多主棒孢SdhB-H278R突变位点AS-real-time PCR 定量检测体系的建立

doi:10.3864/j.issn.0578-1752.2018.24.006

Abstract

Abstract:

【Objective】 The objective of this study is to establish a rapid and efficient allele specific real-time PCR (AS-real-time PCR) method for quantitative detection of the H278R mutation in the SdhB associated with Corynespora cassiicola in cucumber, and to verify the effect. 【Method】 A total of 24 single conidial strains of C. cassiicola were isolated from Daxing, Beijing. The method of mycelial growth inhibition was used to determine the EC₅₀ value to boscalid. Mycelium growth rate, spore outputs and pathogenicity of 4 susceptible (S) and 8 resistant (R) strains were measured in vitro. Primer pair Cc-SdhB-F/R was used to detect the sequence of SdhB and the base change of SdhB. Based on the sequencing results in SdhB by Cc-SdhB-F/R primers, specific primer pair B-H278R-2F/2R14 and internal primers B-H278R-TY-F/R were designed. An AS-real-time PCR reaction system was established and optimized. The specificity, sensitivity and melt curve of the system were also evaluated. DNA and spore suspension containing different proportions of H278R mutation were detected with the optimized reaction system, respectively. 【Result】 The sensitive frequency of C. cassiicola to boscalid was 66.67% in 24 strains and the EC₅₀ values ranged from 0.057 to 0.563 μg·mL ^-1. The resistance frequency of C. cassiicola to boscalid was 33.33% and the EC₅₀ values ranged from 5.395 to 11.710 μg·mL ^-1. Resistant and susceptible strains only significantly differed in terms of mycelium growth rate, and there was a significant negative correlation between the rate of mycelial growth and EC₅₀ values, but no significant difference in spore outputs and pathogenicity. Sequencing analysis showed that all the resistant strains carried SdhB-H278R mutation. The primers B-H278R-2F/2R14 were of great specificity, the specific fragment was amplified from the DNA of H278R strains, but not from the DNA of other fungal strains. The sensitivity of ordinary AS-PCR was 91 pg·μL ^-1, while that of AS-real-time PCR was 9.1 pg·μL ^-1, which was 10 times higher than that of ordinary AS-PCR. The standard curve established by AS-real-time PCR showed a fine linear relationship between ΔCT value and lg of H278R frequency, the correlation coefficient of the standard curve was 0.9857 and with high amplification efficiency (92.59%). The absorption peak of melting curve was single. The internal reference primers B-H278R-TY-F/R and the specific primer B-H278R-2F/2R14 had a single specific peak at 87.81℃and 91.62℃, respectively. The result of mixtures DNA and mixtures spore suspension to verify the standard curve showed a fine linear relationship between expected percentage and detected percentage (R ²=0.9998 and R ²=0.9922). As the proportions of H278R mutation in DNA and spore suspension decreased, the accuracy of the system gradually increased, and the detection limit was 5%. 【Conclusion】 An efficient, quantitative, and rapid AS-real-time PCR detection system was established for the detection of SdhB-H278R mutation site, which can provide a theoretical basis for the SDHIs resistance management.

Key words: cucumber, Corynespora cassiicola, AS-real-time PCR, resistance, SdhB, boscalid

SUN BingXue,SHI YanXia,ZHU FaDI,XIE XueWen,CHAI ALi,LI BaoJu. Establishment of AS-real-time PCR for Quantitatively Detecting the H278R Allele in the SdhB Associated with Corynespora cassiicola in Cucumber[J].Scientia Agricultura Sinica, 2018, 51(24): 4647-4658.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2018.24.006

https://www.chinaagrisci.com/EN/Y2018/V51/I24/4647

Figures/Tables 13

Fig. 1

Table 1

Fig. 2

Table 2

Sensitivity of tested strains"

