significantly inhibit rubber tree Other crop pests FOC4,12DP06 and 12DP116 are also had obvious suppression by the growth of pathogen HDHL, Hz and HbYN58 Effect.

Table 8 is the antagonism data result of 9 days：Hb13-3-2 trichoderma has notable antagonistic effect to 4 kinds of pathogen；Wherein 86.7%-90% is reached to the suppression ratio of rubber tree rubber bar spore mycete and anthrax.To banana blight bacteria and yampi anthrax The suppression ratio of pathogenic bacteria is also more than 50%.So Hb13-3-2 is multi-functional trichoderma.

The result of the test that pathogen and trichoderma strain Hb13-3-2 inoculate simultaneously demonstrates again that Hb13-3-2 is withered to Fructus Musae Pathogenic bacteria FOC4 and the notable antagonism of excellent spore mould fallen leaves pathogenic bacteria YN49.Fig. 9 is Hb13-3-2 bacterial strain and Fructus Musae on PDA plate Wilt FOC4 and rubber bar spore mould fallen leaves 6 days result of the test photos of pathogenic bacteria YN49 opposite culture.In Fig. 9, I A is Fructus Musae The wilt FOC4 single culture situation of 6 days；B shows the repressed situation of FOC4 for opposite culture.In Fig. 9 II, A is rubber The tree excellent spore mould fallen leaves pathogenic bacteria YN49 single culture situation of 6 days；B shows the repressed situation of YN49 for opposite culture.

2nd, Hb13-3-2 is in vitro to the suppression 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 for inoculating truffle, observe in the feelings with the presence of Antagonistic Fungi Under condition, the suppressed situation of infection ability to blade for the anthrax.As shown in Figure 10, in Figure 10 (I), A and C is Hb13-3-2 to result Inoculate with anthrax pathogen 12DP06 simultaneously；B is only to inoculate anthrax 12DP06.In Figure 10 (II), A is Hb13-3-2 and anthrax Pathogen 12DP116 inoculates simultaneously；B is only to inoculate anthrax 12DP116.Under 28 degree, dark culturing 4 days, take pictures.Result table In the presence of the bright Hb13-3-2 in Antagonistic Fungi, compared with the control, there is the withered and yellow area of damaged blade of Antagonistic Fungi less, explanation Anthrax pathogen 12DP06 and 12DP116 significantly reduces it was demonstrated that trichoderma Hb13-3-2 suppresses charcoal to the pathogenic of yampi blade The infecting potential of cellulitis bacterium, can suppress pathogen infecting on blade.Test is repeated 3 times, and result is close.

Embodiment 7, Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2 CGMCC No.13163 produce and wave The property sent out gaseous compound and this gas are tested to the inhibitory action of pathogenic fungi

1st, prepare PDA plate with the culture dish of a diameter of 9cm, and the one piece of diameter of central authorities' inoculation on each flat board is about The Hb13-3-2 of 4mm size cultivates the truffle of 2 days, cultivates 4 days for 28 DEG C, after it covers with flat board substantially, that is, is used for activity volatilization Property gaseous compound test.

2nd, using the method similar to opposite culture, pathogen truffle (rubber tree Arisaema balansae Engl. is first inoculated on culture medium flat plate Pathogenic bacteria B01, B05, anthracnose of rubber trees bacterium HDHL, yampi anthrax bacteria 12DP06), wait its size be 3cm about when, according still further to Following methods co-culture.

3rd, take the Hb13-3-2 flat board that step 1 is cultivated 4 days, gently lower section length is had the ware bottom and 4 of Hb13-3-2 bacterium colony PDA plate ware bottom (central authorities are with the pathogenic fungi truffle inoculated) back-off that kind connects pathogenic fungi, is used together Parafilm " M " sealing co-cultures；And to tip upside down on the PDA plate ware at the ware bottom not cultivating Hb13-3-2 and pathogenic fungi End button, is compared with parafilm " M " sealing co-cultivation, three repetitions, 28 DEG C of light culture are observed after 10 days, according to reality together The method applying example 6 calculates bacteriostasis rate.

As shown in figure 11, in A-D in Figure 11, right figure is respectively and is produced by Hb13-3-2 the downtrod growing state of pathogen The Rubber Red Root Disease bacterium B05 (A and B in Figure 11) of liveliness proof escaping gas material inhibitory effect, 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) colony growth situation, In A-D in Figure 11, left figure is the growth shape not adding a cover the comparison at ware bottom (adding a cover aseptic ware bottom) cultivating Hb13-3-2 Condition.It can be seen that all growths for examination pathogenic fungi are all suppressed, wherein to Rubber Red Root Disease bacterium (protecting booth separation strains B01) disease Bacteria strain suppression ratio highest, shows that bacterial strain Hb13-3-2 can produce the life that active volatile gaseous matter suppresses pathogenic fungi Long.

