Foot rot resistance diagnostic marker and foot rot resistance diagnostic method using the same

Abstract

To provide a novel foot rot resistance diagnostic marker that can diagnose the resistance of sweet potato to foot rot disease, and a foot rot resistance diagnostic method using the same.SOLUTION: Polyphenols contained in sweet potato stems are used as a foot rot resistance diagnostic marker.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to a diagnostic marker for base rot resistance that is effective for selecting sweet potato varieties resistant to base rot, and a method for diagnosing base rot resistance using the same.

[0002] Since its outbreak in Okinawa Prefecture was confirmed in November 2018, sweet potato base rot has been spreading to sweet potato producing areas nationwide. Base rot is a soil disease caused by a filamentous fungus called Diaporthe destruens (hereafter referred to as "base rot fungus") and occurs mainly in seedbeds, fields and during storage of tuberous roots. When sweet potatoes are infected with base rot, the stems at the soil edge turn black, and as the symptoms progress, the above-ground parts such as leaves and stems wither, followed by symptoms of rotting of the tuberous roots, which causes growth inhibition, reduced yield and reduced quality.

[0003] Furthermore, if infected sweet potato residues remain in the soil in a field, the root rot fungus will overwinter and cause secondary infection in newly planted sweet potato seedlings in the spring. Therefore, as shown in Patent Document 1, a cultivation method has been proposed in which the soil is sprayed with an aqueous solution of hypochlorous acid, and after the seedlings are planted, the above-ground parts including the seedlings are sprayed with the same solution multiple times to disinfect the seedlings and soil. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2022-158726 A (pages 4 to 7, Figure 1) Summary of the Invention [Problem to be solved by the invention]

[0005] However, in the cultivation method of Patent Document 1, not only bacteria that adversely affect the growth of seedlings, such as base rot bacteria, but also bacteria that have a positive effect on the growth of seedlings are sterilized by the effect of the hypochlorous acid aqueous solution, so after the hypochlorous acid aqueous solution is sprayed, a plant activator must be sprayed above ground within one week from the next day to allow bacteria that have a positive effect on the growth of seedlings to settle, and there is a problem that the larger the field size, the higher the work cost. Innovative pesticides and soil conditioners for preventing the occurrence of base rot have not yet been developed, and the development of sweet potato varieties that are resistant to base rot is an urgent task.

[0006] Generally, the development of disease-resistant varieties involves repeated processes of disease resistance testing and the creation of hybrid strains of varieties with low disease susceptibility, followed by taste tests and component analysis. Varieties that are resistant to the disease and have high market value are selected and registered. However, there is a problem in that selection of sweet potato varieties resistant to base rot is time-consuming because there is little genetic information available and genetic breeding approaches are difficult to use. Therefore, there is a demand for new technologies to diagnose base rot resistance in sweet potato.

[0007] The present invention has been made in response to these problems, and aims to provide a novel diagnostic marker for base rot resistance that can diagnose resistance to sweet potato base rot, and a method for diagnosing resistance to base rot using the same. [Means for solving the problem]

[0008] In order to solve the above problems, the diagnostic marker for root rot resistance of the present invention comprises: It is characterized by being a polyphenol contained in sweet potato stems. According to this feature, the resistance to root rot of sweet potato can be diagnosed by measuring the polyphenol content using polyphenols contained in sweet potato stems as a diagnostic marker for resistance to root rot.

[0009] The polyphenol is characterized as being at least one of chlorogenic acid, 3,4-dicaffeoylquinic acid, and 3,5-dicaffeoylquinic acid contained in the stem. According to this feature, the resistance to base rot of sweet potatoes can be reliably diagnosed by measuring the polyphenol content using at least one of chlorogenic acid, 3,4-dicaffeoylquinic acid, and 3,5-dicaffeoylquinic acid, which account for the majority of polyphenols contained in sweet potato stems and show a high correlation with the incidence of base rot, as a diagnostic marker for base rot resistance.

[0010] The polyphenol is characterized as being chlorogenic acid. According to this feature, by using chlorogenic acid as a diagnostic marker for root rot resistance, it is possible to diagnose root rot resistance of sweet potato inexpensively and stably.

[0011] The method for diagnosing root rot resistance of the present invention comprises the steps of: A method for diagnosing root rot resistance using polyphenols contained in sweet potato stems as a diagnostic marker for root rot resistance, A measuring step of measuring the polyphenol content in sweet potato stems; The method is characterized by comprising a diagnostic step of comparing the polyphenol content measured in the measurement step with the polyphenol content in the stem of a reference variety of sweet potato cultivated under the same cultivation conditions as the sweet potato, and diagnosing root rot resistance based on the obtained comparison value. According to this feature, the polyphenol content in the stems of sweet potatoes can be used as a diagnostic marker for root rot resistance, and the accuracy of diagnosing root rot resistance in sweet potatoes can be improved by comparing the polyphenol content in the stems of the sweet potatoes to be diagnosed with the polyphenol content in the stems of a reference variety of sweet potato grown under the same cultivation conditions.

