阅读说明：本技术 一种鉴定野生稻抗稻瘟病性的方法 (Method for identifying rice blast resistance of wild rice ) 是由 兰波 杨迎青 黄俊宝 李湘民 于 2019-10-30 设计创作，主要内容包括：本发明提供的一种鉴定野生稻抗稻瘟病性的方法,包括以下步骤：1)叶片的取样；2)把剪好的叶片平置于培养装置上,每个培养皿放置4段叶片；放入光照培养箱内培养,24-26℃,相对湿度100％,黑暗培养24小时,然后光照培养,每天24小时光照,光照强度3400Lux照射12h,1310Lux照射12h,培养6-10天后调查发病情况；3)发病调查及标准：调查病斑大小,并根据对照品种,判定抗感,病级分5级,0-2级的为抗病,3-4级的为感病；本发明优化了接种稻瘟病菌的方法,解决了可用于移植的宿根很有限,只能满足接种几个稻瘟病菌生理小种的需求；且操作繁复,只能一组叶片进行一次鉴定操作的缺陷。本发明通过采用培养装置,大大简化了操作步骤。(The invention provides a method for identifying blast resistance of wild rice, which comprises the following steps: 1) sampling the leaves; 2) the cut leaves are horizontally placed on a culture device, and 4 sections of leaves are placed in each culture dish; culturing in light incubator at 24-26 deg.C and relative humidity of 100% in dark for 24 hr, culturing in light for 24 hr, irradiating at 3400Lux for 12 hr and 1310Lux for 12 hr, and culturing for 6-10 days to investigate disease incidence; 3) investigation and criteria of onset: investigating the size of the lesion, and judging the disease resistance according to the control variety, wherein the disease grade is 5, the disease resistance grade is 0-2, and the disease susceptibility grade is 3-4; the invention optimizes the method for inoculating the rice blast germs, solves the problem that the ratoon roots which can be used for transplantation are very limited and can only meet the requirement of inoculating a plurality of physiological races of the rice blast germs; and the operation is complicated, and only one group of blades can carry out one-time identification operation. The invention greatly simplifies the operation steps by adopting the culture device.)

1. A method for identifying blast resistance of wild rice, comprising the steps of:

1) sampling of the leaves: cutting leaves of wild rice and susceptible control variety Lijiang Xinjiang black rice in a greenhouse, putting the cut leaves into a fresh-keeping device for fresh keeping, bringing the leaves back to a laboratory, and cutting the leaves into 5cm segments;

2) the cut leaves are horizontally placed on a culture device, and 4 sections of leaves are placed in each culture dish; culturing in an illumination incubator at 24-26 deg.C and relative humidity of 100% in the dark for 24 hr, then culturing in the illumination incubator for 24 hr, irradiating with illumination intensity of 3400Lux for 12 hr and 1310Lux for 12 hr, observing the humidity of the filter paper every day to maintain the humidity in the culture dish and relative humidity of 100%, and examining the disease condition after culturing for 6-10 days;

(3) investigation and criteria of onset: investigating the size of the lesion, and judging the disease resistance according to the control variety, wherein the disease grade is 5, the disease resistance grade is 0-2, and the disease susceptibility grade is 3-4;

the culture device in the step 2) comprises an outer cover and 9 groups of culture dish assemblies, wherein the 9 groups of culture dish assemblies are fixedly connected to form a well shape, 9 sleeve assemblies are arranged at the lower part of the outer cover, each sleeve assembly comprises 5 sleeves, and 2.5uL of spore suspension is filled in each sleeve; the bottom of the pipe body is provided with a conical water dropper, the water dropper is formed by solidifying sterile water, and the top of the pipe body is provided with a top cover connected with an outer cover; the culture dish assembly comprises a pair of glass partition plates arranged in a cross shape, a circular culture dish body and a square fixing frame, wherein the glass partition plates are installed in the culture dish body, adaptive filter paper is installed at the bottom of the culture dish body, and the fixing frame is installed outside the culture dish body; a liquid storage cavity is formed in the glass partition plate, an ice block formed by the solidification of sterile water is arranged in the liquid storage cavity, and the bottom of the liquid storage cavity is communicated with the culture dish body; a fixing plate hinged with the glass partition plate is fixed in the glass partition plate, and 5 through holes which correspond to the sleeves one by one are formed in the fixing plate so that the sleeves can be conveniently arranged in the through holes.

