Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Materials

1. Obtain tobacco varieties.
   NOTE: For this experiment "Beinhart1000-1" (a selection of Beinhart 1000) (BH) and "Xiaohuangjin1025" (XHJ) were obtained from the National Medium-term Genbank of the Tobacco Germplasm Resource of China. BH is resistant, whereas XHJ is susceptible to P. nicotianae infection¹⁶. A field isolate of P. nicotianae race 0, which was preserved in the Tobacco Research Institute of the Chinese Acade.......

Discussion

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.......

Materials

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