Assays to Assess Virulence of Xanthomonas axonopodis pv. manihotis on Cassava
木薯中地毯草黄单胞菌毒性的实验评估
(*contributed equally to this work) 发布: 2015年07月05日第5卷第13期 DOI: 10.21769/BioProtoc.1522 浏览次数: 10539
评审: Zhaohui LiuAnonymous reviewer(s)
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The authors used this protocol in:
Nov 2014
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Abstract
Cassava (Manihot esculenta) is a root crop that provides calories for people living in more than 100 tropical and subtropical countries and serves as a raw material for processing into starch and biofuels as well as feed for livestock (Howeler et al., 2013). Xanthomonas axonopodis pv. manihotis (Xam), the causal agent of cassava bacterial blight (CBB), can cause extensive crop damage (reviewed in Lopez et al., 2012; Lozano, 1986). Bacterial movement, growth in planta and the ability to cause disease symptoms are all important measures of bacterial fitness and plant susceptibility to CBB. Here we present a protocol for visualizing the movement of Xam within the plant. We also provide a detailed method of assaying bacterial growth in the cassava leaf midvein, and bacterial growth and disease symptom development in the leaf apoplast. These methods will be important tools for determining Xam strain pathogenicity and for developing cassava varieties that are resistant to CBB.
Keywords: Cassava (木薯)
Xanthomonas axonopodis (地毯草黄单胞菌)
Growth assay (生长测定)
Bacteria (细菌)
Plant disease (植物病害)
Materials and Reagents
- Cassava plants, cultivar TMS60444 (see Note 1)
- Xam strains (grown 48 h on NYGA plates + antibiotic selection)
- MgCl2
- Peptone (Thermo Fisher Scientific, catalog number: BP1420-500 )
- Yeast extract (Thermo Fisher Scientific, catalog number: BP1422-500 )
- Glycerine
- Agar (Thermo Fisher Scientific, catalog number: BP1423-500 )
- NYGA (see Recipes)
Equipment
- 28 °C incubator
- 5 ¾” glass disposable Pasteur pipet (Thermo Fisher Scientific, catalog number: 13-678-20B ), with the tip broken and filed to make a smooth 2 mm diameter tip (Figure 3A)
- 2 in. x 4 in. cardboard covered with Parafilm
- Mini-Beadbeater-96 (Biospec Products, catalog number: 1001 )
- 3 mm glass beads (Thermo Fisher Scientific, catalog number: 11-312A )
- 100 x 15 mm petri dishes
- Spectrophotometer (Pharmacia Biotech Ultrospec 3000 )
- Approximately 0.6 cm2 cork borer
- 1.7 ml Posi-Click tubes (Denville, catalog number: C-2170 )
- Single edge razor blade (Garvey Products Inc., catalog number: 40475 )
- 1 ml needleless syringe (BD, catalog number: 309659 )
- 96 well plate reader for measuring luminescence (PerkinElmer, catalog number: 2104-0010 )
- 96 well OptiPlate, black (PerkinElmer, catalog number: 6005270 )
- Dark room, x-ray imaging cassettes with film, ring stand
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Cohn, M., Shybut, M., Dahlbeck, D. and Staskawicz, B. (2015). Assays to Assess Virulence of Xanthomonas axonopodis pv. manihotis on Cassava . Bio-protocol 5(13): e1522. DOI: 10.21769/BioProtoc.1522.
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分类
微生物学 > 微生物-宿主相互作用 > 细菌
植物科学 > 植物免疫 > 病害生物测定
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