CN102286383A - Talaromyces flavus and application of same in prevention of plant pathogens - Google Patents
Talaromyces flavus and application of same in prevention of plant pathogens Download PDFInfo
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Abstract
The invention provides a talaromyces flavus, and also relates to the application of the talaromyces flavus in the inhibition of a variety of plant pathogens. The preservation number of the talaromyces flavus qw12 is CGMCC5067, the talaromyces flavus colony is round, the texture is flocculent and fluffy or flocculent, and the front of the colony is yellow; the conidiophores have penicilli, and the spore is spherical; the primordium is nearly cylindrical, big and curved; the gymnothecium is spherical or nearly spherical, the ascus is spherical, and the ascospore is spherical or nearly spherical and unicellular. The antagonistic bacterium provided by the invention can effectively inhibit the growth of valsa sordida, rhizoctonia solani, corn sheath blight fungi, curvularia lunata, colletotrichum gloeosporioides, fusarium graminearum, alternaria alternate, cryphonetria parasitica, colletotrichum nicotianae and other plant pathogens, effectively prevent plant diseases, reduce the dosage of chemical pesticide and the accumulation of the chemical pesticide in soil and help to increase the quality and yield of economic crops in China, and therefore economic and social benefits are remarkable.
Description
Technical field
The present invention relates to microorganism and biological control field, relate in particular to the basket bacterium of a kind of yellow and the control phytopathogen application.
Background technology
At present, the general method of controlling plant disease is to use chemical pesticide, yet the life-time service chemical pesticide can cause pathogenic bacteria to develop immunity to drugs and reduce the protection effect of chemical agent, and the life-time service chemical agent can cause pesticide residue to exceed standard and environmental pollution simultaneously.Biological control is a kind of new way of controlling plant disease, and it mainly is the antagonistic action of utilizing between biocontrol microorganisms and the plant pathogenic microorganisms, suppresses the growth of phytopathogen.Peanut is China's main economic crops, and its output and relationship between quality national economy.Owing to the increase in demand to peanut, the cultivated area deficiency causes the phenomenon of peanut continuous cropping generally to take place in recent years, and peanut is again one of crop of not anti-continuous cropping.Disease increases the weight of after the peanut continuous cropping, output descends.At present, be badly in need of in the production a kind of effectively, the new technology of the Plant diseasess such as control peanut of safety, environmental protection.
Summary of the invention
At existing environmental pollution of chemical pesticide control in the prior art and disease problems such as serious taking place, the purpose of this invention is to provide a kind of yellow basket bacterium that can effectively prevent and treat the various crop pathogenic bacteria, also relates to its application aspect the multiple pathogenic bacteria of inhibition.Basket bacterium separates from 4 years peanut rhizosphere soils of continuous cropping yellow of the present invention.Use biocontrol microorganisms of the present invention to prevent and treat crop pest, can reduce the consumption of chemical pesticide and the accumulation in soil thereof, prevent the pollution of the environment, help improving quality and the output of crop, economic benefit and social benefit are very remarkable.
For achieving the above object, the present invention adopts following technical proposals to be achieved:
The invention provides the basket bacterium qw12 of a kind of yellow, its deposit number is: CGMCC 5067.
The basket bacterium bacterium colony of described yellow circle, neat in edge, quality is cotton-shaped double velvet-like or cotton-shaped, and bacterium colony is positive yellow, and center, the back side is orange-yellow, and yellow on every side, conidiophore betides aerial hyphae, penicillus, spore sphere.The basket mushroom cystocarp of described yellow is a gymnothecium, and its bag is interweaved by loose in various degree mycelia and forms, and is spherical or subsphaeroidal; Original hase is closely cylindric, big and crooked; The ascus sphere includes 8 thecaspores; Thecaspore is spherical or subsphaeroidal, is clear to yellow, and is unicellular.
The present invention also provides the bio-feritlizer of the blue shape bacterium preparation of described yellow.
The application of the basket bacterium of described yellow in the control phytopathogen also is provided.
Further improvement to technical scheme: described plant is peanut, tomato, willow, corn, apple, wheat, tobacco and Chinese chestnut.
