CN103190303A - Inoculation method of Ustilago scitaminea - Google Patents
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Abstract
本发明公开了一种甘蔗黑穗病菌接种方法,包括以下步骤:将甘蔗黑穗病菌孢子悬浮液点接接种于蔗种芽体表面,然后保湿放置16-24h。其中,孢子悬浮液的浓度为106个/ml,保湿时先用润湿的卫生纸缠裹后再用塑料纸盖着放置到阴凉处。本发明方法使用方便,有效提高了感病品种的发病率,能使感病品种的发病率达到80%以上,可以有效区分不同甘蔗品种对病害的抗感性,从而为深入研究甘蔗黑穗病抗病育种和生理小种分化研究创造良好的条件。The invention discloses a sugarcane smut inoculation method, which comprises the following steps: spot-inoculate the sugarcane smut spore suspension on the surface of sugarcane buds, and then keep it moist for 16-24 hours. Among them, the concentration of the spore suspension is 10 6 /ml. When moisturizing, first wrap it with wet toilet paper and then cover it with plastic paper and place it in a cool place. The method of the invention is easy to use, effectively improves the incidence rate of susceptible varieties, can make the incidence rate of susceptible varieties reach more than 80%, and can effectively distinguish the resistance and sensitivity of different sugarcane varieties to diseases, thereby providing a basis for further research on sugarcane smut resistance. Create good conditions for disease breeding and physiological race differentiation research.
Description
技术领域technical field
本发明属于农业技术领域,具体涉及一种甘蔗黑穗病菌接种方法。The invention belongs to the technical field of agriculture, and in particular relates to a method for inoculating sugarcane smut.
背景技术Background technique
甘蔗黑穗病是甘蔗生产过程中的重要病害之一,被称为甘蔗的“癌症”。该病最早是在1877年南非纳塔尔发现,随后在亚洲和非洲的大部分国家有所报道。1940年在美洲阿根廷发现后,西半球也发生了该病害(Schenck S,Pear H M,Liu Z,et al.Genetic variation of Ustilagoscitaminea pathotypes in Hawaii evaluated by host range and AFLP marker[J].Sugar CaneInternational,2005,23:15-19.)。20世纪70-90年代,夏威夷、弗罗里达、摩洛哥、澳大利亚等国家、地区也相继有该病害发生的报道(Byther R S,Steiner G W,Wismer C A.Newsugarcane 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 ISSCTConger,New Delhi,India,1999:333-336.)。中国于1932年在广州发现该病害,之后各地也都出现该病。特别是近年来,随着蔗种无性繁殖栽培技术的推广,蔗种频繁引进调运,蔗田长期连作同一品种以及宿根蔗年限的逐渐延长,造成甘蔗黑穗病在我国广西、云南、广东、福建、海南等省蔗区病害发生日趋严重,造成的损失多达10-30%,严重的宿根蔗几乎绝收。Sugarcane smut is one of the important diseases in the production process of sugarcane, known as the "cancer" of sugarcane. The disease was first discovered in Natal, South Africa in 1877, and subsequently reported in most countries in Asia and Africa. After it was discovered in Argentina in America in 1940, the disease also occurred in the western hemisphere (Schenck S, Pear H M, Liu Z, et al. Genetic variation of Ustilagoscitaminea pathotypes in Hawaii evaluated by host range and AFLP marker[J].Sugar Cane International, 2005 , 23:15-19.). From the 1970s to the 1990s, Hawaii, Florida, Morocco, Australia and other countries and regions also reported the occurrence of the disease (Byther R S, Steiner G W, Wismer C A. Newsugarcane 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 ISSTConger,New Delhi,India,1999: 333-336.). China discovered the disease in Guangzhou in 1932, and the disease also appeared in various places afterwards. Especially in recent years, with the popularization of sugarcane vegetative propagation techniques, frequent introduction and transportation of sugarcane varieties, long-term continuous cropping of the same variety in sugarcane fields, and the gradual extension of the life of ratoon cane, resulting in the occurrence of sugarcane smut in Guangxi, Yunnan, Guangdong, and Fujian. The occurrence of diseases in sugarcane areas in provinces such as my country, Hainan, etc. is becoming more and more serious, causing losses of up to 10-30%, and severe rat root sugarcane is almost completely lost.
