CN113388662B - Method for identifying tropical resistance of different banana varieties to wilt disease No. 4 physiological races by using tissue culture seedlings - Google Patents
Method for identifying tropical resistance of different banana varieties to wilt disease No. 4 physiological races by using tissue culture seedlings Download PDFInfo
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Abstract
The invention discloses a method for identifying the tropical resistance of different banana varieties to wilt disease No. 4 physiological races by using tissue culture seedlings, belonging to the field of agricultural production and plant protection. The method comprises the steps of preparation of rooting seedlings, preparation of spore suspension inoculation, pathogen inoculation and disease investigation. The method directly adopts the leaves of the rooted seedlings in the tissue culture bottle to stab and inoculate the pathogenic bacteria without replacing the tissue culture bottle, has simple operation, reduces the processes of seedling hardening and plant culture compared with the traditional root-damaging inoculation of field plants or greenhouse plants, avoids the influence of environmental temperature and humidity on the identification result, and has accurate and reliable identification result; the invention screens the tissue culture seedlings in the indoor tissue culture bottle for disease resistance, the condition is easy to control, the identification period is greatly shortened, the speed is high, and a large amount of manpower, material resources and space can be saved.
Description
Technical Field
The invention relates to a method for identifying disease resistance of plants, in particular to a method for identifying the resistance of different banana varieties to wilt disease No. 4 physiological race tropical type by using tissue culture seedlings, belonging to the field of agricultural production and plant protection.
Background
Bananas are one of the most important commercial and food crops in tropical and subtropical countries. However, global banana industry development is being seriously threatened by wilt. Banana vascular wilt is a fungal disease of vascular bundle necrosis caused by infection of Fusarium oxysporum cubeba (Fusarium oxysporum. sp. cubense), and has the advantages of wide distribution, fast diffusion, strong destructiveness and no effective prevention and control measures. In particular, No. 4 physiological race tropical type (T)R4) The harm is particularly serious, and a great number of banana plantations are caused by TR4And is lost. The asexual propagation and the single genetic basis of bananas increase the application difficulty of breeding disease-resistant germplasm resources by the traditional method, and the selection of the disease-resistant germplasm resources in the existing banana resources is used for preventing and controlling TR4Is very critical. Therefore, the selection and storage of disease-resistant banana germplasm resources can effectively prevent and treat T in YunnanR4Has strategic significance.
For a long time, banana breeders widely collect a large amount of banana germplasm resources from home and abroad and identify the disease resistance of the banana germplasm resources. A large number of researches show that the effective method for identifying the disease resistance scientifically, accurately and quickly identifies the aroma coke germplasm resources to the TR4The resistance degree of the strain is a key link for improving the disease-resistant breeding efficiency and accelerating the breeding process.
The banana vascular wilt is a soil-borne destructive disease of bananas and is the most destructive disease of banana diseases. Screening and cultivating the banana variety with high disease resistance is the most effective method for preventing and treating the banana wilt at present. At present, the research on disease-resistant varieties of banana wilt is reported, the resistance evaluation in seedling stage is carried out, the indoor identification and the field resistance evaluation by inoculating pathogenic bacteria through wound roots of potted seedlings are the disease-resistant identification of inoculating pathogenic bacteria to field plants, and the two methods are the most common methods for resistance evaluation and are also important methods for judging the resistance of bananas. There are few reports on simple, rapid and effective methods for identifying disease resistance. Development or selection of disease resistant varieties is a useful and cost effective method, and therefore, there is also a need for a reliable and rapid screening method for resistance in order to allow rapid and unambiguous discrimination between resistant and susceptible varieties. The evaluation of the greenhouse blight resistance is to use the tissue culture seedling to train the seedling to survive and inoculate the seedling in the greenhouse through the injured root of the potted seedling, the period is long, the occupied area is large, other uncontrollable growth conditions and other organisms exist, cross infection can be caused, and the resistance identification result is influenced. The evaluation of field resistance usually takes more than 9 months to complete the evaluation of the whole period, and the banana plants are large and have large occupied area. The test result is influenced by factors such as uneven distribution of banana wilt pathogens in soil, field water and fertilizer management, climate condition change, environmental condition change existing in a test field and the like.