序号 Number	菌株编号 Strain code	采集时间 Collection time	采集地 Collection area	EC₅₀ (μg·mL^-1)	毒力回归方程Toxicity regression equation (y=)	相关系数 Coefficient (R²)	突变类型Mutation type	敏感性 Sensitivity
1	HG17031006-1	2017	北京Beijing	9.261	1.2509x+3.7941	0.9995	+	R
2	HG17031006-2	2017	北京Beijing	5.925	1.1082x+4.1659	0.9788	+	R
3	HG17031006-3	2017	北京Beijing	0.482	0.4848x+5.1535	0.9899	-	S
4	HG17031006-4	2017	北京Beijing	0.227	0.4565x+5.2941	0.9910	-	S
5	HG17031006-5	2017	北京Beijing	5.395	0.6544x+4.4397	0.9276	+	R
6	HG17031007-1	2017	北京Beijing	0.337	0.4846x+5.2290	0.9905	-	S
7	HG17031007-2	2017	北京Beijing	0.057	1.6824x+7.0839	0.9777	-	S
8	HG17031007-3	2017	北京Beijing	0.261	2.1962x+4.9202	0.9905	-	S
9	HG17031007-4	2017	北京Beijing	0.383	0.4912x+5.2047	0.9331	-	S
10	HG17031007-5	2017	北京Beijing	0.221	0.5069x+5.9325	0.9746	-	S
11	HG17031008-1	2017	北京Beijing	7.585	0.9496x+4.1644	0.9998	+	R
12	HG17031008-2	2017	北京Beijing	0.535	1.5481x+5.8387	0.9219	-	S
13	HG17031008-3	2017	北京Beijing	6.089	1.0695x+4.1608	0.9998	+	R
14	HG17031008-4	2017	北京Beijing	7.408	1.0990x+4.0752	0.9961	+	R
15	HG17031008-5	2017	北京Beijing	7.309	1.3476x+3.8337	0.9967	+	R
16	HG17031009-1	2017	北京Beijing	0.495	1.1440x+5.6638	0.9038	-	S
17	HG17031009-2	2017	北京Beijing	0.469	1.4358x+6.3270	0.9929	-	S
18	HG17031009-3	2017	北京Beijing	0.550	1.1084x+5.9661	0.9944	-	S
19	HG17031009-4	2017	北京Beijing	0.484	0.5298x+5.1666	0.9791	-	S
20	HG17031009-5	2017	北京Beijing	11.710	0.6667x+4.2859	0.9154	+	R
21	HG17031010-1	2017	北京Beijing	0.216	0.5158x+5.3436	0.9991	-	S
22	HG17031010-2	2017	北京Beijing	0.563	1.3191x+5.6942	0.9225	-	S
23	HG17031010-4	2017	北京Beijing	0.296	0.6165x+5.3264	0.9897	-	S
24	HG14102524-4	2014	河北Hebei	0.316	2.9056x+3.5592	0.9945	-	S
25	HG17031010-5	2017	北京Beijing	0.274	1.0630x+5.7384	0.9129	-	S
26	HG14102430-1	2014	河北Hebei	22.371	0.9039x+3.7312	0.9652	SdhB-H278Y	R
27	HG15050729-5	2015	辽宁Liaoning	3.954	0.9434x+4.4106	0.9960	SdhB-I280V	R
28	HG14102415-1	2014	河北Hebei	1.111	0.8091x+4.4362	0.9967	SdhB-P199S	R
29	HG15050701-1	2015	辽宁Liaoning	19.526	0.7921x+4.0567	0.9967	SdhC-P73S	R