甘蔗黑穗病最明显特征是病蔗梢头具有一条向下内卷的黑色鞭状物,长几厘米至数十厘米不等。在适宜的环境条件下,感染蔗株在病菌侵染2-4个月开始形成黑穗,病菌侵染6-7个月黑穗长度可达到顶峰(Riley L T,Jubb T F,Croft B J.First Outbreak of Sugarcane Smut inAustralian[J].Proc.X X III ISSCT Conger,New Delhi,India,1999:333-336.)。其主要借气流、雨水、灌溉水、人事活动等在田间进行传播扩散。The most obvious feature of sugarcane smut is a black whip that rolls downwards at the tip of the diseased sugarcane, ranging from a few centimeters to tens of centimeters long. Under suitable environmental conditions, infected sugarcane plants begin to form smuts 2-4 months after the infection of the pathogen, and the length of the smut can reach the peak after 6-7 months of the infection (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 uses airflow, rainwater, irrigation water, personnel activities, etc. to spread and spread in the field.
目前甘蔗种质资源及品种抗性鉴定方法主要有田间自然感病鉴定和人工接种鉴定。田间自然鉴定周期长、占用土地面积大、鉴定材料数量有限,而且由于田间的自然病原分布不均匀、气候及人事活动等条件变化常常会影响鉴定结果的真实性、稳定性和准确性。人工接种鉴定快速、鉴定材料数量不受限制,且环境条件可人为控制,既可以满足大规模筛选抗源材料的需要,也可以利用鉴别寄主对该病菌的生理小种分化情况进行研究。但现有的人工接种方法接种品种发病率不高(包括感病品种)、鉴定结果的稳定性差、准确性低,直接制约了人工接种方法的应用。At present, the identification methods of sugarcane germplasm resources and variety resistance mainly include field natural susceptibility identification and artificial inoculation identification. Field natural identification takes a long period, occupies a large area of land, and the number of identification materials is limited. Moreover, the authenticity, stability and accuracy of the identification results are often affected by the uneven distribution of natural pathogens in the field, climate and personnel activities and other conditions. Artificial inoculation identification is fast, the number of identified materials is not limited, and the environmental conditions can be controlled artificially. It can not only meet the needs of large-scale screening of anti-source materials, but also use the identification of hosts to study the physiological race differentiation of the pathogen. However, the existing artificial inoculation method has low incidence of inoculated varieties (including susceptible varieties), poor stability and low accuracy of identification results, which directly restrict the application of artificial inoculation method.
发明内容Contents of the invention
本发明的目的在于提供一种用于甘蔗黑穗病抗病性鉴定效果好的甘蔗黑穗病菌接种方法。The object of the present invention is to provide a sugarcane smut inoculation method with good effect for identification of sugarcane smut disease resistance.
为实现上述目的,本发明采用如下技术方案:To achieve the above object, the present invention adopts the following technical solutions:
一种甘蔗黑穗病菌接种方法,包括以下步骤:将甘蔗黑穗病菌孢子悬浮液点接接种于甘蔗种子芽体表面,然后保湿放置16-24h。A sugarcane smut inoculation method comprises the following steps: spot-inoculate the sugarcane smut spore suspension on the surface of sugarcane seed buds, and then keep it moist for 16-24 hours.
上述方法中,采集甘蔗黑穗病鞭,收集甘蔗黑穗病菌孢子,制成孢子悬浮液,所得甘蔗黑穗病菌孢子悬浮液的浓度为106个/ml。In the above method, the sugarcane smut whip is collected, the sugarcane smut spores are collected, and a spore suspension is prepared, and the obtained sugarcane smut spore suspension has a concentration of 10 6 spores/ml.
