阅读说明：本技术 一种菠萝泛菌引起的玉米细菌性条纹病的鉴定方法 (Method for identifying corn bacterial stripe disease caused by Pantoea ananatis ) 是由 张楠 李高科 胡建广 于永涛 李武 谢利华 李光玉 于 2020-04-30 设计创作，主要内容包括：本发明公开一种菠萝泛菌引起的玉米细菌性条纹病的鉴定方法,涉及作物抗病育种技术领域。该方法包括以下步骤：培育玉米苗；病原菌扩繁；病原菌接种；抗性鉴定。本发明接种利用注射器的针头沿着玉米叶片的叶脉划线,制造伤口,协助病原菌入侵,并模拟发病的环境,使菠萝泛菌在玉米叶片成功发病。本发明的鉴定方法操作简单,可在室内完成,重复性好,效率高；且不受季节、外部环境的影响,不但保证了病原菌的数量与生长状态,做到了精准接种、鉴定,而且可在短时间内对玉米材料进行抗性鉴定,大大缩短了鉴定时间,有助于玉米自交系对细菌性条纹病的快速鉴定,应用价值大。(The invention discloses an identification method of corn bacterial stripe disease caused by Pantoea ananatis, and relates to the technical field of crop disease-resistant breeding. The method comprises the following steps: cultivating corn seedlings; pathogenic bacteria propagation; inoculating pathogenic bacteria; and (5) resistance identification. According to the invention, the inoculation utilizes the needle of the injector to line along the veins of the corn leaves, so that wounds are made, the invasion of pathogenic bacteria is assisted, and the pathogenic environment is simulated, so that Pantoea ananatis successfully attacks the corn leaves. The identification method is simple to operate, can be finished indoors, and is good in repeatability and high in efficiency; the method is not influenced by seasons and external environments, not only ensures the quantity and the growth state of pathogenic bacteria and realizes accurate inoculation and identification, but also can identify the resistance of the corn material in a short time, greatly shortens the identification time, is beneficial to the quick identification of the corn inbred line on the bacterial stripe disease, and has high application value.)

1. A method for identifying corn bacterial stripe disease caused by Pantoea ananatis is characterized by comprising the following steps: the method comprises the following steps:

(1) cultivating corn seedlings: sterilizing and sowing corn seeds; culturing until three leaves are in the first heart stage to obtain corn seedlings;

(2) expanding propagation of pathogenic bacteria, namely expanding propagation of corn bacterial stripe disease pathogenic bacteria Pantoea ananatis in L B culture medium, eluting with clear water, centrifugally collecting, and adjusting the concentration of the pathogenic bacteria to 10⁷Adding 0.2-0.5% of Tween 20 according to the total volume to prepare Pantoea ananatis suspension;

(3) and (3) pathogen inoculation: sucking the Pantoea ananatis suspension by using a sterile syringe, pushing the syringe to enable the bacteria liquid to flow out of the needle head, marking on the heart leaves by using the needle head, and uniformly smearing the Pantoea ananatis suspension on the wound;

(4) and (3) resistance identification: after the inoculated corn plants are cultivated for 72 hours, the resistance of the corn material is identified according to the expansion condition of the leaf spots; if necrosis occurs at the wound, the no diffusion phenomenon of necrotic spots is high resistance, and is marked as HR; if the necrotic spot at the wound has a small amount of spread phenomenon, the disease is marked as R; if the spread range of the necrotic spots is large, the shrinkage of the leaves is an infection and is marked as S; if the necrotic spots spread to the whole leaf, the leaf will wilting or even withering into a high feeling, and the result is marked as HS.

2. The method for identifying bacterial streaking disease in maize of Pantoea ananatis as claimed in claim 1, wherein:

the corn in the step (1) is sweet corn.

3. The method for identifying bacterial streaking disease in maize of Pantoea ananatis as claimed in claim 1 or 2, wherein:

the cultivation environment in the step (1) is set to be 28-30 ℃, the humidity is 60 +/-5%, the illumination time is 16 hours, the illumination intensity is 20000-30000L x, the dark time is 8 hours, and the illumination intensity is 0L x.

