KR102042899B1 - The mass inoculation system for selecting resistant varieties of Lettuce Wilt and the selection method of resistant varieties using the same - Google Patents

Abstract

The present invention relates to the construction of a large inoculation system for screening varieties resistant to lettuce wilting disease, and to a method for selecting wilted disease resistant lettuce varieties objectively and efficiently using the same. Lettuce wilting disease resistant varieties screening method according to the present invention can be screened for about 300 to 400 varieties resistant to wilted disease in the same pathogen suspension and conditions can be screened efficiently, and the resistance varieties by judging the resistance to score In the screening process, objective results can be obtained.

Description

The mass inoculation system for selecting resistant varieties of Lettuce Wilt and the selection method of resistant varieties using the same}

The present invention relates to the construction of a large inoculation system for screening varieties resistant to lettuce wilting disease, and to a method for selecting wilted disease resistant lettuce varieties objectively and efficiently using the same.

Lettuce ( Lactuca sativa L.) is an annual herbaceous herbaceous plant, derived from L. serriola , a wild lettuce that grows near the Mediterranean Sea, and is known as a crop grown before BC and grown worldwide in various varieties. have. It is estimated to have been introduced in India and China about 1,400 years ago in Korea, and is considered to be a representative crop widely consumed as a sesame vegetable with perilla leaves or other vegetables. In 2012, it was reported that the world's lettuce cultivation area was 1,116 thousand ha, and the production volume was 24,946 thousand tons, and the cultivated area of lettuce grown in Korea was about 4,000 ha as of 2012, totaling about 110,000 tons. It has been. In addition, Korea is known as the world's 20th largest lettuce producer, as it can be easily consumed through small-scale cultivation at home.

Lettuce wilt (Fusarium wilt) is a plant pest caused by the pathogenic fungus Fusarium oxysporum , which occurs not only in Korea, but also in Europe, the United States, Japan, and Brazil. If the pathogen survives in the soil for a long time and becomes dense, it can become infected through the catheter and root of the lettuce and cause symptoms throughout the plant. Lettuce wilting disease occurs during the entire growth period of lettuce cultivation, but mainly from the middle of the growth, the vitality is deteriorated from the lower leaves, yellowing and gradually withered with the characteristics. Sick abandonment can be diminished, but in warmer, dry cultivation environments, the entire tree dries up and significantly reduces production. Since the lettuce wilted disease spreads as the fungus is infected, the damage occurs at the planting grounds of facility cultivation, and the degree of damage is affected by various factors such as planting season, susceptibility of varieties, density of pathogens in soil, and race. Has been reported to be able to spread.

Therefore, in order to increase productivity in lettuce cultivation, the control measures against lettuce wilting disease may be important. To this end, farmers are trying to control lettuce wilted disease by rotation (rotation) or soil disinfectant using disinfectant.However, as the house cultivation has increased recently, the lettuce cultivation temperature has been maintained at the optimal temperature for onset. Damage is reported. Accordingly, in Korea, efforts have been made to select varieties that are resistant to lettuce wilting disease and to prevent wilting disease.

As a method for screening varieties resistant to lettuce wilting disease , a study was conducted to identify cultivars that show susceptibility to Fusarium oxysporum in lettuce cultivars that have wilted disease in cultivated farms. Patent document 1) has been reported, and has been reported for a method for selecting the resistant varieties of wilted disease by the variety of lettuce (non-patent document 2). In addition, studies were conducted to evaluate the resistance of lettuce varieties to lettuce varieties using irrigation, spraying, or soaking to search for genetic resources resistant to Lettuce Germplasm, a representative pest such as lettuce wilting disease. It was also reported (nonpatent literature 3). However, the resistant varieties selection method has a disadvantage in that the plants must be grown in an independent culture environment for breeding, so that the number of varieties that can be grown at the same time is limited, so that objective and efficient selection is not possible.

Accordingly, the present inventors have tried to develop a method for selecting a resource having resistance to wilt disease at the same time efficiently for a variety of lettuce varieties, devised an inoculation phase that can screen a total of 400 resources at once, The present invention was completed by establishing optimum conditions for selecting varieties having resistance to lettuce wilting disease.

KR 10-2009-0085725 A (2009.08.10) KR 10-1499759 B1 (2015.03.16)

 Jinyoung Kim, et al. "Induction of lettuce wilting disease by Fusarium oxysporum f. Sp. Lactucae and comparison of susceptibility between varieties." Plant Disease Research 14.2 (2008): 79-84.  Myung-Hee Chun, et al. "Selection of wilting disease resistant varieties by lettuce varieties." Proceedings of the Korean Society for Horticultural Science Conference Abstract (2012): 97-97.  Kim Sang-kyu, et al. "Exploring Lettuce-resistant Gene Sources." Korean Journal of International Agricultural Development 28.1 (2016): 92-100.

