CN103340156A - Novel strain of lyophyllum decastes - Google Patents
Novel strain of lyophyllum decastes Download PDFInfo
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- CN103340156A CN103340156A CN201310098139XA CN201310098139A CN103340156A CN 103340156 A CN103340156 A CN 103340156A CN 201310098139X A CN201310098139X A CN 201310098139XA CN 201310098139 A CN201310098139 A CN 201310098139A CN 103340156 A CN103340156 A CN 103340156A
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Abstract
The invention relates to a novel strain of lyophyllum decastes: Finc-LD-4, with a preservation number being CCTCC NO: M 2013008. The Finc-LD-4 strain has characteristics of good stipe length and uniformity in per plant, flat and lotus-shaped pileus, good anti-competitor capability and concentrated harvesting stage, and is suitable for industrialized popularization and planting.
Description
[technical field]
The present invention relates to the edible mushroom technical field, or rather, relate to the new bacterial strain of a kind of Lyophyllum decastes.
[background technology]
Lyophyllum decastes (Latin title Lyophyllum decastes), be subordinate to Basidiomycotina, Hymenomycetes, Agaricales, white mushroom section, belong to from the pleat umbrella, be to eat the good wild edible fungus that Mycota is competitively studied cultivation both at home and abroad, the Lyophyllum decastes bacterial context is plump fine and smooth, A sweety scent assails the nostrils, the crisp cunning of mouthfeel, delicious flavour, nutritious, and to the repair of human body cell with having demonstrated great superiority aspect treatment cardiovascular disease, anti-ageing the waiting for a long time, be a kind of famous and precious edible mushroom that eats the medicine dual-purpose.Since 2006, Lyophyllum decastes began a small amount of artificial cultivation and achieving success in Japan.
[summary of the invention]
Characteristics such as the technical problem that the present invention solves provides the new bacterial strain Finc-LD-4 of a kind of Lyophyllum decastes, and this bacterial strain has stem length high conformity in the individual plant, and anti-assorted bacterium ability is strong, and picking time is concentrated, suitable batch production popularizing planting.
The present invention is achieved by the following technical solutions, and the new bacterial strain Finc-LD-4 of Lyophyllum decastes of the present invention is by gathering the wild resource in Yunnan, through domesticating and cultivating, obtaining through screening system again.This bacterial strain has been preserved in Chinese typical culture collection center on January 8th, 2013, address: Chinese Wuhan City Wuhan University, its preserving number is CCTCC NO:M2013008, and its biology specific name is Lyophyllum decastes Finc-LD-4(Lyophyllum decastes Finc-LD-4).
The new bacterial strain Finc-LD-4 of Lyophyllum decastes of the present invention is characterised in that:
1, morphological feature
The mycelia of Finc-LD-3: aerial hyphae is dense; Cap: diameter is 3 ± 2cm, and it is light yellow that color is, circle, and there is tiny fine hair on the cap surface, and cap is flat, and very easily parachute-opening is patellate, and quality is tender and crisp, frangible; Lamella: light yellow, the lamella section is crescent; Stem: length is 6～15cm, and stem top is pale yellow, and the bottom is pure white, near the stem interior solid consolidation at cap place, away from cap place stem hollow, stem has scale, scale main give birth in the middle and upper part near cap, it is irregular to distribute, stem clusters, and sees also Fig. 1, Fig. 2.
2, biological property
The nutrient component source: wood chip, corncob, rice bran, wheat bran, corn flour, cotton seed hulls, soybean skin etc., wherein, water content 64%～66% (percentage by weight, down together).
Cultivate and cultivation condition: the mycelium cultivation temperature is 20～25 ℃, humidity 75%, CO
2Concentration 2000～3500ppm, cultivation cycle 50～70 days.Suitable fruiting temperature is 15～18 ℃, humidity 93%～100%, CO
2Concentration 1000～2000ppm, preceding 1～7 day, intensity of illumination 10～50Lux, 8～32 days illumination intensity 200～300Lux in back, every day, clearance-type was opened layer bracket lamp stimulation fruiting, and the cultivation cycle is about 32 days.
3, genetics characteristic
The genomic DNA of Lyophyllum decastes Finc-LD-4 bacterial strain carries out pcr amplification through universal primer ITS1 and ITS4, obtains the specific DNA segment that size is about 691bp, and this fragment is checked order, and sequence information is referring to sequence table.
