CN103642699A - Fungus capable of capturing conidium of mulberry powdery mildew - Google Patents
Fungus capable of capturing conidium of mulberry powdery mildew Download PDFInfo
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- CN103642699A CN103642699A CN201310608814.9A CN201310608814A CN103642699A CN 103642699 A CN103642699 A CN 103642699A CN 201310608814 A CN201310608814 A CN 201310608814A CN 103642699 A CN103642699 A CN 103642699A
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Abstract
本发明公开了一株对桑白粉菌分生孢子具有捕食作用的真菌pseudozymaaphidisCNm2012，保藏号为CCTCCNO：M2013563。该菌株分离自桑树叶面，是一种类酵母真菌，在土豆平板培养基上该菌的菌落颜色呈粉红色，中间部分平滑类似于酵母的菌落，但边缘长有菌丝；在萨氏液体培养基中培养7天，其菌体梭状，大小不一，胞内含有许多油滴状物质，随着生长长出菌丝；菌株的生长适温为20-30℃，pH6.5-7.5；菌株的18SrDNA序列1416bp，NCBI序列号为KF443200，28SrDNA序列627bp，序列号为KF443201，ITSrDNA序列784bp，序列号为KF443199。该菌可通过捕食桑白粉菌分生孢子对桑白粉病进行生物防治，并且对桑树以及家蚕没有致病性。
The invention discloses a fungus pseudozymaaphidis CNm2012 which can prey on the conidia of powdery mildew Morus alba, and the preservation number is CCTCCNO: M2013563. The strain is isolated from the leaves of mulberry trees and is a yeast-like fungus. The colony of the fungus is pink in color on the potato plate medium, and the middle part is smooth like a colony of yeast, but there are hyphae growing on the edge; cultured in Sabouraud liquid Cultivated in medium for 7 days, the bacteria are fusiform, with different sizes, and there are many oil drop-like substances in the cells, and mycelium grows with the growth; the growth temperature of the strain is 20-30 ℃, pH6.5-7.5; The 18SrDNA sequence of the strain is 1416bp, the NCBI sequence number is KF443200, the 28SrDNA sequence is 627bp, the sequence number is KF443201, the ITSrDNA sequence is 784bp, the sequence number is KF443199. The fungus can biologically control mulberry powdery mildew by preying on the conidia of powdery mildew mulberry, and has no pathogenicity to mulberry and silkworm.
Description
Technical field
The invention belongs to biocontrol of plant disease field, be specifically related to a strain and mulberry powdery mildew conidium there is to the fungi of predation
pseudozyma aphidiscNm2012.
Background technology
Mulberry tree is a kind of important cash crop, except its leaf can be as raising silkworm, and its edible fruit use, and for extracting anthocyanidin, its root, stem also can be used as medicine.Mulberry powdery mildew is by the fungus-caused a kind of mulberry tree disease of Ascomycotina gang pyrenomycetes Erysiphales Erysiphaceae Phyllactinia, be injured mulberry leaf because of nutriture value value difference, low quality, thereby edible its silkworm physique dies down, easily bring out flacherie, cause cocoon amount and the cocoon shell weight of silkworm few.The outburst of mulberry powdery mildew causes huge financial loss to whole silkworm and mulberry industry, therefore how to prevent and treat the focus that mulberry powdery mildew has become sericulture there to pay close attention to.In order to prevent and treat mulberry powdery mildew, use in a large number chemical agent for a long time, not only cause drug residue, also caused the generation of pathogenic bacteria of drug-resistant strain.Therefore finding the new method tool of preventing and treating Powdery Mildew is of great significance.Biological control method is the focus of Recent study, and biological control is mainly to utilize antagonism, and competition, or predation (parasitic or saprophytic) effect control pathogenic bacteria, there is not yet correlative study report both at home and abroad but utilize at present microbial preparation to prevent and treat mulberry tree disease.
Summary of the invention
For the use that the reduces chemical pesticide impact on the pollution of environment and Biosafety, the invention provides a strain and mulberry powdery mildew conidium is there is to the fungi of predation
pseudozyma aphidiscNm2012, prevents and treats mulberry tree Powdery Mildew by the conidial action mode of predation mulberry powdery mildew.
