水稻稻瘟病和纹枯病抗性鉴定方法

doi:10.11983/CBB20100

植物学报 ›› 2020, Vol. 55 ›› Issue (5): 577-587.DOI: 10.11983/CBB20100

水稻稻瘟病和纹枯病抗性鉴定方法

贺闽¹, 尹俊杰¹, 冯志明², 朱孝波¹, 赵剑华², 左示敏²^,^*(), 陈学伟¹^,^*()

¹西南作物基因资源发掘与利用国家重点实验室(筹), 水稻研究所, 四川农业大学, 成都 611130
²植物功能基因组学教育部重点实验室/江苏省作物基因组学和分子育种重点实验室, 扬州大学农学院, 扬州 225009;

收稿日期:2020-05-28 接受日期:2020-07-21 出版日期:2020-09-01 发布日期:2020-09-03
通讯作者: 左示敏,陈学伟
作者简介:xwchen88@163.com
*E-mail: smzuo@yzu.edu.cn;
基金资助:
国家自然科学基金(31825022);国家自然科学基金(31871920);国家自然科学基金(31872858);江苏省重点研发计划(现代农业)(BE2019339)

Methods for Evaluation of Rice Resistance to Blast and Sheath Blight Diseases

Min He¹, Junjie Yin¹, Zhiming Feng², Xiaobo Zhu¹, Jianhua Zhao², Shimin Zuo²^,^*(), Xuewei Chen¹^,^*()

¹State Key Laboratory of Exploration and Utilization of Crop Gene Resources in Southwest China, Rice Research Institute, Sichuan Agricultural University, Chengdu 611130, China
²Key Laboratory of Plant Functional Genomics of the Ministry of Education/Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, College of Agriculture, Yangzhou University, Yangzhou 225009, China

Received:2020-05-28 Accepted:2020-07-21 Online:2020-09-01 Published:2020-09-03
Contact: Shimin Zuo,Xuewei Chen

摘要/Abstract

摘要： 水稻(Oryza sativa)是世界上最重要的粮食作物, 但稻瘟病和纹枯病等病害严重危害水稻的产量和品质, 给我国乃至全球粮食安全带来巨大威胁。鉴定水稻抗病资源、克隆抗病基因、揭示抗性机理并在育种中加以利用, 对抵御水稻病害和保障粮食安全具有十分重要的作用。准确评价水稻资源的抗病性, 是开展抗病机理研究和育种生产应用的关键环节。该文详述了水稻幼苗期人工喷雾接种、分蘖期和孕穗期田间注射接种与离体叶片戳伤接种的稻瘟病抗性鉴定方法, 以及水稻分蘖期田间接种、孕穗期温室接种和离体茎秆接种的纹枯病抗性鉴定方法, 以期为同行鉴定水稻资源、开展抗病理论和应用研究提供参考。

关键词: 水稻, 稻瘟病, 纹枯病, 抗病性鉴定

Abstract: Rice is the most important crop in the world. However, rice blast caused by Magnaporthe oryzae and sheath blight caused by Rhizoctonia solani are two of diseases, which threaten both yield and quality of rice most severely. To ensure food security, it is very important to identify disease-resistant rice germplasm, clone disease resistant genes, uncover the molecular basis and apply them in rice breeding program. Accurate evaluation of the disease resistance of rice is fundamental to both uncover disease resistance mechanism and improve resistance in rice breeding. Here, we describe the common methods for evaluating rice blast disease resistance by spraying inoculation of seedlings with M. oryzae, injection inoculation at rice tillering and booting stage, and punch inoculation of detached rice leaves. We also describe the methods for evaluating rice sheath blight disease resistance by field inoculation with R. solani at rice tillering stage, greenhouse inoculation at rice booting stage, and inoculation of rice detached-stems in growth chamber. We believe these methods could provide useful protocols for colleagues who aim to identify rice disease-resistant resources, dissect the underlying molecular mechanism and breed elite rice varieties with improved disease resistance.

Key words: rice, blast, sheath blight, resistance evaluation

贺闽, 尹俊杰, 冯志明, 朱孝波, 赵剑华, 左示敏, 陈学伟. 水稻稻瘟病和纹枯病抗性鉴定方法. 植物学报, 2020, 55(5): 577-587.

