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Protocol for RYMV Inoculation and Resistance Evaluation in Rice Seedlings
水稻幼苗RYMV接种和抗性评估方法   

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Molecular Plant Microbe Interactions
Dec 2013

Abstract

Rice yellow mottle virus (RYMV), a mechanically transmitted virus that causes serious damage to cultivated rice plants, is endemic to Africa. Varietal selection for resistance is considered to be the most effective and sustainable management strategy. Standardized resistance evaluation procedures are required for the identification and characterization of resistance sources. This paper describes a protocol for mechanical inoculation of rice seedlings with RYMV and two methods of resistance evaluation – one based on a symptom severity index and the other on virus detection through double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA).

Keywords: Rice (水稻), RYMV (RYMV), Inoculation (接种), Resistance (抗性), ELISA (ELISA), Symptoms (症状)

Background

RYMV is a major biotic constraint for rice production in Africa (Séré et al., 2013) and has been reported in most rice-growing countries in Africa and Madagascar. It is not transmissible through seed (Konaté et al., 2001; Allarangaye et al., 2006) but by insect vectors (particularly beetles) and by contact during agricultural operations (Bakker, 1974; Traoré et al., 2006), especially while transplanting seedlings from seedbeds to the field. The virus is highly stable and capable of multiplying at high concentrations in its rice and wild Poaceae (Bakker, 1974) hosts.

Monitoring of RYMV incidence in seedbeds and varietal selection are the most efficient and sustainable ways of managing RYMV. There are two phenotypes of resistance – partial resistance, characterized by a delay in the appearance of symptoms (Albar et al., 1998), and high resistance, characterized by the absence of virus detection using DAS-ELISA (Ndjiondjop et al., 1999). Although partial resistance is widely distributed among Oryza sativa japonica varieties, only a few varieties from the cultivated rice species O. sativa and O. glaberrima express a high level of resistance to RYMV. Three major resistance genes – RYMV1, RYMV2 and RYMV3 – have been reported (Ndjiondjop et al., 1999; Thiémélé et al., 2010; Pidon et al., 2017).

Evaluation of the level of resistance to RYMV in rice varieties or lines and the comparison of the outcomes of different experiments require the use of a standardized protocol. This paper describes such a protocol which is based on DAS-ELISA and symptom severity. Reference accessions (susceptible, partially and highly resistant) have been included in the protocol to enable the drawing of reliable conclusions by comparing the test entry with reference materials. The number of plants tested for each variety or line is, however, not specified as it depends on the genetic material being tested and the objective of the experiment.

Materials and Reagents

  1. Fontainebleau sand (VWR, catalog number: VWRC27460.295 )
  2. Latex gloves
  3. 2 ml microcentrifuge tubes (Dominique DUTSCHER, catalog number: 033297 )
  4. 5 mm-steel beads (Brammer)
  5. 500 ml-wash bottle
  6. Plates 96 wells (VWR, catalog number: 735-0083 )
  7. 10, 20, 200, and 1,000 μl tips
  8. Paper towel
  9. Rice seeds, including control accessions (susceptible, partially and highly resistant)
  10. RYMV-infected rice leaves (fresh, dried or frozen at -20 °C)
  11. Carborundum 0.037 mm used as an abrasive (VWR, catalog number: 22540.298 )
  12. Liquid nitrogen (if possible)
  13. RYMV antibody (wide spectrum polyclonal antibody against purified RYMV virions – prepared as described in N’Guessan et al. (2000) and Afolabi et al. (2009), or DSMZ, catalog number: AS-0732 or RT-0732 )
  14. Alkaline phosphatase-conjugated polyclonal antibody against RYMV (prepared as described in Clark and Adams (1977), or DSMZ, catalog number: RT-0732 )
  15. Skimmed milk powder
  16. Substrate pNPP (para NitroPhenylPhosphate, Sigma-Aldrich, catalog number: N9389 )
  17. Polyvinylpyrrolidone (PVP-40), MW 40000 (VWR, catalog number: 26616.184 )
  18. Potassium phosphate (KH2PO4) (Sigma-Aldrich, catalog number: P0662 )
  19. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
  20. MilliQ water
  21. Sodium carbonate (Na2CO3) (Sigma-Aldrich, catalog number: S7795 )
  22. Sodium hydrogen carbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761 )
  23. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  24. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  25. Tween 20 (Sigma-Aldrich, catalog number: P1379 )
  26. Diethanolamine (Fisher Scientific, catalog number: 10131470 )
  27. Hydrochloric acid (HCl) 10 N
  28. Phosphate inoculation buffer (0.1 M pH 7.2) (see Recipes)
  29. Buffers for ELISA test (see Recipes)
    1. Buffer PBST 10x
    2. Coating buffer pH 9.6
  30. Substrate buffer pH 9.8 (see Recipes)

