Improvement of the Micro-chamber Inoculation Method for Determination of Rice Seedling Resistance to Sheath Blight (Rhizoctonia solani)

doi:10.3969/j.issn.1001-7216.2015.01.012

Abstract

Abstract:

The sheath blight resistance in rice belongs to a typical quantitative trait, and most of the rice cultivars show low levels of resistance to the disease with small differences. The inoculation and scoring system for rice sheath blight is an important basis for breeding resistant rice cultivars. The micro-chamber method for determination of rice responses to the sheath blight pathogen Rhizoctonia solani was improved by inoculation to rice seedlings with similar growth vigour under controlled illumination, temperature and humidity conditions in a plant growth chamber. The rice cultivars YSBR1, Teqing, Taijing 394, Nipponbare and Lemont showed high to low levels of sheath blight resistance , which was consistent with the results of field inoculation in adult rice plants. RT-PCR analysis showed that the expression of four rice genes related to disease resistance was induced in inoculated seedlings and adult plants. Based on the height of the pulvinus of the second leaf from the top and the straightened seedling height the sheath blight scores ranged from 2.82 to 8.54 and from 1.20 to 3.39, respectively. The scoring method based on the height of the pulvinus led to similar disease scores for both seedlings and adult plants, and thus is more suitable for the micro-chamber inoculation system.

Key words: rice, sheath blight, Rhizoctonia solani, micro-chamber screening method, disease resistance-related genes

摘要：

水稻对纹枯病的抗性属于典型的数量性状,多数栽培品种的抗病水平较低,并且抗性差异较小。水稻纹枯病接种和抗性评价体系是培育抗病品种的重要基础。利用植物生长箱的控温、控光和控湿条件以及生长势相对一致的水稻秧苗,对苗期纹枯病微室接种技术进行了改进。试验品种苗期的纹枯病抗性从高到低依次为YSBR1、特青、泰粳394、日本晴和Lemont,并且与大田成株期接种鉴定的结果一致。RT-PCR分析显示,苗期和成株期接种纹枯病菌均诱导4个水稻抗病相关基因的表达。通过“叶枕高”和“苗挺高”计算的各品种苗期病级变幅分别为2.82~8.54级和1.20~3.39级。前者与大田成株期的病级变幅一致,因而“叶枕高”病级计算方法更适用于微室接种鉴定体系。

关键词: 水稻, 纹枯病, Rhizoctonia solani, 微室法, 抗病相关基因

CLC Number:

Guo-juan XU, Zheng-jie YUAN, Shi-min ZUO, Xue-biao PAN, Zhi-long WANG, Shao-hong QU. Improvement of the Micro-chamber Inoculation Method for Determination of Rice Seedling Resistance to Sheath Blight (Rhizoctonia solani)[J]. Chinese Journal OF Rice Science, 2015, 29(1): 97-105.

徐国娟, 袁正杰, 左示敏, 潘学彪, 王志龙, 瞿绍洪. 水稻苗期纹枯病抗性鉴定微室接种技术的改良[J]. 中国水稻科学, 2015, 29(1): 97-105.

Figures/Tables 10

Fig. 1. Inoculation of the sheath blight pathogen (R. solani) to rice seedlings in a plant growth chamber using a micro-chamber method. A, R. solani on PDA after three days of culture. B, Rice plants inoculated with R. solani; C, Rice plants inoculated with R. solani grown in a plant growth chamber.

Table 1 Sampling method from Taijing 394 plants infected with R. solani.

接种时间 Time after inoculation/h	取样长度 Length of sampled tissue/cm	取样植株数 Number of sampled plants
0	1	10
0.5	1	10
12	1	9
24	1	9
48	2	6
72	2	6
108	2	6

Fig. 2. Illustration of the height of the pulvinus of the second leaf from the top and the straightened seedling height. H1, The height of the pulvinus of the second leaf from the top; H2, The straightened seedling height.

Table 2 Primers for RT-PCR analysis of rice sheath blight resistance genes.

