CN103190303A - Inoculation method of Ustilago scitaminea - Google Patents
Inoculation method of Ustilago scitaminea Download PDFInfo
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- CN103190303A CN103190303A CN2013101294169A CN201310129416A CN103190303A CN 103190303 A CN103190303 A CN 103190303A CN 2013101294169 A CN2013101294169 A CN 2013101294169A CN 201310129416 A CN201310129416 A CN 201310129416A CN 103190303 A CN103190303 A CN 103190303A
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- ustilago scitaminea
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Abstract
The invention discloses an inoculation method of Ustilago scitaminea. The method includes the steps of inoculating Ustilago scitaminea spore suspension to the surface of sugarcane buds by spot inoculation and keeping in the wet place for 16-24h. The pore suspension is 106/ml in concentration. During moisture preservation, the inoculated sugarcane buds are wrapped with humid tissue and are then placed at a shade place after being covered with plastic paper. The inoculation method is convenient to use, incidence rate of susceptible varieties is increased effectively, up to more than 80%, sense resistance of the different sugarcane varieties to Ustilago scitaminea can be distinguished effectively, and accordingly good conditions are created for the intensive study on breeding for disease resistance to Ustilago scitaminea and the study on physiological race differentiation.
Description
Technical field
The invention belongs to agricultural technology field, be specifically related to a kind of ustilago scitaminea bacteria inoculation method.
Background technology
Culmicolous smut of sugar cane is one of important disease in the sugarcane production process, is called as " cancer " of sugarcane.This disease is to find in Natal, South Africa in 1877 the earliest, and the most of country in Asia and Africa reports to some extent subsequently.1940 after America Argentina finds, this disease (Schenck S has also taken place in the Western Hemisphere, Pear H M, Liu Z, et al.Genetic variation of Ustilago scitaminea pathotypes in Hawaii evaluated by host range and AFLP marker[J] .Sugar Cane International, 2005,23:15-19.).The 70-90 age in 20th century, report (the Byther R S that countries such as Hawaii, Florida, Morocco, Australia, area also have this disease to take place in succession, Steiner G W, Wismer C A.New sugarcane diseases reported in Hawaii[J] .Sugarcane Pathologists'Newsletter, 1971,7:18-21.; Riley L T, Jubb T F, Croft B J.First outbreak of sugarcane smut in Australia[J] .Proc.XXIII ISSCT Conger, New Delhi, India, 1999:333-336.).China found this disease in 1932 in Guangzhou, and this disease also all appears in various places afterwards.Particularly in recent years, popularization along with sugarcane kind vegetative propagation culture technique, the sugarcane kind is frequently introduced allocation and transportation, the prolongation gradually of the same kind of the long-term continuous cropping in sugarcane field and the stubble cane time limit, cause culmicolous smut of sugar cane to be on the rise in province's sugarcane district diseases such as China Guangxi, Yunnan, Guangdong, Fujian, Hainan, the loss that causes reaches 10-30%, and serious stubble cane almost has no harvest.
The obvious characteristic of culmicolous smut of sugar cane is that the disease sugarcane tip of a branch has an involute whiplike thing of black downwards, and long several centimetres to tens of centimetres are not waited.Under the adapt circumstance condition, infect the sugarcane strain and began to form dust-brand at infection process 2-4 month, infection process 6-7 month dust-brand length can reach peak (Riley L T, Jubb T F, Croft B J.First Outbreak of Sugarcane Smut in Australian[J] .Proc.X X III ISSCT Conger, New Delhi, India, 1999:333-336.).It mainly borrows air-flow, rainwater, irrigation water, occurrences in human life activity etc. to propagate diffusion in the field.
Sugarcane germ plasm resource and varietal resistance authentication method mainly contain the susceptible naturally evaluation in field and artificial infection evaluation at present.Field nature length qualification cycle, big, the expert evidence limited amount of land occupation area, and because the conditions such as natural cause of disease skewness, weather and occurrences in human life activity in field change authenticity, stability and the accuracy that usually can influence qualification result.Artificial infection is identified fast, expert evidence quantity is unrestricted, and environmental condition can artificially control, and both can satisfy the needs of extensive screening anti-source material, also can utilize the differential host that the biological strain differentiation situation of this germ is studied.But the existing artificial infection method inoculation kind incidence of disease not high (comprising susceptible variety), the poor stability of qualification result, accuracy are low, have directly restricted the application of artificial infection method.