上述方法中,保湿时先用润湿的吸水材料缠裹后再用不透气材料覆盖放置到阴凉处。较好的,采用半湿润的卫生纸轻轻缠裹后放置在阴凉处用塑料纸盖着过夜(16-24h)。In the above method, when moisturizing, first wrap it with a wet water-absorbing material and then cover it with an air-tight material and place it in a cool place. Better, lightly wrap it with semi-moist toilet paper, place it in a cool place and cover it with plastic paper overnight (16-24h).
上述方法中,较好的,利用移液枪进行点接操作,孢子悬浮液的用量为20μl。In the above method, preferably, a pipette gun is used for the spotting operation, and the amount of the spore suspension is 20 μl.
本发明方法在甘蔗黑穗病抗病性鉴定中的应用。Application of the method of the invention in identification of sugarcane smut resistance.
本发明方法使用方便,有效提高了感病品种的发病率,能使感病品种的发病率达到80%以上,可以有效区分不同甘蔗品种对病害的的抗感性,从而为深入研究甘蔗黑穗病抗病育种和生理小种分化研究创造良好的条件。The method of the invention is easy to use, effectively improves the incidence rate of susceptible varieties, can make the incidence rate of susceptible varieties reach more than 80%, and can effectively distinguish the resistance and susceptibility of different sugarcane varieties to diseases, so as to further study sugarcane smut Create good conditions for disease resistance breeding and physiological race differentiation research.
具体实施方式Detailed ways
下面结合实施例对本发明做进一步的说明,但本发明的保护范围并不限于此。The present invention will be further described below in conjunction with the examples, but the protection scope of the present invention is not limited thereto.
实施例1Example 1
1、收集海南蔗区甘蔗黑穗病鞭,并在相对凉爽、干燥、无风的地方收集病菌孢子,用无菌水悬浮孢子,用涡旋混合器振荡后,用血球计数板调孢子悬浮液浓度为106个孢子/ml,备用。1. Collect the sugarcane smut whip in the sugarcane area of Hainan, and collect the spores of the disease in a relatively cool, dry, and windless place, suspend the spores with sterile water, shake them with a vortex mixer, and adjust the spore suspension with a hemocytometer The concentration is 10 6 spores/ml, for later use.
2、常规人工接种:把种苗浸泡在一个装有孢子悬浮液(孢子悬浮液浓度106个孢子/ml)的大容器中,10分钟后取出放在阴凉处用塑料纸盖着保湿过夜(16小时)。2. Conventional artificial inoculation: Soak the seedlings in a large container with spore suspension (spore suspension concentration 10 6 spores/ml), take it out after 10 minutes and put it in a cool place and cover it with plastic paper to keep it moist overnight ( 16 hours).
本发明的人工接种方法:用移液枪吸取20微升孢子悬浮液点接于种苗芽体表面,然后用润湿的卫生纸轻轻缠裹之后放在阴凉处用塑料纸盖着保湿过夜(16小时)。Artificial inoculation method of the present invention: draw 20 microliters of spore suspensions with pipette gun and spot-connect on the surface of the seedling bud body, then gently wrap with moistened toilet paper and then put it in a cool place and cover it with plastic paper to keep it moist overnight ( 16 hours).
其中,种苗采用生产上的常规种苗ROC22。Wherein, the seedling adopts the conventional seedling ROC22 in production.
3、将步骤2得到的两组种苗以完全随机区组设计种植。试验小区5m行长,20个段/行;三次重复。3. Plant the two groups of seedlings obtained in step 2 with a completely randomized block design. The length of the test plot is 5m, 20 sections/row; three repetitions.
4、评价。在种植5个月后,计数每行上总丛数和感染丛数。计算发病率。不同接种方法甘蔗黑穗病的发病情况见表1。发病率=感染丛数/总丛数×100%。4. Evaluation. Five months after planting, the total number of clumps and the number of infected clumps on each row were counted. Calculate the incidence rate. The incidence of sugarcane smut with different inoculation methods is shown in Table 1. Incidence rate = number of infected clusters/total number of clusters × 100%.