It is an object of the present invention to provide a method for screening lettuce wilted disease resistant varieties and a mass inoculation system for the same.

Still another object of the present invention is to provide a mass inoculation phase for selection of wilted disease resistant varieties for use in the mass inoculation system.

In order to achieve the above object, the present invention (a) a tank that can accommodate the treatment liquid; And (b) a cover having an inoculation port, wherein the inoculation port of the cover has a diameter of 60 to 80 mm, and each inoculation port is disposed at intervals of 20 to 30 mm with respect to the center, withering disease resistance. Provide a large inoculation phase for selection of varieties.

The present invention also provides a method for screening lettuce wilted disease resistant varieties, comprising the following steps i) to iv):

(i) transplanting lettuce seedlings with soil removed after soil germination into the inoculation cover of the inoculation of claim 1;

(ii) adding a Fusarium Oxysporum f. sp. Lactucae spore suspension to the inoculation tank of claim 1 to soak the roots of lettuce hair growth;

(iii) hydroponically growing lettuce hair growth to observe the degree of disease of lettuce wither; And

(iv) evaluating the observed wilting disease symptoms in 0 to 9 steps to screen for wilting disease resistant varieties.

In a preferred embodiment of the present invention, the nanny lettuce in step i), it may be to use a nanny grown for 1 to 3 weeks after soil germination.

In a preferred embodiment of the present invention, the implantation of step i) may be to transplant the lettuce hairs 1 to 5 per inoculation of the inoculation cover.

In a preferred embodiment of the present invention, the concentration of the spore suspension of step ii) may be a suspension of spores of Fusarium Oxysporum f. Sp. Lactucae at 10 ⁴ to 10 ⁸ cfu / ㎖ .

In one preferred embodiment of the present invention, the hydroponic cultivation of step iii) may be performed for 24 hours to 72 hours.

Accordingly, the present invention provides a mass inoculation system for selecting varieties resistant to lettuce wilting disease, and provides a method for selecting lettuce wilting disease resistant varieties using the same. Lettuce wilting disease resistant varieties screening method according to the present invention can be screened for about 300 to 400 varieties resistant to wilted disease in the same pathogen suspension and conditions can be screened efficiently, and the resistance varieties by judging the resistance to score In the screening process, objective results can be obtained.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows a diagram of a large inoculation phase for selection of wilted disease resistant varieties of the present invention.
Figure 2 shows the process of transplanting and cultivating the lettuce on a large inoculation screening of the wilted disease resistant varieties of the present invention.
Figure 3 shows the treatment and inoculated control group inoculated lettuce wilting pathogen using root immersion method.
Figure 4 shows the results of observing the symptoms of wilted ill according to varieties after cultivating lettuce by applying the wilted ill mass inoculation system.
Figure 5 is a result of comparing the results of the wilt test performed three times in total.
Figure 6 shows the statistical analysis of the classification of the withering disease indicators according to the lineage in all varieties.
Figure 7 shows the results of the distribution grouped according to the withering indicator in all varieties.

Hereinafter, the terms of the present invention are defined.

As used herein, the term “resistant variety” refers to a variety that is highly resistant to climate, disease, chemicals, and the like. In this case, "resistance" is also called disease resistance (disease resistance), which means that the property of the plant is less likely to get disease when invaded by the pathogen. In general, disease resistance includes factors such as structural characteristics of plants, biochemical reactions of plants, for example, when plants produce substances that are toxic to pathogens or inhibit the development of pathogens. Can be exercised. The intensity of resistance exerted varies from host-pathogen system, and even in the same host-pathogen system, it may vary depending on the growth stage of the host, differences in organs or tissues that are invaded, nutritional conditions of the host, and weather conditions. .

As used herein, the term “inoculation” refers to injecting a pathogen into a human body, a medium, or other animals or animals for the prevention, diagnosis, treatment, or biological experiment of disease. In the present invention, in order to test and select the varieties resistant to lettuce wilted pathogens, the lettuce wilted pathogens is shown the process of injecting into lettuce plants.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for screening lettuce wilted disease resistant varieties, comprising the following steps i) to iv):

(i) transplanting the lettuce seedlings with soil removed after soil germination into the inoculation cover inoculation;

(ii) adding a Fusarium Oxysporum f. sp. Lactucae spore suspension to the inoculation bath to soak the roots of lettuce hair growth;

(iii) hydroponically growing lettuce hair growth to observe the degree of disease of lettuce wither; And

(iv) evaluating the observed wilting disease symptoms in 0 to 9 steps to screen for wilting disease resistant varieties.

