The following experiment introduces an effective inoculation and precise sampling method for Phytophthora capsici on black pepper plants. Black pepper, which is a typical woody vine, is an economically important spice crop. Black pepper production is mostly affected by root rot disease caused by Phytophthora capsici, and has seriously influenced the industrial development.
An effective inoculation and a precise sampling system for Phytophthora capsici on black pepper plants is important for the study of plant-pathogen interaction. The main goal of this study is to demonstrate a detailed methodology in which the basal head of black pepper was inoculated with Phytophthora capsici, and also provide a reference for the inoculation of woody vine plants. This method provides a better way to solve the instability brought by the traditional inoculation method, such as soil drench or root dipping.
Experimental procedures. The cuttings of black peppers are planted in the controlled environment for two months. Subculture the Phytophthora capsici, and incubate for five days under 25 degrees Celsius.
On the day of infection, the agar blocks are covered on the three holes sticked by the needle. And finally, characterize disease symptoms. Preparation of black pepper cutting plants for infection.
Cut orthotropic branches in 40 centimeter lengths, and 0.5 centimeter of diameter, and removed plagiotropic branches at the lower nodes by sterile knife and secateur. The cut branch should contain two nodes and five leaves at the top to reduce water loss. Trim the upper part to a flat cut and the lower part to an oblique cut.
The rooting substrate of black pepper cutting was prepared into a mixture, containing soil and animal manure, such as cow dung, sheep dung, et cetera, at a ratio of one to one. The soil was autoclaved at about 121 degrees Celsius for 20 minutes. Use the breeding bags of 30 centimeter diameter and 50 centimeter height to place the rooting substrates.
Prepare a slope of 50 degrees to fill up the substrates. The cuttings were placed at an angle of 50 degrees, with more than three nodes below the substrate. They were covered with the rooting medium and the auxiliary part should be stick to the medium.
Finally, the cuttings were fully infiltrated. Straight cutting was adopted to insert the cutting. Preparation of Phytophthora capsici cultures.
Choose disease-free potatoes. Clean up the potatoes and cut into small squares. Place 200 grams of potatoes in 800 milliliters of double distilled water, and boiled for 20 minutes.
Then filter the mixture. Add 20 grams dextrose. Fill the mixture to one liter with double distilled water.
Add 15 grams of agar powder in the filtrate. Dispense the medium into flasks, and seal using Parafilm. Autoclave the medium at 121 degree Celsius for 20 minutes.
Calculated medium of potato agar was dispensed into the Petri dish plates, and cooling on the sterile bench for solidification. The inoculation needle was sanitized by heating using alcohol-burning lamp, and cooling for several seconds. Take small pieces of fungus samples using sterile inoculation and place on PDA.
The mycelium covers about three quarters of the plate. Cultures are ready for further experiment. Infection of black peppers.
The location of five centimeter away from the surface of the substrate was considered as the incubating spot. The incubating location should not be far away from the root, aiming to avoid prolonging the infective time. Detailed steps of pinpricking are as follows.
A mycelium pellet of Phytophthora capsici was collected using a stopper borer. Notably, newly developed mycelium pellets on the edge of the plate were preferentially selected because of their higher infective activities. Sterilize a stem using 75%ethanol and take three triangular pores at the sterilized position using the inoculation needle.
Three triangular pores at the inoculation location were stuck using the inoculation needle, and mycelial pellets were covered on the three pores. After eight hours, the pores turned black in general. Dilation was enlarged with the prolongation of incubation.
After seven to ten days, the leaves turned yellow and drop off, until black pepper were died. Presentative results are as follows. After infection, the symptoms appear on the black pepper plants, including leaf yellowing and wilting, xylem browning, and vessel blacking.
Most of the genes expressed differently after inoculation with Phytophthora capsici, compared with the control group. The histopathological analysis of infected tissues has demonstrated Phytophthora capsici colonized in the xylem.Conclusion. In this study, pinpricking the basal head was used to make it damaged to provide a effective inoculation, and the doubling system in black pepper.
Meanwhile, this protocol also represents a more efficient way to provide a reference for incubating with-Our method can give more time and operate more briefly. This protocol provides better result, which enables a strong interaction between one plant and the soil-borne pathogen. Which is visible and a convenient to detect the dynamic process between plants and their pathogens.
In our method that we begin to pinprick the basal head of the black pepper plant to make it damage them. Then, the damaged area is covered with the Phytophthora capsici directly, and kept the moisture. So that the pathogen could infected the plant.
We also provides a promising way for the study of the mode of action between plants and other soil-borne plant pathogens in agricultural precision breeding.