The new bacterial strain of Finc-LD-4 of the present invention, has stem length high conformity in the individual plant, the fruit body exterior quality is good, characteristics such as anti-assorted bacterium ability is strong, has reduced the production management difficulty, and picking time is concentrated, reduced the manual work intensity of production link widely, and long fresh-keeping period, per unit area yield height, suitable batch production popularizing planting.
[description of drawings]
Fig. 1 is the fruit body outward appearance picture of Lyophyllum decastes Finc-LD-4 bacterial strain of the present invention.
Fig. 2 is Lyophyllum decastes Finc-LD-4 bacterial strain phylogenetic tree of the present invention.
[embodiment]
Below by specific embodiment the present invention is described in further detail.
The domestication of embodiment 1 Finc-LD-4 of the present invention
Present embodiment has illustrated the domestication process of Lyophyllum decastes Finc-LD-4, and concrete steps are as follows:
1) preparation medium
PDA medium: every 1L medium potato 200g, glucose 20g, agar 20g.
2) collection of resources
In September, 2007, in 1800～3000 meters height above sea level areas of Yunnan Binchuan Jizu Shan Mountain, gather wild resource 4 strains, take back the laboratory, organize separation, be inoculated on the PDA medium, obtain to treat 4 of naturalized strains, numbering is respectively FLD-A, FLD-B, FLD-C, FLD-D.
3) strain domestication
It is numerous to use the PDA medium to expand the FLD-D in 4 bacterial strains that obtain, and 22 ℃, humidity 70%, gas concentration lwevel is less than 3000ppm, dark culturing.
To the cultivation of going down to posterity respectively of above-mentioned 4 bacterial strains, numerous 20 of each expansion was inoculated after 12～15 days, chose two the fastest test tube subcultures of a bacterium and expanded numerous, be seeded on the PDA medium, inoculate after 12～15 days, get again and send out two the fastest test tube subcultures of bacterium and expand numerously, repeat 10 circulations, to send out bacterium speed very fast to expanding numerous offspring's test tube, send out the neat homogeneous of bacterium speed, finish preliminary domestication work, and with liquid nitrogen cryopreservation bacterial strain is preserved in academy of agricultural sciences, Shanghai City edible mushroom research institute.
4) factory formula screening
Through the screening of many wheel formula tests, obtain female the kind and the cultivated species prescription, wherein,
Mother culture media: wood chip 50%(percentage by weight, down with), rice bran 25%, wheat bran 15% and corn flour 10%, water content is 60%～62%.
The cultivated species medium: wood chip 15%, corncob 25%, cotton seed hulls 25%, rice bran 10%, wheat bran 20% and corn flour 5%, water content is 64%～66%.
5) obtain aimed strain: by a series of domestication breeding work, obtain a Lyophyllum decastes bacterial strain, it has stem length high conformity in the individual plant, anti-assorted bacterium ability is strong, the cultivation link does not need earthing, characteristics such as concentrates picking time, has reached the purpose of domestication breeding strain excellent.This bacterial strain is named as " Finc-LD-4 ".
In being preserved in Chinese typical culture collection center on January 18th, 2013, its preserving number is CCTCC NO:M2013008 to the new bacterial strain Finc-LD-4 of Lyophyllum decastes of the present invention, and bacterial strain is survived after testing, and classification position is for to belong to Lyophyllum decastes from the pleat umbrella.
Embodiment 2 carbon sources are to the influence of Lyophyllum decastes mycelial growth
For trying carbon source: glucose, sucrose and soluble starch.
The basal medium of test carbon source: magnesium sulfate 0.5g, potassium dihydrogen phosphate 1g, peptone 2g, agar 20g add water to 1000ml.
In basal medium, add respectively for trying a kind of of carbon source, addition is 20g, pack into behind the mixing in the 250ml triangular flask, every bottle of 150ml puts into 121 ℃ of sterilizations of autoclave 30min, treats to cool off slightly the back and falls dull and stereotyped, the about 20ml medium of every plate, each dull and stereotyped central authorities connects the bacterium piece that a diameter is 5mm, and each processing arranges 5 repetitions, places under 25 ℃ of constant temperatures continuous culture 20 days.Came divided by the interval fate with the difference of twice test number with right-angled intersection method mensuration colony diameter, and be the mycelial growth rate of average every day in per 7 days.