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

Rubber tissue cultured seedling in incubation, the later stage occur pollution rate constantly raise, growth retardation, developmental disorder, old The phenomenons such as seedling, the survival rate transplanted after not only reducing, commodity selling rate and price, and increased cost (see Figure 12 Middle A).During the young plant of completely healthy and strong to the one suitable transplanting of growth just having formed cotyledon, stem, root from plumule, The pollution of different bacterium that this new individual all can meet with and dead.Deprotect the one-tenth of this new individual with probiotic bacteria Hb13-3-2 Long, can effectively prevent and treat the pollution of other miscellaneous bacterias, and promote its healthy Fast Growth, thus by the industry development of rubber seedling There is important function.

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.It is inoculated into September from September 5 in 2016 29 totally 24 days, profit With heat grind 7-33-97 from root clone embryo from sprout gradually form healthy individuals during the young plant that is contaminated such as scheme A (1-12) shown in, followed by figure A in pollution young plant on the same day immediately inoculation Hb13-3-2 nutritional solution (connect in each test tube Plant Hb13-3-2 conidium nutritional solution 1ml, concentration is 10⁷Individual/ml), take pictures after 24 days handled after contaminated materials whether It is rescued into work.It is with the configuration of PDB+1/2MS inorganic salt solution in each test tube inoculation Hb13-3-2 conidium nutritional solution The conidium of new culture, and constant volume is 10⁷Individual/ml.Result as shown in figure 12, is in the test material of different growth phases (1-12 of figure A), Hb13-3-2 process can kill contaminated bacteria.The result of the test of the present invention shows that Hb13-3-2 can be effective Rubber seedling of preventing and remedying pollution, so as to avoid death, reduces economic loss.

The growth of rubber seedling whether can be promoted to investigate Hb13-3-2, from June 21 in 2016 or 27 days to August 12 days Common 40-50 days.We grind 7-33-97 rubber tissue cultured seedling from the relatively uniform heat of growth potential and have carried out second and third time two Secondary inoculation test, in each test tube for the treatment of group, inoculation Hb13-3-2 conidium quantity is 10⁷The nutritional solution 1ml of individual/ml (uses PDB+1/2MS inorganic salt solution is prepared), add equivalent conidial without Hb13-3-2 in each test tube of matched group PDB+1/2MS inorganic salt solution.

As shown in figure 13, Figure 13 promotes B, C, D and the figure in the Test Drawing I of rubber test tube seedling growth for Hb13-3-2 to result A in II, B, C, D are treatment groups；E in A in figure I and figure II, F, G are the growing state of comparison strain test tube seedling.In Figure 13 I B, C, D plant height and newly fluffy leaf substantially increase, and Hb13-3-2 is processed averagely increases by 1.78 ± 1.51cm than comparison plant height；In Figure 13 II Newly fluffy leaf expedite the emergence of early than comparison, Hb13-3-2 is processed averagely increases by 0.76 ± 0.83cm than comparison plant height.The test of the present invention Result shows that Hb13-3-2 has obvious growth-promoting functions to rubber seedling, increases the vigor of rubber seedling, therefore can apply to rubber The strong seedling culture of Seedling.Note：The growth conditions of the test tube seedling of different batches are different, but the situation of 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) fluffy leaf ground is extracted out, 20-30 days are differed between fluffy and fluffy.The morning that a therefore new fluffy leaf occurs also implies that ahead of time at least 6-7 days time.

In order to better profit from trichoderma Hb13-3-2, we devise in trichoderma Hb13- according to production practices demand Under the protection of 3-2, the lid 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 first opening culture test tube, is because opening lid 3-4 days, rubber seedling will be subject to Pollute and reduce survival rate and growth potential.It is to open lid to September 12 days totally 10 days (each from September 2 in 2016 in the present invention Hb13-3-2 conidium nutritional solution 1ml inoculated by test tube, and concentration is 10⁷Individual/ml (is prepared with PDB+1/2MS inorganic salt solution), Contrast test tube Seedling is to open simultaneously, and only inoculation equivalent is without Hb13-3-2 conidial PDB+1/2MS inorganic salt solution). Result of the test is under the protection of trichoderma Hb13-3-2, opens lid 10 days and also will not be polluted by other miscellaneous bacterias, and It is more more vibrant than comparison plant strain growth that Hb13-3-2 processes plant, and grow fresh and tender fluffy leaf (as 1,2,3 and 4 in Figure 14) and 4 Strain comparison strain (as in Figure 14 5,6,7 and 8) has all been contaminated.This result shows：Under the protection of trichoderma Hb13-3-2, Test tube seedling can exempt the invasion of other miscellaneous bacterias, can obtain the exercise of more preferable light, temperature, water, gas in advance, reach transplanting in advance Requirement.And a dozen sons of uncapping of contrast test tube Seedling are then subject to the invasion of various miscellaneous bacterias in environment to affect its growth promoter.In Figure 14 The situation of 5,6,7 and 8 pollutions.Therefore this measure can be applied aborning, transplant first 20 days in sand bed and add Trichoderma spp. bacterium solution, beat Uncap son, carry out practicing Seedling and strong seedling culture and without worrying to be also actually the young plant pollution problem that can occur again.

