CN109609588A - A kind of accurate identification method of planting Sugarcane white leaf disease resistance - Google Patents
A kind of accurate identification method of planting Sugarcane white leaf disease resistance Download PDFInfo
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Abstract
A kind of accurate identification method of planting Sugarcane white leaf disease resistance, comprising: cut and infected the juicing of Sugarcane white leaf phytoplasma sugarcane stem, 10 times of amount sterile waters is added to prepare inoculation liquid；Sugarcane smut sugarcane stem of participating in the experiment is cut into stem with bud, and flowing running water impregnates 48h, 50 DEG C of hot water treatment 2h, pesticide-germicide Medicament soaked seed 10min；Coated plastic film be inoculated with 25 DEG C at moisturizing for 24 hours；Inoculation material is planted in 20-30 DEG C of insect prevention hot-house culture built in plastic barrel；Diseased plant rate is investigated, carries out disease resistance evaluation by 1-5 grades of grade scales.The method of the present invention creates a set of precisely efficient planting Sugarcane white leaf Resistance Identification method for the first time, provides technical support for the anti-hoja blanca breeding of sugarcane；The inoculation of sugarcane kind coated plastic film is sprayed by cause of disease inoculation liquid, has morbidity habitat conditions, it is as a result objective, true, reliable；It is inoculated with fast and convenient, strong operability, work efficiency are high, and Sugarcane white leaf morbidity is significant after inoculation, high sensitivity, favorable reproducibility.
Description
Technical field
The invention belongs to crop resistance identification technology fields, and in particular to a kind of planting Sugarcane white leaf disease resistance is accurate
Identification method.
Background technique
Sugarcane white leaf (Sugarcane white leaf, SCWL) is first former by Sugarcane white leaf plant on China sugarcane
The important new expression of quarantine risk caused by body (Sugarcane white leaf phytoplasma), diffusion sprawling is very
Rapidly, China's sugarcane production and development are seriously threatened.Sugarcane white leaf in 1954 for the first time Northern Thailand Nan Bangfu find,
The countries in Southeast Asia such as India, Pakistan, Sri Lanka, Japan, Laos, Burma, Vietnam, Philippine are betided extensively.
Sugarcane white leaf phytoplasma (Sugarcane white leaf phytoplasma) is cell-free wall prokaryotic micro-organisms, Shang Buneng
In artificial medium in vitro culture, the incubation period is longer, and shortest is about 30d (minority), and generally 2-3 months, some was even long
For 1 year.
SCWL is a kind of extremely hazardous phytoplasma disease, can cause crushing disaster to sugarcane.It falls ill in Thailand SCWL
Rate reaches 5%-35%, brings lose more than 20,000,000 dollars every year.In New Guinea, which causes cultivar glug to receive
Loss up to 100%, causes very big economic loss to locality.2012 Nian Liwen phoenix etc. detect discovery in Baoshan, Yunnan for the first time
SCWL phytoplasma, occurring area has expanded to 20,000 mu or more at present；2013-2017, successively Yunnan Province Lincang Geng Maxian,
The sugarcane districts such as Zhenkang County, the county Shuan Jiang, the county Cang Yuan, Linxiang District and Pu'er county Xi Meng detect SCWL phytoplasma, at present generation area face
Product in extension sprawling increasingly and aggravates situation more than 300,000 mu.After sugarcane infects SCWL phytoplasma, plant height, stem diameter, at stem rate
It is substantially reduced or reduces again with single stem, the significantly underproduction is caused to subtract sugar.Serious cane yield 6-7 tons per acre of new plant cane of field, the
Destruction is effectively caused without receipts because of SCWL harm within 2 years.SCWL is very rapid in Yunnan Sugarcane Area extension sprawling, gives Yunnan sugar industry
Development brings severe disaster threat, such as cannot get effective prevention and control, can may also bring to sugarcane districts such as China Guangxi province, Guangdong Province
Potential threat.
Breeding and popularization disease-resistant variety are that the most economical effective method of prevention and control Sugarcane Disease and most potential green are raw
State Control Technology.Sugarcane white leaf is the new expression that Quick Extended gets up in recent years, not yet establishes Sugarcane white leaf resistance mirror so far
Determine method.How the hoja blanca disease resistance of accurate efficient evaluations sugar cane breed, which is current cane breeding man breeding resistant variety, faces
A technical bottleneck.In view of China's Sugarcane white leaf increasingly extend sprawling and aggravate situation, therefore be badly in need of exploitation establish it is a set of
Precisely, efficiently it is anti-to fill up domestic Sugarcane white leaf for Sugarcane white leaf Resistance Identification method, the development for pushing breeding for disease resistance to work
Sick breeding research blank.