2. The method for identifying blast resistance of wild rice as claimed in claim 1, wherein the concentration of the spore suspension is 10X 10⁶Per m²。

3. The method for identifying blast resistance of wild rice as claimed in claim 1, wherein the thickness of the bottom end face of said sleeve is 3 to 5 mm.

4. The method for identifying blast resistance of wild rice as claimed in claim 3, wherein the top of said sleeve is adhered to the bottom of said outer cover by ice water coagulation.

5. The method for identifying blast resistance of wild rice as recited in claim 1, 2, 3 or 4, wherein said culture dish body side wall comprises an inner cone protruding outward and an inner borneol fitting to said inner cone; the inner cone and the inner borneol form a hollow cylinder.

6. The method of identifying wild rice blast resistance as recited in claim 5, wherein the thickness of the top of the inner cone is greater than the thickness of the bottom.

7. The method for identifying blast resistance of wild rice as claimed in claim 1 or 2 or 3 or 4, wherein said fixing plate is hinged with a glass partition plate by a hinge.

Technical Field

The invention relates to the field of rice blast resistance detection, in particular to a method for identifying rice blast resistance of wild rice.

Background

At present, rice blast is one of the most major diseases of global rice, and can cause yield loss of 11% -30% to rice, rice loss is up to hundreds of millions of kilograms, and worldwide loss is about 1.57 billion dollars annually. Practice proves that the utilization of the resistant variety is one of the most economic and effective measures for preventing and treating rice blast, and also meets the requirements of green food for human. However, due to the diversity and degeneration of physiological races of Pyricularia oryzae, the resistance of the resistant varieties deteriorates remarkably after continuous use for 3-5 years. Therefore, the method for searching and screening stable and durable disease-resistant varieties, especially for exploring and utilizing durable resistance resources from old local varieties and wild rice resources, is an economic and effective measure for preventing and treating rice blast, and is also one of the main ways for ensuring stable and high yield of rice.

Whether conventional breeding or molecular breeding, the research on the genes stored in different rice varieties and the discovery of new genes thereof are key steps. The rice blast resistance identification method comprises 2 methods: (1) and (5) artificial inoculation identification. The method can be divided into (i) anti-spectrum determination according to different inoculation modes of selected strains, namely, single inoculation identification of a plurality of strains: ② the mixed inoculation identification of a plurality of strains. (2) And (5) naturally inducing identification. At present, the rice blast resistance of wild rice is mainly identified by planting perennial roots in pots and using new leaves grown from the perennial roots for identification. But the ratoon that can be used for transplanting is very limited, and can only meet the requirement of inoculating a plurality of physiological races of rice blast germs; and the operation is complicated, and only one group of blades can carry out one-time identification operation.

Disclosure of Invention

Technical problem to be solved

The invention aims to provide a method for identifying blast resistance of wild rice, which solves the problem that the existing identification of blast resistance of wild rice is mainly carried out by pot-divided planting of perennial roots and identification by using new leaves grown from the perennial roots. But the ratoon that can be used for transplanting is very limited, and can only meet the requirement of inoculating a plurality of physiological races of rice blast germs; and the operation is complicated, and only one group of blades can be used for identifying the operation defects once.

(II) technical scheme

In order to solve the technical problems, the invention provides a method for identifying blast resistance of wild rice, which comprises the following steps:

1) sampling of the leaves: cutting leaves of wild rice and susceptible control variety Lijiang Xinjiang black rice in a greenhouse, putting the cut leaves into a fresh-keeping device for fresh keeping, bringing the leaves back to a laboratory, and cutting the leaves into 5cm segments;

2) the cut leaves are horizontally placed on a culture device, and 4 sections of leaves are placed in each culture dish; culturing in an illumination incubator at 24-26 deg.C and relative humidity of 100% in the dark for 24 hr, then culturing in the illumination incubator for 24 hr, irradiating with illumination intensity of 3400Lux for 12 hr and 1310Lux for 12 hr, observing the humidity of the filter paper every day to maintain the humidity in the culture dish and relative humidity of 100%, and examining the disease condition after culturing for 6-10 days;

