阅读说明：本技术 一种芋软腐病菌接种方法 (Alcasia odorata soft rot germ inoculation method ) 是由 颜梅新 黄伟华 于 2019-11-01 设计创作，主要内容包括：本发明公开了一种芋软腐病菌接种方法,属于农业技术领域,利用移液器吸取芋软腐病菌悬浮液,滴至放置于直径15cm培养皿的芋球茎切片组织表面,室温保湿,盖上皿盖。其中芋软腐病菌悬浮液浓度为1×10<Sup>7</Sup>个/mL,芋软腐病菌悬浮液接种量为10uL,5-10个菌株每片芋切片组织；所测试芋球茎切片组织为长5-10cm、宽5-10cm、厚度1-2cm的芋球茎组织,采用吸水的棉花团置于直径15cm培养皿中芋组织周围保湿。本发明接种芋软腐病菌后发病快、显著、可操作性强、致病性测试周期短,从而提高芋软腐病菌致病性缺陷突变体的筛选效率,为芋软腐病菌致病性相关基因鉴定及机理研究提供技术支持,具有重要意义。(The invention discloses a taro soft rot pathogen inoculation method, which belongs to the technical field of agriculture, and is characterized in that a taro soft rot pathogen suspension is sucked by a liquid transfer device, is dripped to the surface of a taro corm section tissue placed in a culture dish with the diameter of 15cm, is moisturized at room temperature, and is covered with a dish cover. Wherein the concentration of the soft rot pathogen suspension is 1 × 10 7 The inoculation amount of the soft rot germ suspension is 10uL per mL, and each slice tissue of each strain is 5-10; the tested taro bulb slice tissue is taro bulb tissue with the length of 5-10cm, the width of 5-10cm and the thickness of 1-2cm, and the water-absorbing cotton cluster is placed in a culture dish with the diameter of 15cm for moisture preservation. After the soft rot pathogen of taro is inoculated, the disease attack is quick and obvious, the operability is strong, and the pathogenicity test period is short, so that the screening efficiency of the pathogenic defect mutant of taro soft rot pathogen is improved, technical support is provided for identification of the pathogenicity-related gene of taro soft rot pathogen and mechanism research, and the method has important significance.)

1. A taro soft rot germ inoculation method is characterized in that: the method comprises the following steps: sucking the soft rot germ suspension by using a pipette, dripping the soft rot germ suspension on the surface of a taro corm section tissue placed in a culture dish, preserving moisture at room temperature, and covering a dish cover.

2. The method of inoculating soft rot of taro as claimed in claim 1, wherein: the soft rot pathogen suspension comprises a soft rot pathogen wild strain suspension and a soft rot pathogen mutant strain suspension.

3. The method of inoculating soft rot of taro as claimed in claim 2, wherein: the concentrations of the soft rot pathogen wild strain suspension and the soft rot pathogen mutant strain suspension are 1 multiplied by 107/mL, the inoculation amount is 10uL, and each slice tissue of taro of 5-10 strains can be inoculated.

4. The method of inoculating soft rot of taro as claimed in claim 2, wherein: the preparation process of the soft rot pathogen wild strain suspension and the soft rot pathogen mutant strain suspension comprises the following steps:

suspending the rotten tissue of the disease standard sample in sterile water, coating the tissue on an LB culture medium, and culturing for 2d at 28 ℃; separating to obtain thallus, suspending in sterile water, diluting, plating on LB culture medium, and culturing at 28 deg.C for 2d to obtain single colony; culturing the soft rot germs in an LB liquid culture medium for 1d in a shaking table at 28 ℃ and at 200rpm to obtain a suspension of the wild type strains of the soft rot germs; respectively culturing the soft rot germ mutant strains obtained by Tn5 in LB liquid medium at 28 deg.C in shaking table at 200rpm for 1d to obtain suspension of soft rot germ mutant strains.

5. The method of inoculating soft rot of taro as claimed in claim 1, wherein: the culture dish is a culture dish with the diameter of 15cm, and the absorbent cotton ball is placed around the taro tissue in the culture dish with the diameter of 15cm for moisture preservation.

