CN108486215A - A method of the anti-red streak sugar cane breed of identification - Google Patents
A method of the anti-red streak sugar cane breed of identification Download PDFInfo
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- CN108486215A CN108486215A CN201810190507.6A CN201810190507A CN108486215A CN 108486215 A CN108486215 A CN 108486215A CN 201810190507 A CN201810190507 A CN 201810190507A CN 108486215 A CN108486215 A CN 108486215A
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Abstract
The present invention relates to a kind of methods of the anti-red streak sugar cane breed of identification, including sugarcane red streak Bacteria culturing and suspension bacteria liquid preparation, cane seedling cultivation, pathogen inoculated identification, Disease investigation.The identification method of the present invention is not influenced by external environmental condition, as a result more stable, more reliable, actual conditions of the experimental result close to field artificial infection Resistance Identification method, resistance level of the sugar cane breed to red streak can intuitively be shown, the Resistance Identification period can be effectively shortened, time saving and energy saving compared with Field inoculation Resistance Identification, operability is strong, easy to operate, it is low to experimental facilities requirement, it is easy to be used by breeding units.The method of the anti-red streak sugar cane breed of identification of the present invention provides technical support for the screening anti-red streak kind of sugarcane, has significant application value to the anti-red streak breeding of sugarcane.
Description
Technical field
The present invention relates to a kind of methods of Sugarcane Disease Resistance Identification, and in particular to a kind of anti-red streak sugar cane breed of identification
Method, belong to crop disease Resistance Identification technical field.
Background technology
Sugarcane red streak（red stripe disease）It is a kind of bacterial disease generally occurred, in Major Sugarcane kind
Planting country has generation, China's sugarcane district also generally to occur, and causes to seriously affect to the sustainable development of Sugarcane Industry and sugar industry.
Sugarcane red streak symptom has 2 type of leaf streak and top rot, can individually occur, can also occur simultaneously.Leaf streak type：It is big multiple
It is born in tender leaf middle part of blade, close to the position of middle arteries.Water stain shape an IOU issued by a post office is most started to behave as, rear an IOU issued by a post office both ends rapidly develop growth
For strip red to bronzing scab.The incidence tissue of blade and health tissues boundary are apparent.The width of streak is 0.5 mm~4
Mm, long tens millimeters are arrived blade overall length.Streak sometimes 2 or it is a plurality of connection become band, certain kinds may extend to leaf sheath.Top rot
Type：Disease symptom is old leaf yellow, withers, and early stage disease symptom is that the vascular bundle near sugarcane production point reddens, rots, after
Further develop, leads to the formation of the big pocket of internode, the later stage causes lobus cardiacus to rot, and draws in the leaf sheath that can be enclose from the outside
Out, and there is a kind of special rank smell.The destructive disease that top rots to be formed can cause serious economic loss.
Sugarcane red streak is initially believed to be by Pseudomonas rubrilineans（Pseudomonas rubrilineans
Stapp）Cause, Gram's staining reaction is feminine gender, and morphological feature is no gemma, is in rod-shaped, size is 0.7 ~ 1.6 μm, is had
Single polar flagella has thin-walled pod film.Then, Pseudomonas rubrilineans rename as oat lactic acid bacteria oat subspecies
（Acidovorax avenae subsp. avenae, Aaa）.Sugarcane red streak bacterium is propagated in field mainly by wind, rain, in tide
It is easy morbidity under conditions of damp-warm syndrome is warm, the possibility very little propagated by sugarcane kind or mechanical path.
Since sugarcane is asexually propagated crop, crop rotation region is few for many years, long-term continuous cropping plantation, and single varietiesization are tight
Weight results in the constantly accumulation of sugarcane pest and disease damage and aggravates；In addition there is plantation throughout the year, provided to pest and disease damage propagation good
Habitat condition.One of an important factor for Sugarcane Disease is influence sugarcane yield and sugar, also seriously affects Leifsonia performance, contracts
The short perennial root time limit, or even cause variety deterioration, and disease is once generation, even if producing little effect if with chemical control.Selection and breeding and
It is most economical Sugarcane Disease prevention and control, most efficient method and a kind of green ecological Control Technology to promote disease-resistant variety, but so far
Not yet establish quick, efficient, accurate sugar cane breed red streak Resistance Identification method.The usual Field inoculation Resistance Identification period
It is long, big by environmental influence, therefore, it is imperative to establish a kind of method of the anti-red streak sugar cane breed of Rapid identification.