Further improvement to technical scheme: peanut net blotch, tomato early blight bacterium, willow rotten pathogenic bacteria, dry thread Pyrenomycetes, corn sheath blight fungus, the curved spore leaf spot fungi of corn, apple anthrax bacteria, Valsa mali, ring rot of apple bacterium, fusarium graminearum, tobacco brown spot pathogen, chestnut epidemic disease germ and the former bacterium of Colletotricum destructivum.
Further improvement to technical scheme: the basket bacterium of described yellow to the inhibiting rate of pathogenic bacteria at 43%-94%.
Further improvement to technical scheme: yellow basket bacterium living bacteria count is 1 * 10
8-1 * 10
9The cfu/ gram.
Compared with prior art, advantage of the present invention and positively effect are: antagonism bacterium provided by the invention can effectively suppress the willow rotten pathogenic bacteria, dry thread Pyrenomycetes, the corn sheath blight fungus, corn is bent the spore leaf spot fungi, apple anthrax bacteria, fusarium graminearum, tobacco brown spot pathogen, the growth of chestnut epidemic disease germ and Colletotricum destructivum bacterium, use the antagonism bacterium to be the effective controlling plant diseases of the microbial inoculum of effective constituent, and can reduce consumption and its accumulation in soil of chemical pesticide, very important for preventing the pollution of the environment, simultaneously, also help improving the quality and the output of China's peanut and other crops, economic benefit and social benefit are very remarkable.
Description of drawings
Fig. 1 shows that yellow basket bacterium is to the restraining effect of willow rotten pathogenic bacteria among the present invention.
Fig. 2 shows that yellow basket bacterium is to the restraining effect of dry thread Pyrenomycetes among the present invention.
Fig. 3 shows that yellow basket bacterium is to the restraining effect of corn sheath blight fungus among the present invention.
Fig. 4 shows that yellow basket bacterium is to the restraining effect of the curved spore leaf spot fungi of corn among the present invention.
Fig. 5 shows that yellow basket bacterium is to the restraining effect of apple anthrax bacteria among the present invention.
Fig. 6 shows that yellow basket bacterium is to the restraining effect of fusarium graminearum among the present invention.
Fig. 7 shows that yellow basket bacterium is to the restraining effect of tobacco brown spot pathogen among the present invention.
Fig. 8 shows that yellow basket bacterium is to the restraining effect of chestnut epidemic disease germ among the present invention.
Fig. 9 shows that yellow basket bacterium is to the restraining effect of Colletotricum destructivum bacterium among the present invention.
Figure 10 shows that yellow basket bacterium is wound in the miliary damping-off mycelia among the present invention.
Figure 11 shows that yellow basket bacterium makes Colletotricum destructivum bacterium hyphal cell protoplasma concentrate the cell ghostization among the present invention.
Figure 12 shows that yellow basket bacterium makes fusarium graminearum hyphal cell ghostization among the present invention.
Figure 13 shows that yellow basket bacterium makes the distortion of tobacco brown spot pathogen mycelia, cell ghostization among the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments technical scheme of the present invention is described in further detail.
Embodiment 1: antimicrobial short of money separates
Discover the continuous cropping of Laixi City, Shandong Province after 4 years peanut rhizosphere soil and continuous cropping 2 years compare with 7 years peanut rhizosphere soils, phytopathogen kind and quantity significantly reduce, the peanut continuous cropping obstacle obviously alleviates, output and quality improve, and adopt the soil isolation by dilution method that continuous cropping 4 years peanut rhizosphere soils in Laixi City, Shandong Province are separated.Champac shape bacterium of the present invention promptly is to separate the peanut rhizosphere soil in 4 years from continuous cropping to obtain, and by with isolate monospore purifying, with 2 the face-off inoculations on the PDA flat board of frequently seen plants pathogenic bacteria, carries out dull and stereotyped antagonistic effect respectively, and final screening obtains.
Embodiment 2, antimicrobial short of money are identified
1, antimicrobial morphology short of money is identified
It is as follows to test used substratum:
(1) Cha Shi agar (CA): SODIUMNITRATE 3.0g, dipotassium hydrogen phosphate 1.0g, Repone K 0.5g, magnesium sulfate heptahydrate 0.5g, iron vitriol 0.01g, sucrose 30g, agar 15g, distilled water 1000mL, heating makes its dissolving, last constant volume is to 1000mL, 121 ℃ of 15min that sterilize.
