一种可靠的分离疫霉属水霉、产生孢子囊和游动孢子以及感染木豆植株的实验方法

Mamta Sharma; Raju Ghosh

doi:10.21769/BioProtoc.1706

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A Reliable Method for Phytophthora cajani Isolation, Sporangia, Zoospore Production and in Planta Infection of Pigeonpea

一种可靠的分离疫霉属水霉、产生孢子囊和游动孢子以及感染木豆植株的实验方法

Mamta Sharma email

Raju Ghosh

发布: 2016年01月20日第6卷第2期 DOI: 10.21769/BioProtoc.1706 浏览次数: 12101

评审: Samik BhattacharyaShahin S. AliAnonymous reviewer(s)

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Abstract

Pigeonpea (Cajanus cajan L.) is an important legume crop of rainfed agriculture. High levels of protein in pigeonpea make it a valuable protein source for developing countries. Phytophthora blight caused by Phytophthora cajani (P. cajani) is a potential threat to pigeonpea (Cajanus cajan L.) production, affecting the crop irrespective of cropping system, cultivar grown and soil types (Pande et al., 2011; Sharma et al., 2006). The primary mode of infection of P. cajani is sporangium and zoospore. Therefore, sensitive and reliable methods for zoospore production and estimating infection severity are desirable in case of Phytophthora blight of pigeonpea (Sharma et al., 2015). Here we present a protocol for isolation of P. cajani from infected plants, sporangia and zoospore production and in planta infection technique of pigeonpea seedlings. These methods will be important tool to devise a platform for rapid and reliable screening against Phytophthora blight disease of pigeonpea as well as for host x pathogen x environment interaction studies.

Keywords: Phyophthora cajani (phyophthora cajani)

Pigeonpea (木豆)

Phytophthora stem blight (大豆茎枯病)

Inoculation technique (接种技术)

Materials and Reagents

Cotton (Jaycot Industries)
Glass slide (75 x 25 x 1.35 mm) (Blue star)
Parafilm (Sigma-Aldrich, catalog number: P7793 )
Petri dish (100 x 19.5 mm) (Borocil, catalog number: 5550300 )
Phytophthora cajani isolate ICPC 1 (NCBI, GenBank Acc, catalog number: 10534 )
Pigeonpea seedlings, cultivar ICP 7119
Agar (HiMedia Laboratories, catalog number: RM201 )
Ampicillin (Srlchem, catalog number: 61314 )
Calcium carbonate (CaCO₃) (HiMedia Laboratories, catalog number: GMR 397 )
Dextrose (HiMedia Laboratories, catalog number: GRM077 )
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 472301 )
Ethanol
Pentachloronitrobenzene (PCNB) (Purity: ≥ 94%) (Sigma-Aldrich, catalog number: P2205 )
Pimaricin (Sigma-Aldrich, catalog number: P9703 )
Rifampicin (Sigma-Aldrich, catalog number: R7382 )
Sodium hypochlorite (Scribd Inc., Qualigens, catalog number: 27905 )
Sterilized pond water
Sterilized water
Sterilized river sand
V8 Agar media (HiMedia Laboratories, catalog number: M638 )
Vermiculite (locally available)
Tomato juice
Tomato juice agar (see Recipes)
V8 juice agar (see Recipes)
PARP solutions (see Recipes)
Tomato broth (see Recipes)

Equipment

Autoclave (Tomy Seiko)
Beaker (100 and 400 ml) (Borocil, catalog number: 1000D16 and 1000D23 )
Conical flask (50 ml) (Borocil, catalog number: 4060012 )
Cork borer (approximately 5 mm diameter)
Greenhouse (25-28 °C)
Haemocytometer (1/400 mm² and 1/10 mm deep) (Sigma-Aldrich)
Hot air oven (Thermo Fisher Scientific)
Incubator (Temperature range: 5-60 °C, humidity: 0-100%) (Tomy Percival)
Inoculating needle
Horizontal laminar flow clean benches (Esco Micro Pte, Labculture, model: LHC-4C )
Light microscope (OLYMPUS)
Measuring cylinder (100 ml) (Borocil, catalog number: 3024016 )
Plastic tray (30 x 48 x 10 cm)
Test tube (18 x 150 mm) (Borocil, catalog number: 9820U06 )

Procedure

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如何引用

Sharma, M. and Ghosh, R. (2016). A Reliable Method for Phytophthora cajani Isolation, Sporangia, Zoospore Production and in Planta Infection of Pigeonpea. Bio-protocol 6(2): e1706. DOI: 10.21769/BioProtoc.1706.