Min He, Junjie Yin, Zhiming Feng, Xiaobo Zhu, Jianhua Zhao, Shimin Zuo, Xuewei Chen. Methods for Evaluation of Rice Resistance to Blast and Sheath Blight Diseases. Chinese Bulletin of Botany, 2020, 55(5): 577-587.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB20100

https://www.chinbullbotany.com/CN/Y2020/V55/I5/577

图/表 12

表1 稻瘟病菌和纹枯病菌生长培养所需相关试剂配方

Table 1 Reagent formulation used for culturing Magnaporthe oryzae and Rhizoctonia solani

Solution	Composition	Amount
20× Nitrate salts (1 L)	NaNO₃	120 g
	KCl	10.4 g
	MgSO₄.7H₂O	10.4 g
	KH₂PO₄	30.4 g
Vitamin solution (1 L)	Biotin	0.1 g
	Pyridoxin	0.1 g
	Thiamine	0.1 g
	Riboflavin	0.1 g
	p-aminobenzoic acid	0.1 g
	Nicotinic acid	0.1 g
Trace elements (100 mL)	ZnSO₄.7H₂O	2.2 g
	H₃BO₃	1.1 g
	MnCl₂.4H₂O	0.5 g
	FeSO₄.7H₂O	0.5 g
	CoCl₂.6H₂O	0.17 g
	CuSO₄.5H₂O	0.16 g
	Na₂MoO₄.2H₂O	0.15 g
CM agar medium (1 L, pH6.5)	Glucose	10 g
	Peptone	2 g
	Yeast extract	1 g
	Casamino acids	1 g
	20× Nitrate salts	50 mL
	Vitamin solution	1 mL
	Trace elements	1 mL
	Agar	15 g
Oat tomato agar (1 L)	Oatmeal	40 g, collect the liquid filter after boiling
	Fresh tomato juice	150 mL
	Agar	15 g
PDA medium (1 L)	Potato dextrose broth	24 g
PDA medium (1 L)	Agar	20 g

图1 水稻幼苗期接种稻瘟病菌进行抗性鉴定及典型病斑 (A) 苗床上分小区播种水稻种子; (B) 生长2周的幼苗期水稻; (C) CM平板上的稻瘟病菌菌落; (D) 番茄燕麦产孢平板上生长的稻瘟病菌; (E) 对水稻小区喷雾接种孢悬液; (F) 接种6天后稻瘟病的发病情况; (G) 水稻叶片上产生的典型纺锤形病斑(Bar=1 cm)。

Figure 1 Spraying inoculation at rice seedling stage for evaluation of blast disease resistance and typical rice blast symptom (A) Sowing rice seeds on seedbeds; (B) Rice seedlings grown for two weeks; (C) Magnaporthe oryzae colony grown on CM agar medium; (D) M. oryzae sporulation on oat tomato agar medium; (E) Inoculation of rice seedlings by spraying spore suspension; (F) Development of blast lesions six-day post inoculation; (G) Typical blast lesions formed on rice leaves (Bar=1 cm).

表2 水稻幼苗期叶瘟调查的分级标准

Table 2 Grades of blast severity of rice seedlings

Disease score	Resistance level	State of disease
0	High resistance (HR)	No blast lesion
1	Resistance (R)	Only needle-shaped brown lesion s- pots
2	R	Slightly large brown spots with diameter less than 1 mm
3	Medium re- sistance (MR)	Round or oval gray lesion which shows brown edge and appear diameter between 1-2 mm
4	Medium su- sceptibility (MS)	Typical spindle lesion which has leng- th between 1-2 cm. The lesion is us- ually confined between two veins of a leaf and the lesion area occupies less than 2.0% of a leaf
5	MS	Typical spindle lesion whose area occupies the leaf area between 2.1%- 10.0%
6	Susceptibility (S)	Typical spindle lesion whose area occupies the leaf area between 10.1%- 25.0%
7	S	Typical spindle lesion whose area occupies the leaf area between 25.1%- 50.0%
8	High sus- ceptibility (HS)	Typical spindle lesion whose area occupies the leaf area between 50.1%- 75.0%
9	HS	Typical spindle lesion whose area oc- cupies more than 75.1% of the leaf area

图2 水稻分蘖盛期注射接种稻瘟病菌及叶瘟病状 (A) 分蘖盛期注射接种叶鞘; (B) 叶瘟病斑。

Figure 2 Evaluation of leaf blast disease resistance by injection inoculation at rice tillering stage (A) Inoculation of rice sheath by injection of Magnaporthe oryzae spore suspension at the stage of tillering; (B) Blast disease symptoms in the rice leaf.

图3 水稻孕穗期注射接种稻瘟病菌及穗颈瘟病状 (A) 水稻孕穗期注射接种; (B) 穗颈瘟病状。

Figure 3 Evaluation of neck blast disease resistance by injection inoculation at rice booting stage (A) Inoculation by injection of Magnaporthe oryzae spore suspension at the stage of booting; (B) Blast disease symptoms in the rice spike.