Summary of the invention
Sugarcane production is seriously threatened for quarantine new expression Sugarcane white leaf and lacks the production of method of resistance identification again
Industry technical problem, the present invention provide a kind of accurate, efficient planting Sugarcane white leaf method of resistance identification.
A kind of technical solution of accurate identification method of planting Sugarcane white leaf disease resistance provided by the present invention, including it is following
Step:
(1) inoculation liquid is prepared: being cut the sugarcane sugarcane stem for having infected Sugarcane white leaf phytoplasma, is obtained by squeezing the sugarcane stem
Sugarcane white leaf phytoplasma sugarcane juice is carried, is incorporated as carrying the sterile water dilution of 10 times of Sugarcane white leaf phytoplasma sugarcane juice volume amounts
It mixes, is filtered with double gauze, filtrate is cause of disease inoculation liquid, ready-to-use；
(2) Sugarcane smut of participating in the experiment processing: Sugarcane smut of participating in the experiment is cut into 1-2 bud sugarcane stem section of band, flows originally in room temperature
After impregnating 48h in water, with 50 DEG C of ± 0.5 DEG C of hot water treatment 2h, then the sugarcane stem section described in pesticide-germicide mixed liquid dipping
10min；
(3) coating inoculation: the Sugarcane smut of participating in the experiment handled well is placed in plastic film sprinkling inoculation liquid, is stirred in spray
It participates in the experiment Sugarcane smut, a part of the plastic foil is covered into the Sugarcane smut of participating in the experiment after having sprayed, moisturizing is for 24 hours at 25 DEG C；
(4) participate in the experiment the plantation of Sugarcane smut: be packed into plastic barrel be bucket inner volume 2/3 matrix, participating in the experiment after inoculation
Sugarcane smut is planted in plastic barrel, every part of 3-4 repetition of Sugarcane smut of participating in the experiment, and kind is implanted with to the plastic barrel for Sugarcane smut of participating in the experiment
It is placed in 20-30 DEG C of insect prevention greenhouse and cultivates observation；To feel the sugar cane breed of hoja blanca for susceptible check variety, with anti-hoja blanca
Sugar cane breed is disease-resistant check variety；
(5) Disease investigation: after inoculation plantation 30d, investigation records is respectively participated in the experiment Sugarcane smut diseased plant percentage, until susceptible right
According to kind diseased plant percentage stablize until；
(6) disease resistance evaluation: white by 1-5 grades of grading evaluation criterias progress sugarcanes according to Sugarcane smut diseased plant rate of respectively participating in the experiment
Leaf disease disease resistance evaluation, wherein 1-5 grades of grading evaluation criterias successively indicate to Sugarcane white leaf phytoplasma highly resistance, it is disease-resistant, in
Resistant, susceptible, high sense, diseased plant rate range mutually should be 0%-3.0%, 3.1%-10.0%, 10.1%-20.0%, 20.1%-
40.0%, 40.1%-100%.
Pesticide-germicide mixed liquor described in step (2) is that 70% Diacloden water dispersant 18.75g and 50% carbendazim are wettable
Property pulvis 18.75g mixing after with 15kg sterile water be made into pesticide-germicide mixed liquor.
The inoculation of coating described in step (3) are as follows: the Sugarcane smut of participating in the experiment handled well using power spraye by 1000kg
With the ratio of 15kg inoculation liquid, inoculation liquid is uniformly sprayed to the Sugarcane smut of participating in the experiment for being placed in plastic film.
Matrix described in step (4) is mixed by disinfection soil and disinfection well-rotted farmyard manure by 3:1 volume ratio.
Susceptible check variety described in step (4) is Guangdong sugar 86-368, and disease-resistant check variety is Guangdong sugar 83-88.
Beneficial effects of the present invention:
1, a set of accurate, efficient planting Sugarcane white leaf method of resistance identification is created for the first time, has filled up domestic sweet
Sugarcane hoja blanca breeding for disease resistance studies blank, provides key technology support for China's Sugarcane white leaf breeding for disease resistance.Utilize this hair
Bright method can realize to sugarcane quality germplasm, common hybrid parent and new varieties (being) anti-hoja blanca it is quick, precisely comment
Valence, by the practical accuracy and Breeding Efficiency for improving breeding for disease resistance, for anti-hoja blanca Screening of Germplasm and production kind of a choosing
It selects and is of great significance and realistic function.
