This work was financially supported by the National Key R&#38;D Program of China (2020YFD1001200), the China Agriculture Research System (CARS-11), the specific research fund of The Innovation Platform for Academicians of Hainan Province (YSPTZX202154), the Natural Science Foundation of Hainan Province of China (321RC652), and the Natural Science Foundation of China (No. 31601626).

1. Preparation of black pepper cutting plants for infection
<ol>
	<li>Take a five-node cutting, approximately 40 cm long with a diameter of 0.5 cm, from a healthy and vigorously growing orthotropic branch of black pepper using a disinfected pruning knife or secateurs. Prune the lower three nodes of the plagiotropic branches, with the upper two nodes left with approximately 10 leaves intact.</li>
	<li>Prepare rooting substrate containing soil and animal manure (cow dung or sheep dung) at a ratio of 1:1....

<ol>
	<li>Gordo, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+sequencing+of+black+pepper+root+transcriptome.">High-throughput sequencing of black pepper root transcriptome.</a> BMC Plant Biology. 12 (1), (2012).</li><li>Leonian, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+and+fruit+blight+of+Peppers+caused+by+Phytophthora+capsici+sp.+Nov.">Stem and fruit blight of Peppers caused by Phytophthora capsici sp. Nov.</a> Phytopathology. 12 (9), 401-408 (1922).</li><li>Ding, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priming+maize+resistance+by+its+neighbors:+Activating+1,4-benzoxazine-3-ones+synthesis+and+defense+gene+expression+to+alleviate+leaf+disease.">Priming maize resistance by its neighbors: Activating 1,4-benzoxazine-3-ones synthesis and defense gene expression to alleviate leaf disease.</a> Frontiers in Plant Science. 6, 830 (2015).</li><li>Fonseca, C. E. L., Vianda, D. R., Hansen, J. L., Pell, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associations+among+forage+quality+traits,+vigor,+and+disease+resistance+in+alfalfa.">Associations among forage quality traits, vigor, and disease resistance in alfalfa.</a> Crop Science. 39 (5), 1271-1276 (1999).</li><li>Altier, N. A., Thies, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+resistance+to+Pythium+seedling+disease+in+Alfalfa+using+a+culture+plate+method.">Identification of resistance to Pythium seedling disease in Alfalfa using a culture plate method.</a> Plant Disease. 79 (4), 341-345 (1995).</li><li>Pratt, R. G., Rowe, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+simplified+leaf+inoculation+procedures+for+identification+of+quantitative+resistance+to+Sclerotinia+trifoliorum+in+Alfalfa+seedling.">Evaluation of simplified leaf inoculation procedures for identification of quantitative resistance to Sclerotinia trifoliorum in Alfalfa seedling.</a> Plant Disease. 82 (10), 1161-1164 (1998).</li><li>Hao, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+transcriptome+sequencing+of+black+pepper+(Piper+nigrum+L.)+and+an+analysis+of+genes+involved+in+phenylpropanoid+metabolism+in+response+to+Phytophthora+capsici.">De novo transcriptome sequencing of black pepper (Piper nigrum L.) and an analysis of genes involved in phenylpropanoid metabolism in response to Phytophthora capsici.</a> BMC Genomics. 17 (1), 1-14 (2016).</li><li>Dong, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+inoculation+and+classification+of+maize+ear+rot+caused+by+Fusarium+verticillioides.">Field inoculation and classification of maize ear rot caused by Fusarium verticillioides.</a> Bio-protocol. 8 (23), 3099 (2018).</li><li>English, J. T., Laday, M., Bakonyi, J., Schoelz, J. E., &#201;rsek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+molecular+characterization+of+species+hybrids+derived+from+induced+fusion+of+zoospores+of+Phytophthora+capsica+and+Phytophthora+nicotianae.">Phenotypic and molecular characterization of species hybrids derived from induced fusion of zoospores of Phytophthora capsica and Phytophthora nicotianae.</a> Mycological Research. 103 (8), 1003-1008 (1999).</li><li>Chatterjee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidant+activity+of+some+phenolic+constituents+from+green+pepper+(Piper+nigrum+L.)+and+fresh+nutmeg+mace+(Myristica+fragrans).">Antioxidant activity of some phenolic constituents from green pepper (Piper nigrum L.) and fresh nutmeg mace (Myristica fragrans).</a> Food Chemistry. 101 (2), 515-523 (2007).</li><li>Pfender, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+sporangia+and+release+of+zoospores+by+Phytophthora+megasperma+in+soil.">Production of sporangia and release of zoospores by Phytophthora megasperma in soil.</a> Phytopathology. 67 (5), 657-663 (1977).</li><li>Nagila, A., Schutte, B. J., Sanogo, S., Idowu, O. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chile+pepper+sensitivity+to+mustard+seed+meal+applied+after+crop+emergence.">Chile pepper sensitivity to mustard seed meal applied after crop emergence.</a> HortScience. 56 (2), 1-7 (2021).</li><li>Lamour, K. H., Stam, R., Jupe, J., Huitema, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oomycete+broad-host-range+pathogen+Phytophthora+capsica.">The oomycete broad-host-range pathogen Phytophthora capsica.</a> Molecular Plant Pathology. 13 (4), 329-337 (2012).</li><li>Hardham, A., Gubler, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarity+of+attachment+of+zoospores+of+a+root+pathogen+and+pre-alignment+of+the+emerging+germ+tube.">Polarity of attachment of zoospores of a root pathogen and pre-alignment of the emerging germ tube.</a> Cell Biology International Reports. 14 (11), 947-956 (1990).</li></ol>

In this study, the basal head was pinpricked to damage and provide an effective inoculation system in the black pepper plant. Mycelial pellets then covered the three holes to retain moisture and enable the pathogen to infect the plant well. Following inoculation, the leaves turned yellow and dropped off and the inoculated plants died. No lesions developed in the control plants. Most genes expressed differently following inoculation with Phytophthora capsici in comparison to the control group. Fungal diseases are...