Bacterial strain of the present invention is separated from mulberry leaf surface, this bacterial strain has been deposited in Wuhan City, Hubei Province Wuhan University (postcode: 430072 on November 10th, 2013, phone: 027-68754052) Chinese Typical Representative culture collection center, be called for short CCTCC, bacterial strain preserving number is CCTCC NO:M 2013563.
Inoculation, in potato solid medium, is cultivated 4 days for 28 ℃, level and smooth like yeast in the middle of occurring on substratum, edge grows the pink bacterium colony of mycelia, microscopic examination, thalline shuttle shape, not of uniform size, there is oil droplet shape material in thalline central authorities, along with growth grows mycelia different in size.Bacterial strain 18SrDNA sequence 1416bp, sequence number is KF443200; 28SrDNA sequence 627bp, sequence number is KF443201; ITSrDNA sequence 784bp, sequence number is KF443199.According to morphology and molecular biological characteristics, bacterial strain is accredited as
pseudozyma aphidis, bacterial strain CNm2012.
Of the present invention
pseudozyma aphidisthe separation of CNm2012, purifying and protecting method are, to on potato substratum that find mulberry powdery mildew conidium to have the bacterial strain of predation to be applied to contain mycillin, cultivate, picking culture purifying on pure medium is cultivated, culture condition: 20-30 ℃, PH 6.5-7.5, then purifying bacterial strain is transferred to inclined-plane and carries out conservation.
The present invention prevents and treats mulberry powdery mildew in the mode of predation, and test shows bacterial strain
pseudozyma aphidiscNm2012 does not have pathogenic to mulberry tree and silkworm.
accompanying drawing explanation:
fig. 1,
pseudozyma aphidisthe morphological specificity of CNm2012 thalline
28 ℃, under the rotating speed of 180r/min, in Sa Shi liquid nutrient medium, shaking table is cultivated 7 days
pseudozyma aphidisthe micro-structure diagram of CNm2012 thalline, graduated scale is 20 μ m.
fig. 2,
pseudozyma aphidisthe phylogenetic analysis tree of CNm2012 bacterial strain based on ITS rDNA
Choose
pseudozymathe ITS rDNA sequence of 14 kinds that belong to is compared, and according to closing on method constructing system, grows evolutionary tree, in parantheses, is the sequence number of bacterial strain ITS rDNA sequence.
fig. 3,
pseudozyma aphidiscNm2012 bacterial strain is to the conidial predation of mulberry powdery mildew
Utilize inverted microscope CNm2012 bacterial strain and mulberry powdery mildew spore mixed solution to be carried out to the microgram of track up gained per hour, graduated scale is 20 μ m.
fig. 4,the mulberry powdery mildew of take is nutrition
pseudozyma aphidisthe growth curve chart of CNm2012 bacterial strain
By the mulberry powdery mildew conidium co-cultivation of CNm2012 bacterial strain and different concns, every the light absorption value of 12 hours its OD600 of inspection by sampling (every group of mulberry powdery mildew conidium suspension of all usining concentration is separately as blank).
fig. 5,the microscopic examination of field test prevention effect
At mulberry leaf, infect mulberry leaf and the surperficial microscopic morphology of contrast mulberry leaf after one week that mulberry powdery mildew was processed with CNm2012 bacterial strain in early days, A is the mulberry leaf surface that CNm2012 bacterial strain was processed, and B is contrast mulberry leaf surfaces, and graduated scale is 100 μ m.
Advantage of the present invention: bacterial strain of the present invention
pseudozyma aphidisthe field of CNm2012 is used and is compared free from environmental pollution with chemical pesticide or produce poisoning, also can not cause the generation of pathogenic bacteria of drug-resistant strain; Bacterial strain is not harsh to the requirement of growth conditions, and growth rapidly, is easily prepared and produces microbial inoculum; Bacterial strain can significantly suppress the increase of mulberry powdery mildew scab number, significantly reduces conidial generation, diffusion and transmission of infection, is discharged into environment and can brings into play lasting preventive and therapeutic effect.
Embodiment
embodiment 1 pseudozyma aphidisseparated and the evaluation of CNm2012
1. the isolation and purification of bacterial strain
(1) separation.Substratum: potato 200g, glucose 20g, agar 18g, mycillin 3g, single water 1L that steams.Will be in observing powdery mildew conidium germination process, discovery to it, have the bacterial strain of predation to be applied to 28 ℃ of constant temperature culture on substratum.