4. The method for identifying bacterial streaking disease in maize of Pantoea ananatis as claimed in claim 1 or 2, wherein:

the pathogenic bacteria propagation method in the step (2) specifically comprises the following steps:

a. streaking corn bacterial stripe disease pathogenic bacteria Pantoea ananatis on L B solid culture medium, and culturing;

b. selecting a single colony of pantoea ananatis on a scribed L B solid culture medium, placing the single colony in a L B liquid culture medium, carrying out primary culture, carrying out primary propagation, sucking a bacterial liquid obtained by primary propagation in a volume ratio of 1-500: 1000, carrying out secondary culture in a L B liquid culture medium to obtain a bacterial liquid obtained by secondary propagation, centrifuging the bacterial liquid obtained by secondary propagation, collecting pathogenic bacteria, and using sterilized ddH₂O, cleaning twice, and centrifuging again to obtain pathogenic bacteria; by ddH₂O resuspending the pathogenic bacteria, adjusting the concentration of the pathogenic bacteria to 10⁷And adding 0.2-0.5% of Tween 20 according to the total volume, wherein the Tween is used as an inoculation liquid, namely the pantoea ananatis suspension.

5. The method for identifying bacterial streaking disease in maize of Pantoea ananatis as claimed in claim 4, wherein:

the culture condition in the step a is that the culture is carried out for 12-16 h at 28-30 ℃;

the condition of primary culture in the step b is 28-30 ℃, and the culture is carried out for 10-12 h at 150-200 rpm;

the second culture in the step b is carried out under the conditions of 28-30 ℃ and 150-200 rpm for 10-12 h;

and c, centrifuging and re-centrifuging in the step b for 5-10 min at 8000-10000 rpm.

6. The method for identifying bacterial streaking disease in maize of Pantoea ananatis as claimed in claim 1 or 2, wherein:

the injector in the step (3) is a 1m L injector;

and (4) scribing on the heart lobes in the step (3) is to scribe along veins of the heart lobes.

7. The method for identifying bacterial streaking disease in maize of Pantoea ananatis as claimed in claim 6, wherein:

marking 1-5 wounds along the veins of the heart lobes by marking lines on the heart lobes in the step (3);

the wound length of the scribing in the step (3) is 1-1.5 cm.

8. The method for identifying bacterial streaking disease in maize of Pantoea ananatis as claimed in claim 7, wherein:

marking 3-5 wounds along the veins of the heart lobes on the heart lobes by marking lines on the heart lobes in the step (3);

the length of the scribed wound in step (3) is 1 cm.

9. The method for identifying bacterial streaking disease in maize of Pantoea ananatis as claimed in claim 1 or 2, wherein:

the cultivation environment in the step (4) is set to be 35-38 ℃, the humidity is 90 +/-5%, the illumination time is 16 hours, the illumination intensity is 20000-30000L x, the dark time is 8 hours, and the illumination intensity is 0L x.

10. The use of the method for identifying bacterial streaking disease in maize of any one of claims 1 to 9, which is caused by Pantoea ananatis, in breeding for bacterial streaking disease resistance in maize.

Technical Field

The invention relates to the technical field of crop disease-resistant breeding, in particular to an identification method of corn bacterial stripe disease caused by Pantoea ananatis.

Background

Sweet corn is of origin in america, a subspecies of the corn species, and is controlled by one or more naturally recessive mutant genes in the endosperm starch synthesis pathway. The sweet corn has the characteristics of grains, fruits and vegetables, has unique flavor and good palatability, contains higher proteins, amino acids, vitamins and mineral substances, and has high nutritional value (the current research and development situations and development prospects of fresh-eating corn such as Zhang Ying, Li Shi, Munan and the like [ J ]. coarse cereal crops, 2007,27(5): 373-375.). With the increase of national economy and the adjustment of dietary structure of consumers, the planting area and the demand of sweet corn in China are continuously increased. However, because of its variety characteristics and high sugar content, sweet corn is harmed by various microorganisms in the field.

Bacterial streaking was first discovered in the united states in 1960, mostly in the middle and east of the united states, and warm, humid climates contributed to the development of disease. Long and narrow parallel green-fading disease spots appear on leaves at the early stage of plant disease attack, and the disease spots at the later stage become brown and die, so that the photosynthesis of the plants is seriously influenced. Before 2016, bacterial streaking caused by Pantoea ananatis (Pantoea ananatis) occurred occasionally in the Guangdong sweet corn growing area. Pathogenic bacteria in soil accumulate year by year due to continuous cropping of sweet corn all the year round, the incidence rate of bacterial stripe disease rises year by year after 2016, the incidence degree becomes more serious, and the quality and the yield of the sweet corn are seriously influenced. Therefore, a successful and efficient disease identification system for the bacterial stripe disease is established, resistance identification is carried out on the existing planting resources, and the screened disease-resistant sweet corn material has important application value.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a method for identifying the corn bacterial stripe disease caused by Pantoea ananatis. The method can efficiently and stably identify the bacterial streak disease.