Equipment

  1. Greenhouse or plant growth chamber
  2. Mortars and pestles
  3. Tissue lyser (QIAGEN, model: TissueLyser II )
  4. 10, 20, 200, and 1,000 μl volume pipettes
  5. 200 μl volume 8-channel pipette
  6. Vortex
  7. pH meter
  8. Microcentrifuge
  9. Microplate reader with 405 nm filter (TECAN, model: Infinite M200 Pro )

Procedure

  1. Plant and virus preparation
    1. Grow rice plants under the following greenhouse conditions during the whole experiment - approximately 12 h of light, 28 ± 1°C (day) and 25 ± 1°C (night), and 75% relative humidity. Perform inoculation at the 2 to 3-leaves development stage (Figure 1), roughly 2 weeks after inoculation (WAI). Apply a suitable fertilizer to the soil for optimal growth.


      Figure 1. Two weeks old IR64 plants, ready for inoculation

    2. Use the following recommended rice varieties as checks: IR64, Bouake 189 or BG90-2 (susceptible O. sativa varieties); Azucena or Moroberekan (partially resistant O. sativa controls); accessions with any of the high resistance genes (RYMV1, RYMV2 or RYMV3; Thiémélé et al., 2010; Orjuela et al., 2013; Pidon et al., 2017) (resistant controls).
    3. Propagate RYMV isolates on any of the susceptible control accessions – it is highly recommended to always use the same accession. Mechanically inoculate the plants (as described below) and harvest the leaves at 3 WAI when symptoms reach the 5 to 7 severity level (the symptoms severity scale is described below). Infected leaves may be kept at -20 °C for several months or years.

  2. Inoculation
    1. Cut the RYMV-infected leaves to be used as the source of inoculum into 0.5-1 cm pieces. Add Fontainebleau sand (about 200 mg to 1 g of leaves). Grind the mixture in a mortar with 0.1 M phosphate inoculation buffer, pH 7.2 (10 ml of buffer to 1 g of leaves) (Recipe 1; see Video 1). A minimum of 100 µl of inoculum per plant is required.

      Video 1. Preparation of RYMV inoculum from infected rice leaves

    2. Add two pinches of carborundum and mix with a pestle.
    3. Using gloves, rub leaves twice with ground inoculum as shown in Video 2. Keep at least 5 plants of the susceptible check variety as a non-inoculated control.
    4. After 5 min, rinse the leaves with water to eliminate the excess carborundum (see Video 2).

      Video 2. Mechanical inoculation of RYMV on rice leaves

  3. Symptoms observation
    1. Observation for symptoms can start between 10 and 15 days after inoculation (DAI), when symptoms first appear on susceptible control plants. The date of appearance of symptoms can be monitored on each plant on a daily basis. Symptoms are observed on newly emerged leaves.
    2. The symptom severity scale on the leaves (Figure 2) is derived from the Standard Evaluation System for Rice (IRRI, 2002) but does not consider a reduction in plant height and delayed flowering:
      1 = Leaves green (no apparent symptoms);
      3 = Leaves green but with sparse dots or streaks;
      5 = Leaves green or pale green with mottling;
      7 = Leaves pale yellow or yellow;
      9 = Leaves turn orange or show necrosis and sometimes die.