Invention content
For the blank of China's sugarcane red streak Resistance Identification method, the purpose of the present invention is to provide a kind of easy, fast
Speed, the method for accurately identifying anti-red streak sugar cane breed.
To achieve the above object, the technical method that the present invention uses is as follows：
The method of the anti-red streak sugar cane breed of a kind of identification of the present invention, it is characterised in that authentication step is as follows：
（1）It is prepared by sugarcane red streak Bacteria culturing and suspension bacteria liquid
Sugarcane red streak pathogen is activated twice on NA solid mediums using plate streak, picking single bacterium colony is trained in NB
Bacterium solution is centrifuged removal supernatant, 10 is diluted to sterile water by 28 DEG C of shaking table cultures 12 hours in nutrient solution8 CFU/mL is configured to
Sugarcane red streak pathogen suspension bacteria liquid, it is spare；
（2）Cane seedling is cultivated
Each sugarcane kind is broken apart by chopping into simple bud seedling stem, by 50 DEG C of lukewarm water hot water add 0.1 ~ 0.2% carbendazim handle 2 hours, then
Transplanting plantation is spare when sugarcane plant grows to 3 ~ 4 leaf complete leaf to seedling culture hole plate；
（3）Pathogen inoculated identification
By step（1）The side that obtained sugarcane red streak pathogen suspension bacteria liquid is combined by stab inoculation and spray inoculation
Method is inoculated into step（2）The sugarcane plant leaf of each kind；Transparent plastic freshness protection package bagging, plant is used to protect after inoculation
Wet 48 hours, condition of culture was 28-32 DEG C, illumination 16 hours, humidity 80-90%；
（4）Disease investigation
After inoculation 3 days, start to count sugar cane breed red streak morbidity severity and incidence, it is primary every investigation in 7 days, it investigates altogether
Four times, sugar cane breed red streak resistance level is evaluated according to disease index, disease index≤30% is the sugarcane product of anti-red streak
Kind.
Wherein, step（1）The preparation method of the NA solid mediums：Take 10 g of peptone, 3 g of powdered beef, sodium chloride 5
G, 15 g of agar powder is dissolved in 900 ml pure water, is settled to 1 L after adjusting pH=7.3, is then sterilized 20 minutes at 121 DEG C, when
It is cool to 45-55 DEG C when, ampicillin and vancomycin is added, makes final concentration of 75 mg/L of ampicillin, vancomycin
Final concentration of 25 mg/L；
Step（1）The preparation method of the NB culture solutions：10 g of peptone, 3 g of powdered beef, 5 g of sodium chloride are taken, 900 ml are dissolved in
In pure water, it is settled to 1 L after adjusting pH=7.2, is then sterilized 20 minutes at 121 DEG C, when cool to when being less than 55 DEG C, ammonia benzyl is added
Penicillin and vancomycin make final concentration of 75 mg/L of ampicillin, final concentration of 25 mg/L of vancomycin.
The advantage of the invention is that：
1, identification method of the invention is not influenced by external environmental condition, as a result more stable, more reliable, experimental result close to
The actual conditions of field Inoculation Method identification sugarcane red streak morbidity, can intuitively show sugar cane breed to red streak
Resistance level.
2, identification method of the invention can effectively shorten the Resistance Identification period, the time saving province compared with Field inoculation Resistance Identification
Power, operability is strong, easy to operate, requires experimental facilities low, the easy use of each breeding units.
3, identification method of the invention provides technical support for the screening anti-red streak kind of sugarcane, is educated to the anti-red streak of sugarcane
Kind has significant application value.
Specific implementation mode
In order to the present invention is furture elucidated rather than limitation the present invention with reference to specific embodiments to the present invention make into
The detailed elaboration of one step.
Embodiment 1, a kind of method of the anti-red streak sugar cane breed of identification, include the following steps：
1, prepared by sugarcane red streak Bacteria culturing and suspension bacteria liquid
Separation obtains pathogen oat lactic acid bacteria from the sugar cane breed GT42 red streak disease leaf samples that Nanning sugarcane district acquires
Oat subspecies（Acidovorax avenae subsp. avenae）Strains A AGT1 adopts the bacterial strain on NA solid mediums
After being activated twice with plate streak, single bacterium colony is chosen 28 DEG C in NB culture solutions, 200 rpm shaken cultivations 12 hours, is received
Collect thalline, it is 10 to adjust bacterial concentration with sterile water8CFU/mL is configured to sugarcane red streak pathogen suspension bacteria liquid.