[0012] The reference variety is characterized in that it is Tamaakane. According to this feature, by using the polyphenol content of Tamaakane, which has particularly high resistance to base rot among base rot-resistant varieties, as the comparison subject, the accuracy of diagnosing base rot resistance of sweet potato can be improved.

[0013] The measuring step is characterized in that the total polyphenol content in the stem is measured. This feature makes it possible to easily diagnose the resistance of sweet potato to root rot.

[0014] In the diagnostic process, the total polyphenol content in the stem is compared with the total polyphenol content in the stem of the reference variety, Tamaakane, and if the comparison value obtained is 1.05 or less, the variety is diagnosed as having high resistance to root rot. According to this feature, the accuracy of diagnosing sweet potato root rot resistance can be further improved. [Brief description of the drawings]

[0015] [Figure 1] FIG. 2 is a photograph showing cross-sections of stems of sweet potato seedlings (two varieties) from 7 days to 35 days after inoculation of the stems with root rot fungus in Experiment 1 of an example of the present invention. [Diagram 2] FIG. 2 is a graph showing the disease severity (35 days after inoculation) of root rot fungus on the stems of sweet potato seedlings (8 varieties) in Experiment 1 of the Examples. [Diagram 3] FIG. 2 is a graph showing the total polyphenol content in the stems of sweet potato seedlings (13 varieties) measured by the Folin-Ciocalteu method in Experiment 2 of the Examples. [Figure 4] FIG. 2 is a graph showing the correlation between the total polyphenol content in the stems of sweet potato seedlings (five varieties) measured by the Folin-Ciocalteu method and the severity of disease in Experiment 2 of the Examples. [Diagram 5] FIG. 2 is a graph showing the polyphenol composition in the stems of sweet potato seedlings (13 varieties) measured by HPLC in Experiment 2 of the Examples. [Figure 6] 1 is a graph showing the correlation between the contents of three types of phenylpropanoid compounds in the stems of sweet potato seedlings (five varieties) measured by HPLC and the disease severity in Experiment 2 of the Example, where (a) is for chlorogenic acid, (b) is for 3,4-dicaffeoylquinic acid, and (c) is for 3,5-dicaffeoylquinic acid. [Figure 7] FIG. 13 is a photograph showing the changes in root rot fungus cultured for 7 days in a total of four types of polyphenol-containing media containing chlorogenic acid and tannic acid in Experiment 3 of the Example. [Figure 8] FIG. 8 is a graph showing the change in mycelial area during 7 days of culturing root rot fungus in the four types of polyphenol-added media shown in FIG. 7. [Figure 9] FIG. 1 is a graph showing the total polyphenol content in the stems of the reference cultivar Tamaakane and the KNI×TA lines (KNI×TA1-15) in Experiment 4 of the Examples. [Figure 10] FIG. 13 is a graph showing the disease severity (35 days after inoculation) of root rot fungus on the stems of sweet potato seedlings (three varieties) and KNI×TA lines (KNI×TA1 to 15) in Experiment 4 of the Examples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The diagnostic marker for root rot resistance and the method for diagnosing root rot resistance using the same according to the present invention will be described below with reference to examples. EXAMPLES

[0017] [Experiment 1: Evaluation of root rot resistance in sweet potato seedlings] To evaluate the resistance of sweet potato seedlings to base rot, cuttings of eight sweet potato varieties (Konashi-shin, Tama-Akane, Takakei 14, Beniharuka, Benimasari, Silk Sweet (registered trademark), Tanegashima Murasaki, and Kyushu 111 (Starch Queen)) that were cultivated from July to October 2022 and grew to about 30 cm were used as sweet potato seedlings. Note that "Konashi-shin," "Tama-Akane," and "Benimasari" are varieties that have been evaluated as having "strong" or "slightly strong" resistance to base rot fungi by the National Agriculture and Food Research Organization Kyushu Okinawa Agricultural Research Center (hereinafter referred to as "NARO") in the "2021 Edition Sweet Potato Base Rot Prevention Manual."

[0018] In addition, six types of strains A to F collected in Kagoshima and Miyazaki prefectures were used as root rot pathogens. Strains A to C were collected from fields in Kagoshima prefecture, and strains D to F were collected from diseased individuals in Miyazaki prefecture.

[0019] To infect sweet potato seedlings with base rot, each sweet potato seedling was inoculated three times by wounding with the tip of a toothpick containing six types of base rot bacteria. The disease condition was observed up to five weeks after inoculation and evaluated using a six-level disease condition index (0 to 5) shown in Table 1.

[0020] [Table 1]

[0021] The disease severity (average disease severity) was calculated from the disease severity index 35 days after inoculation using the following formula. Note that, as controls, a correction coefficient was set using "Beniharuka," which was rated "weak" for resistance to root rot fungi in a survey by the National Agriculture and Food Research Organization, and "Koshin," which was rated "slightly strong."