2, the present invention wraps up the coating inoculation side of plastic film by cause of disease inoculation liquid sprinkling sugarcane kind (Sugarcane smut of participating in the experiment)
Formula identifies sugar cane breed hoja blanca disease resistance have and meet the habitat conditions that Sugarcane white leaf infects morbidity, can be straight
Sight shows sugar cane breed to the resistance level of Sugarcane white leaf, and qualification result is objective, true, reliable.
3, inoculation liquid of the invention does not have to be separately cultured, and directly acquires and prepares from field, inoculation method is fast and convenient, can grasp
The property made is strong, inoculation work efficiency is high.Sugarcane white leaf morbidity is significant after inoculation, high sensitivity, favorable reproducibility.Therefore, convenient to large quantities of
Amount material is identified and is screened, and provides more widely disease-resistant variety and anti-source materials for breeding and production departments.
Specific embodiment
1, it is inoculated with cause of disease and inoculation liquid is prepared
In Yunnan Province Lincang, the selection of Geng Maxian Sugarcane white leaf diseased region has the high sense kind of typical hoja blanca symptom
The sugarcane stem that Guangdong sugar 86-368 sugarcane strain detects screening phytoplasma containing Sugarcane white leaf through nest-type PRC makees inoculation cause of disease.Pass through before inoculation
The sugarcane stem that inoculation cause of disease is made in squeezing, which obtains, carries Sugarcane white leaf phytoplasma sugarcane juice, is incorporated as carrying Sugarcane white leaf phytoplasma sugarcane
The sterile water of 10 times of juice volume amounts, which dilutes, to be mixed, and is filtered with double gauze, and filtrate is Sugarcane white leaf phytoplasma inoculation liquid, existing
With current.
Typical case's hoja blanca symptom is that diseased plant blade shows as food value of leaf softness, albefaction, and tiller increased significantly.The whole leaf having
Albefaction, some are in striated albefaction, and some is in mottling albefaction.
The nest-type PRC is detected as conventional method, and referring to Li Wenfeng, WANG XIAOYAN, Huang Yingkun waits the important disease of sugarcane point
Sub- detection technique plant protection, 2016,42 (5): 125-130 is specifically included that
First time PCR amplification:
Using the universal primer of amplification phytoplasma 16S rDNA to P1/P7, in the nucleotide sequence of primer P1 such as sequence table
Shown in SEQ ID NO:1, the nucleotide sequence of primer P7 is as shown in SEQ ID NO:2 in sequence table.
0.2g Sugarcane Leaves are taken, liquid nitrogen grinding powdering is added, leaf is extracted using DNA Kit plant DNA extraction kit
Piece total DNA, specific steps are operated according to kit specification, albumen/nucleic acids instrument identification of dna quality are used after extraction, with P1/
P7 is that primer carries out the 1st PCR amplification, and PCR reaction system is 25 μ L: 1 μ L, 10 × PCR buffer2.5 μ of total genomic dna
L, P1 (20 μm of ol/L) 1 μ L, P7 (20 μm of ol/L) 1 μ L, MgCl2(25mmol/L) 2.0 μ L, dNTPs (10mmol/L) 2.0 μ L,
Taq enzyme (5U/ μ L) 0.2 μ L, ddH2O 15.3μL.1st PCR amplification program are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s,
55 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 recycle；Last 72 DEG C of extensions 10min.
Second of PCR amplification:
Using universal primer to R16F2n/R16R2, SEQ ID NO in the nucleotide sequence of primer R16F2n such as sequence table:
Shown in 3, the nucleotide sequence of primer R16R2 is as shown in SEQ ID NO:4 in sequence table.
2nd 25 μ L of PCR reaction system: taking the 1st pcr amplification product of 1 μ L (30 times of dilution) to be used as template DNA,
R16F2n (20 μm of ol/L) 1 μ L, R16R2 (20 μm of ol/L) 1 μ L, other reagents and its dosage are the same as the 1st PCR amplification.2nd time
PCR amplification program are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 recycle；Most
72 DEG C of extension 10min afterwards.
It takes the 2nd pcr amplification product of 10 μ L in electrophoresis on 1.0% Ago-Gel, is seen with BIO-RAD gel imaging system
It examines, differentiate as a result, amplifying 1240bp band is that the SCWL positive contains Sugarcane white leaf phytoplasma, do not amplify 1240bp item
Band is SCWL feminine gender without Sugarcane white leaf phytoplasma.
2, for examination Sugarcane smut processing
For trying Sugarcane smut: Guangdong sugar 60, is full of and educates 91-59, Guangdong sugar 93-159, ROC22, cloud sugarcane 86-161, cloud ROC25
Totally 10 different sugar cane breeds make experimental cultivar by sugarcane 03-194, ROC10, Liucheng 05-136, cloud sugarcane 05-51.
