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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. 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.
P. nicotianae is destructive to many solanaceous crops. It can cause tobacco "black shank"1, potato foliar and tuber rot2, tomato and sweet pepper crown and root rot3, and Goji collar and root rot4. P. nicotianae can attack all parts of tobacco plants, including the roots, stems, and leaves at any growing stage5. The most common symptom of the disease is the black base of the stalk. The roots are initially visible as water-soaked and then become necrotic, and the leaves show large circular lesions5. T....
1. Materials
4-week-old plants of the resistant variety BH and susceptible variety XHJ were challenged with P. nicotianae using the method presented in this article. The experiment was designed with three replicates, each with 8 plants per group. P. nicotianae infection of the two tobacco varieties, BH and XHJ, is presented in Figure 2. At 3 days post inoculation, for XHJ, stem lesions covered approximately one-half of the stem girth, and one-half of the leaves were slightly wilted; in .......
Multiple resistance sources have been used to improve P. nicotianae resistance in cultivated tobacco. Single dominant R genes, Php and Phl, have been introgressed from Nicotiana plumbaginifolia and Nicotiana longiflora, respectively10. The cigar tobacco variety Beinhart 1000 has the highest reported level of quantitative resistance to P. nicotianae13. Multiple interval mapping experiments have suggested that at least six.......
This research was funded by the National Natural Science Foundation of China (31571738) and the Agricultural Science and Technology Innovation Program of China (ASTIP-TRIC01).
....Name | Company | Catalog Number | Comments |
(NH4)2SO4 | Sinopharm | 10002917 | Analytical Reagent |
(NH4)6 Mo7O24•2 H2O | Sinopharm | XW131067681 | Analytical Reagent |
1.5 ml Safe-lock Microcentrifuge Tubes | Eppendorf | 30120086 | Used for Sample Extarction |
2 ml Safe-lock Microcentrifuge Tubes | Eppendorf | 30120094 | Used for Sample Extarction |
Agar | MDBio, Inc | 9002-18-0 | Materials of Culture Medium |
Analytical Balance | AOHAOSI | AX2202ZH | Equipment |
Autoclave | Yamatuo | SQ510C | Equipment |
Autoclave | YAMATUO | SQ510C | Equipment |
Beaker | Bio Best | DHSB-2L | Materials of Culture Medium |
Biological Incubator | JINGHONG | SHP-250 | Equipment |
Ca(NO3)2•4 H2O | Sinopharm | 80029062 | Analytical Reagent |
CaCl2 | Sinopharm | 10005817 | Analytical Reagent |
CuSO4•5 H2O | Sinopharm | 10008218 | Analytical Reagent |
Electromagnetic Oven | Bio Best | DHDCL | Equipment |
FeSO4•7 H2O | Sinopharm | 10002918 | Analytical Reagent |
Filter Paper | Bio Best | DHLZ-9CM | Material |
Fluorescence Ration PCR Instrument | Roche | LightCycler96 | Equipment |
Gauze | Bio Best | 17071202 | Materials of Culture Medium |
H3BO3 | Phytotechnology | B210-500G | Analytical Reagent |
Hemocytometer | Solarbio | 17072801 | Material for disease-resistant identification |
K2SO4 | Sinopharm | 10017918 | Analytical Reagent |
KNO3 | Sinopharm | 10017218 | Analytical Reagent |
KT Foam Sheet | Bio Best | DHKTB | Material for Seedling |
Low Constant Incubator | Jinghong | SHP-250 | Equipment |
Measuring Cylinder | Bio Best | DHBLLT-1000ML | Materials of Culture Medium |
MgSO4•7 H2O | Sinopharm | 10013080 | Analytical Reagent |
Microscope | ECHO | RVL-100-G | Equipment |
MnCl2•4 H2O | Sinopharm | G5468154 | Analytical Reagent |
Na2-EDTA | Sinopharm | G21410-250 | Analytical Reagent |
NaH2PO4•2 H2O | Sinopharm | 20040717 | Analytical Reagent |
NH4NO3 | Sinopharm | B64586-100g | Analytical Reagent |
Oatmeal | Bio Best | DHYMP-1.5KG | Materials of Culture Medium |
Petri Dish | Bio Best | DHPYM-9CM | Material for disease-resistant identification |
Pipettor | THERMO | S1 | Equipment |
Potting | Bio Best | DHYCXHP-12CM | Material for Seedling |
Potting Soil | Bio Best | DHYMJZ-50L | Seedling Material |
Punch | Bio Best | DHDKW | Material |
qRT-PCR Plate | Monad | MQ50401S | qRT-PCR Plate |
SYBR Green Premix Pro Taq HS qPCR Kit | Accurate Biology | AG11718 | PCR Reagent |
Toothpick | Bio Best | DHYQ-900 | Material |
Total RNA Kit II | Omega | R6934-01 | PCR Reagent |
TransScript® II One-Step gDNA Removal and cDNA Synthesis SuperMix | Transgen | AH311-02 | PCR Reagent |
Trays | Bio Best | DHYMTP-90G | Material for Seedling |
Vermiculite | Bio Best | DHZS | Seedling Material |
Water Purification System | HEAL FORCE | HSE68-2 | Equipment |
ZnSO4•7 H2O | Sinopharm | 10024018 | Analytical Reagent |
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