(3) investigation and criteria of onset: investigating the size of the lesion, and judging the disease resistance according to the control variety, wherein the disease grade is 5, the disease resistance grade is 0-2, and the disease susceptibility grade is 3-4;

the culture device in the step 2) comprises an outer cover and 9 groups of culture dish assemblies, wherein the 9 groups of culture dish assemblies are fixedly connected to form a well shape, 9 sleeve assemblies are arranged at the lower part of the outer cover, each sleeve assembly comprises 5 sleeves, and 2.5uL of spore suspension is filled in each sleeve; the bottom of the pipe body is provided with a conical water dropper, the water dropper is formed by solidifying sterile water, and the top of the pipe body is provided with a top cover connected with an outer cover; the culture dish assembly comprises a pair of glass partition plates which are arranged in a cross shape, a circular culture dish body and a square fixing frame, wherein the glass partition plates are installed in the culture dish body, and the fixing frame is installed outside the culture dish body; the bottom of the culture dish body is provided with adaptive filter paper, a liquid storage cavity is arranged in the glass partition plate, an ice block formed by the solidification of sterile water is arranged in the liquid storage cavity, and the bottom of the liquid storage cavity is communicated with the culture dish body; a fixing plate hinged with the glass partition plate is fixed in the glass partition plate, and 5 through holes which correspond to the sleeves one by one are formed in the fixing plate so that the sleeves can be conveniently arranged in the through holes. The invention optimizes the method for inoculating the rice blast germs, solves the problem that the ratoon roots which can be used for transplantation are very limited and can only meet the requirement of inoculating a plurality of physiological races of the rice blast germs; and the operation is complicated, and only one group of blades can carry out one-time identification operation. By adopting the culture device, the invention greatly simplifies the operation steps, is convenient and can complete 9 groups of sample adding tests at one time. The dripper of the invention is made by 5 microliter of sterile ice.

Preferably, the spore suspension has a concentration of 10X 10⁶Per m². The invention adopts spore suspension liquid with higher concentration, the preparation is convenient, the preparation precision is higher, and the spore suspension liquid is dripped into the leaves after being mixed with sterile water formed by the dripper after melting.

Preferably, the thickness of the end face at the bottom of the sleeve is 3-5 mm.

Preferably, the top of the sleeve is adhered to the bottom of the outer cover through ice water solidification.

Preferably, the side wall of the culture dish body comprises an inner cone protruding outwards and an inner borneol matched with the inner cone; the inner cone and the inner borneol form a hollow cylinder. The invention adopts the inner cone and the inner borneol, the inner borneol is immersed in the filter paper after being melted, and the inner cone forms a refraction surface convenient for light refraction, so that light is concentrated on the blade, and the light weakening caused by the fixing plate is avoided.

Preferably, the thickness of the top of the inner cone is greater than the thickness of the bottom.

Preferably, the fixed plate is hinged with the glass partition plate through a hinge seat.

(III) advantageous effects

The method for identifying the blast resistance of the wild rice has the following advantages:

1. the invention optimizes the method for inoculating the rice blast germs, solves the problem that the ratoon roots which can be used for transplantation are very limited and can only meet the requirement of inoculating a plurality of physiological races of the rice blast germs; and the operation is complicated, and only one group of blades can carry out one-time identification operation. By adopting the culture device, the invention greatly simplifies the operation steps, is convenient and can complete 9 groups of sample adding tests at one time. The dripper of the invention is made by 5 microliter of sterile ice.

Drawings

FIG. 1 is a top view of a culture dish assembly of example 1 of the method of identifying blast resistance of wild rice of the present invention;

FIG. 2 is a sectional view of a culture dish assembly of example 1 of the method of identifying blast resistance of wild rice of the present invention;

FIG. 3 is a structural diagram of a fixing plate in example 1 of the method for identifying blast resistance of wild rice of the present invention.

1. The culture dish comprises an outer cover, 2, a culture dish component, 3, a sleeve component, 4, a sleeve, 5, a water dropper, 6, a glass partition plate, 7, a culture dish body, 8, a fixing frame, 9, filter paper, 10, a liquid storage cavity, 11, an ice block, 12, a fixing plate, 13, a through hole, 14, an inner cone, 15, inner borneol, 16, a refraction surface, 17 and a hinged support.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The lesions in the invention are classified as:

level 0: no disease spots;

level 1: dark brown, no visible centers of lesions

And 2, stage: lesions of 0.1 cm diameter, small, no significant centers:

and 3, level: lesions of 0.2 cm diameter, small, no significant centers:

4, level: the lesion is large, forming hyphae and conidia.

In the description of the present invention, "a plurality" means two or more unless otherwise specified; the terms "upper", "lower", "left", "right", "inner", "outer", "front", "rear", "head", "tail", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are only for convenience in describing and simplifying the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed in a particular orientation, and be operated, and thus, should not be construed as limiting the invention.