The tissue culture seedling is a main supply mode of banana seedlings, the growth cycle of the tissue culture seedling is short, the propagation speed is high, the environmental conditions can be manually controlled, the seedling raising time of disease resistance evaluation can be reduced, the uncontrollable conditions of the environment can be changed, and the influence of external bacteria on the identification result can be reduced.
Patent No. CN201610839062.0 discloses a rapid identification method of lily tissue culture seedling blight resistance. The method comprises the steps of preparation of an inoculated spore suspension, preparation of lily tissue culture seedlings to be tested, inoculation, disease condition investigation and classification, wherein the tissue culture seedlings are directly subjected to water culture inoculation and identification, a water culture solution contains a large number of elements, trace elements, ferric salt and organic matters, and fusarium oxysporum lily specialized spore suspension is added into the water culture solution. Although the invention adopts tissue culture rooted seedlings, pathogenic bacteria are added into the hydroponic liquid during inoculation, and the hydroponic liquid is added with nutrient solution, is not sealed and is in an open environment.When the method is inoculated in the aqueous solution, pathogenic bacteria grow slowly, seedlings grow slowly, and the identification period is long. In addition, nutrient solution besides pathogenic bacteria is added in the inoculation process, and the inoculation is in an open environment, so that interference of other bacteria besides the pathogenic bacteria can be caused, and the identification result is influenced. The inventor utilizes the similar method to inoculate the banana rooting tissue culture seedlings TR4Pathogenic bacteria infect the whole culture bottle quickly to cause death of tissue culture seedlings, and the pair T of tissue culture seedlings of different banana varieties cannot be judgedR4Of (3). This is probably a result of the rapid growth of the hyphae of the pathogenic bacteria, which hampers the growth of the root system.
Patent No. CN201811602441.3 discloses a method for identifying resistance of banana varieties. The invention transfers the rooted seedling into sterilized vermiculite under greenhouse condition, and then the root is inoculated with pathogenic bacteria. The method has long identification period and large floor area, and has the possibility of causing cross infection due to other uncontrollable growth conditions and other organisms, thereby influencing the resistance identification result.
At present, in a tissue culture bottle, a method for identifying the tropical resistance of different banana varieties to the wilt disease No. 4 physiological race by using rooting seedlings without seedling hardening is not reported.
Disclosure of Invention
In order to solve the technical problem, the invention provides a method for identifying the tropical resistance of different banana varieties to No. 4 physiological races of blight by using tissue culture seedlings without seedling hardening.
The technical scheme of the invention is as follows:
a method for identifying the tropical resistance of different banana varieties to the wilt disease No. 4 physiological race by using tissue culture seedlings comprises the following steps:
1) preparing tissue culture seedlings of different banana varieties for propagation culture of rooted seedlings to obtain cluster buds with relatively consistent growth; cutting the cluster buds into single plants, and transferring the single plants to a rooting culture medium to obtain rooted seedlings with relatively consistent growth;
2) preparation of spore suspension for inoculation the physiological race tropical strain # 4 of Fusarium wilt (T) will be stored at-20 deg.CR4) Activating and coating on PDA solid culture medium at constant temperature of 25 deg.CCulturing for 4-8 days until the mycelia overgrow the culture medium and no pollution, punching a cake with diameter of 7mm on the culture medium with a puncher, inoculating into PDB liquid culture medium, culturing at 28 deg.C and 160r/min on a light constant temperature culture shaking table for 3 days, filtering the bacterial liquid with 3 layers of sterile gauze, counting with a blood count plate, adjusting the spore concentration to 1 × 10 4 1X 10 per mL 6 Per mL;