Black pepper (Piper nigrum L.) is a woody climber and one of the most important spice crops. It is known as the "King of Spices"1 and is cultivated in over 40 countries and regions throughout Asia, Africa, and Latin America. Phytophthora root rot is the most devastating disease of black pepper, and is caused by the oomycete Phytophthora capsici. This pathogen also infects cucurbits, eggplants, chili peppers, and tomatoes2,3. With black pepper, an entire crop can sometimes be decimated by this disease. The expansion of pepper planting areas is restricted as a result of the unavailability of resistant varieties, which has significantly hindered the development of the Chinese black pepper industry. An effective inoculation and a precise sampling system for Phytophthora capsici on black pepper plants are essential for studying this plant-pathogen interaction.
The identification and screening of resistance in germplasm resources is the basic requirement for researching the pathogenicity of the pathogen and the breeding and utilization of resistant varieties. A widely used approach is to use a variety of identification methods based on plant species and pathogen groups. Current identification methods include population identification, individual identification, organ identification, tissue identification, cell identification, biochemical identification, and molecular identification, which have been developed in recent years4,5. There has been success in these areas, but there are also many problems. No matter which method is chosen, the basic requirements of plant resistance identification are consistent, including clear objectives, reliable results, and methods that are simple, rapid, and easy to standardize. This principle must also be followed in the identification of black pepper resistance.
In natural field conditions, the identification of disease resistance can be influenced by many environmental factors. Therefore, it was proposed that detached leaves and irrigated roots be used in the laboratory to identify disease resistance. Young leaves from healthy plants were inoculated in vitro in the laboratory, and the diseased leaves area was measured by inoculating the pathogen in order to identify the disease resistance of plants6. However, in vitro leaf inoculation can only be used for general resistance identification and not for molecular interaction studies. Despite this, the disease-resistant status often presents in the irrigated root inoculation, causing uncertainty in the follow-up study of molecular breeding for disease resistance. Therefore, fast and simple indoor detection methods are essential. This study aims to provide a method for resistance identification in the laboratory.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar powder</td>
			<td>Solarbio</td>
			<td>A8190</td>
			<td></td>
		</tr>
		<tr>
			<td>Clean bench</td>
			<td>Haier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Xilong Scientific</td>
			<td>15700501</td>
			<td></td>
		</tr>
		<tr>
			<td>High temperature sterilizing oven</td>
			<td>Zaelway</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish plates</td>
			<td>Biosharp</td>
			<td>BS-90-D</td>
			<td></td>
		</tr>
	</tbody>
</table>

an effective inoculation method for phytophthora capsici on black pepper plants

Piper nigrum L. (black pepper) is a typical woody vine that is an economically important spice crop across the world. Black pepper production is significantly impacted by root rot disease caused by Phytophthora capsici, which has seriously influenced the industry development as a "choke point" problem. However, the molecular genetic mechanism of resistance in black pepper is unclear, leading to slow progress in the development of new black pepper varieties. An effective inoculation and precise sampling system for Phytophthora capsici on black pepper plants is essential for studying this plant-pathogen interaction. The main aim of this study is to demonstrate a detailed methodology where the basal head of black pepper is inoculated with Phytophthora capsici, while also providing a reference for the inoculation of woody vine plants. The basal head of the black pepper plant was pinpricked to damage it, and mycelial pellets covered the three holes to retain the moisture so the pathogen could infect the plant well. This method provides a better way of solving the instability that is caused by traditional inoculation methods including soil drench or root dipping. It also provides a promising means for studying the mode of action between plants and other soil-borne plant pathogens in agricultural precision breeding.

Figure 1 shows the symptoms of black pepper leaves following P. capsici inoculation. Figure 2 shows the symptoms of black pepper stems following P. capsici inoculation. The pathogen infected the black pepper at the basal stem; symptoms including leaf yellowing, wilting appearing, xylem browning, and vessel blacking gradually appearing.&#160;Figure 3 shows most of the genes expressed differently after inoculation wi...

Watch this Scientific Journal Video about An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants at JoVE.com

An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants

Pinpricking the basal head of the black pepper plant is a brief and time-saving method to damage it. Here, we provided detailed steps with a video for infecting black pepper plants.

An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants

Piper nigrum L. (black pepper) is a typical woody vine that is an economically important spice crop across the world. Black pepper production is ...

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.

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3.1K visualizações.  Chinese Academy of Tropical Agricultural Science (CATAS). O experimento a seguir introduz uma inoculação eficaz e um método de amostragem preciso para Phytophthora capsici em plantas de pimenta preta. A pimenta-do-reino, que é uma videira amadeirada típica, é uma cultura de especiarias economicamente importante. A produção de pimenta-do-reino é afetada principalmente pela doença da podridão radicular causada pelo Phytophthora capsici, e influenciou seriamente o desenvolvimento industrial.Uma inoculação eficaz e um sistema de amos...