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. Autoclave the rooting substrate at 121 &#176;C for 20 min.</li>
	<li>Insert the cuttings into the rooting substrate at an angle of approximately 50&#176;, with the third node just touching the surface of the substrate and the axillary bud on this node above the substrate. 
	NOTE: The bag used here has the following dimensions: height of 40-60 cm, diameter of 25-30 cm.</li>
	<li>Pour 10-20 L of water over the roots of the plant. Place the cuttings in a greenhouse with 90% shade at a temperature of 25-30 &#176;C for rooting and growth.</li>
</ol>
2. Propagation of Phytophthora capsici (P. capsici)
NOTE: A stock of Phytophthora capsici culture is maintained in the plant protection laboratory of the Spice and Beverage Research Institute, Chinese Academy of Tropical Agricultural Sciences7.
<ol>
	<li>Brush and clean the potato tubers under running tap water and then cut 200 g of potatoes into cubes of 1 cm3. Place some of the cubes into a beaker containing 800 mL of double-distilled water (ddH2O) and boil for 20 min.</li>
	<li>Filter the broth through double gauze using gravity filtration. Prepare potato dextrose agar (PDA) by adding 20 g of dextrose and 15 g of agar to the filtrate and topping the volume up to 1 L with ddH2O. Autoclave the mixture at 121 &#176;C for 20 min8.</li>
	<li>Pour 20 mL of the sterilized PDA in liquid form into a 9 cm diameter round Petri dish inside a laminar air flow hood. Leave the PDA plates with lids open inside the laminar air flow hood overnight as a means of preventing condensation.</li>
	<li>Use an inoculation loop to pick up mycelia from Phytophthora capsici stock inside a test tube. Place the inoculum with the mycelial side in contact with the PDA in a Petri dish.</li>
</ol>
3. Infection of black pepper
<ol>
	<li>Incubation
	<ol>
		<li>Identify an area 5 cm above the substrate surface and near to the roots on the stem for inoculation.</li>
		<li>Core out a disk of mycelia 0.5 cm in diameter at the growing edge of the Phytophthora capsici culture on PDA in a Petri dish using a stopper borer.</li>
		<li>Damage the stem using a syringe needle and make three holes in a triangular pattern at the selected inoculation area. Cover each hole with a mycelial disk. Position the holes close to each other to ensure the wounded area is fully covered with the mycelial disks.</li>
		<li>Cover the mycelial disks with sterilized moistened cotton pads as a means of preventing drying. Tie the pad onto the stem with a polyethylene strip to maintain the position of the inoculating disks. 
		NOTE: At 8 h after inoculation, the inoculated holes turned black and the lesion extended as time passed. The leaves turned yellow and dropped off, and the inoculated plant died 7-10 days after inoculation. No lesions developed in the control plants. Most of the genes expressed differently following inoculation with Phytophthora capsici in comparison to the control group. Histopathological analysis of the infected tissues demonstrated that Phytophthora capsici colonized in the xylem.</li>
	</ol>
	</li>
	<li>Sample the plant materials of interest and store at -80 &#176;C in liquid nitrogen for use in subsequent studies. 
	NOTE: Liquid nitrogen, plastic bags, marker pens, branch shears, and other materials were prepared prior to the experiments.</li>
	<li>After the specific plant materials are sampled for use, autoclave all the remaining plant materials, left-over Phytophthora capsici culture and culture medium, and all the instruments and labware used in this inoculation work.</li>
</ol>

<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><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 ...

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Research

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Genetics

3.2K Views.  Chinese Academy of Tropical Agricultural Science (CATAS). 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.

Video: An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.1 Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut2. However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.
Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is ...