3) inoculating pathogenic bacteria to the banana tissue culture seedlings by adopting a blade pricking inoculation method, selecting rooted seedlings with relatively consistent growth height of 7-8cm and 5-6 unfolded leaves, opening a tissue culture bottle in a sterile workbench to avoid pollution, pricking upper skins at two sides of a main vein of the 4 th or 5 th leaf of the rooted seedlings by using a disposable sterile injector once respectively, and not pricking lower skins, then sucking 10 mu L of spore suspension liquid in the step 2) by using a pipettor respectively, and dripping the spore suspension liquid at the wound on the leaf to avoid the bacterial liquid from sliding down as much as possible; resealing the tissue culture bottle with the inoculated germs, and placing the tissue culture bottle at the temperature: 25-28 ℃, photoperiod: 12h light 12h dark, light intensity: culturing and observing in a 1600-2000Lux tissue culture room;
4) grading disease conditions, namely grading the disease degree of the inoculated leaves according to the following grade 0-4 disease condition grading standards, wherein the disease condition grading standards are as follows: level 0: no disease; level 1: the lesion spots occupy less than 25% of the area of the inoculated leaves; and 2, stage: the lesion spots account for 25 to 50 percent of the area of the inoculated leaves; and 3, level: the lesion spots account for 50 to 75 percent of the area of the inoculated leaves; and 4, stage 4: the lesion spots account for more than 75% of the area of the inoculated leaves;
5) and (4) disease investigation: investigating disease index according to the disease grading standard at 7 days after inoculation in step 3), recording disease incidence and disease index, and dividing banana germplasm or variety into T pairs according to the average disease index of tested banana tissue culture seedlings of different banana varietiesR4, the average disease index is the average value of the disease indexes of three repeated experiments;
the disease index is calculated according to the following formula:
the disease index ∑ (number of diseased plants at each stage × corresponding stage value)/(total number of investigated plants × highest stage value) × 100.
Furthermore, the rooting seedling in the step 1) is obtained by opening a culture bottle in a sterile working platform, taking out the banana proliferated seedling by using forceps, separating cluster buds with the height of 3-4cm into single plants, transferring the single plants into a rooting culture medium, transferring 5 plants in each bottle of culture medium, and culturing for 15-20 days.
Preferably, the proliferation medium: MS +6-BA 3.0mg/L + NAA 0.2g/L + sucrose 30g/L + agar 5 g/L.
Preferably, the rooting medium: MS + NAA 0.4g/L + sucrose 30g/L + agar 5 g/L.
Preferably, the culture conditions of the tissue culture seedling are as follows: the temperature is 25-28 ℃, the photoperiod is 12h, the light is 12h and the darkness is 12h, and the light intensity is 1600-.
Preferably, the PDA solid culture medium in the step 2): peeled potato 200g + glucose 20g + agar 15 g.
Preferably, the PDB liquid medium of step 2): peeled potato 200g and glucose 20g, adding water to a constant volume of 1000 mL.
Because banana vascular wilt is a soil-borne destructive disease of bananas, when pathogenic bacteria are inoculated, the traditional wilt inoculation method is root or soil inoculation, and both field inoculation and greenhouse seedling exercising inoculation. The method of inoculation through roots is closer to the TR4 infected environment, and the plants are more susceptible to infection. Methods by leaf inoculation are less studied.
Compared with the prior art, the invention has the beneficial effects that:
1) the method utilizes tissue culture seedlings in the tissue culture stage to inoculate TR4 to identify the disease resistance of different banana varieties, and lays a foundation for evaluating the disease resistance of banana germplasm resources or developing a technology for comparing the pathogenicity of different bacterial strains. Through the verification of disease resistance of the same fusarium wilt strain and the same inoculation concentration under a greenhouse condition, the result is found to be consistent with the disease resistance of the test result, and the method provides application values for obtaining the banana germplasm resources with the disease resistance, selecting proper resistant banana germplasm resources and storing the banana germplasm resources in a production area, applying the disease resistant germplasm resources and screening disease resistant genes.
2) The method adopts the leaf puncture inoculation identification, punctures the leaves by using the injector, has uniform inoculation conditions, uniform morbidity and is simple and easy to implement; in addition, the inoculation culture is carried out in a culture bottle sealed environment, the inoculated pathogenic bacteria are single, the influence of other pathogenic bacteria or external environmental conditions is avoided, and the identification result is reliable.