(2) purifying.Sabouraud medium: glucose 40g, yeast extract paste 10g, peptone 10g, agar powder 36g, single water 1000ml, PH 6.5-7.5 of steaming.Picking culture is streak inoculation bacterial strain on this substratum, 28 ℃ of constant temperature culture.
(3) conservation.Potato substratum: potato 200g, glucose 20g, agar 18g, single water 1L that steams.The bacterial strain of purifying is preserved at this substratum ramp.
2. the morphology of bacterial strain and molecular biological characteristics
(1) morphological feature
pseudozyma aphidisthe bacterium colony of CNm2012 is usually two condition: the starchiness of middle portion pinkiness or butteriness, there is the mycelia that is radial growth at edge.Its early growth period is as yeast: two ends budding, usually sympodium breeding.Mycelia is transparent have every; every near the appearance of the other branch of stalk shape that blastoconidium is become by fusiform and ellipse is cylindric, usually there will be short aerial hyphae chain, some aerial hyphaes have branch; what have does not have, and aerial hyphae chain is all comprised of fusiform ramoconidium.The appearance of aerial hyphae usually makes its bacterium colony occur fine hair, or looks like one deck bloom of having put on.Along with the aging of bacterial strain, bacterium colony seems to have water chestnut line shape.
(2) molecular biological characteristics
Purifying inoculation in Sa Shi liquid nutrient medium (glucose 4g, yeast extract paste 1g, peptone 1g, single water 100ml, PH6.5-7.5 of steaming), 20-30 ℃, 150-200r/min, shaking culture 10-20 days.Next 5000 leave the heart 5 minutes, abandon supernatant liquor, and then with aseptic water washing three times, purging method be resuspended, centrifugal, abandon liquid.Then in baking oven 40 ℃ dry 10-15 hour, then ground with liquid nitrogen, extract thalline genome.
ITS primer pair is
ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′)
ITS4 (5′-TCCTCCGCTTATTGATATGC-3′)，
26S primer pair is
NL-1(5'-GCATATCAATAAGCGGAGGAAAA-3') NL-4(5'-GGTCCGTGTTTCAAGACGG-3')，
18S primer pair is
NS1（5’- GTAGTCATATGCTTGTCTC- 3’）
NS6 （5’ -- GCATCACAGACCTGTTATTGCCTC 3’）
By the method for PCR, increase, obtain ITS rDNA sequence 784bp, 18S rDNA sequence 1416bp, 28S rDNA sequence 627bp.In NCBI, apply BLAST software these three sequences compared, and build evolutionary tree based on ITSrDNA, the phylogenetic tree building according to contiguous method show this bacterial strain with
pseudozyma aphidisgather is one (Fig. 2).
According to morphology and the molecular biological characteristics of bacterial strain, bacterial strain CNm2012 is accredited as
pseudozyma aphidis.
embodiment 2bacterial strain
pseudozyma aphidisthe microscopic examination of CNm2012 to mulberry powdery mildew conidium predation
Take potato 100g, glucose 10g, agar 9g, single water 500ml that steams, PH6.5-7.5, inserts in pressure kettle 120 ℃ of sterilizings 30 minutes by the substratum configuring.Bacterial strain CNm2012 is activated on this PDA plate.Then with the triangular flask of 500ml, bacterial strain is fermented, the substratum used that ferments is sabouraud medium, and its formula is: glucose 4g, yeast extract paste 1g, peptone 1g, single water 100ml, PH6.5-7.5 of steaming.Fermentation condition is: temperature 20-30 ℃, 180r/min, ferments 4 days.The bacterium liquid 1000 fermenting is left to the heart 5 minutes and removes supernatant liquor, then use aseptic water washing three times, purging method be resuspended, centrifugal, abandon liquid.The resulting thalline of above-mentioned steps is mixed with powdery mildew conidium, be resuspended in sterilized water, it is carried out to tracing observation (Fig. 3).