(2) the dense liquid storage 10mL of Cha Shi yeast extract paste agar (CYA): Cha Shi, dipotassium hydrogen phosphate 1.0g, yeast extract paste 5g, sucrose 30g, agar 15g, distilled water 1000mL, heating makes its dissolving, and last constant volume is to 1000mL, 121 ℃ of 15min that sterilize.
The dense liquid storage of Cha Shi: SODIUMNITRATE 30g, Repone K 5g, magnesium sulfate heptahydrate 5g, iron vitriol 0.1g, water 100mL, heating makes its dissolving, and last constant volume is to 100mL.
(3) brewer's wort agar (WA): brewer's wort adds 2% agar after adding water move to 10 degree Beaume again, 121 ℃ of sterilization 15min.
(4) potato-agar glucose (PDA): remove skin potato 200g, be cut into small pieces, add water 1000mL, boil 10min, filtered through gauze adds 20g agar, 20g glucose, and reheat makes its fusing, and last constant volume is to 1000mL, 121 ℃ of 20min that sterilize.
The inoculation that screens to the PDA flat board, is placed 28 ℃ of fixed temperature and humidity incubators to be inverted and cultivates, observe the strain growth situation.The bacterium colony circle, quality is cotton-shaped double velvet-like or cotton-shaped, and conidiophore betides aerial hyphae, penicillus, spore sphere.Ascoma is a gymnothecium, and its bag is interweaved by loose in various degree mycelia and forms, and is spherical or subsphaeroidal; Original hase is closely cylindric, big and crooked, and the ascus sphere includes 8 thecaspores; Thecaspore is spherical or subsphaeroidal, is clear to yellow, and is unicellular.
The yellow blue shape bacteria strain qw12 that screens is carried out culture presevation, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC); Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Preservation date: on July 22nd, 2011; Yellow blue shape bacteria strain qw12 deposit number is: CGMCC No.5067; The classification called after of yellow blue shape bacteria strain: yellow blue shape bacterium Talaromyces flavus.
2, antimicrobial Molecular Identification short of money
This example comes it is carried out Molecular Identification according to antimicrobial ITS sequence short of money.Adopt the CTAB method to extract the DNA of bacterial strain.DNA with bacterial strain is a template, and ITS1 is: 5 '-TCCGTAGGTGAACCTGCGG-3 ' (SEQ ID No:2) and ITS4 be: 5 '-TCCTCCGCTTATTGATATGC-3 ' (SEQ ID No:3) is the ITS sequence of primer amplification bacterial strain.After amplified production reclaims test kit and reclaims purifying with glue, connect and transform, choose positive colony and be sent to Beijing Nuo Sai genome company and check order.By with GenBank in existing ITS sequence carry out the kind that the Blast similarity is relatively determined bacterial strain.In conjunction with the morphological feature and the ITS comparison result of bacterial strain, bacterial strain is defined as yellow basket bacterium the most at last.
The GenBank number of landing: JN602366, sequence following (SEQ ID No:1):
TCCGTAGGTG AACCTGCGGA AGGATCATTA CCGAGTGCGG GCCCTCGCGG
CCCAACCTCC CACCCTTGTC TCCTATACAC CTGTTGCTTT GGCGGGCCCA
CCGGGGCCAC CTGGTCGCCG GGGGACGCAC GTCCCCGGGC CCGCGCCCGC
CGAAGCGCGC TGTGAACCCT GATGAAGATG GGCTGTCTGA GTACTATGAA
AATTGTCAAA ACTTTCAACA ATGGATCTCT TGGTTCCGGC ATCGATGAAG
AACGCAGCGA AATGCGATAA GTAATGTGAA TTGCAGAATT CCGTGAATCA
TCGAATCTTT GAACGCACAT TGCGCCCCCT GGCATTCCGG GGGGCATGCC
TGTCCGAGCG TCATTTCTGC CCTCAAGCAC GGCTTGTGTG TTGGGTGTGG
TCCCCCCGGG GACCTGCCCG AAAGGCAGCG GCGACGTCCG TCTGGTCCTC
GAGCGTATGG GGCTCTGTCA CTCGCTCGGG AAGGACCTGC GGGGGTTGGT
CACCACCACA TTTTACCACG GTTGACCTCG GATCAGGTAG GAGTTACCCG
CTGAACTTAA GCATATCAAT AAGCGGAGGA
The bacteriostatic action of embodiment 3, yellow basket bacterium.