      Figure 2. Symptom severity scale on rice leaves

  4. DAS-ELISA
    Harvest samples of the last emerged leaves between 10 and 15 DAI. The samples can be used fresh, rapidly frozen at -20 °C, or dried in a herbarium.
    For sample preparation with tissue lyser (QIAGEN), place 25 mg of fresh material (or 10 mg of dried material) in a 2 ml tube with a metal bead. Put the tubes in liquid nitrogen for 5 min. Grind in tissue lyser for 30 sec at an oscillation frequency of 30 Hz. Add 500 μl of 1x PBST. Vortex vigorously. Alternatively, grind the same quantity of samples with pestle and mortar with 500 µl of 1x PBST and then transfer to a 2 ml tube. Centrifuge for 5 min at 5,200 x g. Keep the samples for up to 24 h at 4 °C or frozen at -20 °C before using them for the ELISA test.
    1. Coating
      Dilute antibodies directed against purified RYMV virions in the coating buffer (Recipe 2) to a concentration of 1 µg/ml IgG or according to the dilution factor recommended by the provider. Add 100 μl of antibodies solution per well, except in the wells at the edges of the plate which should be filled to avoid edge effect. Cover the plate and incubate for 2 h at 37 °C in a moist environment (box with moist paper).
    2. Washing
      Discard the coating solution from the wells. Wash the plate by filling the wells with 1x PBST using a wash bottle and decant the washing buffer immediately (for a quick wash) or after 3 min. Proceed successively to a quick wash and three 3 min-washes. Remove residual buffer on a paper towel (Video 3).

      Video 3. DAS-ELISA plate washing

    3. Blocking
      Add 200 μl of 3% (3 g by 100 ml) skimmed milk in 1x PBST per well. Cover the plate and incubate for 1 h at 37 °C.
    4. Washing
      Discard the solution and wash for 3 min with 1x PBST. Remove residual buffer on a paper towel.
    5. Deposit of antigens
      Add 100 µl of the sample supernatant to the wells. Cover the plate and incubate for 2 h at 37 °C. Always include positive controls (usually the RYMV inoculum) and negative controls (PBST buffer alone and non-inoculated plant) among samples. Two technical replicates of both controls and samples are recommended.
    6. Washing
      Discard the sample solution from the wells and remove residual sample solution on a paper towel to avoid inter-well contamination. Proceed to wash with 1x PBST as described in Step C2 above and remove residual buffer on a paper towel.
    7. Addition of enzyme-conjugated antibodies
      Dilute the antibody against RYMV conjugated with alkaline phosphatase in 1x PBST according to the recommendations of the provider or as determined experimentally according to Hill et al. (1981). Add 100 μl of the conjugated antibodies solution per well. Cover the plates and incubate for 2 h at 37 °C.
    8. Washing
      Proceed to wash as described in Step C2 above.
    9. Addition of substrate
      Dissolve pNPP tablets in the substrate buffer (1 mg/ml; Recipe 3). As pNPP is light-sensitive, be careful to keep it in the dark as much as possible. Add 100 μl of substrate solution per well. Incubate in the dark and at room temperature for 1 h. Read the optical density (OD) at 405 nm on a microplate reader.

Data analysis

  1. Symptom severity
    1. The susceptible variety IR64 generally develops the first symptoms 10 to 15 DAI and the severity index rapidly reaches 7 or 9 on newly emerged leaves. The partially resistant variety Azucena develops symptoms about one week after IR64 does and often reaches a severity index of 5. These two varieties generally show clear mottling. On the contrary, other lines, in particular O. glaberrima accessions, can show less specific symptoms that may be difficult to distinguish from chlorosis caused by different abiotic stresses. Highly resistant varieties, such as Gigante, Tog5681, Tog7291 or Tog5307 (Ndjiondjop et al., 1999; Thiémélé et al., 2010; Orjuela et al., 2013; Pidon et al., 2017), do not develop any symptoms except if a resistance-breaking isolate has been used as the source of inoculum or if resistance-breaking variants appear during the experiment. However, the appearance of resistance-breakdown is generally limited to a few plants in a short-term experiment. Therefore, it is advisable to stop the observation for symptoms 4 WAI to reduce the occurrence of resistance-breakdown cases. Figure 3 shows the symptoms of RYMV on the whole plant and its impact on plant development.