3) According to the invention, tissue culture seedlings are directly inoculated and identified in the tissue culture bottle through leaf stabbing, compared with greenhouse plant root damaging inoculation, the influence of environmental temperature and humidity on the identification result is avoided, and the accuracy of the identification result is improved; the invention can easily control the disease resistance screening condition of the tissue culture seedling in the indoor tissue culture bottle, has short identification period and high speed, and can save a large amount of manpower and material resources. The method has the identification period of 22-28 days, the greenhouse root injury inoculation identification period of 3-4 months, and the seedling hardening condition can easily cause the death of the rooted seedlings and occupy space, thereby laying a good technical foundation for accelerating the breeding of disease-resistant banana varieties and having important practical significance and application prospect.
Description of the figures and reference numerals
FIG. 1 is a graph showing the symptoms of banana wilt of leaves after different banana varieties are inoculated with tissue culture seedlings TR41-7 days,
FIG. 2 is 1X 10 6 The disease indexes of different banana germplasm resources under the greenhouse condition under the inoculation concentration of the number of spores/mL TR4 are as follows: A-C represents the T test result of 20-day disease index of different banana germplasm resources; a-b represents the T test result of the 14-day disease index of different banana germplasm resources,
FIG. 3 is 1X 10 6 Bulb anatomy map of each banana germplasm resource under greenhouse condition under the infection of spore number/mL TR4,
FIG. 4 is a graph of the inoculation of banana rooting tissue culture seedlings in tissue culture bottles with TR4 drop, wherein FIG. 4(A) is the case of 3 days of inoculation, and FIG. 4(B) is the case of 5 days of inoculation.
Detailed Description
Materials used
Different banana varieties were tested: as shown in Table 1, all the banana tissue culture seedlings tested were obtained from agricultural biodiversity of agricultural environmental resource institute of agricultural academy of sciences of Yunnan province and proliferated and rooted seedlings provided by banana research institute.
TABLE 1 Banana germplasm resources
Plant material: the Brazil banana rooted seedlings, the Pahang banana rooted seedlings, the Bank sii rooted seedlings and the Mbwazirude rooted seedlings have been cultured and grown in a rooting medium for 20 d.
Pathogenic bacteria to be tested: the laboratory-preserved Yunan wilt disease No. 4 physiological race tropical wild strain TR4 (15-1).
Proliferation culture medium: MS +6-BA 3.0mg/L + NAA 0.2g/L + sucrose 30g/L + agar 5 g/L.
Rooting culture medium: MS + NAA 0.4g/L + sucrose 30g/L + agar 5 g/L.
PDA solid medium: peeled potato 200g + glucose 20g + agar 15 g.
PDB liquid medium: peeled potato 200g and glucose 20g, adding water to a constant volume of 1000 mL.
All media were autoclaved at 121 ℃ for 20 min.
Example 1A method for identifying the tropical resistance of different banana varieties to the wilt disease No. 4 physiological race by using tissue culture seedlings
1.1 cultivation of rooted seedlings of different Banana varieties
Carrying out enrichment culture on tissue culture seedlings of different banana varieties to obtain cluster buds with relatively consistent growth; cutting the cluster buds into single plants, transferring the plants into a rooting culture medium, and transferring 5 plants into each bottle of culture medium for 40 bottles and 200 plants in total.
The culture conditions are as follows: the temperature of the culture room is 25-28 ℃, the photoperiod is 12h, the illumination is 12h, the darkness is 12h, and the illumination intensity is 1600-.
1.2 inoculation method of pathogenic bacteria of banana tissue culture seedlings
Preparing suspension of banana vascular wilt spores by activating TR4 strain stored at-20 deg.C, plating on PDA solid culture medium, culturing at 25 deg.C for about 10 days until mycelia overgrow the culture medium and no pollution is caused, and making use of puncherBeating the fungus cake with diameter of 7mm on the culture medium, inoculating in PDB liquid culture medium, placing on a light constant temperature culture shaking bed, culturing at 28 deg.C and 160r/min for 3 d. Then filtering the bacterial liquid with 3 layers of sterile gauze, counting with a blood counting chamber, adjusting the spore concentration to 1 × 10 4 1X 10 per mL 6 one/mL.
