CN112899342B - Method for identifying pathogenicity of peanut kernel rot - Google Patents

Abstract

The invention discloses a method for identifying pathogenicity of peanut kernel rot, which adopts peanut seed cotyledon as an inoculation material and can visually reflect the pathogenicity of the peanut kernel rot through the morbidity and morbidity of the seed cotyledon. The method has the advantages of simple, rapid and efficient experimental operation, obvious experimental phenomenon, strong intuition and good stability, and can rapidly and efficiently carry out pathogenicity determination and disease resistance identification on a large number of different peanut rot germs and a large number of peanut varieties. The method overcomes the limitations of difficult storage of identification materials, long culture period of plant tissue materials, complex equipment, high requirements on culture conditions, long disease period, low stability and the like in the prior art, and has important significance on pathogenicity identification of peanut rot germs, screening of peanut disease-resistant germplasm resources and disease control.

Description

Method for identifying pathogenicity of peanut kernel rot

Technical Field

The invention belongs to the technical field of identification of pathogenicity of plant pathogens, and particularly relates to a method for identifying pathogenicity of peanut kernel rot.

Background

Peanuts are important oil and economic crops in our country, the underground part of which is susceptible to soil-borne diseases and is directly related to peanut yield and quality. Peanut rot is a main disease in peanut production and poses a significant threat to the yield and quality of peanuts. In recent years, fruit rot diseases in northern peanut production areas tend to be aggravated year after year. The disease is caused by infection of complex pathogens, including various peanut pathogenic fungi, parasitic nematodes, soil mites and the like. The pathogenic cause of the peanut rot is complex, the use of the bactericide often cannot achieve the ideal effect, and the pathogenic bacteria pathogenicity identification, peanut disease-resistant germplasm screening and disease control of the disease are difficult. The establishment of the method for identifying the pathogenicity of peanut fruit rot has important significance for quickly detecting the pathogenicity of peanut fruit rot and identifying the peanut fruit rot resistance germplasm resources, effectively preventing and treating the disease and developing disease-resistant breeding work.

At present, the pathogenicity of peanut kernel rot is mainly determined by a traditional field investigation method, the time and the labor are consumed, a plurality of influencing factors are caused, and the result is unstable; in some methods, peanut shells and peanut kernels are used as materials, and a culture medium is added to inoculate bacteria in the culture medium for determination, so that the problems of complex operation, easy pollution, long period and the like exist (CN 104711317A); the method also has the problems of long period, complex operation, low peanut sensitivity and the like when the measurement is carried out by using different parts of peanut seedlings and culturing tissues in vitro as materials (CN 109750080A). Therefore, the invention provides a simple, rapid, stable and easily-observed pathogenicity identification method aiming at peanut canker and peanut materials, and has important significance for identifying pathogenicity of pathogenic bacteria and resistance of peanuts to the peanut canker.

Disclosure of Invention

The invention aims to provide a method for identifying pathogenicity of peanut fruit rot, which aims to solve the problems of limitation of growth cycle, complex operation, poor stability and the like in the prior art, so as to conveniently determine the pathogenicity of the peanut fruit rot and screen anti-disease peanut germplasm.

In order to achieve the purpose, the technical scheme of the invention is as follows:

(1) obtaining peanut kernel rot germs (separating to obtain the peanut kernel rot germs):

cleaning fruit pods with clear water, cutting peanut shells at the junction of disease and health into small blocks of 3 x 3mm, sterilizing the surfaces of the small blocks, placing the small blocks on a PDA (personal digital assistant) flat plate for culturing for 2-3 days at 25 ℃, selecting fresh tips of colonies, transferring the fresh tips to a new PDA flat plate, obtaining pure culture, selecting hyphae, crushing the hyphae, extracting DNA, performing PCR (polymerase chain reaction) amplification by using a universal primer TTS1/ITS4 in an ITS zone of a fungus, and comparing the obtained product with GenBank after sequencing;

(2) culturing fruit rot pathogenic fungi:

carrying out dark culture on the separated strain on a PDA (personal digital assistant) plate at 25 ℃, cutting mycelium blocks with the size of 4 x 4mm in a fresh area at the edge of a colony after 3 days, and carrying out pathogenicity determination on the peanut material by inoculating the mycelium;