Environment

Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control

Beauveria bassiana is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana may play a role in protecting plants from herbivory and disease. This protocol demonstrates two inoculation methods to establish B. bassiana endophytically in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control. Plants are grown from surface-sterilized seeds for two weeks before receiving a B. bassiana treatment of 108 conidia/ml (or water) applied either as a foliar spray or a soil drench. Two weeks later, the plants are harvested and their leaves, stems and roots are sampled to evaluate endophytic fungal colonization. For this, samples are individually surface sterilized, cut into multiple sections, and incubated in potato dextrose agar media for 20 days. The media is inspected every 2-3 days to observe fungal growth associated with plant sections and record the occurrence of B. bassiana to estimate the extent of its endophytic colonization. Analyses of inoculation success compare the occurrence of B. bassiana within a given plant part (i.e. leaves, stems or roots) across treatments and controls. In addition to the inoculation method, the specific outcome of the experiment may depend on the target crop species or variety, the fungal entomopathogen species strain or isolate used, and the plant's growing conditions.

This protocol demonstrates two inoculation methods to introduce the fungal entomopathogen Beauveria bassiana as an endophyte in the common bean (Phaseolus vulgaris), in preparation for subsequent evaluations of endophytic biological control.

 Beauveria bassiana is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana may play a role in ...

Bioengineering

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in vegetable production annually. Phytophthora capsici is soil-borne and a persistent problem in vegetable fields due to its long-lived survival structures (oospores and chlamydospores) that resist weathering and degradation. The main method of dispersal is through the production of zoospores, which are single-celled, flagellated spores that can swim through thin films of water present on surfaces or in water-filled soil pores and can accumulate in puddles and ponds. Therefore, irrigation ponds can be a source of the pathogen and initial points of disease outbreaks. Detection of P. capsici in irrigation water is difficult using traditional culture-based methods because other microorganisms present in the environment, such as Pythium spp., usually overgrow P. capsici making it undetectable. To determine the presence of P. capsici spores in water sources (irrigation water, runoff, etc.), we developed a hand pump-based filter paper (8-10 &#181;m) method that captures the pathogen&#8217;s spores (zoospores) and is later used to amplify the pathogen&#8217;s DNA through a novel loop-mediated isothermal amplification (LAMP) assay designed for the specific amplification of P. capsici. This method can amplify and detect DNA from a concentration as low as 1.2 x 102 zoospores/mL, which is 40 times more sensitive than conventional PCR. No cross-amplification was obtained when testing closely related species. LAMP was also performed using a colorimetric LAMP master mix dye, displaying results that could be read with the naked eye for on-site rapid detection. This protocol could be adapted to other pathogens that reside, accumulate, or are dispersed via contaminated irrigation systems.

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

We developed a method to detect Phytophthora capsici zoospores in water sources using a filter paper DNA extraction method coupled with a loop-mediated isothermal amplification (LAMP) assay that can be analyzed in the field or in the lab.

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in ...

Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide variety of microorganisms, but studies on soil-borne interactions are still behind those performed on aboveground organs. Although some inoculation strategies for infecting model plants with model root pathogens are described in the literature, it remains difficult to get a comprehensive methodological overview. To address this problem, three different root inoculation systems are precisely described that can be applied to gain insights into the biology of root-microbe interactions. For illustration, Verticillium species (namely, V. longisporum and V. dahliae) were employed as root invading model pathogens. However, the methods can be easily adapted to other root colonizing microbes - both pathogenic and beneficial. By colonizing the plant xylem, vascular soil-borne fungi such as Verticillium spp. exhibit a unique lifestyle. After root invasion, they spread via the xylem vessels acropetally, reach the shoot, and elicit disease symptoms. Three representative plant species were chosen as model hosts: Arabidopsis thaliana, economically important oilseed rape (Brassica napus), and tomato (Solanum lycopersicum). Step-by-step protocols are given. Representative results of pathogenicity assays, transcriptional analyses of marker genes, and independent confirmations by reporter constructs are shown. Furthermore, the advantages and disadvantages of each inoculation system are thoroughly discussed. These proven protocols can assist in providing approaches for research questions on root-microbe interactions. Knowing how plants cope with microbes in the soil is crucial for developing new strategies to improve agriculture.