Adopt the method for 2 face-offs to be inoculated on the PDA flat board with plant pathogenic fungis such as chestnut epidemic disease germ, the curved spore leaf spot fungi of corn, fusarium graminearum, tobacco brown spot pathogen, willow rotten pathogenic bacteria and dry thread Pyrenomycetess respectively the basket bacterium of yellow, the PDA flat board with independent each pathogenic bacteria of inoculation is contrast simultaneously.Place 28 ℃ of fixed temperature and humidity incubators to cultivate, inoculate and measure colony diameter after 3 days, till flat board is covered with in contrast.Calculate bacteriostasis rate, the result is as shown in table 1.
Embodiment 4, mycelia suppress pathological observation
In 26 ℃ of the face-off yellow basket bacterium of cultivation and each pathogenic bacteria on the PDA flat board, each pathogenic bacteria comprises: plant pathogenic fungis such as willow rotten pathogenic bacteria, dry thread Pyrenomycetes, corn sheath blight fungus, the curved spore leaf spot fungi of corn, apple anthrax bacteria, fusarium graminearum, tobacco brown spot pathogen, chestnut epidemic disease germ and Colletotricum destructivum bacterium, and referring to accompanying drawing 1-9.After the basket bacterium of yellow and each pathogenic bacteria were intersected, picking intersection pathogenic bacteria mycelia was observed the interaction of two kinds of mycelia under opticmicroscope, referring to accompanying drawing 10-13.
Yellow basket bacterium mainly is hyperparasitism, competition effect and antibiosis to the antagonism mechanism of above-mentioned 9 kinds of pathogenic bacterias.Yellow basket bacterium is wrapped on the mycelia of pathogenic bacteria or the growth of parallel wavy formula; Make the fracture of germ mycelia, protoplasma concentrates; Make the hyphal cell hollow deformation.In the interaction process of research champac shape bacterium to 9 kinds of pathogenic fungies, find to be wound between mycelia, phenomenons such as mycelia fracture, protoplasma concentrate, mycelia distortion, from picture, can observe out yellow basket bacterium and 9 kinds of interactional phenomenons of pathogenic bacteria clearly.
Embodiment 5, yellow basket fermented liquid are to the effect of pathogenic bacteria
The yellow basket bacterium of inoculation was cultivated 5 days under 28 ℃ of conditions on the PDA inclined-plane, the preparation spore suspension.Get the 1mL spore suspension with liquid-transfering gun and insert and to be equipped with in the 50mL PD liquid nutrient medium triangular flask, 28 ℃ of shaking culture 4 days are as seed liquor.Getting seed liquor 5mL access is equipped with in the triangular flask of 100mL PD liquid nutrient medium, shaking culture is 7 days under 28 ℃ of conditions, fermented liquid obtains no fermented liquid with centrifugal 15 minutes of 8 layers of filtered through gauze rear filtrate 10000r/min, supernatant liquor with the biofilter filtration sterilization of 0.22um.
The dull and stereotyped center inoculation of solid PDA pathogenic bacteria, 28 ℃ are cultured to colony diameter 2cm, add no fermented liquid 0uL, 300uL, 400uL, 500uL respectively at the about 1.5cm of distance colony edge place, continue to cultivate.Observe and write down the growing way of bacterium colony every day, and measure and write down the distance of bacterium colony center, cover with flat board until the contrast bacterium colony to each edge, 3 repetitions, the result is as shown in table 2.
Table 2: various dose champac shape fermented liquid is to the restraining effect of pathogenic bacteria
The result shows, described no fermented liquid has significant inhibitory effect to peanut net blotch, willow rotten pathogenic bacteria, chestnut epidemic disease germ, ring rot of apple bacterium, Valsa mali, tomato early blight bacterium, tobacco brown spot pathogen, plant pathogenic fungis such as corn curved spore leaf spot fungi, fusarium graminearum and miliary damping-off germ, and along with the increase of fermented liquid consumption, to the restraining effect enhancing of pathogenic bacteria.