It is respectively cut into 2 bud sugarcane stem sections (i.e. double bud sections) of band respectively for examination Sugarcane smut, is soaked in room temperature flowing running water
After steeping 48h, with 50 DEG C of ± 0.5 DEG C of hot water treatment 2h, then the sugarcane stem section 10min described in pesticide-germicide mixed liquid dipping；Institute
Stating pesticide-germicide mixed liquor is after 70% Diacloden water dispersant 18.75g and 50% carbendazol wettable powder 18.75g is mixed
Pesticide-germicide mixed liquor is made into 15kg sterile water.
3, coating inoculation
The Sugarcane smut of participating in the experiment handled well is placed in plastic film, the ginseng handled well using power spraye by 1000kg
Inoculation liquid is uniformly sprayed the Sugarcane smut sprinkling of participating in the experiment for being placed in plastic film by the ratio for trying Sugarcane smut 15kg inoculation liquid
Inoculation liquid stirs Sugarcane smut of participating in the experiment in spray, a half mulching of the plastic foil is participated in the experiment Sugarcane smut after having sprayed, and at 25 DEG C
Lower moisturizing is for 24 hours.
4, for examination Sugarcane smut plantation
?Be packed into plastic barrel be bucket inner volume 2/3 matrix, the matrix by disinfection soil and
Sterilize well-rotted farmyard manure by disinfection soil: the volume ratio for sterilizing well-rotted farmyard manure mixes for 3:1, the disinfection soil with disappear
Malicious well-rotted farmyard manure is to be sterilized respectively with boiling a heatable brick bed to soil and well-rotted farmyard manure boiling 1h.Sugarcane smut kind of participating in the experiment after inoculation
It being implanted in the plastic barrel for filling the matrix, every part of Sugarcane smut of participating in the experiment plants 4 barrels i.e. every part and participates in the experiment 4 repetitions of Sugarcane smut,
Kind is implanted with the plastic barrel of Sugarcane smut of participating in the experiment and is placed in by sugarcane stem section totally 8 bud of every barrel of plantation 4 bands, 2 buds, 4 barrels of totally 32 buds
Observation is cultivated in 20~30 DEG C of insect prevention greenhouse；Make susceptible check variety with Guangdong sugar 86-368, Guangdong sugar 83-88 makees disease-resistant reference substance
Kind.
5, Disease investigation
After inoculation plantation 30d, investigation records is respectively participated in the experiment Sugarcane smut diseased plant percentage, investigates 1 time every 15d later, directly
Until the diseased plant percentage of susceptible check variety is stablized.Record project includes: to be inoculated with date, number of emerging, Disease symptoms beginning now
Phase, accumulative morbidity strain number.
6, disease resistance evaluation
Sugarcane is carried out by 1-5 grades of Sugarcane white leaf disease resistance grading evaluation criterias according to Sugarcane smut diseased plant rate is respectively participated in the experiment
Hoja blanca disease resistance evaluation, see Table 1 for details for 1-5 grades of Sugarcane white leaf disease resistance grading evaluation criterias.
Respectively participate in the experiment Sugarcane smut disease resistance it is as shown in table 2: Guangdong sugar 60, is full of and educates 91-59 and show as 5 grades of height ROC25
Sense, Guangdong sugar 93-159, ROC22 show as 4 grades it is susceptible, cloud sugarcane 86-161 show as in 3 grades resist, cloud sugarcane 03-194, ROC10 performance
Disease-resistant for 2 grades, Liucheng 05-136, cloud sugarcane 05-51 show as 1 grade of highly resistance.It respectively participates in the experiment Sugarcane smut Resistance Identification result and field
Natural occurrence situation matches.