      Figure 3. Symptoms of RYMV on the whole rice plant. A. Inoculated IR64 plant (left) compared with a non-inoculated IR64 control (right) one month after inoculation. B-D. Plants with different resistance levels: highly resistant plant with a symptom severity index of 1 (B); susceptible plant with a symptom severity index of 5 (C); susceptible plant with a symptom severity index of 7 (D).

    2. Contrary to what can be observed in other plant/virus pathosystems (Ouibrahim et al., 2014; Poque et al., 2015), mechanical inoculation of RYMV is highly efficient – all susceptible or partially resistant plants are expected to be infected and symptom severity is expected to be homogenous among plants with the same genotype.
    3. The symptoms severity indices corresponding to different observation dates on the same sample can be combined based on their arithmetic mean or on the Area Under Symptom Progression Curve (AUSPC) calculated as follows:

    4. AUSPC = Σ [(Si +Si+1) x (ti+1 – ti)/2]


      where, Si is the symptom severity index observed at date ti.

  2. DAS-ELISA
    1. After the OD reading, subtract the mean of negative controls (PBST) from all values. Calculate the means of technical replicates. Samples are considered positive when they are superior to 0.1 and twice the mean of non-inoculated controls. Table 1 provides an example of ELISA data analysis.

      Table 1. Example of ELISA data analysis with two repeats per sample and four repeats for the PBST control

      *ODcor corresponds to the OD405nm of the sample minus the mean of OD405nm of the PBST controls.
      **Samples are considered positive if the mean (ODcor) is superior to 0.1 and twice the non-inoculated control (0.002 here).

    2. Highly resistant controls are negative, except in the case of resistance-breakdown, whereas Azucena and IR64 are expected to be positive. Susceptible and partially resistant accessions can be differentiated using semi-quantitative analysis but preliminary tests are necessary to identify a dilution range that maximizes differences between samples.

Notes

  1. RYMV is a viral pathogen infecting rice and wild grasses and its manipulation may be restricted by biosafety concerns. Always manipulate the virus in accordance with your country’s legislation.
  2. Resistance or susceptibility depends on specific interactions between plant and viral genotypes and the choice of resistant or susceptible controls may also depend on your RYMV isolates and the objectives of the experiment. If the highly resistant controls show RYMV symptoms before 15 DAI, your inoculum may contain a resistance-breaking genotype (Hébrard et al., 2006; Traoré et al., 2010; Pinel-Galzi et al., 2017; Pinel-Galzi et al., 2016; Hébrard et al., 2018) and would be inappropriate for assessing the resistance phenotype of your material. In addition, to avoid false interpretations, it is highly recommend to check the presence of the resistance allele in each seed batch, using molecular markers linked to resistance alleles (Thiémélé et al., 2010; Orjuela et al., 2013; Pidon et al., 2017).
  3. Infected leaves to be used as a source of inoculum may be kept at -20 °C for several months or years. However multiplication of the virus on susceptible controls or a preliminary DAS-ELISA test is recommended when the source of inoculum is a leaf sample collected in the field or kept under drying conditions.
  4. Incubation at 37 °C for 1 or 2 h during the DAS-ELISA test can be replaced by incubation overnight at 4 °C, provided the same procedure is used between experiments that have to be compared.
  5. If required, background reactions in DAS-ELISA can be reduced by combining the following procedures: (i) add 2% Polyvinylpyrrolidone (PVP-40) to 1x PBST (sample preparation buffer); (ii) add 2% PVP-40 and 1% skimmed milk to the conjugate dilution solution (1x PBST).