Table 2

Fig. 3

Table 4

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Table 5

References 35

[1]	李宝聚, 高苇, 石延霞, 谢学文 . 多主棒孢和棒孢叶斑病的研究进展. 植物保护学报, 2012,39(2):171-176.
	LI B J, GAO W, SHI Y X, XIE X W . Progress in researches on Corynespora leaf spot. Acta Phytophylacica Sinica, 2012,39(2):171-176. (in Chinese)
[2]	于淑晶, 王满意, 田芳, 赵卫光, 边强, 李宝聚 . 黄瓜棒孢叶斑病的防治及抗药性研究进展. 农药, 2014,53(1):7-11.
	YU S J, WANG M Y, TIAN F, ZHAO W G, BIAN Q, LI B J . Progress in research on control of cucumber Corynespora leaf spot and fungicide resistance. Agrochemicals, 2014,53(1):7-11. (in Chinese)
[3]	李良孔, 袁善奎, 潘洪玉, 王岩 . 琥珀酸脱氢酶抑制剂类(SDHIs)杀菌剂及其抗性研究进展. 农药, 2011,50(3):165-169.
	LI L K, YUAN S K, PAN H Y, WANG Y . Progress in research on SDHIs fungicides and its resistance. Agrochemicals, 2011,50(3):165-169. (in Chinese)
[4]	颜范勇, 刘冬青, 司马利锋, 石恒, 胡欣 . 新型烟酰胺类杀菌剂——啶酰菌胺. 农药, 2008,47(2):132-135.
	YAN F Y, LIU D Q, SIMA L F, SHI H, HU X . Boscalid, a novel carboxamide aka anilide class of fungicides. Agrochemicals, 2008,47(2):132-135. (in Chinese)
[5]	AVENOT H, MICHAILIDES T J . Resistance to boscalid fungicide in Alternaria alternata isolates from pistachio in California. Plant Disease, 2007,91(10):1345-1350.
[6]	YIN Y N, KIM Y K, XIAO C L . Molecular characterization of boscalid resistance in field isolates of Botrytis cinerea from apple. Phytopathology, 2011,101(8):986-995. doi: 10.1094/PHYTO-01-11-0016 pmid: 21469935
[7]	AVENOT H F, THOMAS A, GITAITIS R D , JR LANGSTON D B,STEVENSON K L . Molecular characterization of boscalid- and penthiopyrad-resistant isolates of Didymella bryoniae and assessment of their sensitivity to fluopyram. Pest Management Science, 2012,68(4):645-651.
[8]	WANG Y, DUAN Y B, WANG J X, ZHOU M G . A new point mutation in the iron-sulfur subunit of succinate dehydrogenase confers resistance to boscalid in Sclerotinia sclerotiorum. Molecular Plant Pathology, 2015,16(7):653-661.
[9]	MIYAMOTO T, ISHII H, SEKO T, KOBORI S, TOMITA Y . Occurrence of Corynespora cassiicola isolates resistant to boscalid on cucumber in Ibaraki Prefecture, Japan. Plant Pathology, 2009,58(6):1144-1151. doi: 10.1111/j.1365-3059.2009.02151.x
[10]	MIYAMOTO T, ISHII H, STAMMLER G, KOCH A, OGAWARA T, TOMITA Y, FOUNTAINE J M, USHIO S, SEKO T, KOBORI S . Distribution and molecular characterization of Corynespora cassiicola isolates resistant to boscalid. Plant Pathology, 2010,59(5):873-881.
[11]	FURUYA S, SUZUKI S, KOBAYASHI H, SAITO S, TAKAYANAGI T . Rapid method for detecting resistance to a QoI fungicide in Plasmopara viticola populations. Pest Management Science, 2010,65(8):840-843.
[12]	AOKI Y, FURUYA S, SUZUKI S . Method for rapid detection of the PvCesA3 gene allele conferring resistance to mandipropamid, a carboxylic acid amide fungicide, in Plasmopara viticola populations. Pest Management Science, 2011,67(12):1557-1561. doi: 10.1002/ps.2214 pmid: 21674751
[13]	李红霞, 周明国 . 用等位基因特异性寡核苷酸(ASO)-PCR快速检测抗多菌灵的油菜菌核病菌. 中国农业科学, 2004,37(9):1396-1399.