Table 1 carbon source is to the influence of Finc-LD-4 and FLD-D mycelial growth
As shown in Table 1, supply to try carbon source from 3 kinds, FLD-D mycelial growth in the medium that with sucrose is carbon source is the fastest, and it is centimetre/day that average growth rate reaches 0.68cm/d(, down together), next is starch, is glucose at last; Finc-LD-4 of the present invention average growth rate of mycelia in the medium that with sucrose is carbon source is the fastest, has reached 0.68cm/d, and than the fast 0.09cm/d of wild strain, it is slow partially to grow in starch and glucose, and average growth rate is 0.68cm/d and 0.63cm/d.Explanation thus, in the medium of different carbon sources, the average mycelial growth rate of Finc-LD-4 is all fast than wild strain, and sucrose is best, and the uniformity of growth rate is better, and excursion is little.
Embodiment 3 nitrogenous sources are to the influence of Lyophyllum decastes mycelial growth
For trying nitrogenous source: ammonium sulfate, peptone and wheat bran.
The basal medium of test nitrogenous source: magnesium sulfate 0.5g, potassium dihydrogen phosphate 1g, glucose 20g, agar 20g add water to 1000ml.
In the basal medium of measuring the nitrogenous source utilization, add respectively for trying a kind of of nitrogenous source, addition is the absolute mass of 2g(N), pack into behind the mixing in the 250ml triangular flask, every bottled 150ml puts into 121 ℃ of sterilizations of autoclave 30min, treats to cool off slightly the back and falls dull and stereotyped, the about 20ml medium of every plate, each dull and stereotyped central authorities connects the bacterium piece that a diameter is 5mm, and each processing arranges 5 repetitions, places under 25 ℃ of constant temperature culture conditions continuous culture 20 days.Came divided by the interval fate with the difference of twice test number with right-angled intersection method mensuration colony diameter, and be the mycelial growth rate of average every day in per 7 days.
Table 2 nitrogenous source is to the influence of Finc-LD-4 and FLD-D mycelial growth rate
As shown in Table 2, supply to try nitrogenous source from 3 kinds, FLD-D mycelial growth in the medium that with wheat bran is nitrogenous source is the fastest, and average growth rate is 1.74cm/d, is peptone secondly, is ammonium sulfate, only 0.71cm/d at last; And Finc-LD-4 of the present invention average growth rate of mycelia in the medium that with wheat bran is nitrogenous source has reached 1.81cm/d, than the fast 0.09cm/d of wild strain, growth rate is taken second place in peptone, is 1.01cm/d, it is the slowest that ammonium sulfate is that nitrogenous source is then grown, and is 0.80cm/d.Show that thus in the medium of different nitrogen sources, the average mycelial growth rate of Finc-LD-4 is all fast than wild strain, wheat bran is best,
Peptone takes second place, and ammonium sulfate is the poorest.
Embodiment 4 C/N ratios are to the influence of Lyophyllum decastes mycelial growth
Be carbon source with glucose, ammonium sulfate is nitrogenous source, use magnesium sulfate 0.5g, yeast extract 2g, potassium dihydrogen phosphate 1g, agar 20g, add water to 1000ml, preparation C/N ratio (C/N) is respectively 5 kinds of medium of 20:1,30:1,40:1,50:1 and 60:1, in the 250ml triangular flask of packing into, every bottled 120ml, pH is adjusted into 6.0, puts into 121 ℃ of sterilizations of autoclave 30min, treats to cool off slightly the back and falls dull and stereotyped.The about 20ml medium of every plate is the bacterium piece of 5mm at each dull and stereotyped central authorities inoculation one diameter, and the processing of each C/N ratio arranges 5 repetitions, places under 25 ℃ of constant temperatures continuous culture 20 days.Measured colony diameter with the right-angled intersection method in per 5 days, twice test number difference namely draws the mycelial growth rate of average every day divided by the interval fate, the results are shown in Table 3.