[0022]

number

[0023] [result] As shown in Figure 1, cross sections (stem cross sections) of the inoculation site of the base rot fungus were observed under an optical microscope from 7 to 35 days after inoculation. The results showed that "Beniharuka," which was rated as having "weak" resistance to the base rot fungus in a survey by the National Agriculture and Food Research Organization, had completely died from 28 days onwards (see Figure 1(a)), whereas "Konashin," which was rated as having "fairly strong" resistance to the base rot fungus, did not rot (see Figure 1(b)), and clear histological differences in the symptoms of the disease were confirmed.

[0024] Furthermore, as shown in Figure 2, the severity of disease among sweet potato varieties (35 days after inoculation) was low in the base rot resistant varieties "Tamaakane (severity 50.0)" and "Benimasari (severity 52.2)," while the severity was high in the base rot susceptible varieties "Takakei 14gou (severity 82.2)" and "Starchiqueen (severity 81.6)," confirming that there are differences in severity of disease among varieties.

[0025] In addition, it was confirmed that the severity of disease in five cultivars, "Takakei 14," "Beniharuka," "Benimasari," "Konashishin," and "Tamaakane," was ranked approximately the same as the results of a root rot resistance test conducted by the National Agriculture and Food Research Organization using a method different from this one.

[0026] [Experiment 2: Varietal differences in polyphenol content and composition] To investigate the varietal differences in polyphenol content (total polyphenol content, individual polyphenol content) and its composition, samples were used from the stems of 13 sweet potato varieties (Koganesengan, Karayutaka, Quick Sweet, Suzuhokkuri, Aikomachi, Anna, Purple Sweet Road, Tamaakane, Himeayaka, Takakei 14, Beniharuka, Konashin, and Benimasari) planted on May 22, 2022 and collected 40 cm from the root on July 9, 2022.The samples were freeze-dried at -30°C using a freeze dryer (FDU1100, manufactured by Tokyo Rika Kikai (EYELA)) and a square dry chamber for the freeze dryer (DRC-1000, manufactured by Tokyo Rika Kikai (EYELA)), and then powdered.

[0027] The total polyphenol content was measured by the general Folin-Ciocalteu method. In detail, 5 mL of 80% methanol was added to 0.02 g of freeze-dried powder of each sample, and ultrasonic extraction was performed for 15 minutes. The extracted sample obtained by filtering through a 0.22 μm syringe filter was added with phenol reagent (Sigma-Aldrich Japan F9252) and saturated sodium carbonate reagent (WAKO 199-01585), and after leaving it for 30 minutes, the absorbance at 760 nm was measured using a spectrophotometer (Japan Bio-Rad Laboratories Smartspec Plus). The measurement was performed in triplicate for each strain, and the results were calculated as the amount of gallic acid equivalent (mg) per 100 g fresh weight.

[0028] In addition, the content of individual polyphenols was measured by reversed-phase high-performance liquid chromatography (reverse-phase HPLC) using the same extracted samples as those for the measurement of the total polyphenol content described above, using a high-performance liquid chromatograph (Shimadzu Corporation Prominence LC solution system) and an ODS column (GL Sciences ODS-3). The chromatography conditions were 100% ethanol as solvent A, 20 mM KH2PO4 (pH 2.4) as solvent B, column temperature 40°C, detection wavelength 320 nm, flow rate 1.0 mL / min, and binary gradient 85-68%B (0-12 min), 68%B (12-15 min), 50-55%B (15-20 min), 85%B (20-29 min). Chlorogenic acid, caffeic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid were used as standards (pure standard substances) and were identified by comparing retention times and spectra. All measurements were performed in triplicate, and the results were calculated in mg / 100g FW (see Figure 3).

[0029] The total polyphenols contained in sweet potatoes are seven types: chlorogenic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, caffeic acid, catechin, and epicatechin, and the content varies depending on the variety. Also, some varieties do not contain caffeic acid, catechin, or epicatechin.

[0030] [result] As shown in Figure 3, when the total polyphenol content in sweet potato stems was measured by the Folin-Ciocalteu method, statistical differences were obtained between varieties (Tukey's multiple test indicates a 5% significant difference between different alphabets).

[0031] The total polyphenol content of the five varieties used in the evaluation of root rot resistance in Experiment 1 was 304mg / 100gFW for the root rot susceptible variety "Takakei 14gou," 228mg / 100gFW for "Beniharuka," 150mg / 100gFW for the root rot resistant variety "Benimasari," 124mg / 100gFW for "Tamaakane," and 100mg / 100gFW for "Koshin." There was no significant relationship between the total polyphenol content of the five varieties and the severity of root rot resistance in the evaluation. 2 = 0.7765, a high positive correlation was confirmed (see Figure 4). The dotted line in Figure 4 is an approximation curve for the total polyphenol content of the above five varieties.