	LI H X, ZHOU M G . Rapid identification of carbendazim resistant strains of sclerotinia sclerotiorum using allele-specific oligonucleotide (ASO)-PCR. Siientia Aricutura Sinica, 2004,37(9):1396-1399. (in Chinese)
[14]	MALLIK I, ARABIAT S, PASCHE J S, BOLTON M D, PATEL J S, GUDMESTAD N C . Molecular characterization and detection of mutations associated with resistance to succinate dehydrogenase- inhibiting fungicides in Alternaria solani. Phytopathology, 2014,104(1):40-49. doi: 10.1094/PHYTO-02-13-0041-R pmid: 23901829
[15]	WHEELER I, KENDALL S, BUTTERS J, HOLLOMON D . Detection of benzimidazole resistance in Rhynchosporium secalis using allele-specific oligonucleotide probes. Bulletin OEPP/EPPO Bulletin, 1995,25(1/2):113-116.
[16]	DE MICCOLIS ANGELINI R M, MASIELLO M, ROTOLO C, POLLASTRO S, FARETRA F . Molecular characterization and detection of resistance to succinate dehydrogenase inhibitor fungicides in Botryotinia fuckeliana (Botrytis cinerea). Pest Management Science, 2014,70(12):1884-1893.
[17]	LEHNER M S , JÚNIOR T J P, SILVA R A, VIEIRA R F, SCHNABEL G, MIZUBUTI E S G. Fungicide sensitivity of Sclerotinia sclerotiorum: A thorough assessment using discriminatory dose, EC₅₀, high-resolution melting analysis, and description of new point mutation associated with thiophanate-methyl resistance. Plant Disease, 2015,99(11):1537-1543.
[18]	SAMARAS A, MADESIS P, KARAOGLANIDIS G S. Detection of sdhB gene mutations in SDHI-resistant isolates of Botrytis cinereal using high resolution melting (HRM)analysis.Frontiers in Microbiology , 2016, 7: Article 1815. doi: 10.3389/fmicb.2016.01815 pmid: 27895633
[19]	聂燕钗, 王斌, 赵子琴, 周怀谷 . 等位基因特异性PCR技术及其法医学应用. 法医学杂志, 2014,30(4):282-287.
	NIE Y C, WANG B, ZHAO Z Q, ZHOU H G . Allele-specific PCR and its application in forensic science. Journal of Forensic Medicine, 2014,30(4):282-287. (in Chinese)
[20]	DIXON L J, SCHLUB R L, PERNEZNY K, DATNOFF L E . Host specialization and phylogenetic diversity of Corynespora cassiicola. Phytopathology, 2009,99(9):1015-1027. doi: 10.1094/PHYTO-99-9-1015 pmid: 19671003
[21]	杨苗 . 我国蔬菜棒孢叶斑病病原菌多样性研究[D]. 北京: 中国农业科学院, 2013.
	YANG M . Diversity of pathogen of corynespora leaf spot on vegetables in China[D]. Beijing: Chinese Academy of Agricultural Sciences, 2013. ( in Chinese)
[22]	高苇, 李宝聚, 石延霞, 谢学文 . 河北青县黄瓜棒孢叶斑病病原菌种群分化的研究. 华北农学报, 2011,26(5):9-15.
	GAO W, LI B J, SHI Y X, XIE X W . Population differentiation of Corynespora cassiicola in Qing County, Hebei Province. Acta Agriculturae Boreali-Sinica, 2011,26(5):9-15. (in Chinese)
[23]	李长松, 张眉, 李林, 李凡, 齐军山, 徐作珽, 张博 . 山东省黄瓜棒孢叶斑病(褐斑病)病原菌鉴定和防治 . 中国蔬菜, 2009(18):29-33.
	LI C S, ZHANG M, LI L, LI F, QI J S, XU Z T, ZHANG B . Identification of cucumber target leaf spot (brown spot) pathogen and its control .China Vegetables, 2009(18):29-33. (in Chinese)
[24]	KWON M K, KANG B R, CHO B H, KIM Y C . Occurrence of target leaf spot disease caused by Corynespora cassicola on cucumber in Korea. Plant Pathology, 2010,52(3):424.