1.3 inoculation method leaf wounding inoculation method is adopted
Adopting a leaf pricking inoculation method, selecting a rooting seedling which grows relatively uniformly and is 7-8cm high and has 5-6 unfolded leaves, opening a tissue culture bottle in a sterile workbench to prevent the seedling from being polluted, pricking the upper epidermis on the two sides of the main vein of the 4 th or 5 th leaf of the rooting seedling once respectively by using a disposable sterile syringe, and not pricking the lower epidermis, then sucking 10 mu L of the spore suspension liquid by using pipettors respectively, and dripping the spore suspension liquid at the wound on the leaf to avoid the bacterial liquid from sliding down as much as possible. A sample to which sterile water was added was used as a control group. Each banana variety was inoculated with a spore suspension and each treatment was inoculated with 10 banana tissue culture seedlings, each treatment was repeated 3 times. The well inoculated tissue culture bottle is closed again and is placed at the temperature: 25-28 ℃, photoperiod: 12h light 12h dark, light intensity: in the tissue culture room with 1600-2000Lux, the morbidity is counted at 1d, 2d, 3d, 4d, 5d, 6d and 7d after the inoculation of pathogenic bacteria, and the disease index is investigated at 7 d.
1.4 grading of disease conditions
The leaf disease grade was classified into 5 grades, and the corresponding survey criteria are shown in table 2:
TABLE 2 disease investigation grading Standard
As a result:
identification of wilt resistance of rooted seedlings of 4 different banana varieties: the test adopts the leaves of the tissue culture rooted seedlings to inoculate, and the symptoms of the leaves of the disease-resistant banana varieties after being inoculated with pathogenic bacteria show obvious difference, as shown in figure 1. Brazil bananas are susceptible varieties, and the incidence rate of tissue culture rooted seedlings is gradually increased along with the extension of observation time, as shown in table 3. The Brazil banana tissue culture seedlings begin to generate disease symptoms at the 2 nd day after inoculation, and the disease incidence is 60.0 percent; after 3d inoculation, the incidence rate of Brazil bananas reaches 100.0%, and the control does not suffer from the disease. The attack time of Pahang is at 4d, the attack rate is 26.7%, the control does not attack, and the attack rate reaches 60.0% at 7 days after inoculation. The incidence of the disease starts at the 2d of Bank sii, and the incidence rate is 23.3 percent and reaches 86.7 percent at the 7 d. Mbwazirum starts at 4d with a prevalence of only 6.0% and at 7d with a prevalence of 13.3%. Of the 4 banana varieties, the incidence of Brazil banana is up to 100.0% and the incidence is early, followed by Bank sii. The incidence of Pahang and mbwazirum is lower than that of brazil and Banksii, mbwazirum. The test result shows that Brazilian banana and Bank sii are susceptible varieties of blight, and Pahang and Mbwazirum have certain resistance to TR 4. The test results are consistent with the evaluation results of the team in the greenhouse, and are detailed in example 2. The identification method is scientific, and the identification result is credible.
TABLE 3 incidence of wilt disease in tissue culture seedlings of different banana varieties
1.5 disease index
The method for inoculating spore suspension to different banana varieties is carried out according to 1.3, and each treatment is used for inoculating 10 leaves of tissue culture rooting seedlings, and the treatment is repeated for 3 times. The disease index calculation formula is as follows: disease index ∑ (number of diseased plants at each stage × corresponding stage value)/(total number of investigated plants × highest stage value) × 100. Statistical analysis was performed on experimental data using SPSS 16.0 software and differential significance test was performed on TTEST.
On day 7 after inoculation of the pathogenic bacteria, investigation and analysis of disease indexes were performed on tissue-cultured rooted seedlings of 4 different banana varieties, as shown in table 4. The data show that the disease index of the tissue-cultured rooted seedling of the Brazilian banana is 50.67, the Pahang is 15.83, the Bank sii is 32.50, and the Mbwazirude is 3.33. The results show that Brazil banana and Bank sii belong to susceptible varieties, and Pahang and Mbwazirude have stronger resistance to blight. The test result is consistent with the identification result of the team in the greenhouse.