Table 3 C/N ratio is to the influence of the mycelial growth of Finc-LD-4 and FLD-D
As known from Table 3, when C/N ratio increased to 60:1 by 20:1, the mycelial growth rate variation tendency of Finc-LD-4 and two bacterial strains of FLD-D was not obvious, when C/N ratio is 50:1, all reach maximum growth rate, Finc-LD-4 is 0.51cm/d, and uniformity is better, and FLD-D is 0.42cm/d.Show that thus Finc-LD-4 bacterial strain of the present invention is cultivated, and is stronger than the adaptive capacity of wild strain FLD-D in the medium of different C/N ratios, it is faster to send out bacterium speed, simultaneously, the Finc-LD-4 bacterial strain suits to grow in the medium of various C/N ratios, and is stronger to the adaptive capacity of medium.
The influence of the Lyophyllum decastes mycelial growth of embodiment 5pH
Sodium hydroxide solution with 1.0% and 1.0% hydrochloric acid solution are regulated the acid-base value of PDA medium, obtain the pH value and are respectively 6 soda acid gradients of 4.0,5.0,6.0,7.0,8.0 and 9.0.The medium of gained is respectively charged in the 250ml triangular flask, and every bottle of 120ml puts into 121 ℃ of sterilizations of autoclave 30min, and cooling is fallen dull and stereotyped slightly.The dull and stereotyped about 20ml medium of every culture dish, treat to cool off fully afterwards solid plate.During inoculation, with one of the bacterium colony of the aseptic card punch cut-off footpath 5mm of sterilization, be seeded in the dull and stereotyped central authorities of PDA.Same pH condition is done 5 repetitions, places under 25 ℃ of constant temperature culture conditions continuous culture 20 days.Measured colony diameter with the right-angled intersection method in per 5 days, the difference of measuring numerical value twice namely draws the mycelial growth rate of average every day again divided by the interval fate, the results are shown in Table 4.
The influence of the table Finc-LD-4 of 4pH and FLD-4 mycelial growth
As shown in Table 4, the optimal pH of Finc-LD-4 and the required medium of FLD-D all is 8.0, and mycelial growth rate is respectively 0.76cm/d and 0.97cm/d, and Finc-LD-4 is faster slightly than FLD-D growth rate.And, in the medium of identical pH condition, to cultivate, the mycelial growth rate of Finc-LD-4 is all slightly fast than FLD-D.
Embodiment 6 morphological features and biological property
Morphological feature: aerial hyphae is dense; Cap: diameter is 3 ± 2cm, and it is light yellow that color is, circle, and there is tiny fine hair on the cap surface, and cap is flat, and very easily parachute-opening is patellate, and quality is tender and crisp, frangible; Lamella: light yellow, the lamella section is crescent; Stem: length is 6～15cm, and stem top is pale yellow, and the bottom is pure white, and near the stem interior solid consolidation at cap place, away from cap place stem hollow, stem has scale, scale main give birth in the middle and upper part near cap, it is irregular to distribute, stem clusters.
Biological property: nutrient component source: wood chip, corncob, rice bran, wheat bran, corn flour, cotton seed hulls, soybean skin etc., wherein, water content 64%～66% (percentage by weight, down together).
Cultivate and cultivation condition: the mycelium cultivation temperature is 20～25 ℃, humidity 75%, CO
2Concentration 2000～3500ppm, cultivation cycle 50～70 days.Suitable fruiting temperature is 15～18 ℃, humidity 93%～100%, CO
2Concentration 1000～2000ppm, preceding 1～7 day, intensity of illumination 10～50Lux, 8～32 days illumination intensity 200～300Lux in back, every day, clearance-type was opened layer bracket lamp stimulation fruiting, and the cultivation cycle is about 32 days.
Embodiment 7 cultivation comparative trials
Present embodiment passes through from the new bacterial strain Finc-LD-4 of pleat umbrella, with cultivate contrast without habituation of wild bacterial strain FLD-D, investigate the anti-assorted bacterium ability of strains tested, index such as a situation arises, the concentration degree of gathering and per unit area yield, the individual plant fruit body stem length uniformity of the original hase (forming fruit body in the future) under the earthing condition not, be applicable to that to embody this bacterial strain the batch production bottle plants advantage and the characteristics of production.
The cultivated species medium that present embodiment adopts is: wood chip 15%, and corncob 25%, cotton seed hulls 25%, rice bran 10%, wheat bran 20% and corn flour 5%, water content is 64%～66%.