[25]	余玲, 刘慧平, 韩巨才, 张宝俊 . 山西省灰霉菌对啶酰菌胺的敏感性测定. 山西农业大学学报(自然科学版), 2012,32(3):232-234.
	YU L, LIU H P, HAN J C, ZHANG B J . Sensitivity of Botrytis cinerea from Shanxi province to boscalid. Journal of Shanxi Agricultural University (Natural Science Edition), 2012,32(3):232-234. (in Chinese)
[26]	VELOUKAS T, LEROCH M, HAHN M, KARAOGLANIDIS G S . Detection and molecular characterization of boscalid-resistant Botrytis cinerea isolates from strawberry. Plant Disease, 2011,95(10):1302-1307. doi: 10.1094/PDIS-04-11-0317
[27]	史晓晶 . 番茄早疫病菌对啶酰菌胺的抗性检测及抗性机理初探[D]. 太谷: 山西农业大学, 2015.
	SHI X J . Study on sensitivity of Alternaria solani to boscalid and resistant mechanisms[D]. Taigu: Shanxi Agricultural University, 2015. ( in Chinese)
[28]	YANG J H, BRANNEN P M, SCHNABEL G . Resistance in Alternaria alternate to SDHI fungicides causes rare disease outbreak in peach orchards. Plant Disease, 2015,99(1):65-70.
[29]	HORSEFIELD R, YANKOVSKAYA V, SEXTON G, WHITTINGHAM W, SHIOMI K, OMURA S, BYRNE B, CECCHINI G, IWATA S . Structural and computational analysis of the quinone-binding site of complex II (succinate-ubiquinone oxidoreductase): a mechanism of electron transfer and proton conduction during ubiquinone reduction. The Journal of Biological Chemistry, 2006,281(11):7309-7316.
[30]	LALÈVE A, GAMET S, WALKER A, DEBIEU D, TOQUIN V, FILLINGER S . Site-directed mutagenesis of the P225, N230 and H272 residues of succinate dehydrogenase subunit B from Botrytis cinerea highlights different roles in enzyme activity and inhibitor binding. Environmental Microbiology, 2014,16(7):2253-2266. doi: 10.1111/1462-2920.12282 pmid: 24119086225230272
[31]	高苇, 李宝聚, 王万立, 郝永娟, 石延霞 . 土壤中黄瓜棒孢叶斑病病原菌实时荧光定量PCR检测技术研究. 华北农学报, 2014,29(2):71-74.
	GAO W, LI B J, WANG W L, HAO Y J, SHI Y X . Detection of Corynespora cassiicola in soil with real-time quantitative PCR. Acta Agriculturae Boreali-Sinica, 2014,29(2):71-74. (in Chinese)
[32]	陈璐 . 黄瓜细菌性角斑病菌和多主棒孢菌PCR检测技术的建立[D]. 北京: 中国农业科学院, 2014.
	CHEN L . PCR-based specific detection of Pseudomonas syrigae pv. lachrymans and Corynesopora cassiicola[D]. Beijing: Chinese Academy of Agricultural Sciences, 2014. ( in Chinese)
[33]	MICHALECKA M, MALINOWSKI T, BRONIAREKNIEMIEC A, BIELENIN A . Real-time PCR assay with SNP-specific primers for the detection of a G143A mutation level in Venturia inaequalis field populations. Journal of Phytopathology, 2011,159(7/8):569-578. doi: 10.1111/j.1439-0434.2011.01805.x
[34]	LUO Y, MA Z H, MICHAILIDES T J . Quantification of allele E198A in beta-tubulin conferring benzimidazole resistance in Monilinia fructicola using real-time PCR. Pest Management Science, 2007,63(12):1178-1184. doi: 10.1002/ps.1425 pmid: 17912681
[35]	KIANIANMOMENI A, SCHWARZ G, FELSENSTEIN F G, WENZEL G . Validation of a real-time PCR for the quantitative estimation of a G143A mutation in the cytochrome bc₁ gene of Pyrenophora teres. Pest Management Science, 2010,63(3):219-224.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