According to the tracing observation that this bacterial strain and mulberry powdery mildew conidium are mixed, find, the conidial appearance of powdery mildew has attracted bacterial strain
pseudozyma aphidiscNm2012, this bacterial strain is grown round powdery mildew conidium, and utilizes it to breed.
embodiment 3based on mulberry powdery mildew, be nutrition
pseudozyma aphidisthe growth curve of CNm2012 bacterial strain is measured
CNm2012 bacterial strain is mixed into respectively to the suspension of 100ml with the mulberry powdery mildew conidium of different concns, in suspension, including concentration is 10
5the CNm2012 bacterial strain of cfu/ml, concentration is respectively 6 * 10
2cfu/ml, 6 * 10
3cfu/ml, 6 * 10
4the mulberry powdery mildew conidium of cfu/ml.Mixed solution is placed in to 28 ℃, in the shaking table of 180r/min, cultivates, every the light absorption value of 12 hours its OD600 of inspection by sampling (every group of mulberry powdery mildew conidium suspension of all usining concentration is separately as blank).Test arranges 10
5the negative contrast of the CNm2012 aqueous solution of cfu/ml concentration.Result shows: the absorption photometric value of CNm2012 bacterial strain increases (table 1) along with the increase of mulberry powdery mildew conidium concentration, shows
pseudozyma aphidiscNm2012 bacterial strain really using mulberry powdery mildew as nutrition carried out preying on (Fig. 4).
table 1.cNm2012 bacterial strain be take the OD600 value of mulberry powdery mildew as nourishing and growing
embodiment 4bacterial strain
pseudozyma aphidisthe safety testing of CNm2012 to mulberry tree and silkworm
1. the safety testing to silkworm
Take potato 100g, glucose 10g, agar 9g, single water 500ml that steams, PH6.5-7.5, inserts in pressure kettle 120 ℃ of sterilizings 30 minutes by the substratum configuring.Bacterial strain CNm2012 is activated on this PDA plate.Then with fermentor tank, bacterial strain is fermented, the substratum used that ferments is sabouraud medium, and its formula is: glucose 4%, yeast extract paste 1%, peptone 1%, PH6.5-7.5.Fermentation condition is: 28 ℃ of temperature, ferment 4 days.The bacterium liquid 1000 fermenting is left to the heart 5 minutes and removes supernatant liquor, then use aseptic water washing three times, purging method be resuspended, centrifugal, abandon liquid.Then resuspended to it with sterilized water, be configured to following concentration: 10
9cfu/ml, 10
7cfu/ml, 10
5cfu/ml, 10
3cfu/ml.
With the autosexing race silkworm of bluish waves and Dongting Lake, since five ages, silkworm is tested it by feeding method, and concrete test method is as follows:
(1) 540 silkworms of random choose (270 male silkworms, 270 female silkworms), every 30 (15 male silkworms, 15 female silkworms) silkworm one boxes, totally 18 boxes, every 3 boxes are done same processing, do altogether 6 processing, and wherein having three boxes is blank.Every feeding 30g mulberry leaf of every box silkworm, every morning, that carried out respectively 6 processing to mulberry leaf, and the concentration of spraying respectively 10ml is 10
9cfu/ml, 10
7cfu/ml, 10
5cfu/ml, 10
3the CNm2012 bacterial strain of cfu/ml and the sterilized water of 10ml, each processes 3 repetitions, and three remaining boxes are as blank.All the other times are not made the new fresh mulberry leaf of any processing to its feeding, feed every box silkworm 30g mulberry leaf at every turn.
(2) silkworm rising for 5 ages one day is weighed, the silkworm in 6 days 5 ages is weighed.
(3) observe silkworm mortality ratio, the rate of falling ill, observes its cocoon situation and cocoon look, and cocoon weight and cocoon shell weight are weighed.
By observing, find bacterial strain
pseudozyma aphidiscNm2012 can not cause the death of silkworm, can not cause its pathology, compares with negative control, to being placed on small straw bundles to spin cocoons the time of silkworm, cocoon look all has no significant effect, and body weight of silkworm larva, cocoon are weighed and can not produce detrimentally affect, and (table 2, table 3, table 4) can not have a negative impact to its economic benefit.