In order to preferably verify trichoderma of the present invention to the growth-promoting of rubber seedling with the effect prevented and remedied pollution, the present invention devises Following experiment：Grind 7-33-97 test tube seedling from 5 groups of rubber tree heat, different according to its growth potential, it is divided into five groups of A, B, C and D, E, Every group of 40 plants of test tube seedlings, E group is comparison.Wherein A group test tube seedling root, stem, leaf are various, but leaf color obfuscation, growth retardation, there is 4- 5 leaves；B group test tube seedling growing way is poor, and leaf turns to be yellow, and plant is small and weak, has 3-4 piece leaf；The long potential difference of C group test tube seedling, plant 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, simply one, the light stem having.Comparison E group It is respectively to take 10 plants from A-D, totally 40 plants；Add 1ml PDB+1/2MS solution in contrast test tube Seedling, process each addition 1ml in test tube seedling Prepare Hb13-3-2 conidium liquid with PDB+1/2MS solution, spore count is 10⁷Individual/ml.Each test tube seedling processing is placed on greenhouse Grow under interior similarity condition.Process time is totally 24 days 20 days 27 days-October of September in 2016.Result of the test counts as table 9 institute Show：

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

* represents possess significant difference compared with the control

This result shows, processes rubber tree 7-33-97 strain tissue cultured seedling with trichoderma Hb13-3-2 and can substantially reduce dirt Dye rate (from 20% to zero) and mortality rate are from 7% to 1-5%, and significantly promote rubber tree 7-33-97 strain test tube seedling Growth.

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

With Hb13-3-2 conidium nutritional solution, (concentration is 10⁷Individual/ml) inoculate the tissue culture seedling polluting pathogen, connect altogether Plant 100 plants, survival rate is 95.2%.Co-culture respectively 10 days time period, 15 days, after 30 days, randomly select 3 plants of processes respectively The tissue cultured seedling that Hb13-3-2 was processed, first flowing water fully rinses 30 minutes, then through 70% (volumn concentration) alcohol disinfecting 3-5 Minute, then the liquor natrii hypochloritises sterilization 20min being 20% with mass percentage concentration, sterile water wash 5 times, aseptic filter paper blots Afterwards, different tissues cutting or piece are placed on 1/2MS flat board, using same for last sterilized water coated plate as comparison, whether to investigate Sterilization is completely.Flat board is put in 28 DEG C of light culture 2-3 days, observes and count band hyphostroma daily, and record data, and result shows The survival rate of Hb13-3-2 seedling root after treatment is 99 ± 0.21%, and stem is 40 ± 0.11%, and spire and old leaf are 14.37 ± 0.62%.Therefore processed after rubber seedling with Hb13-3-2, rubber seedling can obtain 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 plant of trichoderma harzianum strain and its application

<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

1. Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2, it is in Chinese microorganism strain preservation conservator The preserving number of meeting common micro-organisms center is CGMCC No.13163.

2. Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2CGMCC No.13163 is short of money as phytopathogen Application in antibacterial and/or the application in controlling plant diseases.

3. Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2CGMCC No.13163 is in preparation suppression pathogenic Application in the microbial inoculum of bacterium and/or controlling plant diseases.

4. the application according to Claims 2 or 3 it is characterised in that：Described phytopathogen is Rubber Red Root Disease bacterium, rubber Gum rod spore mould fallen leaves pathogenic bacteria, anthracnose of rubber trees bacterium, banana blight bacteria and/or yampi anthrax bacteria；Described plant is rubber Gum；Described plant disease is Rubber Red Root Disease bacterium, rubber tree rod spore mould fallen leaves pathogenic bacteria, anthracnose of rubber trees bacterium, and Fructus Musae is withered Wither pathogenic bacteria and/or yampi anthrax microbial rubber tree disease.

5. a kind of phytopathogen drip irrigation, its active component is Trichoderma harzianum (Trichoderma harzianum) Hb13- 3-2CGMCC No.13163.

6. a kind of bio-bacterial manure, including Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2CGMCC No.13163.

7. bio-bacterial manure according to claim 6 it is characterised in that：Molybdenum element, zinc unit is also included in described bio-bacterial manure Element and/or copper.

8. bio-bacterial manure according to claim 7 it is characterised in that：Also include in described bio-bacterial manure a great number of elements and/ Or fertilizer.

9. Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2CGMCC No.13163 promote rubber tree seedling, Tissue cultured seedling growth or preventing and treating rubber tree disease, and/or promote rubber seedling, tissue cultured seedling growth or preventing and treating rubber tree disease in preparation Bacteria agent or bio-bacterial manure in application.

10. Trichoderma harzianum (Trichoderma harzianum) Hb13-3-2CGMCC No.13163 is antibacterial in preparation volatility Application in gas；Wherein, escaping gas is to Rubber Red Root Disease bacterium, rubber tree rod spore mould fallen leaves pathogenic bacteria, rubber tree anthrax Pathogenic bacteria, banana blight bacteria and/or yampi anthrax bacteria have fungistatic effect.