(3) preparation of peanut plant material for inoculation

Preparing hulled peanut kernels without disease spots, sterilizing the surfaces of the hulled peanut kernels with 75% ethanol for 30s and 10% sodium hypochlorite for 3-5min, washing the surface of the hulled peanut kernels with sterile water for 4 times, transferring the hulled peanut kernels into the sterile water for soaking, peeling the peanut kernels after soaking the peanut kernels for 36 hours, separating two cotyledons, cutting off growing points between the two cotyledons (removing growing points such as germs and the like), and keeping the cotyledons, wherein each cotyledon is an inoculation material;

(4) pathogenicity measuring device and inoculum

The soaked peanut cotyledons are placed in a tray filled with absorbent paper and added with water for moisture preservation in order, and the water amount is less than half of that of the test peanut inoculation material. And (3) carrying out wound on the surface of each peanut cotyledon, scratching a wound with the length of 5mm at the center of the inoculation material by using an operating knife, and attaching the hypha surface to the wound. Covering a preservative film on the tray after inoculation for moisturizing, and culturing in the dark at 25 ℃;

(5) determining the pathogenic strength

And (3) counting infection pathogenic conditions, and judging the pathogenicity according to the severity and incidence of the disease, wherein the severity grading standard of the peanut rot disease is as follows:

level 0: the disease is not developed;

level 1: generating lesion spots on the inoculated part, wherein the size of the lesion spots does not exceed 1/4 of the area of the cotyledon;

and 3, level: the size of the lesion spots does not exceed the area of the cotyledon 1/4-1/2;

and 5, stage: the size of the lesion exceeds the area of the cotyledon 1/2;

and 7, stage: the disease is developed in the whole cotyledon.

The invention has the advantages that:

the traditional field investigation method has the limitations of time and labor consumption, numerous influencing factors, unstable result and the like, and the method for identifying peanut seedling tissue in vitro, fresh peanut seed and peanut shell and peanut kernel in the prior art has relatively long test period, complex operation and easy pollution, and has definite advantages aiming at the defects of the prior art:

(1) compared with fresh peanut fruits and peanut plants, the dry peanut seeds have the advantages of easy acquisition and easy preservation; the cotyledon of the dry peanut seeds is used as the inoculated plant material after disinfection, soaking and peeling, the preparation of the inoculated plant material only needs 2 days, and the preparation method has the advantages of convenience and quickness in preparation of the inoculated material.

(2) The seeds are milky white after being peeled, the phenotype of the cotyledon disease spots is clear after inoculation, and the pathogenicity of the peanut rot pathogen can be intuitively reflected according to the disease severity and the disease rate of the seed cotyledon.

(3) The method has the advantages that the incidence time of the inoculated peanut cotyledons is short, obvious scabs can be observed in only 4 days, and the experimental period is short; the inoculation method is simple, strong in stability and high in efficiency.

(4) The inoculated material is only required to be placed in a moisturizing device, and water is added for moisturizing culture, so that a culture medium or a culture solution is not required to be added, and the pollution is not easy to occur.

(5) The method has the advantages of simple, rapid and efficient experimental operation, obvious experimental phenomenon, strong intuition and good stability, and can rapidly and efficiently carry out pathogenicity determination and disease resistance identification on a large number of different peanut rot germs and a large number of peanut varieties. The method overcomes the limitations of the prior art that the identification methods of the field, the pot culture, the seedling, the fresh fruit, the fruit shell and the like are limited by the difficulty in preservation of plant materials, long culture period of plant tissue materials, complicated inoculation process and device, high requirement on culture conditions, long disease period and the like, and has important significance for identifying the pathogenicity of peanut fruit rot bacteria, screening peanut disease-resistant germplasm resources and preventing and controlling diseases.

Drawings

Fig. 1 is a colony morphology of the isolated fusarium Fusarium.solani, Fusarium.oxysporum, Fusarium.Equiseti;

FIG. 2 shows the test material consisting of peanut pod, peanut kernel cotyledon and seedling cotyledon of peanut line Luheihua 01 inoculated with Pythium irregulare;

FIG. 3 shows that the cotyledons of peanut kernels after being soaked for different periods of time are used as test materials to be inoculated with the fruit rot germs;

FIG. 4 shows that the cotyledons of peanut kernels of different varieties including Ji flower 311, Hua Yu 9515, Hua Yu 33, Hua Yu 20 and Lu Black flower 01 are used as test materials to inoculate the fruit rot bacteria for pathogenicity determination;

in the figure, JH331, HY9515, HY33, HY20 and LHH01 represent Ji flower 311, Hua Yu 9515, Hua Yu 33, Hua Yu 20 and Lu Black flower 01, respectively;

FIG. 5 shows pathogenicity assays of 4 different strains of peanut rot disease inoculated into peanut kernel cotyledons.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. However, the specific experimental procedures referred to in the following examples were carried out in a conventional manner or under the conditions recommended by the manufacturer's instructions unless otherwise specified.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. The reagents and materials used are commercially available, unless otherwise specified.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are exemplary only.