Summary of the invention
The object of the present invention is to provide a kind of for the good ustilago scitaminea bacteria inoculation method of culmicolous smut of sugar cane disease resistance identification result.
For achieving the above object, the present invention adopts following technical scheme:
A kind of ustilago scitaminea bacteria inoculation method may further comprise the steps: ustilago scitaminea bacteria spore suspension point is inoculated in sugarcane seed sprout surface, preserves moisture then and place 16-24h.
In the said method, gather the culmicolous smut of sugar cane whip, collect the ustilago scitaminea bacteria spore, make spore suspension, the concentration of gained ustilago scitaminea bacteria spore suspension is 10
6Individual/ml.
In the said method, cover with gas impermeable material again after swathing with wetting absorbent material earlier when preserving moisture and be placed into shady and cool place.Preferably, adopt semi-humid toilet paper to swathe the back placement gently and be covered with spend the night (16-24h) in the cool with plastic paper.
In the said method, preferably, utilize liquid-transfering gun to put and connect operation, the consumption of spore suspension is 20 μ l.
The application of the inventive method in the culmicolous smut of sugar cane disease resistance is identified.
The inventive method is easy to use, effectively improved the incidence of disease of susceptible variety, the incidence of disease of susceptible variety is reached more than 80%, can effectively distinguish different sugar cane breeds to disease anti-perception, thereby create good condition for further investigation culmicolous smut of sugar cane breeding for disease resistance and biological strain differentiation research.
Embodiment
The present invention is described further below in conjunction with embodiment, but protection scope of the present invention is not limited to this.
Embodiment 1
1, collect sugarcane district, Hainan culmicolous smut of sugar cane whip, and collect the germ spore in nice and cool, dry, calm relatively place, with the sterile water spore that suspends, after the turbine mixer vibration, transferring spore suspension concentration with blood counting chamber is 10
6Individual spore/ml, standby.
2, conventional artificial infection: seedling is immersed in spore suspension (spore suspension concentration 10 is housed
6In the bulk container of individual spore/ml), take out after 10 minutes and be placed on shady and cool place and be covered with spend the night (16 hours) of preserving moisture with plastic paper.
Artificial infection method of the present invention: draw 20 microlitre spore suspension points with liquid-transfering gun and be connected to seedling sprout surface, be placed on shady and cool place after swathing gently with wetting toilet paper then and be covered with spend the night (16 hours) of preserving moisture with plastic paper.
Wherein, seedling adopts the conventional seedling ROC22 on producing.
3, two groups of seedlings that step 2 obtained are planted with the completely random block design.Experimental plot 5m is capable long, 20 sections/OK; Repeat for three times.
4, estimate.After plantation 5 months, count total clump number and infection clump number on every row.Calculate the incidence of disease.The incidence of different vaccination method culmicolous smut of sugar cane sees Table 1.The incidence of disease=infection clump number/total clump number * 100%.
A situation arises for table 1 different vaccination method culmicolous smut of sugar cane
Table 1 is the result show: utilize the incidence of disease of three repetitions of artificial infection method of the present invention to be respectively 85%, 80%, 85%, and average out to 83.33%, and the incidence of disease of three repetitions of conventional artificial infection method is respectively 15%, 20%, 15%, average out to 16.67%.Show that artificial infection method of the present invention has improved the incidence of disease of inoculation kind greatly.
Embodiment 2
1, it is high anti-to expand in advance numerous ustilago scitaminea bacteria differential host NCO376(half a year), the anti-microspecies 2 of NCO310(, sense microspecies 1), F134(feel microspecies 2), F173(is high feels) and Hainan sugarcane district main breed ROC22, standby.
2, different places, different cultivar are gathered the culmicolous smut of sugar cane whip from Hainan sugarcane district, collect the black tassel bacteria spore, and with the sterile water spore that suspends, after the turbine mixer vibration, transferring spore suspension concentration with blood counting chamber is 10
6Individual spore/ml, numbering, sample kind and numbering see Table 2.
3, draw 20 microlitre spore suspension points with liquid-transfering gun and be connected to seedling sprout surface, be placed on shady and cool place after swathing gently with wetting toilet paper then and be covered with spend the night (20 hours) of preserving moisture with plastic paper.