table 2.the mulberry leaf that different concns is processed are raised the silkworm weightening finish significance of difference (LSD check, trapezoidal method)
table 3.the mulberry leaf that different concns is processed are raised the silkworm time variance analytical table of being placed on small straw bundles to spin cocoons
table 4.the mulberry leaf that different concns is processed are raised family's cocoon shell weight significance of difference (LSD check, trapezoidal method)
2. the safety testing of pair mulberry tree
Take potato 100g, glucose 10g, agar 9g, single water 500ml that steams, PH6.5-7.5, inserts in pressure kettle 120 ℃ of sterilizings 30 minutes by the substratum configuring.Bacterial strain CNm2012 is activated on this PDA plate.Then with fermentor tank, bacterial strain is fermented, the substratum used that ferments is sabouraud medium, and its formula is: glucose 4%, yeast extract paste 1%, peptone 1%, PH6.5-7.5.Fermentation condition is: 28 ℃ of temperature, ferment 4 days.The bacterium liquid 1000 fermenting is left to the heart 5 minutes and removes supernatant liquor, then use aseptic water washing three times, purging method be resuspended, centrifugal, abandon liquid.Then resuspended to it with sterilized water, be configured to following concentration: 10
9cfu/ml, 10
7cfu/ml, 10
5cfu/ml, 10
3cfu/ml.
Random choose 18 strain mulberry treies, one group of every three strain, does same processing for every group, does altogether 6 processing, and each is processed and sprays respectively 10 of 30ml
9cfu/ml, 10
7cfu/ml, 10
5cfu/ml, 10
3cfu/ml concentration
pseudozyma aphidiscNm2012 and sterilized water, remaining one is treated to blank.Observe the variation of mulberry leaves, stem every day.
By the lasting observation of 4 weeks, bacterial strain
pseudozyma aphidiscNm2012 does not all have infected sign to mulberry leaves, stem, does not all find illness.
embodiment 5bacterial strain
pseudozyma aphidisthe field test of CNm2012 to the effect of mulberry powdery mildew
Take potato 100g, glucose 10g, agar 9g, single water 500ml that steams, PH6.5-7.5, inserts in pressure kettle 120 ℃ of sterilizings 30 minutes by the substratum configuring.Bacterial strain CNm2012 is activated on this PDA plate.Then with fermentor tank, bacterial strain is fermented, the substratum used that ferments is sabouraud medium, and its formula is: glucose 4%, yeast extract paste 1%, peptone 1%, PH6.5-7.5.Fermentation condition is: 28 ℃ of temperature, ferment 4 days.The bacterium liquid 1000 fermenting is left to the heart 5 minutes and removes supernatant liquor, then use aseptic water washing three times, purging method be resuspended, centrifugal, abandon liquid.Then resuspended to it with sterilized water, be configured to 10
7the suspension of cfu/ml concentration.
The initial stage of infecting powdery mildew at mulberry leaf is sprayed onto the bacterium liquid of this concentration the pros and cons of mulberry leaf, after one week, compares with negative control, and the conidium of mulberry leaf surface powdery mildew obviously reduces (Fig. 5).
When processing three days, the mulberry leaf of processing with CNm2012 wettable powder are compared with negative control, and the rising tendency of its scab number has the phenomenon of remarkable minimizing.To processing rear new infection blade ratio, add up demonstration: dispenser the 7th day, in contrast, reach in 30.90%, the growth of CNm2012 is 11.24%, but the sick leaf growth of derosal is 19.47%, preventive effect is better than derosal.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543116A (en) * | 2016-02-03 | 2016-05-04 | 北京市农林科学院 | Microzyme strain and application thereof in disease control after fresh fruit picking |
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| CN103096717A (en) * | 2010-04-14 | 2013-05-08 | 拜耳知识产权有限责任公司 | Fungicidal combinations of dithiino-tetracarboxamide derivatives and microorganisms or isoflavones |
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2013
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| CN103096717A (en) * | 2010-04-14 | 2013-05-08 | 拜耳知识产权有限责任公司 | Fungicidal combinations of dithiino-tetracarboxamide derivatives and microorganisms or isoflavones |
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| 邱欣: "一株捕食桑白粉菌分生孢子的类酵母真菌的发现与研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543116A (en) * | 2016-02-03 | 2016-05-04 | 北京市农林科学院 | Microzyme strain and application thereof in disease control after fresh fruit picking |
| CN105543116B (en) * | 2016-02-03 | 2021-06-04 | 北京市农林科学院 | Yeast and application thereof in disease control after fresh fruit picking |
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Application publication date: 20140319 |