[0032] Thus, based on the correlation between total polyphenol content and the incidence of base rot, it can be inferred that the higher the total polyphenol content in sweet potato stems, the more susceptible they are to base rot, and the lower the total polyphenol content, the higher the resistance to base rot.

[0033] Furthermore, as shown in Figure 5, the results of measuring the content of individual polyphenols (7 types) contained in sweet potatoes by HPLC confirmed that the majority (80% or more) of the polyphenols contained in the stems of sweet potatoes in all 13 varieties were made up of phenylpropanoid compounds, chlorogenic acid, 3,4-dicaffeoylquinic acid, and 3,5-dicaffeoylquinic acid. Phenylpropanoid compounds are ester-bonded compounds of quinic acid and caffeic acid, and are synthesized in plants via the shikimic acid pathway.

[0034] As shown in Figure 6(a), the chlorogenic acid content of the five cultivars used in the evaluation of root rot resistance in Experiment 1 was 65.8 mg / 100 g FW for the root rot susceptible cultivar "Takakei 14gou," 26.5 mg / 100 g FW for "Beniharuka," 13.0 mg / 100 g FW for the root rot resistant cultivar "Benimasari," 20.8 mg / 100 g FW for "Tamaakane," and 17.9 mg / 100 g FW for "Koshin." There was a significant correlation between the chlorogenic acid content of the five cultivars and the severity of root rot resistance in the evaluation. 2 = 0.7006, a high positive correlation was confirmed. The dotted lines in Figure 6(a) are approximate curves relating to the chlorogenic acid contents of the above-mentioned five varieties.

[0035] As shown in Figure 6(b), the 3,4-dicaffeoylquinic acid content was 99.2mg / 100gFW in the root rot susceptible cultivar "Takakei 14gou," 51.8mg / 100gFW in "Beniharuka," 34.4mg / 100gFW in the root rot resistant cultivar "Benimasari," 13.8mg / 100gFW in "Tamaakane," and 16.3mg / 100gFW in "Konashishin." There was a significant correlation between the 3,4-dicaffeoylquinic acid content of the five cultivars and the severity of root rot resistance in the root rot resistance evaluation. 2 The highest positive correlation of 0.7722 was confirmed. The dotted lines in Figure 6(b) are approximate curves for the 3,4-dicaffeoylquinic acid contents of the five varieties mentioned above.

[0036] As shown in Figure 6(c), the 3,5-dicaffeoylquinic acid content was 70.0mg / 100gFW in the root rot susceptible cultivar "Takakei 14gou," 29.7mg / 100gFW in "Beniharuka," 25.2mg / 100gFW in the root rot resistant cultivar "Benimasari," 16.6mg / 100gFW in "Tamaakane," and 15.3mg / 100gFW in "Konashishin." There was a significant correlation between the 3,5-dicaffeoylquinic acid content of the five cultivars and the severity of root rot resistance in the root rot resistance evaluation. 2 = 0.6708, a high positive correlation was confirmed. The dotted lines in Figure 6(c) are approximate curves relating to the 3,5-dicaffeoylquinic acid content (mg / 100gFW) of the five varieties mentioned above.

[0037] Thus, based on the correlation between the content of the three phenylpropanoid compounds, which account for the majority of polyphenols in sweet potato stems as measured by HPLC, and the severity of base rot, it can be inferred that the higher the content of the three phenylpropanoid compounds in sweet potato stems, the more susceptible they are to base rot, and the lower the content of the three phenylpropanoid compounds, the higher their resistance to base rot.

[0038] In addition, since the polyphenol content of sweet potato stems increases toward the tip, it is preferable to sample a portion of the stem from the base to a depth of 30 to 40 cm and then measure the polyphenol content.

[0039] [Experiment 3: Effect of polyphenols on the in vitro growth of root rot fungi] To investigate the effect of polyphenols on the growth of root rot bacteria in vitro, chlorogenic acid (Fujifilm Wako Pure Chemical Industries, Ltd.), which was found to have a high positive correlation with the severity of root rot in Experiment 2, and tannic acid (Fujifilm Wako Pure Chemical Industries, Ltd.), another representative polyphenol, were used. Chlorogenic acid was used as the standard because it is the least expensive and most readily available of the three phenylpropanoid compounds mentioned above. The test bacteria used were the root rot bacteria strain E, which was collected from the diseased seedlings of "Tanegashima Murasaki" used in the above-mentioned root rot resistance test and subcultured for one week at 30°C. The basic medium used was SPDA medium (sweet potato decoction + glucose 5% + gellan gum 3% + streptomycin sulfate (30mg / 100mg)).

[0040] Two types of polyphenol-added media (SPDA+chlorogenic acid medium, SPDA+tannic acid medium) were prepared by adding either chlorogenic acid or tannic acid to the above basic medium through a sterile filter, and two types of polyphenol-added media (chlorogenic acid medium, tannic acid medium) were prepared by replacing sweet potato decoction of the above basic medium with distilled water as a control, and either chlorogenic acid or tannic acid was added through a sterile filter to the medium. The polyphenol (chlorogenic acid, tannic acid) was added at seven concentrations of 0, 20, 40, 80, 120, 160, and 200 mg per 100 g of medium, and the culture was performed three times for each.