基因缩写 Gene symbol	参考文献 Reference	基因登录号 Gene ID	引物序列 Primer sequence(5'-3')
Actin	Jain et al.(2006)^[22]	Os03g50885	F:CCCTCCTGAAAGGAAGTACAGTGT
			R:GTCCGAAGAATTAGAAGCATTTCC
E2F-related protein	Magyar(2008)^[23]	Os03g13050	F:ACGTCACGCACGTTCATTAGCA
			R:GACCGAATAAATGGCTGCTGGT
OsPR1b	Agrawal et al.(2000)^[24]	Os01g28450	F:GCGTCTTCATCACATGCAACTA
			R:ACCTGAAACAGAAAGAAACAGAGG
PBZ1	Kim et al.(2011)^[25]	Os12g36880	F:CAAATTCTCGTGGCGTTTGAGTC
			R:CGGCAGCATTCACAATGATTTTC
PDR (pleiotropic drug	Crouzet et al.(2003)^[26]	Os01g42370	F:TGGTGGAGATGGTACTGCTGGA
resistance protein)			R:ATGATGGCAAAGCCAAAGAGGA

Fig. 3. Symptoms of sheath blight disease on five rice cultivars inoculated by micro-chamber method (9 days after inoculation). A, YSBR1, resistant; B, Teqing, moderately resistant; C, Taijing 394, moderately susceptible; D, Nipponbare, susceptible; E, Lemont, highly susceptible.

Table 3 Multiple comparisons of sheath blight scores among five rice cultivars by the disease rating method based on the height of the pulvinus of the second leaf from the top.

品种 Cultivar	平均病级 Average disease score	显著水平 Significance level
品种 Cultivar	平均病级 Average disease score	5%	1%
Lemont	8.54	a	A
日本晴Nipponbare	6.12	b	B
泰粳394Taijing 394	4.52	c	C
特青Teqing	3.48	d	D
YSBR1	2.82	d	D

Table 4 Multiple comparisons of sheath blight scores among rice cultivars by the disease rating method based on the height of straightened seedling.

品种 Cultivar	平均病级 Average disease score	显著水平 Significance level
品种 Cultivar	平均病级 Average disease score	5%	1%
Lemont	3.39	a	A
日本晴Nipponbare	2.82	b	B
泰粳394Taijing 394	2.04	c	C
特青Teqing	1.52	d	D
YSBR1	1.20	d	D

Fig. 4. Expression levels of resistance-related genes in the seedlings of rice cultivar Taijing 394 induced by R.solani inoculation. HPI,Hours post-inoculation.

Table 5 Multiple comparisons of sheath blight scores among four rice cultivars inoculated with R.solani at the adult plant stage in field.

品种 Cultivar	成株期平均病级 Average disease score of adult plants	5%显著水平 5% significance level	1%显著水平 1% significance level	苗期平均病级 Average disease score of seedlings
Lemont	8.09	a	A	8.54
日本晴Nipponbare	7.55	a	A	6.12
泰粳394Taijing 394	4.68	b	B	4.52
YSBR1	2.65	c	C	2.82

Fig. 5. RT-PCR analysis of expression of resistance-related genes in the adult plants of rice cultivars YSBR1 and Lemont inoculated with R.solani in field. Y0, Y6, Y10 and Y20, YSBR1 plants 0 h, 6 h, 10 h and 20 h post-inoculation, respectively; L0, L6, L10 and L20, Lemont plants 0 h, 6 h, 10 h and 20 h post-inoculation, respectively.