This protocol presents a detailed summary of strategies to inoculate plant roots with soil-borne microbes. Exemplified for the fungi Verticillium longisporum and Verticillium dahliae, three different root infection systems are described. Potential applications and possible downstream analyses are highlighted, and advantages or disadvantages are discussed for each system.

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide ...

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...

Biology

Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance

Black shank, caused by the oomycetes Phytophthora nicotianae, is destructive to tobacco, and this pathogen is highly pathogenic to many solanaceous crops. P. nicotianae is well adapted to high temperatures; therefore, research on this pathogen is gaining importance in agriculture worldwide because of global warming. P. nicotianae-resistant varieties of tobacco plants are commonly screened by inoculation with oat grains colonized by P. nicotianae and monitoring for the disease symptoms. However, it is difficult to quantify the inoculation intensity since accurate inoculation is crucial in this case. This study aimed to develop an efficient and reliable method for evaluating the resistance of tobacco to infection with P. nicotianae. This method has been successfully used to identify resistant varieties, and the inoculation efficiency was confirmed by real-time PCR. The resistance evaluation method presented in this study is efficient and practical for precision breeding, as well as molecular mechanism research.

Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance

Here, a protocol is presented for the efficient and accurate screening of tobacco genotypes for Phytophthora nicotianae resistance in seedlings. This is a practical approach for precision breeding, as well as molecular mechanism research.

Black shank, caused by the oomycetes Phytophthora nicotianae, is destructive to tobacco, and this pathogen is highly pathogenic to many solanaceous crops. ...

A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates

Fusarium wilt of watermelon (Citrullus lanatus), caused by Fusarium oxysporum f. sp. niveum (Fon), has reemerged as a major production constraint in the southeastern USA, especially in Florida. Deployment of integrated pest management strategies, such as race-specific resistant cultivars, requires information on the diversity and population density of the pathogen in growers' fields. Despite some progress in developing molecular diagnostic tools to identify pathogen isolates, race determination often requires bioassay approaches.
Race typing was conducted by root-dip inoculation, infested kernel seeding method, and the modified tray-dip method with each of the four watermelon differentials (Black Diamond, Charleston Grey, Calhoun Grey, Plant Introduction 296341-FR). Isolates are assigned a race designation by calculation of disease incidence five weeks after inoculation. If less than 33% of the plants for a particular cultivar were symptomatic, they were categorized as resistant. Those cultivars with incidence greater than 33% were regarded as susceptible. This paper describes three different methods of inoculation to ascertain race, root-dip, infested kernel, and modified tray-dip inoculation, whose applications vary according to the experimental design.

A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates

Managing Fusarium wilt of watermelon requires knowledge of the pathogen races present. Here, we describe the root-dip, infested kernel seeding, and modified tray-dip inoculation methods to demonstrate their efficacy in race-typing of the pathogenic fungus Fusarium oxysporum f. sp niveum (Fon).

Fusarium wilt of watermelon (Citrullus lanatus), caused by Fusarium oxysporum f. sp. niveum (Fon), has reemerged as a major production constraint in the ...

Analysis of Effect of Compound Salt Stress on Seed Germination and Salt Tolerance Analysis of Pepper (Capsicum annuum L.)