In the method of screening varieties of lettuce wilted disease resistant varieties of the present invention, the "vaccinated phase" includes (a) a tank capable of receiving a treatment liquid; And (b) a cover having an inoculation hole, wherein the inoculation hole of the cover has a diameter of 60 to 80 mm, and each inoculation hole is preferably disposed at an interval of 20 to 30 mm with respect to the center. Specifically, the inoculation port more preferably has a diameter of 50 mm to 70 mm. Given the optimization of the spacing of lettuce implanted on the inoculation, the inoculation openings of the lid on the inoculation for use in the method of the invention have a diameter of 75 mm, each inoculation spaced 25 mm apart from the center. Most preferred.

In the method for screening lettuce wilted disease resistant varieties of the present invention, it is preferable to use the "lettuce lettuce" of the step i) grown for 1 to 3 weeks after the soil germination. Lettuce wilting disease occurs in all growth stages when growing lettuce, but it is known to occur mainly in the mid-growth period, and it is recommended to use the mothers grown for 2 weeks after germination in consideration of the physiological timing of the inoculated plants in order to observe the symptoms in detail. Most preferably, but not limited to.

In the selection method of the lettuce wilted disease resistant varieties of the present invention, the "transplantation" of step i) is to be interpreted to mean the preparation process for immersing the roots in the spore suspension of wilted pathogenic pathogens by moving lettuce lettuce to inoculation Can be. The transplant may be inoculated by injecting 1 to 5 per inoculation port of the inoculation cover, specifically, 3 is most preferably transplanted. At this time, when five or more lettuce hairs are implanted in one inoculation port, too many hairs are implanted in a narrow space, so that spores of the wilt pathogen may not be significantly distributed, and resistance determination may not be effectively performed.

In the method for screening lettuce rot resistant varieties of the present invention, the “spore suspension concentration” of step ii) is suspended from 10 ⁴ to 10 ⁸ cfu / ml of spores of Fusarium Oxysporum f. Sp. Lactucae. One is preferable, and it is more preferable that the concentration is 10 ⁵ to 10 ⁷ cfu / ml. When used in suspending the pathogen spores in a concentration of less than 10 ⁴ cfu / ㎖, mothamyeo exhibit symptoms significantly with the concentration of the pathogen reduced, 10 ⁸ when used at a concentration of cfu / ㎖ excess, due to high concentrations of pathogenic bacteria in accordance with the varieties Since the presence or absence of resistance is not observed significantly, the effect can be reduced. From the viewpoint of effectively determining the physiological relationship between the pathogen and the plant, the concentration of the spore suspension for use in the method of the present invention is most preferably selected to 10 ⁶ cfu / ml.

In the selection method of the lettuce wilted disease resistant varieties of the present invention, the "hydroponic cultivation" of step iii) is preferably carried out for 24 hours to 72 hours, specifically, it is most preferably carried out for 48 hours, but is not limited thereto. . In addition, the temperature conditions of the "hydroponic cultivation" can be applied without limitation if the temperature range known in the art that lettuce wilted disease can occur, it is most preferably carried out by adjusting the temperature to 30 ℃, but not limited to this It doesn't work.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Preparation of lettuce test varieties and preparation of pathogens

First, in order to select varieties having resistance to lettuce wilting disease, test varieties were selected among the published lettuce varieties. Selected varieties were a total of 469 varieties of the following [Table 1]. The selected varieties were sown each by 3 points, and lettuce seedlings were used two weeks after germination.

List of lettuce varieties used for lettuce wilting mass inoculation system System name Tree tree (point)

Domestic breeding species
(allele mining set /
171 points total) Butterhead 4 Crisp head 6
leaf
(Leafy) Native species 5 A stock plane 16 Don't bite 17 Apron 21 Surplus 12 Romain 8 Stem 9 US USDA
(Lettuce germplasm collection /
298 points total) Butterhead 123 Crisphead 62 Leaf 53 Romaine 53 Stem 7

Next, Fusarium Oxysporum f. Sp. Lactucae, a fungal pathogen of lettuce wilting disease, was cultured in a solid medium to prepare as a pathogen. The prepared pathogens were prepared by suspending the cultured cells in water to prepare a pathogen inoculum at a concentration of 10 ⁶ cfu / ml.

Established mass inoculation system for screening lettuce wilted disease resistant resources

<2-1> Lettuce wilting disease mass inoculation phase design

In the present invention, in the process of selecting lettuce resources having resistance to lettuce wilting disease, a large inoculation phase was designed to establish a method for screening multiple varieties simultaneously objectively and with high efficiency.

As shown in Figs. 1 and 2, the large inoculation phase was designed to be composed of a water tank having a width, length, and height of 510 mm × 510 mm × 60 mm, and a water cover covering the same. The tank cover had a total of 400 cover holes (inoculation holes), and the diameter of each inoculation hole was set equal to 75 mm. The interval between each inoculation hole was set to have a 25 mm interval with the center point of the inoculation hole as the mood.