6. The method of inoculating soft rot of taro as claimed in claim 1, wherein: the section tissue of the taro corm is 5-10cm long, 5-10cm wide and 1-2cm thick.

7. The method of inoculating soft rot of taro as claimed in claim 1, wherein: the pipettor is a laboratory pipettor, and the measuring range is 100 uL.

8. The method of inoculating soft rot of taro as claimed in claim 1, wherein: the method also comprises the steps of marking the taro bulb slice tissues by using a label, checking the disease after inoculation for 24 hours, investigating for 1 time every 24 hours, and counting the disease incidence rate of taro soft rot within 48 hours.

Technical Field

The invention relates to the technical field of agriculture, in particular to a soft rot germ inoculation method for taro.

Background

Taro [ Colocasia esculenta (L.) Schott ] belongs to the genus taro of the family of the Araceae, is a perennial root herbaceous plant, native to china and india. Taro has high nutritive value, the bulb of the taro is rich in nutrient components such as starch, vitamins, amino acid and the like, and the taro is a vegetable and grain crop widely cultivated all over the world and also an optional biological energy crop, and a plurality of varieties also have medicinal value. The Zhujiang river basin is the most planted in China, and due to subtropical regions in Guangxi, the climate conditions are very suitable for planting taros, so that the method becomes one of main production areas of national taros, wherein the Lipu taros planted in Guangxi Lipu is particularly famous and becomes a national geographical sign to protect agricultural products. According to statistics, 20 ten thousand mu of taro (mainly areca taro) is planted in Guangxi province, wherein the areca taro planted in only Lipu county and Hezhou city exceeds 8 ten thousand mu, the average yield per mu reaches 2000 kg, the total output reaches 12 ten thousand tons, the annual total output value exceeds 8 hundred million yuan, and a solid foundation is laid for the economic stability and development promotion of rural areas in two cities.

In recent years, due to the development of special crops and the favor of people on taro products, the planting area of taros is greatly increased, and the trend of two vigorous supplies and demands appears. However, the varieties available for cultivation in the taro producing areas are few, the main cultivated varieties in all areas are local varieties formed by artificial and natural selection in all areas, the disease incidence rate is high, and the taro soft rot is serious. The etiological agent is reported to be Erwinia carotovora subsp. carotovora (Jones) Bersey et al, which is called Erwinia carotovora and is of the carrot soft rot pathotype, and belongs to the bacteria. In addition, because the soft rot of taro prevents taro crops from being continuously planted, the land can not be fully utilized, the planting cost is increased, the yield and the market demand are influenced, and the soft rot of taro becomes one of the important restriction factors which hinder the development of the taro industry at present.

At present, the pathogenicity determination of taro is carried out by a large amount of mutants and the inoculation of taro is carried out by a large amount of mutants. At present, no report is found about a method for determining the pathogenicity of the taro. In order to improve the screening efficiency of the soft rot pathogen pathogenic defect mutant, technical experts of Guangxi Zhuang autonomous region agricultural science and academic institutions develop the research work of soft rot pathogen inoculation of taro, so that a soft rot pathogen inoculation method is formed, the screening efficiency of the soft rot pathogen pathogenic defect mutant of taro is improved, and the development of the taro industry is promoted.

Disclosure of Invention

The invention aims to provide a taro soft rot pathogen inoculation method. The method has the advantages of fast and obvious morbidity after the soft rot pathogen is inoculated, strong operability and short pathogenicity test period, thereby improving the screening efficiency of the soft rot pathogen pathogenic defect mutant and providing technical support for the pathogenic related gene identification and mechanism research of the soft rot pathogen.

A soft rot germ inoculation method comprises the following steps: sucking the soft rot germ suspension by using a pipette, dripping the soft rot germ suspension on the surface of a taro corm section tissue placed in a culture dish with the diameter of 15cm, preserving moisture at room temperature, and covering a dish cover.

The pipette is a laboratory pipette, and the measuring range is 100 uL.