The test adopts the leaves of tissue culture rooted seedlings to inoculate, and the symptoms of the leaves of different banana varieties inoculated with pathogenic bacteria are shown in figure 1. No symptom is shown in the 1d and 4 strains inoculated. The 2d Brazilian banana leaf is seriously attacked, the scab of the Brazilian banana is dark brown at the initial stage, the scab is circular or elliptical, yellow halos are arranged around the scab, white hypha is attached to the scab, and the scab at the later stage gradually expands towards the leaf edge and is irregular. The disease spot is yellow and gray brown after 7 days of Pahang inoculation of fusarium wilt, and the disease spot is slower to spread compared with Brazilian banana varieties. The symptoms of Bank sii show that the disease spots are round or oval, black brown and necrotic, have yellow halos, and the disease spots also gradually spread to the leaf margin in the later period and have white hypha. The disease condition of the Mbwazirude is the lightest, the lesion is round and yellow brown, and the lesion does not expand in the later period.
TABLE 4 disease indexes of different banana variety tissue culture seedlings to fusarium wilt bacteria (TR4)
Example 2 method for identifying different banana varieties for resistance to TR4 in greenhouse using tissue culture seedlings
Testing banana germplasm resources: the method is introduced from the international biodiversity research center, banana germplasm resources stored in agricultural academy of sciences in Yunnan province are utilized, and the inoculated strains are the same strains by utilizing the disease resistance identification result of the same banana germplasm resources in a greenhouse.
2.1 Banana seedling culture
The selected tissue culture seedlings of the banana germplasm resources before inoculation are cultured in a matrix coconut chaff in a greenhouse for about 6 weeks, and are used for artificial inoculation of TR4 after the seedlings grow to 10cm, wherein each batch of germplasm is inoculated with more than 20 plants, and 2 batches of germplasm are respectively inoculated in 7 months in 2019 and 8 months in 2019.
2.2 cultivation of TR4 spores
TR4 stored at-80 ℃ was inoculated onto PDA plates and incubated at 28 ℃ in the dark for 7 days. Conidia on the PDA plate were eluted with sterile water, and the solution was poured into a conical flask containing 300mL of PDA liquid medium and shake-cultured at 25 ℃ and 150rpm for 3 days. Filtering with two layers of gauzeAdjusting spore concentration to 1 × 10 with blood counting plate under microscope 6 Number of spores/mL.
The PDA culture medium configuration method comprises the following steps: peeling potato 200g, cutting into small pieces, boiling with 1000mL water for 15min, filtering with four layers of gauze to remove potato residue, adding glucose 20g and agar powder 20g, adding water to a constant volume of 1000mL, and autoclaving at 121 deg.C for 20 min. The PDA liquid culture medium is a PDA culture medium without agar powder.
2.3 Artificial inoculation method
Inoculating by adopting a root-damaging bacterium soaking method: taking out banana seedlings cultured in coconut chaff, orderly taking out and arranging the banana seedlings to be inoculated according to germplasm resources, trimming the root systems of the banana seedlings to the length of about 5cm by using scissors, and then immersing the roots in spore suspension (the concentration is respectively 1 multiplied by 10) 6 Number of spores/mL), the suspension was stirred once every five minutes. Taking out the soaked banana seedlings after 30min, and transplanting the banana seedlings into pots (1 × 10 in each pot) 6 Spore number/g inoculation amount for root irrigation), the weight of the mixed matrix soil in the pot is 250g, and the total number of the inoculated spores is 2.5 multiplied by 10 respectively 8 Number of spores.
2.4 disease index survey
Disease index was investigated at 14 days and 20 days after inoculation, respectively, and the disease index was investigated by bulb dissection according to a 0-4 grade scale. And (3) data analysis: disease index ∑ (number of diseased plants at each stage × corresponding stage value)/(total number of investigated plants × highest stage value) × 100. Statistical analysis was performed on experimental data using SPSS 16.0 software and differential significance test was performed on TTEST.
2.5 evaluation of disease resistance of Banana germplasm resources
The disease resistance evaluation reference of different germplasm resources divides the resistance of banana germplasm resources to TR4 into 6 different levels according to the 20-day disease index, and the levels are respectively plant immunity (I): i is 0; high Resistance (HR): 0< HR < 20; disease resistance (R): r is more than or equal to 20 and less than 40; anti-Medium (MR): MR <60 is more than or equal to 40; infection (S): s is more than or equal to 60 and less than 80; high feeling (HS): HS is more than or equal to 80 and less than or equal to 100.