To determine the salt tolerance and physiological mechanism of pepper (Capsicum annuum L.) at the germination stage, the Hongtianhu 101 and Xinxiang 8 varieties, which have large differences in salt tolerance, are employed as the study materials. Six mixed salt concentrations of 0, 3, 5, 10, 15, and 20 g/L derived using equal molar ratios of Na2CO3, NaHCO3, NaCl, CaCl2, MgCl2, MgSO4, and Na2SO4 are used. To determine their effects, the related indexes of seed germination, seedling growth, and physiology are measured, and salt tolerance is comprehensively evaluated using membership function analysis. The results show that as the mixed salt concentration increases, the germination potential, germination index, germination rate, seed germination vigor index, root length, and root fresh weight of the two cultivars significantly decrease, whereas the relative salt rate gradually increases. The hypocotyl length and fresh weight aboveground increase first and then decrease, while the malondialdehyde (MDA), proline (Pro) content, catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activity decrease and then increase. The germination potential, germination index, germination rate, seed germination vigor index, root length, root fresh weight, MDA and Pro content, and CAT activity of the Hongtianhu 101 seeds are higher than those of Xinxiang 8 for all salt concentrations employed here. However, hypocotyl length, fresh weight aboveground, and relative salt rate are lower in Hongtianhu 101 than in Xinxiang 8. The comprehensive evaluation of salt tolerance reveals that the total weighted values of the two membership function indexes increase first and then decrease as the mixed salt concentration increases. Compared with 5 g/L, which has the highest membership function value, the index under salt concentrations of 3 g/L, 10 g/L, and 15 g/L decreases by 4.7%-11.1%, 25.3%-28.3%, and 41.4%-45.1%, respectively. This study provides theoretical guidance for the breeding of salt-tolerant varieties of pepper and an analysis of the physiological mechanisms involved in salt tolerance and salt-tolerant cultivation.

Analysis of Effect of Compound Salt Stress on Seed Germination and Salt Tolerance Analysis of Pepper Capsicum annuum L.

The paper below presents a protocol for measuring seed germination, seedling growth, and physiological indexes of two pepper varieties with salinity tolerance differences in response to six mixed salt concentrations. This protocol can be used to evaluate the salt tolerance of pepper varieties.

To determine the salt tolerance and physiological mechanism of pepper (Capsicum annuum L.) at the germination stage, the Hongtianhu 101 and Xinxiang 8 ...

Optimized Bioassay for Entomopathogenic Fungi Against Mustard Aphid

Assessment of Aphidicidal Effect of Entomopathogenic Fungi against Parthenogenetic Insect, Mustard Aphid, Lipaphis erysimi (Kalt.)

The mustard aphid (L. erysimi) is a pest that infests various cruciferous crops and transmits plant viruses. To achieve eco-friendly pest management, entomopathogenic fungi (EPF) are potential microbial control agents for controlling this pest. Therefore, virulence screening of EPF isolates under Petri dish conditions is necessary before field application. However, the mustard aphid is a parthenogenetic insect, making it difficult to record data during Petri dish experiments. A modified system for detached-leaf bioassays was developed to address this issue, using a micro-sprayer to inoculate conidia onto aphids and prevent drowning by facilitating air-drying after spore suspension. The system maintained high relative humidity throughout the observation period, and the leaf disc remained fresh for over ten days, allowing parthenogenetic reproduction of the aphids. To prevent offspring buildup, a process of daily removal using a painting brush was implemented. This protocol demonstrates a stable system for evaluating the virulence of EPF isolates against mustard aphids or other aphids, enabling the selection of potential isolates for aphid control.

Author Spotlight: Eco-Friendly Pest Management Using Entomopathogenic Fungi Against Mustard Aphids 

This protocol presents an optimized detached-leaf bioassay system for evaluating the effectiveness of entomopathogenic fungi (EPF) against the mustard aphid (Lipaphis erysimi (Kalt.)), a parthenogenetic insect. The method outlines the data collection process during Petri dish experiments, enabling researchers to consistently measure the virulence of EPF against mustard aphids and other parthenogenetic insects.

The mustard aphid (L. erysimi) is a pest that infests various cruciferous crops and transmits plant viruses. To achieve eco-friendly pest management, ...

An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants

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A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates

Analysis of Effect of Compound Salt Stress on Seed Germination and Salt Tolerance Analysis of Pepper (Capsicum annuum L.)

Assessment of Aphidicidal Effect of Entomopathogenic Fungi against Parthenogenetic Insect, Mustard Aphid, Lipaphis erysimi (Kalt.)