References 29

[1]	Lee F N, Rush M C.Rice sheath blight: A major rice disease.Plant Dis, 1983, 67(7): 829-832.
[2]	Pan X B, Rush M C, Sha X Y, et al.Major gene, nonallelic sheath blight resistance from the rice cultivars Jasmine 85 and Teqing.Crop Sci, 1999, 39(2): 338-346.
[3]	Zou J H, Pan X B, Chen Z X, et al.Mapping quantitative trait loci controlling sheath blight resistance in two rice cultivars(Oryza sativa L.). Theor Appl Genet, 2000, 101(4): 569-573.
[4]	Jia Y L, Correa-Victoria F, McClung A, et al. Rapid determination of rice cultivar responses to the sheath blight pathogen Rhizoctonia solani using a micro-chamber screening method.Plant Dis, 2007, 91(5): 485-489.
[5]	Savary S, Castilla N P, Elazegui F A, et al.Direct and indirect effects of nitrogen supply and disease source structure on rice sheath blight spread.Phytopathol, 1995, 85(9): 959-965.
[6]	Lee F N, Moldenhauer K A K, Gibbons J W, et al. Rice blast and sheath blight evaluation results for the 2002 uniform regional rice nursery//Norman R J, Meullenet J F, Molden hayer K A K. Rice Research Studies. Fayetteville, AR: University of Arkansas Agriculture Experiment Station Research, 2002: 73-84.
[7]	Sha X Y, Zhu L H.Resistance of some rice varieties to sheath blight (ShB).Int Rice Res Newsl, 1990, 15(6): 7-8.
[8]	Li Z K, Pinson S R M, Marchetti M A, et al. Characterization of quantitative trait loci (QTLs) in cultivated rice contributing to field resistance to sheath blight (Rhizoctonia solani).Theor Appl Genet, 1995, 91(2): 382-388.
[9]	Pinson S R M, Capdevielle F M, Oard J H. Confirming QTLs and finding additional loci conditioning sheath blight resistance in rice using recombinant inbred lines.Crop Sci, 2005, 45(2): 503-510.
[10]	左示敏,张亚芳,殷跃军,等. 田间水稻纹枯病抗性鉴定体系的确立与完善. 扬州大学学报:农业与生命科学版,2006,27(4):57-61.
[11]	左示敏,王子斌,陈夕军,等. 水稻纹枯病改良新抗源 YSBR1 的抗性评价. 作物学报,2009,35(4):608-614.
[12]	Cartwright R D, Parsons P E, Sutton E A, et al.Disease monitoring and evaluation of rice cultivars on Arkansas farms//Norman R J, Meullenet J F, Molden hayer K A K. Rice Research Studies. Fayetteville, AR:University of Arkansas Agriculture Experiment Station Research, 2002: 219-228.
[13]	王子斌,左示敏,李刚,等. 水稻抗纹枯病苗期快速鉴定技术研究. 植物病理学报,2009,39(2):174-182.
[14]	Jia Y L, Singh P, Eizenga G C, et al.In vitro identification of cultivar responses to rice sheath blight pathogen Rhizoctonia solani//Norman R J, Meullenet J F, Molden hayer K A K. Rice Research Studies. Fayetteville, AR:University of Arkansas Agriculture Experiment Station Research, 2002: 229-236.
[15]	Prasad B, Eizenga G C.Rice sheath blight disease resistance identified in Oryza spp. accessions.Plant Dis, 2008, 92(11): 1503-1509.
[16]	Jia L M, Yan W G, Zhu C S, et al.Allelic analysis of sheath blight resistance with association mapping in rice. PloS One, 2012, 7(3): e32703. (
[17]	Liu G, Jia Y L, Correa-Victoria F J, et al. Mapping quantitative trait loci responsible for resistance to sheath blight in rice.Phytopathol, 2009, 99(9): 1078-1084.
[18]	Yoshida S, Forno D A, Cock J H, et al.2nd edn.Laboratory Manual for physiological studies of rice. Los Bafios, Philippines:International Rice Research Institute, 1972: 57-63.
[19]	潘学彪,陈宗祥. 不同接种调查方法对抗水稻纹枯病遗传研究的影响. 江苏农学院学报,1997,18(3):27-32.
[20]	王子斌,左示敏,李刚,等. 水稻成株期对纹枯病的抗性表现研究. 吉林农业大学学报,2011,33(2):144-150.
[21]	Singh G, Kumar S, Singh P.A quick method to isolate RNA from wheat and other carbohydrate-rich seeds.Plant Mol Biol Rep, 2003, 21(1): 93.
[22]	Jain M, Nijhawan A, Tyagi A K, et al.Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR.Biochem Biophys Res Commun, 2006, 345(2): 646-651.
[23]	Magyar Z.Keeping the balance between proliferation and differentiation by the E2F transcriptional regulatory network is central to plant growth and development//Bögre L, Beemster G T S. Plant Growth Signaling. Springer-Verlag Berlin and Heidelberg GmbH & Co. K, 2008, 10: 89-105.
[24]	Agrawal G K, Rakwal R, Jwa N S.Rice (Oryza sativa L.) OsPR1b gene is phytohormonally regulated in close interaction with light signals.Biochem Biophys Res Commun, 2000, 278(2): 290-298.
[25]	Kim S G, Kim S T, Wang Y M, et al.The RNase activity of rice probenazole-induced protein1 (PBZ1) plays a key role in cell death in plants.Mol Cells, 2011, 31(1): 25-31.
[26]	Crouzet J, Trombik T, Fraysse Å S, et al.Organization and function of the plant pleiotropic drug resistance ABC transporter family.FEBS lett, 2006, 580(4): 1123-1130.
[27]	Rush M C, Hoff B J, Mellrath W O.A uniform disease rating system for rice disease in the United States. Proc 16th Rice Tech Working Group, Lake Charles, Louisana, USA, 1976: 64.
[28]	Eizenga G C, Lee F N, Rutger J N.Screening Oryza species plants for rice sheath blight resistance.Plant Dis, 2002, 86(7): 808-812.
[29]	Wasano K, Oro S, Kido Y.The syringe inoculation method for selecting rice plants resistant to sheath blight, Rhizoctonia solani Kühn.Japan J Trop Agric, 1983, 27(3): 131-134.