表3 水稻穗颈瘟单穗损失率分级标准

Table 3 Grades for evaluation of single panicle loss by rice blast disease

Disease score	State of disease
0	No disease symptom
1	Blast disease symptom appears in primary or secondary branch of panicle and leads to less than 5.0% yield loss in each spike
3	Blast disease symptom appears in rice rachis or spike neck which leads to 5.1%-20.0% yield loss in each spike
5	Blast disease symptom appears in rice rachis or spike neck which leads to half-shriveled grain and 20.0%-50.0% yield loss in each spike
7	Blast disease symptom appears in rice spike neck which leads to shriveled kernel and 50.0%-70.0% yield loss in each spike
9	Blast disease symptom appears in rice spike neck which leads to more than 70.0% yield loss in each spike

图4 水稻离体叶片的点滴接种及稻瘟病病斑 (A) 采用10 μL移液器枪头对标1.5 cm的刻度戳伤水稻叶片; (B) 戳伤之后漂浮于平皿6-BA溶液中的水稻叶片; (C) 对戳伤叶片进行点滴接种; (D) 点滴接种之后的水稻叶片; (E) 水稻叶片上的病斑(Bar=1 cm)。

Figure 4 Evaluation of blast disease resistance by inoculation of detached rice leaves (A) Punching detached rice leaves using 10 μL pipette tips with 1.5 cm scale; (B) Rice leaves put on the surface of 6-BA solution after punching; (C) Inoculation of punched leaves by spotting spore suspension; (D) Rice leaves inoculated with spore suspension of Magnaporthe oryzae; (E) Blast lesions formed on detached rice leaves (Bar=1 cm).

图5 分蘖期水稻纹枯病的田间木皮物接种鉴定 (A) 0.8 mm厚竖纹木皮; (B) 布满纹枯菌菌丝的木皮接种物(以下简称木皮接种物); (C) 木皮接种物表面菌丝的显微观测; (D) 用镊子将接种物嵌入自上而下第3叶鞘内侧; (E) 水稻分蘖期田间人工接种; (F) 高感水稻品种Lemont接种后11天的病情扩展情况; (G) 高感水稻品种Lemont抽穗后30天的病情扩展情况。

Figure 5 Evaluation of sheath blight disease resistance in field by using wood veneer inoculation method at rice tillering stage (A) Wood veneer with a thickness of 0.8 mm; (B) Wood veneer colonized with mycelia of Rhizoctonia solani (hereafter named wood inoculum); (C) Mycelia of R. solani on wood inoculum observed by micrograph; (D) Wood inoculum was put into inner side of the third leaf sheath from up to down by tweezer; (E) Rice plants in the field at tillering stage used for inoculation; (F) Sheath blight development in the highly susceptible rice variety Lemont at 11 days after inoculation in the field; (G) Disease symptoms of Lemont at 30 days after heading in the field.

表4 田间纹枯病病级的评价标准

Table 4 Rating scales for determining sheath blight disease severity on rice using field inoculation method

Disease sore	Sheath blight severity
0	Plant healthy, no symptoms
1	Lesions mainly restricted to the emerged portion of sixth sheath from top on the culm
1.5	Lesions extending to lower 1/2 of the emerged portion of fifth sheath from top on the culm
2	Lesions extending to upper 1/2 of the emerged portion of fifth sheath from top on the culm
2.5	Lesions extending to lower 1/2 of the emerged portion of fourth sheath from top on the culm
3	Lesions extending to upper 1/2 of the emerged portion of fourth sheath from top on the culm
3.5	Lesions extending to lower 1/2 of the emerged portion of third sheath from top on the culm
4	Lesions extending to upper 1/2 of the emerged portion of third sheath from top on the culm
4.5	Lesions extending to lower 1/2 of the emerged portion of second sheath from top on the culm
5	Lesions extending to upper 1/2 of the emerged portion of second sheath from top on the culm
5.5	Lesions extending to lower 1/4 of the emerged portion of flag leaf sheath from top on the culm
6	Lesions usually coalescing and reaching to lower 1/4 to 1/2 of the emerged portion of flag leaf sheath on the culm
6.5	Lesions usually coalescing and reaching to lower 1/2 to 3/4 of the emerged portion of flag leaf sheath on the culm
7	Lesions usually coalescing and reaching to upper 1/4 of the emerged portion of flag leaf sheath on the culm
7.5	Lesions usually coalescing and reaching to lower 1/2 of flag leaf on the culm, the flag leaf presenting semi-rolling or <50% flag leaf tissues affected
8	Lesions usually coalescing and reaching to upper 1/2 of flag leaf on the culm, more than 50% flag leaf tissues affected
8.5	Panicle rachis and culm with brown streak and becoming light brown, all sheath and leaf tissues dead and drying on the culms, florets in lower 1/3 of panicle often not filling on the culm
9	Panicle rachis and culm dead and becoming dry, severely affected culms lodging, florets in lower 1/3 to 1/2 or more of panicle not filling on the culm