Cultivation stage pollution rate test is taked to cultivate common best cultivation again and carry out in advance earlier, inoculate 128 bottles, is placed in the high pre-incubation chamber of cleanliness factor cultivation then 15 days, go to common culturing room again, total cultivation cycle 64 days, statistics pollution condition during mycelium stimulation, the result consults table 5.
As known from Table 5, the new bacterial strain Finc-LD-4 of the Lyophyllum decastes pollution rate that obtains by domestication is 0.78%, and the wild strain pollution rate is 3.91%.This shows, compare with wild strain that Finc-LD-4 has stronger anti-assorted bacterium infection ability.
Table 5Finc-LD-4 and FLD-D cultivation stage pollute contrast
Original hase generation number is research object with above-mentioned 128 inoculation bottles in the cultivation process, and under the situation of earthing not, a situation arises sees Table 6 for the original hase of Finc-LD-4 and wild strain FLD-D.As known from Table 6, the new bacterial strain Finc-LD-4 of Lyophyllum decastes by domestication obtains compares with wild strain, and original hase generation ratio is higher, and is stronger for the adaptive capacity of soil covering culture not.
A situation arises contrasts for table 6Finc-LD-4 and FLD-D original hase
Same batch of concentration degree of gathering of statistics is subjects with above-mentioned 128 inoculation bottles when gathering, and result of the test is consulted table 7.As known from Table 7, the new bacterial strain Finc-LD-4 of Lyophyllum decastes that obtains by domestication concentrates on first and gathers, and recovery ratio reaches 70.25%, and the gathering based on second batch of FLD-D, recovery ratio is 60.00%.This shows, compare with wild strain that the fruit body of Finc-LD-4 cycle of gathering is more concentrated, and concentrate the date of gathering obviously to shift to an earlier date than wild strain.
Table 7Finc-LD-4 and the FLD-D concentration degree of gathering contrasts
The subjects that is used for per unit area yield mensuration is above-mentioned 128 inoculation bottles, as known from Table 8, the new bacterial strain Finc-LD-4 of Lyophyllum decastes by the domestication acquisition, compare with wild strain, the cultivation per unit area yield is higher, under the situation of same test quantity, Finc-LD-4 has obtained higher gross yield, and total amount of increase in production has reached 30.78%.
Per unit area yield contrast after table 8Finc-LD-4 and FLD-D gather
Embodiment 8 uniformity comparative trials
Finc-LD-4 and the FLD-D back fruit body of gathering is carried out the cap uniformity and stem length consistency analysis, as follows:
As shown in Table 9, Finc-LD-4 bacteria cover diameter average out to 2.6cm, FLD-D bacteria cover diameter average out to 2.8cm, the coefficient of variation of Finc-LD-4 bacteria cover diameter is 33.1%, the coefficient of variation of FLD-D bacteria cover diameter is 37.5%, as seen the relative variability degree of Finc-LD-4 bacteria cover diameter is less, illustrates that the bacteria cover diameter of Finc-LD-4 is more even.
The different Lyophyllum decastes bacterial strain of the table 9 fruit body cap size uniformity
Table 10 shows, Finc-LD-4 stem length average out to 9.2cm, FLD-D stem length average out to 8.9cm, the coefficient of variation of Finc-LD-4 stem length is 22.2%, the coefficient of variation of FLD-D stem length is 36.6%, and the relative variability degree of visible Finc-LD-4 stem length is less, and this explanation Finc-LD-4 stem length of the present invention is more even, the mushroom handle length uniformity of Dan Duogu body is better, instant packed.
The long uniformity of the different Lyophyllum decastes bacterial strain of table 10 fruit body stem
The embodiment 9ITS evaluation of checking order
Present embodiment student on commission worker biotechnology (Shanghai) limited company (hereinafter to be referred as " giving birth to the worker ") carries out, and may further comprise the steps:
Genomic extraction: extract according to giving birth to worker SK1375 fungal gene group DNA extraction agent box specification.Resulting dna solution is placed-20 ℃ of preservations, standby.