The preparation method of the soft rot pathogen suspension comprises the following steps:

suspending the rotten tissue of the disease standard sample in sterile water, coating the tissue on an LB culture medium, and culturing for 2d at 28 ℃; separating to obtain thallus, suspending in sterile water, diluting, plating on LB culture medium, and culturing at 28 deg.C for 2d to obtain single colony; culturing the soft rot germs in an LB liquid culture medium for 1d in a shaking table at 28 ℃ and at 200rpm to obtain a suspension of the wild type strains of the soft rot germs; respectively culturing the soft rot germ mutant strains obtained by Tn5 in LB liquid medium at 28 deg.C in shaking table at 200rpm for 1d to obtain suspension of soft rot germ mutant strains.

The section tissue of the taro corm is 5-10cm long, 5-10cm wide and 1-2cm thick.

The concentrations of the soft rot pathogen wild strain suspension and the soft rot pathogen mutant strain suspension are both 1 × 10⁷The inoculation amount is 10uL, and 5-10 strains can be inoculated to each slice of taro tissue.

The cotton ball which absorbs water is placed in a culture dish with the diameter of 15cm to keep moisture around taro tissues.

The research on the pathogenesis of the soft rot pathogen of taro requires a large amount of mutants to carry out pathogenicity determination on taro and a large amount of taro to be inoculated. However, no report is found on the existing method for determining the pathogenicity of the taro. The method comprises sucking soft rot germ suspension by pipette, dripping onto tissue surface of corm section of taro placed in a culture dish with diameter of 15cm, and keeping moisture at room temperatureAnd the dish cover is covered. The tissue of the taro bulb slice tested by the method is taro bulb tissue with the length of 5-10cm, the width of 5-10cm and the thickness of 1-2cm, and a water-absorbing cotton ball is placed in a culture dish with the diameter of 15cm for moisturizing; the concentration of the soft rot pathogen suspension is 1 multiplied by 10⁷The inoculation amount of the soft rot pathogen suspension is 10uL, 5-10 strains can be inoculated to each slice tissue of taro, the number of the tested mutants is increased, the taro starts to be attacked within 12 hours, the attack symptoms within 24 hours are obvious, the period for screening the pathogenic defect mutants of the soft rot pathogen is shortened, the screening efficiency is improved, the pathogenicity determination (inoculation) result is stable, and the test land and the capital investment are effectively saved. After the soft rot pathogen of taro is inoculated, the disease attack is quick and obvious, the operability is strong, and the pathogenicity test period is short, so that the screening efficiency of the pathogenic defect mutant of the taro soft rot pathogen is improved, and the technical support is provided for the identification of the pathogenicity-related gene of the taro soft rot pathogen and the mechanism research.

After the soft rot of taro is inoculated, the method has the advantages of quick and obvious morbidity, strong operability and short pathogenicity test period, and improves the screening efficiency of the soft rot of taro pathogenic defect mutant.

By adopting the technical scheme, the invention has the following technical effects:

(1) the dasheen soft rot germ suspension is sucked by a liquid transfer device and is dripped to the surface of a dasheen corm slice tissue placed on a culture dish with the diameter of 15cm, so that the chance of infecting the dasheen soft rot germ by the dasheen is increased; the moisture is preserved at room temperature, the container is covered, the disease of some mutants in the test is later than that of the wild type of the soft rot pathogen of the taro, the inoculation result is more stable, and the screening effect of the pathogenic defect mutants of the taro soft rot pathogen is achieved;

(2) a pipettor is adopted to suck the soft rot pathogen suspension liquid of the taro and drip the suspension liquid on the tissue surface of the taro corm slice placed on a culture dish with the diameter of 15cm, the operation is convenient, and the cost is low; the sliced tissue of the taro corm is the taro corm tissue with the length of 5-10cm, the width of 5-10cm and the thickness of 1-2cm (5-8 sliced tissues of the specification can be made by one taro), each sliced tissue of the taro can be inoculated with 5-10 strains, a large number of taro tissues can be inoculated, the number of test mutants is greatly increased, and the screening efficiency of the taro soft rot pathogen defect mutant is improved.