[0041] The root rot fungus was cut into 5 mm diameter pieces along with the medium and placed in the center of the four types of medium and cultured in an incubator at 30°C. The progress after culture (days 3, 5, and 7) was recorded by photographs and the mycelial area was measured using the image analysis software ImageJ.

[0042] [result] As shown in Figures 7(a) and (b), in media containing added chlorogenic acid (SPDA + chlorogenic acid medium, chlorogenic acid medium (control)), the mycelial area was larger than in media containing no added chlorogenic acid (0 mg / 100 g medium), indicating a tendency for the growth of the root rot fungus to be promoted.

[0043] As shown in FIG. 8(a), the mycelium area in the SPDA + chlorogenic acid medium did not differ significantly depending on the concentration of chlorogenic acid added until the third day of culture, but on the seventh day of culture, the mycelium area was 543.3 mm at a concentration of 0 mg of chlorogenic acid. 2 , 835.2 mm at 20 mg 2 It was confirmed that there was a statistically significant difference. It was also confirmed that the mycelial area in the SPDA + chlorogenic acid medium peaked at a chlorogenic acid concentration of 20 mg, and that the mycelial area generally decreased as the chlorogenic acid concentration increased beyond 20 mg.

[0044] Similarly, as shown in FIG. 8(b), the mycelium area in the chlorogenic acid medium did not show any significant difference depending on the concentration of chlorogenic acid added until the third day of culture, but on the seventh day of culture, the mycelium area was 797.3 mm at a chlorogenic acid concentration of 40 mg. 2 It was confirmed that the mycelium area generally decreased as the concentration of chlorogenic acid increased from 40 mg.

[0045] In this way, in media containing added chlorogenic acid (SPDA + chlorogenic acid medium, chlorogenic acid medium (control)), it was confirmed that the addition of chlorogenic acid promoted the growth of root rot bacteria up to a specified concentration (20 to 40 mg / 100 g of medium), and that at higher concentrations the growth of root rot bacteria tended to be inhibited.

[0046] Furthermore, as shown in Figures 8(c) and (d), in media containing tannic acid (SPDA + tannic acid medium, tannic acid medium (control)), the mycelial area was significantly smaller than in media containing no tannic acid (0 mg / 100 g medium), indicating a tendency for the growth of root rot fungi to be inhibited. It was also confirmed that the mycelial area decreased as the concentration of tannic acid increased.

[0047] In this way, it was confirmed that the addition of chlorogenic acid to the medium clearly promoted the growth of the base rot bacteria up to a certain concentration. In other words, it can be assumed that the base rot bacteria can utilize chlorogenic acid up to a certain concentration as a factor necessary for growth, or that it may convert chlorogenic acid into a substance effective for growth. In plants, phenylpropanoid compounds such as chlorogenic acid are synthesized in the shikimic acid pathway, and this synthesis pathway is also an important pathway for the synthesis of aromatic amino acids for fungi. Considering this, it is assumed that the onset of the sweet potato base rot bacteria involves the following fungal growth mechanism. First, when the base (stem) of a sweet potato seedling, which is easily broken, is damaged due to physical damage caused by a typhoon or the like, the sweet potato seedling synthesizes chlorogenic acid and the like in response to injury to increase the polyphenol content of the base and defend itself. It is assumed that the base rot bacteria are attracted to this increase in the polyphenol content of the base and grow by utilizing polyphenols such as chlorogenic acid in some way. It is also speculated that the difference in polyphenol content in the base (stem) at this time affects the difference in the severity of disease among sweet potato varieties.

[0048] As described above, root rot resistance of sweet potato can be diagnosed by measuring the polyphenol content using polyphenols contained in sweet potato stems as a diagnostic marker for root rot resistance.

[0049] In addition, by using all the polyphenols contained in sweet potato stems as diagnostic markers for root rot resistance, the root rot resistance of sweet potato can be diagnosed using a simple measurement method such as the Folin-Ciocalteu method.

[0050] Furthermore, by measuring the polyphenol content using at least one of chlorogenic acid, 3,4-dicaffeoylquinic acid, and 3,5-dicaffeoylquinic acid, which account for the majority of polyphenols contained in sweet potato stems and show a high correlation with the incidence of base rot disease, as a diagnostic marker for base rot resistance, the resistance of sweet potatoes to base rot disease can be stably diagnosed without being influenced by other polyphenols that have a low correlation with the incidence of base rot disease. Note that when the contents of chlorogenic acid, 3,4-dicaffeoylquinic acid, and 3,5-dicaffeoylquinic acid are individually measured, the measurement is performed using a measurement method such as HPLC consisting of a group of high-precision devices, so that the measurement accuracy of the content of each polyphenol is high.