引物名称 Primer name	序列 Sequence	引物长度 Length of primer (bp)	产物长度 Length of product (bp)
Cc-SdhB-F	CACTCTTCTTCGCCATCC	18	1422
Cc-SdhB-R	CATCACACTCACGGTCAC	18	1422
B-H278R-2F	GTGAATACCGCCAGTCCAA	19	244
B-H278R-2R14	CAGTTGAGAATCGCGC	16	244
B-H278R-TY-F	GACCTTTAGGCGAAGTTGC	19	246
B-H278R-TY-R	CTGCTTGTAGAAGAGCGTCAT	21	246

菌株编号 Strain code	敏感性 Sensitivity	菌丝生长速率（11d） Mycelium growth rate (cm·d^-1)	产孢量 Spore outputs (×10⁵/mL)	病斑直径 Lesion diameter (cm)
HG17031007-2	S	0.60±0.09f	0.92±1.38a	0.78±0.09efg
HG17031010-1	S	0.61±0.09f	7.00±4.61e	0.65±0.26de
HG14102524-4	S	0.61±0.01f	3.85±1.99bcd	0.84±0.16fg
HG17031008-2	S	0.49±0.06de	0.33±0.52a	0.38±0.10ab
HG17031006-2	R	0.49±0.04de	5.15±2.41de	0.70±0.26def
HG17031008-4	R	0.45±0.05cd	4.25±2.67bcd	0.75±0.17efg
HG17031006-5	R	0.30±0.03a	2.31±2.06abc	0.56±0.19cd
HG17031008-1	R	0.37±0.02ab	1.33±1.87a	0.45±0.22abc
HG17031008-3	R	0.56±0.08ef	7.46±2.70e	0.91±0.12g
HG17031008-5	R	0.52±0.01de	4.82±2.93cd	0.54±0.05bcd
HG17031006-1	R	0.37±0.02ab	1.85±1.28ab	0.80±0.09efg
HG17031009-5	R	0.40±0.01bc	4.00±2.80bcd	0.33±0.07a

	EC₅₀	生长速率 Growth rate	产孢量 Spore outputs	致病性 Pathogenicity
EC₅₀	1	-0.700*	0.141	-0.21
生长速率Growth rate			0.42	0.457
产孢量Spore outputs				0.51
致病性Pathogenicity				1

预期值Expected percentage (%)		80	40	20	10	5
检测值Detected percentage (%)	DNA	85.95±1.45	44.64±0.69	22.24±0.85	11.86±0.60	6.40±0.24
检测值Detected percentage (%)	孢子Spore	116.45±4.83	48.39±6.15	26.38±4.59	11.86±0.98	6.83±1.43

Establishment of AS-real-time PCR for Quantitatively Detecting the H278R Allele in the SdhB Associated with Corynespora cassiicola in Cucumber