Examples

Separation and identification of peanut kernel rot

Peanut pods with fruit rot are collected from fields in the tobacco station city, and pathogenic bacteria are separated and purified, wherein the specific method comprises the following steps: cleaning fruit pods with clear water, cutting peanut shells at the disease-health junction into small blocks of 3 x 3mm, sterilizing the surfaces of the peanut shells for 30s with 75% ethanol and 3-5min with 10% sodium hypochlorite, culturing the peanut shells on a PDA flat plate at 25 ℃ for 2-3 days, picking fresh tips of bacterial colonies, transferring the fresh tips of the bacterial colonies to a new PDA flat plate, obtaining pure culture, picking up mycelia, crushing the mycelia, extracting DNA, performing PCR amplification by using a universal primer ITS1/ITS4 in a fungus ITS zone, performing sequence comparison in GenBank after sequencing, and identifying and separating strains mainly including fusarium.

Secondly, inoculating fruit rot fungi on different test materials for comparison

Sorani was inoculated with the peanut rot fungus fusarium solani as the test object and the peanut, peanut kernel cotyledon and seedling cotyledon of peanut line LHH01 as the test material (fig. 2):

preparing peanut pods without disease spots and shelled peanut kernels, carrying out surface disinfection by using 75% ethanol for 30s and 10% sodium hypochlorite for 3-5min, washing the peanut pods and shelled peanut kernels with sterile water for 4 times, transferring the peanut pods to the sterile water for soaking treatment, wherein the peanut pods are soaked for 36h, the shelled peanut kernels are soaked for 12h, selecting a part of peanut kernels to be soaked, peeling the peanut kernels, separating two cotyledons of the peanut kernels, removing growth points such as germs and the like, and keeping the cotyledons, wherein each cotyledon is an inoculation material. In addition, healthy cotyledons of peanut seedlings potted in a greenhouse for about 15 days are cut out and used as in vitro inoculation materials. The soaked peanut pods, shelled peanut kernels, peanut kernel cotyledons and peanut seedling cotyledons were then placed in a tray lined with absorbent paper and moistened with water at less than half the amount of water of the test peanut inoculation material. Wounds were made on each inoculated material, and a wound of about 5mm in length was made with a scalpel in the center of the inoculated material. The colonies freshly activated and cultured on the PDA plate were cut into 4 × 4 mm-sized hypha blocks, and the hypha surfaces were attached to the wounds. Covering with preservative film for keeping moisture, and culturing at 25 deg.C in dark. 150 of each inoculation material were prepared, one for each 50, in triplicate, three replicates were set up. Onset was observed starting on day 4 of inoculation (figure 2), the number of diseased peanut material and severity of disease were recorded and the experiment was repeated twice.

After 4 days of inoculation, hyphae are on the surfaces of the fruit pods and the nuts, but no obvious scab exists; then, the treatment of hulling the fruit pods and hulling the kernels is further carried out, and comparison shows that the surfaces of the inoculated materials after hulling the fruit pods and hulling the kernels hardly have disease spots, so that the disease degree cannot be judged. The surfaces of peanut kernel cotyledons and seedling cotyledons are both provided with leaf spots. The cotyledon of the peanut kernel is light, the size of the scab after the disease is developed is similar, the shape is clear, the observation is easy, and the disease development degree can be judged according to the size of the scab. The cotyledon of the seedling is green, the scab after inoculation is not as clear as the cotyledon of the peanut kernel, and the size of the scab among the inoculated material repetitions is greatly different, so that the stability is relatively poor. Therefore, the disease phenotype is clearest and the effect is best after the peanut kernel cotyledon is selected for inoculation, and the peanut kernel cotyledon is adopted for inoculation in the following experiments.