2, sugar cane breed（System）Seedling fostering
Choose good fortune agriculture 09-4095, cloud sugarcane 09-1601, new platform sugar No. 22, osmanthus sugar No. 42, Fujian sugar 11-610,5 different sugarcane product
Kind（System）Seedling stem breaks apart by chopping into simple bud seedling stem, adds 0.1% carbendazim to handle 2 hours by 50 DEG C of lukewarm water hot water, then transplants extremely
72 holes（54 cm × 28 cm）Seedling culture hole plate can also select appropriate size according to actual needs.Each kind inoculation is at least
60 plants.Sugarcane plant carries out bacterium solution inoculation when growing 3 ~ 4 complete leaves.
3, bacterium solution needle thorn and spray inoculation
Using disposable sterilized injector by+1 leaf or gently penetrating tissue 10 times of+2 blade bases, sterile absorbent cotton-wool is then used
It is impregnated with sugarcane red streak pathogen suspension bacteria liquid, and is covered in blade and stabs place, then, then it is sweet with the whole strain of same bacterium solution spraying
Sugarcane plant leaf.Sugarcane plant carries out bagging processing with transparent plastic freshness protection package after inoculation, then puts artificial climate incubator into
It is cultivated, condition of culture is 30 DEG C, 16 h of illumination, humidity 85%.
4, sugar cane breed red streak evaluation of resistance
After inoculation 3 days, every plant of red streak morbidity severity of each sugar cane breed and incidence are instituted an inquiry, and make a record.Often
It is primary every investigation in 7 days, it investigates four times.Sugar cane breed plant red streak morbidity severity is evaluated by 1 standard of table, according to each
Sugar cane breed incidence and every plant of morbidity severity calculate disease index.
1 sugar cane breed plant leaf red streak of table morbidity severity judgment criteria
The disease index calculation formula is：
Disease index=[∑（Morbidity number of degrees × corresponding morbidity number）/（Highest morbidity number of degrees × total investigation
Number）]×100.
Grading evaluation is carried out by morbidity grading standard, morbidity grading standard is shown in Table 2.It is evaluated according to disease index
Sugar cane breed red streak disease resistance, disease index≤30% are the sugar cane breed of anti-red streak.
2 sugarcane red streak varietal resistance opinion rating standard of table
5, investigation finishes, that is, after being inoculated with one month, calculates good fortune agriculture 09-4095, cloud sugarcane 09-1601, new platform sugared No. 22, osmanthus sugar 42
Number, the informal voucher disease disease indexs of Fujian sugar 11-610 sugar cane breeds, respectively 12%, 4%, 1%, 60% and 35%.Resistant reaction type is distinguished
It is disease-resistant for good fortune agriculture 09-4095, cloud sugarcane 09-1601, new platform sugar 22, osmanthus sugar 42 is high sense, and Fujian sugar 11-610 is susceptible.It is real
It is consistent with outdoor field resistance inoculation experiments result to test result.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification should all belong to the covering scope of the present invention.
Claims (3)
1. a kind of method of the anti-red streak sugar cane breed of identification, it is characterised in that authentication step is as follows：
（1）It is prepared by sugarcane red streak Bacteria culturing and suspension bacteria liquid
Sugarcane red streak pathogen is activated twice on NA solid mediums using plate streak, picking single bacterium colony is trained in NB
Bacterium solution is centrifuged removal supernatant, 10 is diluted to sterile water by 28 DEG C of shaking table cultures 12 hours in nutrient solution8 CFU/mL is configured to
Sugarcane red streak pathogen suspension bacteria liquid, it is spare；
（2）Sugar cane breed seedling fostering
Each sugarcane kind is broken apart by chopping into simple bud seedling stem, by 50 DEG C of lukewarm water hot water add 0.1 ~ 0.2% carbendazim handle 2 hours, then
Transplanting plantation is spare when sugarcane plant grows to 3 ~ 4 leaf complete leaf to seedling culture hole plate；
（3）Pathogen inoculated identification
By step（1）The side that obtained sugarcane red streak pathogen suspension bacteria liquid is combined by stab inoculation and spray inoculation
Method is inoculated into step（2）The sugarcane plant leaf of each kind；Transparent plastic freshness protection package bagging, plant is used to protect after inoculation
Wet 48 hours, condition of culture was 28-32 DEG C, illumination 16 hours, humidity 80-90%；
（4）Disease investigation
After inoculation 3 days, start to count sugar cane breed red streak morbidity severity and incidence, it is primary every investigation in 7 days, it investigates altogether
Four times, sugar cane breed red streak resistance level is evaluated according to disease index, disease index≤30% is the sugarcane product of anti-red streak
Kind.