Recipes

  1. Phosphate inoculation buffer (0.1 M pH 7.2)
    Prepare a solution of 0.1 M KH2PO4: 1.36 g in 100 ml of MilliQ water
    Prepare a solution of 0.1 M Na2HPO4: 1.41 g in 100 ml of MilliQ water
    Mix 32 ml of KH2PO4 0.1 M with 68 ml of Na2HPO4 0.1M
    Control pH with a pH meter. It should be pH 7.2
    Store at 4 °C
  2. Buffers for ELISA test
    1. Phosphate Buffer Saline Tween (PBST) concentrated 10x (1 L)
      79.5 g NaCl
      1.9 g KH2PO4
      11.36 g Na2HPO4 anhydrous
      1.93g KCl
      Weigh and dissolve the above chemicals in 900 ml of MilliQ water, pH should be 6.8
      Add 5 ml of Tween 20, Adjust volume to 1 L. Store at room temperature
      For use, dilute 10 times in MilliQ water
    2. Coating buffer pH 9.6 (500 ml)
      0.79 g Na2CO3
      1.425 g NaHCO3
      Weigh and dissolve the above chemicals in 450 ml of MilliQ water, pH should be 9.6
      Adjust the final volume to 500 ml with MilliQ water
      Store at 4 °C
  3. Buffer for substrate (diethanolamine)
    Take 49 ml of diethanolamine, add 400 ml of MilliQ water, mix
    Adjust pH to 9.8 with 10 N HCl
    Complete up to 500 ml with MilliQ water
    Store at 4 °C

Acknowledgments

This protocol was developed from procedures used previously in different laboratories (Fauquet and Thouvenel, 1977; Yassi et al., 1994; Konaté et al., 1997; N'Guessan et al., 2001; Albar et al., 1998). The work was funded by the Global Rice Science Partnership (GRiSP) as a New Frontiers Research program (project MENERGEP). The authors thank Dr. Ajayi for his help in manuscript correction. The authors declare no conflict of interest.