Thirdly, the influence of different time for soaking peanut seeds on the inoculation of fruit rot germs

Preparing plump peanut kernels, sterilizing the surfaces of the peanut kernels with 75% ethanol for 30s and 10% sodium hypochlorite for 3-5min, washing with sterile water for 4 times, peeling, transferring to the sterile water for soaking treatment, taking out after soaking for 0h, 6h, 12h, 24h and 36h respectively, separating two cotyledons, removing growth points such as germs and the like, wherein each cotyledon is an inoculation material. Peanut cotyledons were placed in a tray lined with absorbent paper and moistened with water, the amount of water was less than half of the test peanut cotyledons. A wound with the length of about 5mm is scratched by a scalpel at the center of a peanut cotyledon, a mycelium block with the size of 4 x 4mm is cut from a fusarium solani colony which is freshly activated and cultured on a PDA (personal digital assistant) plate, and the mycelium surface is attached to the wound. Covering with preservative film for keeping moisture, and culturing at 25 deg.C in dark. 150 cotyledons were prepared for each treated peanut, one replicate for each 50, in triplicate. Onset was observed starting on day 4 of inoculation (figure 3), the number of peanut material affected and the severity of the disease were recorded and the experiment was repeated twice.

Through comparison, disease spots are concentrated at an inoculation point after cotyledon inoculation without soaking treatment, the disease severity is difficult to judge, and through comparison of different soaking times, disease spots are most clear and have the largest amplification area after soaking treatment for 36 hours, and the disease severity can be more visually reflected.

Determination of pathogenicity of peanut rot germs on different peanut varieties

Preparing plump peanut kernels of different varieties of Ji flower 311, Hua Yu 9515, Hua Yu 33, Hua Yu 20 and Lu Hei Hua 01, sterilizing the surfaces of the plump peanut kernels for 30s with 75% ethanol and 3-5min with 10% sodium hypochlorite, washing with sterile water for 4 times, peeling, transferring to the sterile water for soaking, taking out after soaking for 36h respectively, separating two cotyledons, removing plumule and other growing points, wherein each cotyledon is an inoculation material. Peanut cotyledons were placed in a tray lined with absorbent paper and moistened with water, the amount of water was less than half of the test peanut cotyledons. And scratching a wound with the length of about 5mm at the center of the peanut cotyledon by using a scalpel to perform trauma. The Fusarium solani colonies freshly activated and cultured on the PDA plate are cut into hypha blocks with the size of 4 x 4mm, and the hypha surfaces are attached to the wounds. Covering with preservative film for keeping moisture, and culturing at 25 deg.C in dark. 150 peanut cotyledons were prepared for each variety, one replicate for each 50, in triplicate. Onset was observed starting on day 4 of inoculation (fig. 4), and the number of diseased peanut material and severity of disease were recorded and the experiment was repeated twice. Through tests, the morbidity of all peanut varieties after inoculation is 100%, which shows that the method has good stability. The experimental result also shows that the same peanut rot disease strain has different pathogenicity on different peanut varieties, LHH01 and HY33 have serious disease and show more susceptible disease, and HY20 has lighter scab and shows relative disease resistance. The degree of peanut flu can be measured by the morbidity and disease index, and the results are shown in table 1.

TABLE 1 statistics of disease resistance of different peanut varieties

Peanut variety	Incidence of disease	Index of disease condition
			LHH01	100％	69.07±1.22
JH311	100％	61.87±1.67
			HY9515	100％	56.80±3.20
HY33	100％	66.67±1.67
			HY20	100％	26.93±3.33

Fifthly, inoculating different peanut rot strains to susceptible variety peanut kernel cotyledon for pathogenicity determination

Preparing plump peanut kernels, sterilizing the surface of the plump peanut kernels with 75% ethanol for 30s and 10% sodium hypochlorite for 3-5min, washing with sterile water for 4 times, peeling, transferring to sterile water for soaking treatment, taking out after soaking for 36h, separating two cotyledons, removing growth points such as germs and the like, wherein each cotyledon is an inoculation material. Peanut cotyledons were placed in a tray lined with absorbent paper and moistened with water, the amount of water was less than half of the test peanut cotyledons. And scratching a wound with the length of about 5mm at the center of the peanut cotyledon by using a scalpel for carrying out trauma. 4 peanut rot strains Fe-3, Fs-9, Fs-17 and Fo-23 which are freshly activated and cultured on a PDA plate, hypha blocks with the size of 4 x 4mm are cut from bacterial colonies, and the hypha surfaces are stuck to wounds. The tray is covered with a preservative film for moisture preservation and is placed in the dark at 25 ℃ for culture. 150 peanut cotyledons were prepared for each variety, one replicate for each 50, in triplicate. Onset was observed starting on day 4 of inoculation (fig. 5), and the number of diseased peanut material and severity of disease were recorded and the experiment was repeated twice. Tests show that the morbidity of different peanut rot strains inoculated with peanut cotyledons is different, the morbidity of Fusarium equiseti Fe-3 is 93.3%, the morbidity of Fusarium solani Fs-9 and Fs-17 is 100% and 95.3%, respectively, and the morbidity of Fusarium oxysporum Fo-23 is 98.7%, so that the method has good disease-causing effect stability. The experimental result also shows that different strains of peanut rot disease have different pathogenicity on the same peanut variety, the Fs-9 and Fs-17 have serious morbidity and stronger pathogenicity after being inoculated with peanut cotyledons, and the Fe-3 has the lightest scab and the weakest pathogenicity after being inoculated with the peanut cotyledons. In addition, the pathogenicity of different strains can be measured by the incidence and disease index, and the results are shown in table 2.