表4 田间纹枯病病级的评价标准

Table 4 Rating scales for determining sheath blight disease severity on rice using field inoculation method

Disease sore	Sheath blight severity
0	Plant healthy, no symptoms
1	Lesions mainly restricted to the emerged portion of sixth sheath from top on the culm
1.5	Lesions extending to lower 1/2 of the emerged portion of fifth sheath from top on the culm
2	Lesions extending to upper 1/2 of the emerged portion of fifth sheath from top on the culm
2.5	Lesions extending to lower 1/2 of the emerged portion of fourth sheath from top on the culm
3	Lesions extending to upper 1/2 of the emerged portion of fourth sheath from top on the culm
3.5	Lesions extending to lower 1/2 of the emerged portion of third sheath from top on the culm
4	Lesions extending to upper 1/2 of the emerged portion of third sheath from top on the culm
4.5	Lesions extending to lower 1/2 of the emerged portion of second sheath from top on the culm
5	Lesions extending to upper 1/2 of the emerged portion of second sheath from top on the culm
5.5	Lesions extending to lower 1/4 of the emerged portion of flag leaf sheath from top on the culm
6	Lesions usually coalescing and reaching to lower 1/4 to 1/2 of the emerged portion of flag leaf sheath on the culm
6.5	Lesions usually coalescing and reaching to lower 1/2 to 3/4 of the emerged portion of flag leaf sheath on the culm
7	Lesions usually coalescing and reaching to upper 1/4 of the emerged portion of flag leaf sheath on the culm
7.5	Lesions usually coalescing and reaching to lower 1/2 of flag leaf on the culm, the flag leaf presenting semi-rolling or <50% flag leaf tissues affected
8	Lesions usually coalescing and reaching to upper 1/2 of flag leaf on the culm, more than 50% flag leaf tissues affected
8.5	Panicle rachis and culm with brown streak and becoming light brown, all sheath and leaf tissues dead and drying on the culms, florets in lower 1/3 of panicle often not filling on the culm
9	Panicle rachis and culm dead and becoming dry, severely affected culms lodging, florets in lower 1/3 to 1/2 or more of panicle not filling on the culm

图6 水稻孕穗期温室木皮物接种的纹枯病抗性鉴定 (A) 生长于花盆中的孕穗期水稻植株; (B) 对孕穗期水稻的叶鞘接种纹枯病菌木皮; (C) 接种后置于温室中的水稻植株; (D) 感病品种接种21天后的纹枯病病状。

Figure 6 Evaluation of sheath blight disease resistance in greenhouse by using wood veneer inoculation method at rice booting stage (A) Rice plants at booting stage growing in flowerpots; (B) Inoculation of rice sheath with Rhizoctonia solani wood inoculum at booting stage; (C) Rice plants growth in greenhouse after inoculation; (D) Disease symptoms of susceptible rice variety at 21 days after inoculation.

图7 水稻离体茎秆纹枯病接种鉴定方法 (A) 接种框架; (B) 使用嵌入法进行木皮接种物接种; (C) 接种后的培养室; (D) 高感品种Lemont接种后3天病斑的扩展情况; (E) Lemont接种7天后病斑的扩展情况。

Figure 7 Methods for evaluation of sheath blight disease resistance by using detached stems of rice (A) Inoculation shelves; (B) Inoculation of detached rice stem by using wood inoculum; (C) Growth chamber containing inoculated plants; (D) Sheath blight disease symptoms of the highly susceptible cultivar Lemont at three days after inoculation; (E) Sheath blight disease symptoms of the highly susceptible cultivar Lemont at seven days after inoculation.

表5 离体茎秆接种鉴定法的纹枯病抗感分类标准

Table 5 Grades of sheath blight resistance levels of rice varieties using detached-stem inoculation method

Relative lesion length (%)	0-0.15	0.16-0.3	0.31-0.45	0.46-0.6	0.61-0.75	0.76-1.0
Resistant level	HR	R	MR	MS	S	HS

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水稻稻瘟病和纹枯病抗性鉴定方法

Methods for Evaluation of Rice Resistance to Blast and Sheath Blight Diseases

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