The traditional corn leaf diseases mostly adopt a leaf surface spraying method. However, the Pantoea ananatis belongs to a bacterial pathogenic bacterium, and cannot form an infection structure such as an attachment cell, an attachment branch and the like, so that the cell wall is broken through and the Pantoea ananatis enters the cell, and therefore, a wound needs to be artificially made to help pathogenic bacteria to invade. Therefore, the inoculation utilizes the needle head of the injector to line along the veins of the corn leaves, so as to make wounds, assist the invasion of pathogenic bacteria and simulate the pathogenic environment, so that the Pantoea ananatis successfully attacks the corn leaves.

The purpose of the invention is realized by the following technical scheme:

a method for identifying corn bacterial stripe disease caused by Pantoea ananatis comprises the following steps:

(1) cultivating corn seedlings: sterilizing and sowing corn seeds; culturing until three leaves are in the first heart stage to obtain corn seedlings;

(2) expanding propagation of pathogenic bacteria, namely expanding propagation of corn bacterial stripe disease pathogenic bacteria Pantoea ananatis (Pantoea ananatis) in L B culture medium, eluting with clear water, centrifugally collecting, and adjusting the concentration of the pathogenic bacteria to 10⁷Adding 0.2-0.5% (preferably 0.2%) of Tween 20 into the mixture according to the total volume to prepare Pantoea ananatis suspension;

(3) and (3) pathogen inoculation: sucking the Pantoea ananatis suspension by using a sterile syringe, pushing the syringe to enable the bacteria liquid to flow out of the needle head, marking on the heart leaves by using the needle head, and uniformly smearing the Pantoea ananatis suspension on the wound;

(4) and (3) resistance identification: after the inoculated corn plants are cultivated for 72 hours, the resistance of the corn material is identified according to the expansion condition of the leaf spots; if necrosis occurs at the wound, the no diffusion phenomenon of necrotic spots is high resistance, and is marked as HR; if the necrotic spot at the wound has a small amount of spread phenomenon, the disease is marked as R; if the spread range of the necrotic spots is large, the shrinkage of the leaves is an infection and is marked as S; if the necrotic spots spread to the whole leaf, the leaf will wilting or even withering into a high feeling, and the result is marked as HS.

The corn in the step (1) is preferably sweet corn; further preferred is sweet corn variety Yuetween No. 28, sweet corn inbred line group 1-1 or sweet corn inbred line GQ-1, but not limited thereto.

The disinfection in the step (1) is sequentially carried out by alcohol and NaClO; the specific disinfection steps are as follows:

soaking corn seeds for 12-16 h at 28-30 ℃ (preferably 28 ℃), so that the seeds are fully swelled; soaking the imbibed seeds in 70-75% alcohol for 5-10 min (preferably 5min), and performing surface disinfection; seeds were treated with sterilized ddH₂Cleaning for three times; after cleaning, soaking the seeds in 10% NaClO for 20-30 min (preferably 30min), and then disinfecting again; pouring out the NaClO solution and using the sterilized ddH₂And washing for three times.

The cultivation environment in the step (1) is set to be 28-30 ℃, the humidity is 60 +/-5%, the illumination time is 16 hours, the illumination intensity is 20000-30000L x, the dark time is 8 hours, and the illumination intensity is 0L x.

Further, the incubation environment in step (1) is set at 28 ℃, humidity 60 +/-5%, illumination time 16 hours, illumination intensity 30000L x, dark time 8 hours and illumination intensity 0L x.

The pathogenic bacteria propagation method in the step (2) specifically comprises the following steps:

a. streaking Pantoea ananatis (Pantoea ananatis) which is a pathogen of corn bacterial stripe disease on L B solid culture medium, and culturing;

b. selecting a single colony of pantoea ananatis on a scribed L B solid culture medium, placing the single colony in a L B liquid culture medium, carrying out primary culture, carrying out primary propagation, sucking a bacterial liquid obtained by primary propagation in a volume ratio of 1-500: 1000 (preferably 1: 1000) to carry out secondary culture in a L B liquid culture medium to obtain a bacterial liquid obtained by secondary propagation, centrifuging the bacterial liquid obtained by secondary propagation, collecting pathogenic bacteria, and using sterilized ddH₂O, cleaning twice, and centrifuging again to obtain pathogenic bacteria; by ddH₂O resuspending the pathogenic bacteria, adjusting the concentration of the pathogenic bacteria to 10⁷And adding 0.2-0.5% (preferably 0.2%) of Tween 20 into the mixture according to the total volume, wherein the Tween 20 is used as an inoculation solution, namely the pantoea ananatis suspension.