<2-2> Inoculation of Lettuce withered pathogen by root immersion method

To screen for varieties resistant to lettuce wilting disease, pathogens were inoculated into lettuce varieties using a mass inoculation system.

On the large-scale inoculation designed in Example <2-1>, the lettuce hairs at 2 weeks after germination were transplanted. The lettuce husks were removed with water to remove the soil, and then three lettuce husks of the same variety were transplanted to each inoculation. Then, the Fusarium oxis forum pathogen inoculum was inoculated by dipping the roots of lettuce nursery for a total of 48 hours in a tank on the inoculation at a concentration of 10 ⁶ cuf / ml. After immersion initiation, the cultivation temperature was maintained at 30 ° C, and light irradiation and shading were repeated (light: darkness) at 12 hour intervals to observe the symptoms on each of the lettuce hairs. Control experiments were inoculated and cultivated lettuce nanny under the same conditions by filling the tank with water instead of pathogen inoculum. The observed symptom was given a score by dividing the degree of wilting appearing in the pathogen treatment group into a total of nine stages according to the steps of the following [Table 2]. For accurate observation, the same experiment was repeated three times on independent inoculations, and then the scores determined in each experiment were averaged and used as an indicator of the withering of lettuce varieties.

Scorecard for judging the wilting of lettuce wilting score Sickness 0 0% lying down One 25% lay down 2 50% lay down 3 75% lay down 4 100% lying down 5 25% withering 6 50% wither 7 75% wither 8 100% withering 9 Test

As a result, as shown in Figures 3 to 5, it was confirmed that the symptom of lettuce wilting in the experimental group (treatment) inoculated pathogens compared to the control group not inoculated pathogens (Fig. 3). When the same lettuce varieties were judged to be wilted compared to the control group, the varieties clearly exhibiting wilting symptoms compared to the control group (scores 6 to 9), the varieties showing slightly wilting symptoms compared to the control group (scores 4 to 5) and the control group Varieties (scores 0 to 3) that did not show wilting symptoms at similar levels were selected (FIG. 4).

Specifically, when the target varieties 425, 347, and 319 were compared, the 425 varieties were all withered with 0, indicating that they were significantly resistant to withering pathogens. The wilting disease indices were 5, 2, and 5, respectively, indicating that they had some resistance to wilting pathogens. In comparison, the number 319 varieties with the withering index 7, 6 and 7, it was confirmed that the susceptibility to withering pathogens (Fig. 5).

As shown in Table 3, FIG. 6 and FIG. 7 when these results are statistically synthesized. A total of 301 subject lettuce varieties were found to have an average wilting disease index of 4.78, with a standard deviation of 2.43 (Table 3). It was confirmed that there could be a significant difference in resistance to wilting disease in each strain. When judging the resistance and susceptibility of wilt disease depending on the strain, it was confirmed that the cabbage lettuce contains a large number of susceptibility to wilt disease, followed by butterhead lettuce, leaf lettuce, romaine lettuce and stem lettuce. It was confirmed that the resistance to wilting disease (Fig. 6). Varieties representing the lower 10% of the indicators were selected for all varieties, and about 30 points were secured as a candidate group for withering disease (FIG. 7).

Statistical analysis result of wilting disease index according to wilting disease mass inoculation system Average 4.78 Standard Deviation 2.43 Std err mean 0.14 Top 95% Average 5.06 Lower 95% average 4.51 Total 301.00

Claims (6)

delete (i) transplanting lettuce seedlings with soil removed after soil germination into the inoculation cover inoculation,
The inoculation phase is a water tank that can accommodate the treatment liquid; And a cover having an inoculation hole, wherein the diameter of the inoculation hole is 60 to 80 mm, and each inoculation hole is disposed at an interval of 20 to 30 mm with respect to the center.
(ii) adding 10 ⁴ to 10 ⁶ cfu / ml of Fusarium Oxysporum f. sp. Lactucae spore suspension to the inoculation tank of step (i) to soak the roots of lettuce hair growth;
(iii) hydroponicly cultivating lettuce hair growth for 24 to 72 hours to observe the extent of lettuce wilting disease; And
(iv) evaluating the observed wilting disease symptoms in steps 0 to 9, and selecting wilted disease resistant varieties.
Selection method of lettuce wilted disease resistant varieties.
The method of claim 2, wherein the lettuce nursery in step (i) is characterized by using nurseries grown for 1 to 3 weeks after soil germination.
The method of claim 2, wherein the transplanting of the step (i) is characterized in that the lettuce nursery transplants 1 to 5 per inoculation port of the inoculation cover, lettuce wilted disease resistant varieties.

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