The results are shown in Table 5:
TABLE 5 evaluation of resistance of banana germplasm resources in greenhouse by No. 4 physiological microspecies strains
Inoculation of 1X 10 6 The bulb dissection was investigated at TR414 days and 20 days of spore count/mL (fig. 2, fig. 3). The average disease indexes of the disease-resistant germplasm resource Pahang after being inoculated for 14 days and 20 days are respectively 10.42 and 28.57; the disease indexes of the Brazil bananas as the susceptible germplasm resource are 43.75 and 79.43. The results show that the inoculation data is reliable and can be used for further analysis. The analysis of the disease indexes of different banana germplasm resources inoculated with TR4 shows that the disease index of Brazil banana is obviously higher than that of other banana germplasm resources after inoculation for 14 days, and the disease indexes of other banana germplasm resources have no obvious difference. Disease indexes of Brazil bananas and Bank sii are obviously higher than those of Mbwazirude and disease-resistant germplasm resource Pahang after 20 days of inoculation. Disease index range of disease-resistant germplasm resources Pahang and Mbwazirude inoculated for 14 days is 3.13-18.75, disease index range of inoculated for 20 days is 20.31-29.27, and the classification is R. The data results show that 1X 10 cells were inoculated 6 Under the condition of TR4 of spore number/mL, Mbwazirude, Pahang is the disease-resistant germplasm resource of Yunnan strain TR 4. In conclusion, the test results of the method are consistent with the test results of pot culture and field tests reported in the literature. Simultaneously with 1X 10 inoculation in the greenhouse 6 The wild type strain TR4 of Yunnan fusarium wilt with spore number/mL has consistent investigation disease index results, the Mbwazirum and Pahang are disease-resistant germplasm resources of TR4, and the Brazilian banana and Bank sii are disease-sensitive germplasm resources.
EXAMPLE 3 inoculation of TR4 Strain added dropwise to the injured root of the rooted tissue culture seedling in the tissue culture bottle
The method comprises the following steps: (1) after the rooted seedlings are cultured for 15 to 20 days, 300 mu L of the rooting seedlings with the concentration of 1 multiplied by 10 are added into a tissue culture bottle 6 Spore number/mL bacterial liquid, and performing a test in a sterile workbench; (2) the tissue culture bottle with the inoculated strain is closed again and is placed at the temperature: 25-28 ℃, photoperiod: 12h of illumination, 12h of darkness and 1600-2000Lux illumination intensity, and the results are shown in figure 4.
As a result: as shown in FIG. 4(A), 3 days after the inoculation of TR4 strain, the medium was flooded with mycelia; as shown in fig. 4(B), the seedlings died after 5 days, but no yellow leaf symptoms were evident, and could not be determined to be caused by TR4 infestation. The result shows that the banana rooting seedling is not suitable for being inoculated with TR4 by adopting the method.
Claims (7)
1. A method for identifying the tropical resistance of different banana varieties to the wilt disease No. 4 physiological race by using tissue culture seedlings comprises the following steps:
1) preparation of rooted seedlings: carrying out enrichment culture on tissue culture seedlings of different banana varieties to obtain cluster buds with relatively consistent growth; cutting the cluster buds into single plants, and transferring the single plants to a rooting culture medium to obtain rooted seedlings with relatively consistent growth;
2) preparation of the inoculum spore suspension: activating and coating No. 4 physiological race tropical strain (TR4) of wilt disease stored at-20 deg.C on PDA solid culture medium, culturing at 25 deg.C for 4-8d, beating cake with diameter of 7mm on the culture medium with hole puncher when the mycelium is full of the culture medium and has no pollution, inoculating in PDB liquid culture medium, culturing at 28 deg.C and 160r/min on illumination constant temperature culture shaking bed for 3d, filtering with 3 layers of sterile gauze, counting with blood cell counting plate, adjusting spore concentration to 1 × 10 4 one/mL to 1X 10 6 Per mL;