表1 不同接种方法甘蔗黑穗病的发生情况Table 1 Occurrence of sugarcane smut with different inoculation methods
表1结果表明:利用本发明的人工接种方法的三个重复的发病率分别为85%、80%、85%,平均为83.33%,而常规人工接种方法的三个重复的发病率分别为15%、20%、15%,平均为16.67%。表明本发明的人工接种方法大大提高了接种品种的发病率。Table 1 result shows: utilize the three repeated sickness rates of artificial inoculation method of the present invention to be respectively 85%, 80%, 85%, be 83.33% on average, and three repeated sickness rates of conventional artificial inoculation method are respectively 15% %, 20%, 15%, with an average of 16.67%. Show that artificial inoculation method of the present invention has improved the sickness rate of inoculation variety greatly.
实施例2Example 2
1、提前半年扩繁甘蔗黑穗病菌鉴别寄主NCO376(高抗)、NCO310(抗小种2,感小种1)、F134(感小种2)、F173(高感)及海南蔗区主栽品种ROC22,备用。1. Proliferate sugarcane smut half a year in advance to identify hosts NCO376 (highly resistant), NCO310 (resistant to race 2, susceptible to race 1), F134 (susceptible to race 2), F173 (highly susceptible) and main planters in Hainan sugarcane area Variety ROC22, spare.
2、从海南蔗区不同地方、不同栽培品种上采集甘蔗黑穗病鞭,收集黑穗病菌孢子,用无菌水悬浮孢子,用涡旋混合器振荡后,用血球计数板调孢子悬浮液浓度为106个孢子/ml,编号,样品品种及编号见表2。2. Collect sugarcane smut whip from different places and different cultivars in Hainan sugarcane area, collect smut spores, suspend the spores with sterile water, shake with a vortex mixer, and adjust the concentration of the spore suspension with a hemocytometer It is 10 6 spores/ml, number, sample species and number are shown in Table 2.
3、用移液枪吸取20微升孢子悬浮液点接于种苗芽体表面,然后用润湿的卫生纸轻轻缠裹之后放在阴凉处用塑料纸盖着保湿过夜(20小时)。3. Use a pipette to draw 20 microliters of spore suspension and apply it on the surface of the seedling bud, then gently wrap it with wet toilet paper, put it in a cool place and cover it with plastic paper to moisturize overnight (20 hours).
4、利用步骤3的方法分别将孢子悬浮液接种于甘蔗黑穗病菌鉴别寄主及ROC22上,每个编号为一个组合。之后以完全随机区组设计种植在试验小区。试验小区的规格为5m行长,20个段/行。4. Use the method in step 3 to inoculate the spore suspension on the identification host of sugarcane smut and ROC22 respectively, and each number is a combination. Afterwards, they were planted in experimental plots in a completely randomized block design. The specification of the test plot is 5m row length, 20 sections/row.
5、评价。种植5个月后计数每行上总丛数和感染丛数。6个月后再对宿根进行评价,宿根评价两次。根据感染百分率进行评价分级(评价分级见表5)。发病率=感染株数/总株数×100%。5. Evaluation. The total number of clumps and the number of infected clumps on each row were counted 5 months after planting. The perennials were evaluated 6 months later and the perennials were evaluated twice. Evaluation grades were carried out according to the percentage of infection (see Table 5 for evaluation grades). Incidence rate = number of infected strains/total number of strains × 100%.