TABLE 2 pathogenicity statistics of different strains

The above-mentioned embodiments are merely preferred embodiments of the present invention, which are merely illustrative and not restrictive, and it should be understood that other embodiments may be easily made by those skilled in the art by replacing or changing the technical contents disclosed in the specification, and therefore, all changes and modifications that are made on the principle of the present invention should be included in the scope of the claims of the present invention.

Claims (5)

1. The method for identifying the pathogenicity of peanut rot bacteria is characterized by comprising the following steps:

(1) separating peanut kernel rot germs: collecting peanut pods with fruit rot from the field, and separating and purifying pathogenic strains;

(2) culturing fruit rot pathogenic fungi: culturing the separated strain on a PDA plate at 25 ℃ in the dark, cutting mycelium blocks with the size of 4 x 4mm in a fresh area at the edge of a colony after 3 days, and inoculating the mycelium to peanut materials for pathogenicity determination;

(3) preparation of peanut plant material for inoculation: preparing hulled peanut kernels without disease spots, sterilizing, transferring to sterile water for soaking for 36 hours, peeling, separating two cotyledons of the peanut kernels, cutting off growing points between the two cotyledons, and keeping the cotyledons, wherein each cotyledon is an inoculation material;

(4) inoculating bacteria: the soaked peanut kernels and cotyledons are placed in a tray filled with absorbent paper and added with water for moisture preservation in order, and the water amount is less than half of that of the test peanut inoculation material; carrying out wound on the cotyledon surface of each peanut kernel, and pasting a hypha surface on the wound; covering a preservative film on the tray for moisturizing, and culturing in the dark at 25 ℃;

(5) judging the pathogenicity: and (4) counting infection pathogenic conditions, and judging the pathogenicity according to the severity and the morbidity of the disease.

2. The method for identifying the pathogenicity of peanut rot bacteria according to claim 1, wherein the specific steps of separating and purifying the pathogenic strains are as follows: cleaning fruit pods with clear water, cutting peanut shells at the junction of disease and health into small blocks of 3 x 3mm, sterilizing the surfaces of the small blocks, placing the small blocks on a PDA (personal digital assistant) plate for culturing for 2-3 days at 25 ℃, selecting fresh tips of colonies, transferring the fresh tips to a new PDA plate, obtaining pure culture, selecting hyphae, crushing the hyphae, extracting DNA, performing PCR (polymerase chain reaction) amplification by using universal primers ITS1/ITS4 in an ITS zone of fungi, performing sequencing, comparing the obtained products in GenBank, and identifying and separating strains.

3. The method for identifying the pathogenicity of peanut rot bacteria as claimed in claim 1, wherein the peanut kernel sterilization in step (3) is performed by surface sterilization with 75% ethanol for 30s, surface sterilization with 10% sodium hypochlorite for 3-5min, and washing with sterile water for 4 times.

4. The method for identifying the pathogenicity of peanut rot fungi according to claim 1, wherein the method for treating the surface of cotyledon of each peanut kernel in the step (4) by wound treatment comprises the following steps: a5 mm wound was scribed at the center of the inoculated material with a scalpel.

5. The method of claim 1, wherein the severity grading criteria for peanut rot disease are:

level 0: the disease is not developed;

level 1: generating lesion spots on the inoculated part, wherein the size of the lesion spots does not exceed 1/4 of the area of the cotyledon;

and 3, level: the size of the lesion is 1/4-1/2 of the area of the cotyledon;

and 5, stage: 1/2 the size of the lesion spot exceeds the area of the cotyledon;

and 7, stage: the disease is developed in the whole cotyledon.

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