1 1-5 grades of Sugarcane white leaf disease resistance grading evaluation criteria of table
Resistance Identification result of the different Sugarcane smuts of table 2 to Sugarcane white leaf
Sequence table
<110>Sugarcane Inst., Yunnan Prov. Agriculture Academy
<120>a kind of accurate identification method of planting Sugarcane white leaf disease resistance
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aagagtttga tcctggctca ggatt 25
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
cgtccttcat cggctctt 18
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<212> DNA
<213>artificial sequence (Artificial Sequence)
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gaaacgactg ctaagactgg 20
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tgacgggcgg tgtgtacaaa ccccg 25
Claims (5)
1. a kind of accurate identification method of planting Sugarcane white leaf disease resistance, which comprises the following steps:
(1) inoculation liquid is prepared: being cut the sugarcane sugarcane stem for having infected Sugarcane white leaf phytoplasma, is carried by squeezing the sugarcane stem
Sugarcane white leaf phytoplasma sugarcane juice, the sterile water dilution for being incorporated as carrying 10 times of Sugarcane white leaf phytoplasma sugarcane juice volume amounts are mixed
It is even, it is filtered with double gauze, filtrate is cause of disease inoculation liquid, ready-to-use；
(2) Sugarcane smut of participating in the experiment processing: Sugarcane smut of participating in the experiment is cut into 1-2 bud sugarcane stem section of band, in room temperature flowing running water
After impregnating 48h, with 50 DEG C of ± 0.5 DEG C of hot water treatment 2h, then the sugarcane stem section 10min described in pesticide-germicide mixed liquid dipping；
(3) coating inoculation: the Sugarcane smut of participating in the experiment handled well is placed in plastic film sprinkling inoculation liquid, stirs and participates in the experiment in spray
A part of the plastic foil is covered the Sugarcane smut of participating in the experiment after having sprayed by Sugarcane smut, and moisturizing is for 24 hours at 25 DEG C；
(4) participate in the experiment the plantation of Sugarcane smut: be packed into plastic barrel be bucket inner volume 2/3 matrix, the sugarcane of participating in the experiment after inoculation
Material is planted in plastic barrel, every part of 3-4 repetition of Sugarcane smut of participating in the experiment, and the plastic barrel that kind is implanted with Sugarcane smut of participating in the experiment is placed in
Observation is cultivated in 20-30 DEG C of insect prevention greenhouse；To feel the sugar cane breed of hoja blanca for susceptible check variety, with the sugarcane of anti-hoja blanca
Kind is disease-resistant check variety；
(5) Disease investigation: after inoculation plantation 30d, investigation records is respectively participated in the experiment Sugarcane smut diseased plant percentage, until susceptible reference substance
Until the diseased plant percentage of kind is stablized；
(6) disease resistance evaluation: Sugarcane white leaf is carried out by 1-5 grades of grading evaluation criterias according to Sugarcane smut diseased plant rate is respectively participated in the experiment
Disease resistance evaluation, wherein 1-5 grades of grading evaluation criterias successively indicate to Sugarcane white leaf phytoplasma highly resistance, it is disease-resistant, in resist, sense
Disease, high sense, diseased plant rate range mutually should be 0%-3.0%, 3.1%-10.0%, 10.1%-20.0%, 20.1%-
40.0%, 40.1%-100%.
2. a kind of accurate identification method of planting Sugarcane white leaf disease resistance according to claim 1, it is characterised in that: step
(2) pesticide-germicide mixed liquor described in is 70% Diacloden water dispersant 18.75g and 50% carbendazol wettable powder 18.75g
Pesticide-germicide mixed liquor is made into 15kg sterile water after mixing.
3. a kind of accurate identification method of planting Sugarcane white leaf disease resistance according to claim 1, it is characterised in that: step
(3) the coating inoculation described in are as follows: the Sugarcane smut 15kg inoculation liquid of participating in the experiment handled well using power spraye by 1000kg
Ratio, inoculation liquid is uniformly sprayed to the Sugarcane smut of participating in the experiment for being placed in plastic film.
4. a kind of accurate identification method of planting Sugarcane white leaf disease resistance according to claim 1, it is characterised in that: step
(4) matrix described in is mixed by disinfection soil and disinfection well-rotted farmyard manure by 3:1 volume ratio.
5. a kind of accurate identification method of planting Sugarcane white leaf disease resistance according to claim 1, it is characterised in that: step
(4) the susceptible check variety described in is Guangdong sugar 86-368, and disease-resistant check variety is Guangdong sugar 83-88.
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| CN114793812A (en) * | 2022-04-13 | 2022-07-29 | 华南农业大学 | Method for identifying sugarcane white streak disease resistance |
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| CN104450862A (en) * | 2014-11-04 | 2015-03-25 | 广西大学 | Sugarcane top rot resistance identification method |
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| CN104450862A (en) * | 2014-11-04 | 2015-03-25 | 广西大学 | Sugarcane top rot resistance identification method |
| CN105211062A (en) * | 2015-11-13 | 2016-01-06 | 汪恒森 | A kind of sugarcane agricultural chemicals preventing Sugarcane white leaf and preparation method thereof |
| CN108486216A (en) * | 2018-03-08 | 2018-09-04 | 福建农林大学 | Resistance Identification method in sugar cane breed informal voucher ward |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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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