(3) Sucking the soft rot germ suspension by using a pipette, dripping the soft rot germ suspension on the surface of a taro corm section tissue placed in a culture dish with the diameter of 15cm, preserving moisture at room temperature, and covering a dish cover. The concentration of the soft rot pathogen suspension is 1 multiplied by 10⁷The inoculation amount of the soft rot germ suspension is 10uL per mL, and each slice tissue of each strain is 5-10; the tested taro bulb slice tissue is taro bulb tissue with the length of 5-10cm, the width of 5-10cm and the thickness of 1-2cm, a water-absorbing cotton cluster is placed around the taro tissue in a culture dish with the diameter of 15cm for moisture preservation, the taro tissue starts to be attacked within 12h, the attack symptoms are obvious within 24h, the screening period of the taro soft rot pathogen pathogenicity defect mutant is shortened, namely the pathogenicity testing period, the screening efficiency is improved, and the screening time is effectively saved.

(4) Sucking the soft rot germ suspension by using a pipette, dripping the soft rot germ suspension on the surface of a taro corm section tissue placed in a culture dish with the diameter of 15cm, preserving moisture at room temperature, and covering a dish cover. The concentration of the soft rot germ suspension is 1 multiplied by 107 per mL, the inoculation amount of the soft rot germ suspension is 10uL, and each slice tissue of each strain is 5 to 10; the tested taro bulb slice tissue is taro bulb tissue with the length of 5-10cm, the width of 5-10cm and the thickness of 1-2cm, and the water-absorbing cotton cluster is placed around the taro tissue in a culture dish with the diameter of 15cm for moisturizing, so that the screening efficiency of the taro soft rot pathogen pathogenic defect mutant is improved, and the test land, the fund and the labor input are saved.

(5) The soft rot of taro has obvious disease after attack, good repeatability, short test period and strong operability. The method can be used for screening the pathogenic defect mutant of the soft rot pathogen of taro and provides technical support for identifying related pathogenic genes and researching mechanism of the soft rot pathogen of taro.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to preferred embodiments. It should be noted, however, that the numerous details set forth in the description are merely for the purpose of providing the reader with a thorough understanding of one or more aspects of the present invention, which may be practiced without these specific details.

The soft rot germ inoculation method comprises the following steps:

preparing taro slice tissues:

taro can be purchased from the market, and the taro corm slice tissue is taro corm tissue with the length of 5-10cm, the width of 5-10cm and the thickness of 1-2 cm; placing the cotton ball with water absorption in a culture dish with diameter of 15cm, and moisturizing.

The soft rot germ suspension is prepared by the following operations:

the taro soft rot disease standard sample is collected from taro soft rot specimens of the academy of agricultural sciences of Guangxi Zhuang nationality and separated.

Suspending the rotten tissue of the disease standard sample in sterile water, coating the tissue on an LB culture medium, and culturing for 2d at 28 ℃; separating to obtain thallus, suspending in sterile water, diluting, plating on LB culture medium, and culturing at 28 deg.C for 2d to obtain single colony; culturing taro soft rot pathogen in LB liquid culture medium at 28 deg.C in shaking table at 200rpm for 1d to obtain thallus suspension; respectively culturing the soft rot germ mutant strains obtained by Tn5 in LB liquid medium at 28 deg.C in shaking table at 200rpm for 1d to obtain suspension of soft rot germ mutant strains.

Pipetting and marking:

sucking 10uL of the wild type taro soft rot pathogen suspension and the mutant suspension by a pipette, injecting the two suspensions to the surface of taro slice tissues respectively, and marking by a pen.

Investigation of soft rot disease of taro:

the disease is checked 24h after inoculation, the earliest disease (symptom) time is 24h, and then the investigation is carried out for 1 time every 24h, and the disease incidence rate of the soft rot of the taro is more than 90% within 48 h.

9 of the wild strains and the normal pathogenic mutant strains of the taro soft rot disease are attacked within 24-48 h after inoculation of 10 strains, namely the taro soft rot disease incidence rate within 48h is 90%.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.