2. a kind of method of anti-red streak sugar cane breed of identification according to claim 1, it is characterised in that step（1）It is described
The preparation method of NA solid mediums：10 g of peptone, 3 g of powdered beef, 5 g of sodium chloride, 15 g of agar powder are taken, 900 ml are dissolved in
In pure water, adjust pH=7.3 after be settled to 1 L, then sterilize 20 minutes at 121 DEG C, when it is cool to 45-55 DEG C when, addition ammonia benzyl
Penicillin and vancomycin make final concentration of 75 mg/L of ampicillin, final concentration of 25 mg/L of vancomycin.
3. a kind of method of anti-red streak sugar cane breed of identification according to claim 1, it is characterised in that step（1）It is described
The preparation method of NB culture solutions：10 g of peptone, 3 g of powdered beef, 5 g of sodium chloride are taken, is dissolved in 900 ml pure water, pH=7.2 are adjusted
After be settled to 1 L, then sterilize 20 minutes at 121 DEG C, when cool to when being less than 55 DEG C, ampicillin and mould through the ages be added
Element makes final concentration of 75 mg/L of ampicillin, final concentration of 25 mg/L of vancomycin.
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| CN201810190507.6A CN108486215A (en) | 2018-03-08 | 2018-03-08 | A method of the anti-red streak sugar cane breed of identification |
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| CN201810190507.6A CN108486215A (en) | 2018-03-08 | 2018-03-08 | A method of the anti-red streak sugar cane breed of identification |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111937622A (en) * | 2020-08-18 | 2020-11-17 | 广西特色作物研究院 | Inoculation method of citrus canker pathogen |
| CN114793812A (en) * | 2022-04-13 | 2022-07-29 | 华南农业大学 | Method for identifying sugarcane white streak disease resistance |
Citations (3)
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| US6423499B1 (en) * | 1999-09-10 | 2002-07-23 | The United States Of America, As Represented By The Secretary Of Agriculture | PCR primers for detection and identification of plant pathogenic species, subspecies, and strains of acidovorax |
| CN102204465A (en) * | 2011-04-20 | 2011-10-05 | 广州甘蔗糖业研究所 | Method for identifying sugarcane smut resistance |
| CN104450862A (en) * | 2014-11-04 | 2015-03-25 | 广西大学 | Sugarcane top rot resistance identification method |
-
2018
- 2018-03-08 CN CN201810190507.6A patent/CN108486215A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6423499B1 (en) * | 1999-09-10 | 2002-07-23 | The United States Of America, As Represented By The Secretary Of Agriculture | PCR primers for detection and identification of plant pathogenic species, subspecies, and strains of acidovorax |
| CN102204465A (en) * | 2011-04-20 | 2011-10-05 | 广州甘蔗糖业研究所 | Method for identifying sugarcane smut resistance |
| CN104450862A (en) * | 2014-11-04 | 2015-03-25 | 广西大学 | Sugarcane top rot resistance identification method |
Non-Patent Citations (3)
| Title |
|---|
| SYED ZIA-UL-HUSSNAIN等: "Isolation and biochemical characterizations of the bacteria (Acidovorax avenae subsp. avenae) associated with red stripe disease of sugarcane", 《AFRICAN JOURNAL OF BIOTECHNOLOGY》 * |
| 刘慧等: "《现代食品微生物学实验技术》", 28 February 2017, 中国轻工业出版社 * |
| 李晓燕等: "甘蔗赤条病病原菌分离与鉴定", 《中国植物病理学会2016年学术年会论文集》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111937622A (en) * | 2020-08-18 | 2020-11-17 | 广西特色作物研究院 | Inoculation method of citrus canker pathogen |
| CN111937622B (en) * | 2020-08-18 | 2022-08-02 | 广西特色作物研究院 | Inoculation method of citrus canker pathogen |
| CN114793812A (en) * | 2022-04-13 | 2022-07-29 | 华南农业大学 | Method for identifying sugarcane white streak disease resistance |
| CN114793812B (en) * | 2022-04-13 | 2023-06-06 | 华南农业大学 | Method for identifying sugarcane white streak resistance |
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