References

  1. Afolabi, S., Akator, S. K., Abo, E. M., Onasanya, A. and Séré, Y. (2009). Production of polyclonal antibodies to various strains of rice yellow mottle virus (RYMV) obtained across different agro-ecological zones in West Africa. Sci Res Essay 4: 306-309.
  2. Albar, L., Lorieux, M., Ahmadi, N., Rimbault, I., Pinel, A., Sy, A. A., Fargette, D. and Ghesquiere, A. (1998). Genetic basis and mapping of the resistance to rice yellow mottle virus. I. QTLs identification and relationship between resistance and plant morphology. Theor Appl Genet 97: 1145-1154.
  3. Allarangaye, M. D., Traoré, O., Traoré, E. V. S., Millogo, R. J. and Konaté, G. (2006). Evidence of non-transmission of Rice yellow mottle virus through seeds of wild host species. J Plant Pathol 88: 307-313.
  4. Bakker, W. (1974). Characterisation and ecological aspects of rice yellow mottle virus in Kenya. Agricultural University, Wageningen, The Netherlands.
  5. Clark, M. F. and Adams, A. N. (1977). Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J Gen Virol 34: 475-483.
  6. Fauquet, C. and Thouvenel, J. (1977). Isolation of the rice yellow mottle virus in Ivory Coast. Plant Disease Report 61: 443-446.
  7. Hébrard, E., Pinel-Galzi, A., Bersoult, A., Siré, C. and Fargette, D. (2006). Emergence of a resistance-breaking isolate of Rice yellow mottle virus during serial inoculations is due to a single substitution in the genome-linked viral protein VPg. J Gen Virol 87(5): 1369-1373.
  8. Hébrard, E., Pinel-Galzi, A., Oludare, A., Poulicard, N., Aribi, J., Fabre, S., Issaka, S., Mariac, C., Dereeper, A., Albar, L., Silue, D. and Fargette, D. (2018). Identification of a hypervirulent pathotype of Rice yellow mottle virus: A threat to genetic resistance deployment in West-Central Africa. Phytopathology 108(2): 299-307.
  9. Hill, J. H., Bryant, G. R. and Durand, D. P. (1981). Detection of plant virus by using purified IgG in ELISA. J Virol Methods 3(1): 27-35.
  10. IRRI (International Rice Research Institute). (2002). Standard evaluation system for rice. Philippines: International Rice Research Institute, Manila, Philippines.
  11. Konaté, G., Sarra, S. and Traoré, O. (2001). Rice yellow mottle virus is seed-borne but not seed transmitted in rice seeds. Eur J Plant Pathol 107: 361-364.
  12. Konaté, G., Traoré, O. and Coulibaly, M. M. (1997). Characterization of rice yellow mottle virus isolates in Sudano-Sahelian areas. Arch Virol 142(6): 1117-1124.
  13. Ndjiondjop, M. N., Albar, L., Fargette, D., Fauquet, C. and Ghesquière, A. (1999). The genetic basis of high resistance to rice yellow mottle virus (RYMV) in cultivars of two cultivated rice species. Plant Disease 83: 931-935.
  14. N’Guessan, P., Pinel, A., Caruana, M. L., Frutos, R., Sy, A., Ghesquière, A. and Fargette, D. (2000). Evidence of the presence of two serotypes of Rice yellow mottle sobemovirus in Côte d’Ivoire. Eur J Plant Pathol 106(2): 167-178.
  15. N’Guessan, P., Pinel, A., Sy, A. A., Ghesquière, A. and Fargette, D. (2001). Distribution, pathogenicity, and interactions of two strains of Rice yellow mottle virus in forested and savanna zones of West Africa. Plant Dis 85: 59-64.
  16. Orjuela, J., Deless, E. F., Kolade, O., Cheron, S., Ghesquiere, A. and Albar, L. (2013). A recessive resistance to Rice yellow mottle virus is associated with a rice homolog of the CPR5 gene, a regulator of active defense mechanisms. Mol Plant Microbe Interact 26(12): 1455-1463.
  17. Ouibrahim, L., Mazier, M., Estevan, J., Pagny, G., Decroocq, V., Desbiez, C., Moretti, A., Gallois, J. L. and Caranta, C. (2014). Cloning of the Arabidopsis rwm1 gene for resistance to Watermelon mosaic virus points to a new function for natural virus resistance genes. Plant J 79(5): 705-716.
  18. Pinel-Galzi, A., Rakotomalala, M., Sangu, E., Sorho, F., Kanyeka, Z., Traoré, O., Sérémé, D., Poulicard, N., Rabenantoandro, Y., Séré, Y., Konaté, G., Ghesquière, A., Hébrard, E. and Fargette, D. (2007). Theme and variations in the evolutionary pathways to virulence of an RNA plant virus species. PLoS Pathog 3(11): e180.
  19. Pinel-Galzi, A., Dubreuil-Tranchant, C., Hebrard, E., Mariac, C., Ghesquiere, A. and Albar, L. (2016). Mutations in Rice yellow mottle virus polyprotein P2a involved in RYMV2 gene resistance breakdown. Front Plant Sci 7: 1779.
  20. Pidon, H., Ghesquiere, A., Cheron, S., Issaka, S., Hebrard, E., Sabot, F., Kolade, O., Silue, D. and Albar, L. (2017). Fine mapping of RYMV3: a new resistance gene to Rice yellow mottle virus from Oryza glaberrima. Theor Appl Genet 130(4): 807-818.
  21. Poque, S., Pagny, G., Ouibrahim, L., Chague, A., Eyquard, J. P., Caballero, M., Candresse, T., Caranta, C., Mariette, S. and Decroocq, V. (2015). Allelic variation at the rpv1 locus controls partial resistance to Plum pox virus infection in Arabidopsis thaliana. BMC Plant Biol 15: 159.
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  24. Traoré, O., Pinel-Galzi, A., Issaka, S., Poulicard, N., Aribi, J., Aké, S., Ghesquiere, A., Séré, Y., Konaté, G., Hébrard, E. and Fargette, D. (2010). The adaptation of Rice yellow mottle virus to the eIF(iso)4G-mediated rice resistance. Virology 408(1): 103-108.
  25. Traoré, O., Traoré, M. D., Fargette, D. and Konaté, G. (2006). Rice seedbeds as a source of primary infection by Rice yellow mottle virus. Eur J Plant Pathol 115: 81-186.
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简介