表2 甘蔗黑穗病菌样品来源Table 2 Sources of sugarcane smut samples
注:样品采集品种为该蔗区种植相对普遍的品种Note: The species collected for the samples are relatively common varieties planted in this sugarcane area
表3 海南蔗区甘蔗黑穗病菌生理小种鉴定结果Table 3 Identification results of physiological races of sugarcane smut in Hainan sugarcane area
表3结果表明,海南蔗区甘蔗黑穗病菌生理小种为小种2,或优势小种为小种2。这为了解海南蔗区甘蔗黑穗病菌生理小种的种类及分布情况,以及对今后海南蔗区甘蔗黑穗病的防控及抗病种质的选育提供了依据。The results in Table 3 show that the physiological race of sugarcane smut in Hainan sugarcane region is race 2, or the dominant race is race 2. This provides a basis for understanding the species and distribution of physiological races of sugarcane smut in the sugarcane area of Hainan, as well as for the prevention and control of sugarcane smut and the selection of disease-resistant germplasm in the sugarcane area of Hainan in the future.
结合试验结果发现利用本发明的人工接种方法的鉴定结果能很好的反应各鉴别寄主抗感性表现,且常规品种ROC22的发病率新植维持在80%左右,而宿根则表现为全部发病,充分说明本发明的人工接种方法的可靠性及稳定性。In conjunction with the test results, it is found that the appraisal results utilizing the artificial inoculation method of the present invention can well reflect each differentiating host's resistance performance, and the new planting rate of the conventional variety ROC22 is maintained at about 80%, while the perennial root shows as all morbidity, sufficient The reliability and stability of the artificial inoculation method of the present invention are illustrated.
实施例3Example 3
1、扩繁转核糖体失活蛋白基因抗病甘蔗SPAP的不同株系SPAP1、SPAP2、……、SPAP7,备用。1. Propagation of different strains SPAP1, SPAP2, ..., SPAP7 of sugarcane SPAP by transfecting ribosomal inactivation protein gene, spare.
2、采集海南蔗区甘蔗黑穗病鞭,收集黑穗病菌孢子。用无菌水悬浮孢子,用涡旋混合器振荡后,用血球计数板调孢子悬浮液浓度为106个孢子/ml,备用。2. Collect sugarcane smut whips and collect smut spores in the sugarcane area of Hainan. Suspend the spores with sterile water, shake with a vortex mixer, adjust the concentration of the spore suspension to 10 6 spores/ml with a hemocytometer, and set aside.
3、用移液枪吸取20微升孢子悬浮液点接于种苗芽体表面,然后用润湿的卫生纸轻轻缠裹之后放在阴凉处用塑料纸盖着保湿过夜(24小时),依照此方法分别接种不同的转基因株系;之后种植于温室大棚(主要起隔离作用)的塑料大盆中,以非转基因株系的品种ROC22为对照。3. Use a pipette gun to draw 20 microliters of spore suspension and place it on the surface of the seedling bud, then gently wrap it with moistened toilet paper, then put it in a cool place and cover it with plastic paper to moisturize overnight (24 hours), according to This method was used to inoculate different transgenic strains respectively; after that, they were planted in large plastic pots in greenhouses (mainly for isolation), and the non-transgenic strain variety ROC22 was used as a control.
4、评价。种植6个月后,计数各株系及对照的总株数及发病株数,发病率=感染株数/总株数×100%,根据感染百分率进行评价分级(分级标准见表5)。4. Evaluation. After 6 months of planting, the total number of plants and the number of diseased plants of each line and the control were counted, and the incidence rate = number of infected plants/total number of plants × 100%, and was evaluated and graded according to the infection percentage (see Table 5 for grading standards).
表4 甘蔗转基因株系对黑穗病的抗病性Table 4 Resistance of sugarcane transgenic lines to smut
表4结果表明,利用本发明的人工接种方法可以很好的评价抗病转基因株系的抗病性。非转基因株系ROC22的发病率达到了79.68%,而所有转基因株系对黑穗病的抗性都有所提高,达到了抗病和中抗的效果。The results in Table 4 show that the disease resistance of the disease-resistant transgenic lines can be well evaluated by using the artificial inoculation method of the present invention. The incidence rate of the non-transgenic line ROC22 reached 79.68%, while the resistance of all transgenic lines to smut was improved, achieving the effect of disease resistance and moderate resistance.
表5 甘蔗黑穗病抗性等级标准Table 5 Sugarcane smut resistance grade standard
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