4, utilize the method for step 3 respectively spore suspension to be inoculated on ustilago scitaminea bacteria differential host and the ROC22, each is numbered a combination.Be planted in the experimental plot with the completely random block design afterwards.The specification of experimental plot is that 5m is capable long, 20 sections/OK.
5, estimate.Plant and count total clump number and infection clump number on every row after 5 months.Perennial root is estimated after 6 months, perennial root is estimated twice again.Estimate classification (estimate classification and see Table 5) according to infecting percentage.The incidence of disease=infection strain number/total strain number * 100%.
Table 2 ustilago scitaminea bacteria sample source
| Sample number into spectrum | The sampling position | Sampling time | The GPS location | Sugar cane breed |
| 1 | Gantry, Dingan County town | 2010-05-14 | N19°32.353；E110°19.441 | ROC22 |
| 2 | Long He town, Dingan County | 2010-05-14 | N19°24.549；E110°16.696 | ROC22 |
| 3 | Maple town, Tunchang County | 2010-05-14 | N19°12.074；E109°58.006 | ROC22 |
| 4 | Hong Mao town, Qiong Zhong county | 2010-05-14 | N18°59.939；E109°37.823 | 00-236 |
| 5 | Shi Yun township, Qiong Zhong county | 2010-05-14 | N19°00.018；E109°35.982 | ROC22 |
| 6 | Yuan Men township, white sand county | 2010-05-14 | N19°07.805；E109°31.204 | ROC22 |
| 7 | Tooth fork town, white sand county | 2010-05-15 | N19°13.630；E109°27.335 | 93-159 |
| 8 | Fu Long township, white sand county | 2010-05-15 | N19°18.485；E109°27.975 | ROC10 |
| 9 | Da An town, white sand county | 2010-05-15 | N19°19.438；E109°26.543 | ROC16 |
| 10 | Qi Fang town, white sand county | 2010-05-15 | N19°17.935；E109°12.468 | ROC22 |
| 11 | Bang Xi town, white sand county | 2010-05-15 | N19°21.593；E109°04.977 | ROC22 |
| 12 | Tian Town in October, Chang River county | 2010-05-16 | N19°17.712；E108°52.849 | 93-159 |
| 13 | Da Po town, Chang River county | 2010-05-16 | N19°19.267；E109°03.383 | ROC22 |
| 14 | The strong town of crow, Chang River county | 2010-05-16 | N19°17.232；E108°48.109 | ROC22 |
| 15 | Hai Wei town, Chang River county | 2010-05-16 | N19°20.186；E108°46.803 | ROC22 |
| 16 | Chang Hua town, Chang River county | 2010-05-16 | N19°16.751；E108°44.544 | ROC22 |
| 17 | Hai Tou town, Danzhou City | 2010-05-17 | N19°29.209；E109°04.209 | ROC22 |
| 18 | Pai Pu town, Danzhou City | 2010-05-17 | N19°39.204；E109°08.880 | ROC22 |
| 19 | Wang Wu town, Danzhou City | 2010-05-17 | N19°38.385；E109°16.979 | ROC22 |
| 20 | East, Danzhou City Cheng Zhen | 2010-05-18 | N19°31.152；E109°34.634 | ROC22 |
| 21 | Bo Hou town, Lingao County | 2010-05-18 | N19°51.645；E109°47.742 | ROC22 |
| 22 | The big Fengzhen in Dengmai County | 2010-05-18 | N19°53.066；E110°02.368 | 00-236 |
| 23 | White lotus town, Dengmai County | 2010-05-18 | N19°51.574；E110°06.743 | ROC22 |
| 24 | Haikou City group list village | 2010-05-18 | N19°57.467；E110°12.338 | ROC22 |
Annotate: it is the general relatively kind of this sugarcane district plantation that sample is gathered kind
Sugarcane district, table 3 Hainan ustilago scitaminea bacteria biological strain qualification result
Table 3 is the result show, Hainan sugarcane district ustilago scitaminea bacteria biological strain is microspecies 2, or dominant races is microspecies 2.This is kind and the distribution situation of understanding sugarcane district, Hainan ustilago scitaminea bacteria biological strain, and provides foundation to the prevention and control of Hainan sugarcane district culmicolous smut of sugar cane and the seed selection of disease-resistant germplasm from now on.