The culture condition in the step a is that the culture is carried out for 12-16 h at 28-30 ℃; further culturing at 30 deg.C for 16 h;

the condition of primary culture in the step b is 28-30 ℃, and the culture is carried out for 10-12 h at 150-200 rpm; further culturing at 30 ℃ and 200rpm for 12 h;

the second culture in the step b is carried out under the conditions of 28-30 ℃ and 150-200 rpm for 10-12 h; further culturing at 30 ℃ and 200rpm for 12 h;

the conditions of the centrifugation and the re-centrifugation in the step b are 8000-10000 rpm centrifugation for 5-10 min; further centrifuging at 8000rpm for 10 min;

l B liquid culture medium comprises 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, and pH of 7.4;

l B solid culture medium comprises 10 g/L of tryptone, 5 g/L of yeast extract, 5 g/L of NaCl, 15-20 g/L of agar, and has pH of 7.4.

The injector in the step (3) is a 1m L injector;

the lineation on the heart lobes in the step (3) is a lineation along veins of the heart lobes;

further, the lineation on the heart lobes in the step (3) is to lineate 1-5 wounds along the veins of the heart lobes; further drawing 3-5 wounds along the veins of the heart leaves;

the wound length of the scribing in the step (3) is 1-1.5 cm; further 1 cm;

the cultivation environment in the step (4) is set to be 35-38 ℃, the humidity is 90 +/-5%, the illumination time is 16 hours, the illumination intensity is 20000-30000L x, the dark time is 8 hours, and the illumination intensity is 0L x, wherein the temperature is set to be 35-38 ℃, the humidity is 90 +/-5%, and the disease occurrence condition of bacterial stripe disease at high temperature and high humidity is simulated.

Further, the cultivation environment in the step (4) is set to 35 ℃, the humidity is 90 +/-5%, the illumination time is 16 hours, the illumination intensity is 30000L x, the dark time is 8 hours, and the illumination intensity is 0L x, wherein the temperature is set to 35 ℃, the humidity is 90 +/-5%, and the disease onset condition of bacterial stripe disease at high temperature and high humidity is simulated.

The application of the identification method of the corn bacterial stripe disease caused by the Pantoea ananatis in corn bacterial stripe disease resistant breeding is also the protection scope of the invention.

Compared with the prior art, the invention has the following advantages and effects:

(1) the identification method specifically comprises the following steps: (1) cultivating corn seedlings; (2) pathogenic bacteria propagation; (3) inoculating pathogenic bacteria; (4) and (5) resistance identification. The invention simulates the most suitable environment (high temperature and high humidity) for the onset of the bacterial stripe disease by illuminating the incubator, causes the leaves to be wounded by the syringe needle, and coats bacterial liquid on the wound to lead pathogenic bacteria to invade, thus leading the corn leaves to successfully attack in the suitable environment. The method is not influenced by seasons and external environments, not only ensures the quantity and the growth state of pathogenic bacteria and realizes accurate inoculation and identification, but also can identify the resistance of the corn material in a short time and greatly shortens the identification time.

(2) The identification method provided by the invention is simple to operate, can be completed indoors, is good in repeatability, high in efficiency and accurate in identification, effectively shortens the time from inoculation to disease attack, is beneficial to the rapid identification of the bacterial stripe disease by the maize inbred line, and has a high application value.

Drawings

FIG. 1 shows interfield disease strains of bacterial streaking.

FIG. 2 shows Pantoea ananatis cultured in L B solid medium according to the present invention.

FIG. 3 is a graph showing the disease onset 72 hours after indoor inoculation of bacterial streak bacteria in example 3.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer.

L B liquid culture medium is prepared by adjusting pH to 7.4 with 10g Tryptone (Tryptone), 5g yeast extract (Yeast), 5g NaCl and NaOH, and adding deionized water to a volume of 1L, and steam sterilizing under high pressure for 20 min.

L B solid medium is prepared by adjusting pH to 7.4 with 10g Tryptone (Tryptone), 5g yeast extract (Yeast), 5g NaCl, 15g agar and NaOH, adding deionized water to 1L, steam sterilizing under high pressure for 20min, and pouring into 10cm petri dish.

The interfield disease strains of bacterial streaking disease are shown in FIG. 1.