In sum, champac shape bacterium provided by the invention has very strong restraining effect to multiple pathogenic bacteria, and by growth parasitic and competition effect inhibition cause of disease mycelia, its meta-bolites also can effectively suppress growth of pathogenic bacteria simultaneously.Can be used for preventing and treating plurality of plant diseases.
Above embodiment is only in order to illustrating technical scheme of the present invention, but not limits it; Although the present invention is had been described in detail with reference to previous embodiment, for the person of ordinary skill of the art, still can make amendment to the technical scheme that previous embodiment is put down in writing, perhaps part technical characterictic wherein is equal to replacement; And these modifications or replacement do not make the essence of appropriate technical solution break away from the spirit and scope of the present invention's technical scheme required for protection.
SEQUENCE LISTING
＜110〉Qingdao Agricultural University
＜120〉basket bacterium of a kind of yellow and the application in the control phytopathogen thereof
<160>3
<170>PatentIn version 3.3
<210>1
<211>580
<212>DNA
<213>Talaromyces flavus
<400>1
tccgtaggtg aacctgcgga aggatcatta ccgagtgcgg gccctcgcgg cccaacctcc 60
cacccttgtc tcctatacac ctgttgcttt ggcgggccca ccggggccac ctggtcgccg 120
ggggacgcac gtccccgggc ccgcgcccgc cgaagcgcgc tgtgaaccct gatgaagatg 180
ggctgtctga gtactatgaa aattgtcaaa actttcaaca atggatctct tggttccggc 240
atcgatgaag aacgcagcga aatgcgataa gtaatgtgaa ttgcagaatt ccgtgaatca 300
tcgaatcttt gaacgcacat tgcgccccct ggcattccgg ggggcatgcc tgtccgagcg 360
tcatttctgc cctcaagcac ggcttgtgtg ttgggtgtgg tccccccggg gacctgcccg 420
aaaggcagcg gcgacgtccg tctggtcctc gagcgtatgg ggctctgtca ctcgctcggg 480
aaggacctgc gggggttggt caccaccaca ttttaccacg gttgacctcg gatcaggtag 540
gagttacccg ctgaacttaa gcatatcaat aagcggagga 580
<210>2
<211>19
<212>DNA
＜213〉artificial sequence
<400>2
tccgtaggtg aacctgcgg 19
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<400>3
Tcctccgctt attgatatgc 20
Claims (8)
1. basket bacterium qw12 of yellow, its deposit number is: CGMCC 5067.
2. the basket bacterium of a kind of yellow according to claim 1 is characterized in that, the basket bacterium bacterium colony of described yellow circle, neat in edge, quality is cotton-shaped double velvet-like or cotton-shaped, and bacterium colony is positive yellow, and center, the back side is orange-yellow, and is yellow on every side, conidiophore betides aerial hyphae, penicillus, spore sphere; Gymnothecium is spherical or subsphaeroidal; Original hase is closely cylindric, and is big and crooked; The ascus sphere includes 8 thecaspores; Thecaspore is spherical or subsphaeroidal, is clear to yellow, and is unicellular.
3. the bio-feritlizer of the blue shape bacterium preparation of yellow according to claim 1.
4. the application of the basket bacterium of yellow according to claim 1 in the control phytopathogen.
5. the application of the basket bacterium of yellow according to claim 4 in the control phytopathogen is characterized in that described plant is peanut, tomato, willow, corn, apple, wheat, tobacco and Chinese chestnut.
6. the application of the basket bacterium of yellow according to claim 4 in the control phytopathogen, it is characterized in that described pathogenic bacteria comprises: peanut net blotch, tomato early blight bacterium, willow rotten pathogenic bacteria, dry thread Pyrenomycetes, corn sheath blight fungus, the curved spore leaf spot fungi of corn, apple anthrax bacteria, Valsa mali, ring rot of apple bacterium, fusarium graminearum, tobacco brown spot pathogen, chestnut epidemic disease germ and the former bacterium of Colletotricum destructivum.
7. the basket bacterium of yellow according to claim 4 is characterized in that in the application of control in the phytopathogen, the basket bacterium of described yellow to the inhibiting rate of pathogenic bacteria at 43%-94%.
8. the application of the basket bacterium of yellow according to claim 4 in the control phytopathogen is characterized in that yellow basket bacterium living bacteria count is 1 * 10
8-1 * 10
9The cfu/ gram.
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