Find to utilize the qualification result of artificial infection method of the present invention can well react the anti-perception performance of each differential host in conjunction with result of the test, and the incidence of disease planting of conventional variety ROC22 maintains about 80%, perennial root then shows as whole morbidities, proves absolutely reliability and the stability of artificial infection method of the present invention.
Embodiment 3
1, the not homophyletic that expands the disease-resistant sugarcane SPAP of numerous commentaries on classics ribosome-inactivating protein gene be SPAP1, SPAP2 ..., SPAP7, standby.
2, gather sugarcane district, Hainan culmicolous smut of sugar cane whip, collect the black tassel bacteria spore.With the sterile water spore that suspends, after the turbine mixer vibration, transferring spore suspension concentration with blood counting chamber is 10
6Individual spore/ml, standby.
3, draw 20 microlitre spore suspension points with liquid-transfering gun and be connected to seedling sprout surface, be placed on shady and cool place after swathing gently with wetting toilet paper then and be covered with spend the night (24 hours) of preserving moisture with plastic paper, inoculate different transgenic lines respectively according to the method; Plant afterwards in the plastic big basin of green house (mainly buffer action), the kind ROC22 that is with the non-transgenic strain is contrast.
4, estimate.Plant after 6 months, count total strain number and the morbidity strain number of each strain system and contrast, the incidence of disease=infection strain number/total strain number * 100% is estimated classification (grade scale sees Table 5) according to infecting percentage.
The sugarcane genetically modified strain of table 4 system is to the disease resistance of smut
Table 4 is the result show, utilizes artificial infection method of the present invention can well estimate the disease resistance of disease-resistant transgenic strain system.The non-transgenic strain is that the incidence of disease of ROC22 has reached 79.68%, and all transgenic lines all increase to the resistance of smut, reached disease-resistant and in anti-effect.
Table 5 culmicolous smut of sugar cane resistance classification standard
| Sugarcane stem cumulative life-incidence | The disease resistance response type | The resistance grade |
| 0%～3% | High anti-(HR) | 1 |
| 4%～6% | Disease-resistant (R) | 2 |
| 7%～9% | Disease-resistant (R) | 3 |
| 10%～12% | In anti-(I) | 4 |
| 13%～25% | In anti-(I) | 5 |
| 26%～35% | Susceptible (S) | 6 |
| 36%～50% | Susceptible (S) | 7 |
| 51%～75% | High sense (HS) | 8 |
| 76%～100% | High sense (HS) | 9 |
Claims (7)
1. a ustilago scitaminea bacteria inoculation method is characterized in that, may further comprise the steps:
Ustilago scitaminea bacteria spore suspension point is inoculated in sugarcane kind sprout surface, preserves moisture then and place 16-24h.
2. a kind of inoculation method of culmicolous smut of sugar cane according to claim 1 is characterized in that the concentration of described spore suspension is 10
6Individual/ml.
3. a kind of ustilago scitaminea bacteria inoculation method according to claim 1 is characterized in that, covers with gas impermeable material after swathing with wetting absorbent material earlier when preserving moisture again and is placed into shady and cool locating.
4. as a kind of ustilago scitaminea bacteria inoculation method as described in the claim 3, it is characterized in that described absorbent material is toilet paper; Described gas impermeable material is plastic paper.
5. a kind of ustilago scitaminea bacteria inoculation method according to claim 1 is characterized in that, utilizes liquid-transfering gun to put inoculation.
6. as a kind of ustilago scitaminea bacteria inoculation method as described in the claim 4, it is characterized in that the consumption of spore suspension is 20 μ l.
7. the application of the arbitrary described ustilago scitaminea bacteria inoculation method of claim 1-6 in the culmicolous smut of sugar cane disease resistance is identified.
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| CN106544314A (en) * | 2016-10-20 | 2017-03-29 | 华南农业大学 | A kind of ustilago scitaminea bacteria teleutospore collects storage practice |
| CN113142002A (en) * | 2021-01-26 | 2021-07-23 | 广西壮族自治区农业科学院 | Sugarcane smut resistance determination method based on Internet of things system |
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Application publication date: 20130710 |