非洲特有的稻黄斑驳病毒(RYMV)是一种机械传播的病毒,对栽培稻株造成严重损害。 品种抗性选择被认为是最有效和可持续的管理策略。 需要标准化的阻力评估程序来识别和表征阻力源。 本文介绍了利用RYMV机械接种水稻幼苗的方案,以及两种抗性评估方法 - 一种基于症状严重性指数,另一种基于双抗体夹心酶联免疫吸附试验(DAS-ELISA)进行病毒检测。

【背景】RYMV是非洲水稻生产的主要生物限制因素(Séréet al。,2013),在非洲和马达加斯加的大多数水稻生产国都有报道。它不能通过种子传播(Konaté等人,2001; Allarangaye等人,2006),而是通过昆虫载体(特别是甲虫)和农业作业期间的接触传播Bakker,1974;Traoré等人,2006),尤其是在将苗床的苗移植到田间时。该病毒非常稳定,能够在大米和野生禾本科(Bakker,1974)宿主中以高浓度繁殖。

监测苗床RYMV发病率和品种选择是管理RYMV最有效和可持续的方式。存在两种表型的抗性 - 部分抗性,其特征在于症状出现延迟(Albar等人,1998)和高抗性,其特征在于使用DAS-ELISA不存在病毒检测(Ndjiondjop et al。,1999)。虽然部分抗性在水稻品种中广泛分布,但栽培水稻品种仅有少数品种O.水稻和 O。 glaberrima 表示对RYMV的高度抗性。已经报道了三种主要的抗性基因--RYMV1,RYMV2和RYMV3。(Ndjiondjop等人,1999; Thiéméléet al。,2010; Pidon et al。,2017)。

评估水稻品种或品系对RYMV的抗性水平以及不同实验结果的比较需要使用标准化方案。本文描述了这种基于DAS-ELISA和症状严重性的方案。参考材料(易感性,部分耐药性和高度耐药性)已被列入方案中,以便通过比较测试条目和参考材料得出可靠的结论。然而,对于每个品种或品系测试的植物数目没有规定,因为它取决于正在测试的遗传物质和实验的目标。

关键字:水稻, RYMV, 接种, 抗性, ELISA, 症状

材料和试剂

  1. Fontainebleau砂(VWR,目录号:VWRC27460.295)
  2. 乳胶手套
  3. 2 ml微量离心管(Dominique DUTSCHER,目录号:033297)
  4. 5毫米钢珠(Brammer)
  5. 500毫升洗瓶
  6. 96孔板(VWR,目录号:735-0083)
  7. 10,20,200和1,000微升的提示
  8. 纸巾
  9. 水稻种子,包括控制种质(易感,部分和高度抗性)
  10. 感染RYMV的水稻叶片(新鲜,干燥或在-20°C冷冻)
  11. 碳化硅0.037毫米用作磨料(VWR,目录号:22540.298)
  12. 液氮(如果可能的话)
  13. RYMV抗体(针对纯化的RYMV病毒粒子的宽谱多克隆抗体 - 按照N'Guessan等人(2000)和Afolabi等人(2009)或DSMZ ,产品目录号:AS-0732或RT-0732)
  14. 针对RYMV的碱性磷酸酶共轭多克隆抗体(按Clark和Adams(1977)或DSMZ,目录号:RT-0732所述制备)
  15. 脱脂奶粉
  16. 底物pNPP(对硝基苯基磷酸酯,Sigma-Aldrich,目录号:N9389)
  17. 聚乙烯吡咯烷酮(PVP-40),MW 40000(VWR,目录号:26616.184)
  18. 磷酸钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P0662)
  19. 磷酸二氢钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S3264)
  20. MilliQ水
  21. 碳酸钠(Na 2 CO 3)(Sigma-Aldrich,目录号:S7795)
  22. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S5761)
  23. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  24. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  25. 吐温20(Sigma-Aldrich,目录号:P1379)
  26. 二乙醇胺(Fisher Scientific,目录号:10131470)
  27. 盐酸(HCl)10 N
  28. 磷酸盐接种缓冲液(0.1 M pH 7.2)(见食谱)
  29. ELISA检测缓冲液(见食谱)
    1. 缓冲PBST 10x
    2. 包被缓冲液pH9.6
  30. 底物缓冲液pH值9.8(见食谱)