The pcr amplification system is: cumulative volume 25 μ L, genomic templates 1 μ L, fungi rDNA universal primer ITS1 sequence is 5'-TCCGTAGGTGAACCTGCGG-3'(10 μ M) 0.5 μ L, the ITS4 sequence is 5'-TCCTCCGCTTATTGATATGC-3'(10 μ M) 0.5 μ L, dNTP mix (10mM each) 0.5 μ L, 10 * Taq reaction buffer, 2.5 μ L, 5U/ μ L Taq0.2 μ L adds water to 25 μ L.The PCR program: pre-94 ℃ of 5mim of sex change, 94 ℃ of 30s, 55 ℃ of 35s, 72 ℃ of 1min, 35 circulations, 72 ℃ are extended 8min.
Adopt conventional sequence measurement that above-mentioned pcr amplification product is checked order.The ITS sequence sequencing result of this test strain Finc-LD-3 sees also sequence table, and its sequence is the dna fragmentation of 691bp.Check in from GenBank, Finc-LD-4 bacterial strain ITS sequence is the highest with the ITS sequence similarity that belongs to Lyophyllum decastes from the pleat umbrella.Simultaneously, from GenBank, check in and the ITS sequence of close to pleat umbrella symbolic animal of the birth year, planting, use the phylogenetic tree that DNAStar MegAlign software building goes out strains tested, see also accompanying drawing 2.
More than describing is embodiments of the invention only, forgives and can understand, and under the prerequisite that does not depart from the present invention's design, all should be included within the technical conceive of the present invention simple modification of the present invention and replacement.
SEQUENCE LISTING
＜110〉Shanghai Finc Bio-tech Inc.
＜120〉Lyophyllum decastes bacterial strain-4
＜130〉ITS sequence
<160>1
<170>PatentIn version3.3
<210>1
<211>691
<212>DNA
<213>Lyophyllum shimeji
<400>1
tccgtaggtg aacctgcgga aggatcatta ttgaataaat ttggctgggc tgttgctggc 60
tcctaggagt atgtgcacgc ctagctccat tttttaccac ctgtgcacct tttgtagacc 120
tggatatctt tcgaggaaac tcggtttgag gactgcggag cgaaagccct agctttcctt 180
acatttccgg tctatgtctt tacatacccc atatgaatgt aacagaatgt cgtttactgg 240
cctttgtgcc tttaatcaaa tacaactttc agcaacggat ctcttggctc tcgcatcgat 300
gaagaacgca gcgaaatgcg ataagtaatg tgaattgcag aattcagtga atcatcgaat 360
ctttgaacgc acctggcgct ccctggtatt ccggggagca tgtctgtttg agtgtcatta 420
aattctcaac ctttccaact tttgcgagtt tggttaggct tggatgtggg gggttgcggg 480
cttcacagaa gtcggctcct ctgaaatgca ttagtgaaac ctttgttgat catctcttgg 540
tgtgataatt atctacgcca ctagagtgtc agcctcaact tagttgagag gtttttgctt 600
ctaatcgtcc tccaaggaca acttctttac tttttgacct caaatcagat aagactaccc 660
gctgaactta agcatatcaa taagcggagg a691
Claims (1)
1. new bacterial strain Finc-LD-4 of Lyophyllum decastes, its deposit number is CCTCC NO:M 2013008.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104350954A (en) * | 2014-02-22 | 2015-02-18 | 江苏菇本堂生物科技股份有限公司 | Lyophyllum decastes cultivation method |
| CN105210676A (en) * | 2015-10-29 | 2016-01-06 | 青岛丰科生物科技有限公司 | Lyophyllum decastes bacterial strain Finc-L6 and cultivation method thereof and application |
| CN105230342A (en) * | 2015-10-22 | 2016-01-13 | 上海丰科生物科技股份有限公司 | Lyophyllum decastes strain, cultivation method and application thereof |
| CN111264303A (en) * | 2020-04-09 | 2020-06-12 | 中华全国供销合作总社昆明食用菌研究所 | Lyophyllum fumosoroseum cultivar and preparation method thereof |
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| CN105210676A (en) * | 2015-10-29 | 2016-01-06 | 青岛丰科生物科技有限公司 | Lyophyllum decastes bacterial strain Finc-L6 and cultivation method thereof and application |
| CN111264303A (en) * | 2020-04-09 | 2020-06-12 | 中华全国供销合作总社昆明食用菌研究所 | Lyophyllum fumosoroseum cultivar and preparation method thereof |
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