[0051] Furthermore, by using chlorogenic acid as a diagnostic marker for root rot resistance, it is possible to diagnose root rot resistance of sweet potato inexpensively and stably.

[0052] Furthermore, the method for diagnosing base rot resistance of the present invention is a method for diagnosing base rot resistance that uses polyphenols contained in the stems of sweet potato as a diagnostic marker for base rot resistance, and includes a measurement step of measuring the polyphenol content in the stems of sweet potato, and a diagnostic step of comparing the polyphenol content measured in the measurement step with the polyphenol content in the stems of a reference variety of sweet potato grown under the same cultivation conditions as the sweet potato, and diagnosing base rot resistance based on the obtained comparison value.By using the content of polyphenols, which are secondary metabolites that change depending on cultivation conditions such as the growth stage of sweet potato, soil (physical properties), soil components, and soil moisture content, as a diagnostic marker for base rot resistance, the accuracy of diagnosing base rot resistance of sweet potato can be improved.

[0053] In addition, by using the polyphenol content of ``Tamaakane,'' a reference variety that has particularly high resistance to base rot among the above-mentioned base rot-resistant varieties, as a comparison target for the diagnostic process, the accuracy of diagnosing sweet potato base rot resistance can be improved.

[0054] For example, based on the results of measuring the total polyphenol content using the Folin-Ciocalteu method in Experiment 2 described above, if we compare the total polyphenol content (124 mg / 100 g FW) of "Tamaakane", which is rated as having a "strong" level of resistance to root rot fungi, the following comparison values ​​are obtained: "Takakei 14", which is rated as having a "weak" level of resistance to root rot fungi, 304 / 124 = 2.45; "Beniharaka", 228 / 124 = 1.84; "Benimasari", which is rated as having a "slightly strong" level of resistance, 150 / 124 = 1.21; and "Konashishin", 100 / 124 = 0.81.

[0055] When the total polyphenol content in sweet potato stems is used as a diagnostic marker for base rot resistance, a comparison value of 1.3 or less compared to the total polyphenol content of "Tamaakane" measured by the Folin-Ciocalteu method can be used to diagnose the plant as having "slightly strong" resistance to base rot, and a value of 1.05 or less can be used to diagnose the plant as having "strong" resistance to base rot.

[0056] In addition, based on the results of measuring the chlorogenic acid content using the HPLC method in Experiment 2 described above, when the chlorogenic acid content (20.8 mg / 100 g FW) of "Tamaakane," which is rated as having a "strong" level of resistance to root rot fungi, is used as a comparison object, the following comparative values ​​are obtained: "Takakei 14," which is rated as having a "weak" level of resistance to root rot fungi, 65.8 / 20.8 = 3.16; "Beniharaka," 26.5 / 20.8 = 1.27; "Benimasari," which is rated as having a "slightly strong" level of resistance, 13.0 / 20.8 = 0.63; and "Konashishin," 17.9 / 20.8 = 0.86.

[0057] When the chlorogenic acid content in sweet potato stems is used as a diagnostic marker for resistance to base rot, the comparison value when used against the chlorogenic acid content of "Tamaakane" measured by HPLC is 1.0 or less, indicating that the sweet potato has "moderately strong" or "strong" resistance to base rot.

[0058] In addition, based on the results of measuring the 3,4-dicaffeoylquinic acid content using the HPLC method in Experiment 2 described above, when the 3,4-dicaffeoylquinic acid content (13.8 mg / 100 g FW) of "Tamaakane," which is rated as having a "strong" level of resistance to root rot fungi, is used as a comparison object, the following comparative values ​​are obtained: "Takakei 14," which is rated as having a "weak" level of resistance to root rot fungi, 99.2 / 13.8 = 7.19; "Beniharaka," 51.8 / 13.8 = 3.75; "Benimasari," which is rated as having a "slightly strong" level of resistance, 34.4 / 13.8 = 2.49; and "Konashishin," 16.3 / 13.8 = 1.18.

[0059] When the 3,4-dicaffeoylquinic acid content in sweet potato stems is used as a diagnostic marker for resistance to root rot, the comparison value when compared with the 3,4-dicaffeoylquinic acid content of "Tamaakane" measured by HPLC is 2.6 or less, indicating that the sweet potato has "moderately strong" or "strong" resistance to root rot.

[0060] In addition, based on the results of measuring the 3,5-dicaffeoylquinic acid content using the HPLC method in Experiment 2 described above, when the 3,5-dicaffeoylquinic acid content (16.6 mg / 100 g FW) of "Tamaakane," which is rated as having a "strong" level of resistance to root rot fungi, is used as a comparison object, the following comparative values ​​are obtained: "Takakei 14," which is rated as having a "weak" level of resistance to root rot fungi, 70.0 / 16.6 = 4.22; "Beniharaka," 29.7 / 16.6 = 1.79; "Benimasari," which is rated as having a "slightly strong" level of resistance, 25.2 / 16.6 = 1.54; and "Konashishin," 15.3 / 16.6 = 0.92.