RichHTML

PDF

Abstract

Cite this article

share this article

Figures/Tables 13

References 35

Related Articles 15

Metrics

Comments

Recommended 0

[1]	ZHANG XiaoLi, TAO Wei, GAO GuoQing, CHEN Lei, GUO Hui, ZHANG Hua, TANG MaoYan, LIANG TianFeng. Effects of Direct Seeding Cultivation Method on Growth Stage, Lodging Resistance and Yield Benefit of Double-Cropping Early Rice [J]. Scientia Agricultura Sinica, 2023, 56(2): 249-263.
[2]	LI QingLin,ZHANG WenTao,XU Hui,SUN JingJing. Metabolites Changes of Cucumber Xylem and Phloem Sap Under Low Phosphorus Stress [J]. Scientia Agricultura Sinica, 2022, 55(8): 1617-1629.
[3]	LI GuiXiang,LI XiuHuan,HAO XinChang,LI ZhiWen,LIU Feng,LIU XiLi. Sensitivity of Corynespora cassiicola to Three Common Fungicides and Its Resistance to Fluopyram from Shandong Province [J]. Scientia Agricultura Sinica, 2022, 55(7): 1359-1370.
[4]	LIU Jiao,LIU Chang,CHEN Jin,WANG MianZhi,XIONG WenGuang,ZENG ZhenLing. Distribution Characteristics of Prophage in Multidrug Resistant Escherichia coli as well as Its Induction and Isolation [J]. Scientia Agricultura Sinica, 2022, 55(7): 1469-1478.
[5]	GUO ZeXi,SUN DaYun,QU JunJie,PAN FengYing,LIU LuLu,YIN Ling. The Role of Chalcone Synthase Gene in Grape Resistance to Gray Mold and Downy Mildew [J]. Scientia Agricultura Sinica, 2022, 55(6): 1139-1148.
[6]	YAN LeLe,BU LuLu,NIU Liang,ZENG WenFang,LU ZhenHua,CUI GuoChao,MIAO YuLe,PAN Lei,WANG ZhiQiang. Widely Targeted Metabolomics Analysis of the Effects of Myzus persicae Feeding on Prunus persica Secondary Metabolites [J]. Scientia Agricultura Sinica, 2022, 55(6): 1149-1158.
[7]	WANG Kai,ZHANG HaiLiang,DONG YiXin,CHEN ShaoKan,GUO Gang,LIU Lin,WANG YaChun. Definition and Genetic Parameters Estimation for Health Traits by Using on-Farm Management Data in Dairy Cattle [J]. Scientia Agricultura Sinica, 2022, 55(6): 1227-1240.
[8]	ZHANG YaLing, GAO Qing, ZHAO Yuhan, LIU Rui, FU Zhongju, LI Xue, SUN Yujia, JIN XueHui. Evaluation of Rice Blast Resistance and Genetic Structure Analysis of Rice Germplasm in Heilongjiang Province [J]. Scientia Agricultura Sinica, 2022, 55(4): 625-640.
[9]	WANG MengRui, LIU ShuMei, HOU LiXia, WANG ShiHui, LÜ HongJun, SU XiaoMei. Development of Artificial Inoculation Methodology for Evaluation of Resistance to Fusarium Crown and Root Rot and Screening of Resistance Sources in Tomato [J]. Scientia Agricultura Sinica, 2022, 55(4): 707-718.
[10]	XIANG MiaoLian, WU Fan, LI ShuCheng, WANG YinBao, XIAO LiuHua, PENG WenWen, CHEN JinYin, CHEN Ming. Effects of Melatonin Treatment on Resistance to Black Spot and Postharvest Storage Quality of Pear Fruit [J]. Scientia Agricultura Sinica, 2022, 55(4): 785-795.
[11]	HU ChaoYue, WANG FengTao, LANG XiaoWei, FENG Jing, LI JunKai, LIN RuiMing, YAO XiaoBo. Resistance Analyses on Wheat Stripe Rust Resistance Genes to the Predominant Races of Puccinia striiformis f. sp. tritici in China [J]. Scientia Agricultura Sinica, 2022, 55(3): 491-502.
[12]	TANG ZiYun,HU JianXin,CHEN Jin,LU YiXing,KONG LingLi,DIAO Lu,ZHANG FaFu,XIONG WenGuang,ZENG ZhenLing. Relationship Between Biofilm Formation and Molecular Typing of Staphylococcus aureus from Animal Origin [J]. Scientia Agricultura Sinica, 2022, 55(3): 602-612.
[13]	LI ZhiLing,LI XiangJu,CUI HaiLan,YU HaiYan,CHEN JingChao. Development and Application of ELISA Kit for Detection of EPSPS in Eleusine indica [J]. Scientia Agricultura Sinica, 2022, 55(24): 4851-4862.
[14]	ZHANG Qi,DUAN Yu,SU Yue,JIANG QiQi,WANG ChunQing,BIN Yu,SONG Zhen. Construction and Application of Expression Vector Based on Citrus Leaf Blotch Virus [J]. Scientia Agricultura Sinica, 2022, 55(22): 4398-4407.
[15]	DU JinXia,LI YiSha,LI MeiLin,CHEN WenHan,ZHANG MuQing. Evaluation of Resistance to Leaf Scald Disease in Different Sugarcane Genotypes [J]. Scientia Agricultura Sinica, 2022, 55(21): 4118-4130.