3) inoculating pathogenic bacteria to the banana tissue culture seedlings: adopting a leaf pricking inoculation method, selecting a rooting seedling with the relatively consistent growth height of 7-8cm and 5-6 unfolded leaves, opening a tissue culture bottle in an aseptic workbench to avoid pollution, pricking the upper epidermis on the two sides of the main vein of the 4 th or 5 th leaf of the rooting seedling once respectively by using a disposable aseptic syringe, and not pricking the lower epidermis, then sucking 10 mu L of spore suspension liquid in the step 2) by using a pipettor respectively, and dripping the spore suspension liquid at the wound on the leaf to avoid the bacterial liquid from sliding down as much as possible; resealing the tissue culture bottle inoculated with the germs, and placing the tissue culture bottle at the temperature: 25-28 ℃, photoperiod: 12h light 12h dark, light intensity: 1600-2000Lux tissue culture indoor culture observation;
4) grading the disease condition: the disease degree of the inoculated leaves is graded according to the following grade 0-4 disease grading standard, and the disease grading standard is as follows: level 0: no disease exists; level 1: the lesion spots occupy less than 25% of the area of the inoculated leaves; and 2, stage: the lesion spots account for 25 to 50 percent of the area of the inoculated leaves; and 3, level: the lesion spots account for 50 to 75 percent of the area of the inoculated leaves; 4, level: the lesion spots account for more than 75% of the area of the inoculated leaves;
5) and (4) disease investigation: investigating disease index according to the disease grading standard at the disease spot size of 7 days after inoculation in the step 3), recording disease incidence and disease index, and dividing banana germplasm or variety pair T according to the average disease index of tested banana tissue culture seedlings of different banana varietiesR4, the average disease index is the average value of the disease indexes of three repeated experiments;
the disease index is calculated according to the following formula:
disease index = Σ (number of diseased plants at each stage × corresponding stage value)/(total number of investigated plants × highest stage value) × 100.
2. The method for identifying tropical resistance of different banana varieties to wilt disease No. 4 physiological races by using tissue culture seedlings according to claim 1, characterized in that: the rooting seedling in the step 1) is obtained by opening a culture bottle in a sterile workbench, taking out the banana proliferated seedling by using forceps, separating cluster buds with the height of 3-4cm into single plants, transferring the single plants into a rooting culture medium, transferring 5 plants into each bottle of culture medium, and culturing for 15-20 days.
3. The method for identifying the tropical resistance of different banana varieties to the wilt disease No. 4 physiological race by using tissue culture seedlings according to claim 2, which is characterized in that: the proliferation culture medium used in the proliferation culture of the step 1): MS +6-BA 3.0mg/L + NAA 0.2g/L + sucrose 30g/L + agar 5 g/L.
4. The method for identifying the tropical resistance of different banana varieties to the wilt disease No. 4 physiological race by using tissue culture seedlings according to claim 2, which is characterized in that: the rooting culture medium in the step 1): MS + NAA 0.4g/L + sucrose 30g/L + agar 5 g/L.
5. The method for identifying tropical resistance of different banana varieties to wilt disease No. 4 physiological races by using tissue culture seedlings according to claim 1, characterized in that: the culture conditions of the tissue culture seedling in the step 1) are as follows: the temperature is 25-28 ℃, the photoperiod is 12h, the illumination is 12h and the darkness is 12h, and the illumination intensity is 1600-2000 Lux.
6. The method for identifying the tropical resistance of different banana varieties to the wilt disease No. 4 physiological race by using tissue culture seedlings according to claim 1, which is characterized in that: the PDA solid culture medium in the step 2): peeled potato 200g + glucose 20g + agar 15 g.
7. The method for identifying the tropical resistance of different banana varieties to the wilt disease No. 4 physiological race by using tissue culture seedlings according to claim 1, which is characterized in that: the PDB liquid culture medium in the step 2): peeled potato 200g and glucose 20g, adding water to a constant volume of 1000 mL.
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| US10334799B1 (en) * | 2018-01-26 | 2019-07-02 | H.J. Heinz Company Brands Llc | Hybrid tomato variety H1765 |
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| US10334799B1 (en) * | 2018-01-26 | 2019-07-02 | H.J. Heinz Company Brands Llc | Hybrid tomato variety H1765 |
| CN109593823A (en) * | 2018-12-26 | 2019-04-09 | 云南省农业科学院农业环境资源研究所 | Banana variety Resistance Identification method |
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