[0061] When the 3,5-dicaffeoylquinic acid content in sweet potato stems is used as a diagnostic marker for resistance to base rot, the comparison value when compared with the 3,5-dicaffeoylquinic acid content of "Tamaakane" measured by HPLC is 1.6 or less, indicating that the sweet potato has "moderately strong" or "strong" resistance to base rot.

[0062] In addition, when the total polyphenol content in the sweet potato stem is measured in the measurement step, the measurement can be performed by the Folin-Ciocalteu method, which is a colorimetric method, so that the base rot resistance of sweet potato can be diagnosed by a simple measurement method. In addition, the base rot resistance diagnostic method using all polyphenols contained in sweet potato stems as a base rot resistance diagnostic marker, i.e., measuring the total polyphenol content by the Folin-Ciocalteu method, shows a high positive correlation with the incidence of base rot disease equivalent to the case where at least one of chlorogenic acid, 3,4-dicaffeoylquinic acid, and 3,5-dicaffeoylquinic acid is used as the base rot resistance diagnostic marker, and therefore has sufficiently high diagnostic accuracy.

[0063] In this way, in sweet potato, which has little genetic information and makes it difficult to use DNA markers, it is possible to easily diagnose root rot resistance in sweet potato by polyphenol marker selection, especially by measuring the total polyphenol content.

[0064] In addition, conventional disease resistance testing in sweet potato breeding requires highly specialized techniques such as isolation facilities and fields, and handling of pathogens, and is time-consuming because there is a limit to the number of samples that can be tested at one time. However, the selection of base rot-resistant varieties using polyphenols as a diagnostic marker for base rot resistance does not require specialized pathological techniques and has the advantage of being able to measure a large number of samples at one time.

[0065] It has been confirmed that sweet potato stems have lower contents of chlorogenic acid, 3,4-dicaffeoylquinic acid and 3,5-dicaffeoylquinic acid, as well as lower contents of tannic acid, compared to leaves. Therefore, in the present invention, by using the polyphenols contained in sweet potato stems as a diagnostic marker for resistance to base rot, it is possible to steadily select varieties resistant to base rot while minimizing the influence of tannic acid, which has a high inhibitory effect on the proliferation of base rot bacteria.

[0066] [Experiment 4: Diagnosis of root rot resistance in KNI×TA lines] In the method for diagnosing base rot resistance of the present invention, the total polyphenol content in sweet potato stems is used as a diagnostic marker for base rot resistance, and the base rot resistance of the KNI×TA line, which is a hybrid line of "Konashishin" and "Tamaakane", is diagnosed. Note that "Tamaakane", which has particularly high resistance to base rot among the above-mentioned base rot resistant varieties, is used as the reference variety in the diagnostic process, and as in the case of the above-mentioned existing sweet potato varieties, a comparative value of 1.05 or less, using the total polyphenol content in the stems of "Tamaakane" measured by the Folin-Ciocalteu method as a comparison target, is used to diagnose the variety as having "strong" base rot resistance.

[0067] [result] As shown in Figure 9, when comparing the total polyphenol content (142mg / 100gFW) of "Tamaakane", which is evaluated as having a "strong" level of resistance to root rot fungi based on the results of measuring the total polyphenol content in the stems using the Folin-Ciocalteu method, the comparison values ​​for "KNI×TA8" were 113 / 142=0.80 and for "KNI×TA7" were 146 / 142=1.03, and comparison values ​​of 1.05 or less were diagnosed as having "strong" resistance to root rot. All comparison values ​​of the KNI×TA lines other than "KNI×TA7" and "KNI×TA8" were greater than 1.05. In addition, there is no data for "KNI×TA1" because it had died at the time of measuring the total polyphenol content.

[0068] Next, as shown in Figure 10, the disease severity (35 days after inoculation) for the KNIxTA line was lowest for "KNIxTA7 (disease severity 1.8)" followed by "KNIxTA8 (disease severity 16.4)". It was confirmed that "KNIxTA7" and "KNIxTA8", which had a comparative value of 1.05 or less, had high resistance to root rot. In addition, for the KNIxTA line, "KNIxTA4", "KNIxTA10", "KNIxTA13", and "KNIxTA3" had lower disease severity than "Tamaakane", but "KNIxTA7" and "KNIxTA8", which had a comparative value of 1.05 or less, were confirmed to have particularly high resistance to root rot.

[0069] In this way, the base rot resistance diagnosis method of the present invention, in the diagnosis step, compares the total polyphenol content in the stem of the sweet potato being diagnosed with the total polyphenol content in the stem of the reference variety, Tamaakane, and diagnoses high base rot resistance when the obtained comparison value is 1.05 or less, thereby further improving the accuracy of diagnosing base rot resistance in sweet potatoes.

