Chinese Bulletin of Botany ›› 2020, Vol. 55 ›› Issue (5): 577-587.DOI: 10.11983/CBB20100
• INVITED PROTOCOLS • Previous Articles Next Articles
Min He1, Junjie Yin1, Zhiming Feng2, Xiaobo Zhu1, Jianhua Zhao2, Shimin Zuo2,*(
), Xuewei Chen1,*()
HomeReceived:2020-05-28
Accepted:2020-07-21
Online:2020-09-01
Published:2020-09-03
Contact:
Shimin Zuo,Xuewei Chen
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[J]. Chinese Bulletin of Botany, 2020, 55(5): 577-587.
| 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 |
| Agar | 20 g |
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 |
| Agar | 20 g |
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).
| 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 |
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 |
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.
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.
| 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 |
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 |
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).
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.
| 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 |
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 |
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.
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.
| 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 |
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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