[0070] Furthermore, the base rot resistance diagnosis method of the present invention can diagnose with high accuracy the resistance to base rot not only in existing sweet potato varieties whose resistance to base rot fungi has been evaluated in surveys by the National Agriculture and Food Research Organization, but also in new hybrid lines such as the KNI x TA line.

[0071] The reference variety used as a comparison target in the diagnostic step of the method for diagnosing root rot resistance of the present invention is not limited to "Tamaakane," but may be "Benihayato," "Okikogane Daichi no Yume," "Kyushu 201," "Koshin," "Benimasari," "Suzuhokkuri," "Michishizuku," etc., which are evaluated as having a "strong" or "slightly strong" level of resistance to root rot fungi. In this case, the threshold value of the comparison value may be appropriately set depending on the reference variety, the type of polyphenol to be measured, or a combination thereof.

[0072] Although the embodiments and modified examples of the present invention have been described above with reference to the drawings, the specific configuration is not limited to these embodiments, and the present invention also includes modifications and additions that do not deviate from the gist of the present invention.

[0073] For example, in the above examples, sweet potato seedlings 48 days after planting were used to measure the polyphenol content, but if the seedlings are planted in early May, which is the general planting time from the seedbed to the field, the method for diagnosing base rot resistance can be applied to sweet potato seedlings 40 to 80 days after planting, and the stability of diagnostic accuracy can be improved. In addition, the method for diagnosing base rot resistance of the present invention can be applied not only to sweet potato seedlings planted in a field, but also to sweet potato seedlings planted in a pot.

[0074] In addition, since sweet potato base rot often occurs in June to August during actual cultivation, the accuracy of diagnosis of base rot resistance can be improved by measuring the polyphenol content between June and August, preferably in July.

[0075] In addition, in the above examples, the total polyphenols contained in sweet potatoes were described as seven types: chlorogenic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, caffeic acid, catechin and epicatechin. However, as long as chlorogenic acid, 3,4-dicaffeoylquinic acid and 3,5-dicaffeoylquinic acid account for the majority of polyphenols, the root rot resistance diagnosis method can also be applied to sweet potatoes containing other polyphenols.

[0076] In the above embodiment, the method for diagnosing base rot resistance was described as comparing the polyphenol content in the stem of a reference variety of sweet potato cultivated under the same cultivation conditions as the sweet potato to be diagnosed, and diagnosing base rot resistance from the obtained comparative value, but the present invention is not limited to this. As long as the polyphenol contained in the stem of sweet potato is used as a diagnostic marker for base rot resistance, the base rot resistance may be diagnosed based on a polyphenol content threshold value for the total polyphenol content or at least one of the contents of chlorogenic acid, 3,4-dicaffeoylquinic acid, and 3,5-dicaffeoylquinic acid. In this case, it is preferable that the polyphenol content threshold value is set appropriately according to the conditions such as the method for measuring the polyphenol content, the growth stage of the sweet potato to be diagnosed, the soil (physical properties), the soil components, and the soil moisture content. [Industrial Applicability]

[0077] The present invention utilizes polyphenols contained in sweet potato stems as a diagnostic marker for base rot resistance, and thus makes it possible to diagnose the resistance of sweet potato to base rot by measuring the total polyphenol content in sweet potato stems, which can lead to shortening the sweet potato breeding period, which is said to take 10 years, and overcoming sweet potato base rot, and has industrial applicability. In addition, the present invention can be applied to tuberous roots (tubers) other than sweet potato stems, and the polyphenol content of tuberous roots may become an important indicator for storage and export.

Claims

1. A diagnostic marker for resistance to sweet potato root rot disease, characterized by being a polyphenol contained in the stems of sweet potatoes.

2. The diagnostic marker for root rot resistance according to claim 1, characterized in that the polyphenol is at least one of chlorogenic acid, 3,4-dicaffeoylquinic acid, and 3,5-dicaffeoylquinic acid contained in the stem.

3. The diagnostic marker for root rot resistance according to claim 1, wherein the polyphenol is chlorogenic acid.

4. A method for diagnosing root rot resistance using polyphenols contained in sweet potato stems as a diagnostic marker for root rot resistance, A measuring step of measuring the polyphenol content in sweet potato stems; and a diagnostic step of comparing the polyphenol content measured in the measurement step with the polyphenol content in the stem of a reference variety of sweet potato grown under the same cultivation conditions as the sweet potato, and diagnosing resistance to root rot based on the obtained comparison value.

5. The method for diagnosing root rot resistance according to claim 4, wherein the reference variety is Tamaakane.

6. 6. The method for diagnosing root rot resistance according to claim 4 or 5, wherein the measuring step comprises measuring a total polyphenol content in the stem.

7. The method for diagnosing resistance to root rot described in claim 6, characterized in that in the diagnostic process, the total polyphenol content in the stem is compared with the total polyphenol content in the stem of the reference variety, Tamaakane, and if the obtained comparison value is 1.05 or less, the root rot resistance is diagnosed as high.

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