设备

  1. 温室或植物生长室
  2. 灰泥和杵
  3. 组织裂解液(QIAGEN,型号:TissueLyser II)
  4. 10,20,200和1,000μl容量移液器
  5. 200μl容量的8通道移液器
  6. 涡流
  7. pH计
  8. 微量离心机
  9. 带有405纳米过滤器的微孔板读取器(TECAN,型号:Infinite M200 Pro)

程序

  1. 植物和病毒制备
    1. 在整个实验过程中,在下列温室条件下种植水稻 - 大约12小时光照,28±1℃(天)和25±1℃(夜间),以及75%相对湿度。接种2周后(WAI),在2至3叶发育阶段(图1)进行接种。
      在土壤中施用合适的肥料以获得最佳生长。


      图1.两周龄的IR64植株,准备接种

    2. 使用以下推荐的水稻品种作为支票:IR64,Bouake 189或BG90-2(易感的水稻品种); Azucena或Moroberekan(部分抗性苜蓿对照);具有任何高抗性基因的材料(RYMV1,RYMV2,RYMV3,Thiémélé等人,2010; Orjuela等人,2013; Pidon等人,2017)(抗性对照)。
    3. 在任何敏感的对照材料中繁殖RYMV分离株 - 强烈建议始终使用相同的材料。机械地接种植物(如下所述),当症状达到5至7级严重程度(症状严重程度如下所述)时以3 WAI收获叶子。受感染的叶子可能会保存在-20°C几个月或几年。

  2. 接种
    1. 切割RYMV感染的叶子,将其用作0.5-1cm片段的接种物来源。加枫丹白露沙(约200毫克到1克叶)。用0.1M磷酸盐接种缓冲液(pH7.2)(10ml缓冲液至1g叶片)研磨混合物(配方1;参见视频1)。
      每个植物至少需要100μl的接种物。

      视频1
    2. 加两片碳化硅,并用杵混合。
    3. 使用手套,如视频2所示,用地面接种物揉搓两次。保留至少5个敏感支票品种作为未接种的对照。
    4. 5分钟后,用水冲洗叶片以除去多余的碳化硅(见视频2)。

      视频2
  3. 症状观察
    1. 当症状首先出现在敏感对照植物上时,观察症状可在接种后10至15天(DAI)开始。日常出现症状的日期可以在每个工厂进行监测。在新出现的叶子上观察到症状。
    2. 叶片的症状严重程度(图2)源自水稻标准评估系统(IRRI,2002),但不考虑植物高度和延迟开花的降低:
      1 =叶绿色(无明显症状);
      3 =叶子绿色,但有点稀疏或条纹;
      5 =留下绿色或淡绿色斑点;
      7 =叶子呈淡黄色或黄色;
      9 =叶子变成橙色或者表现出坏死,有时会死亡。


      图2.水稻叶片的症状严重程度

  4. DAS-ELISA
    最后收获的样品在10至15天之间出现。样品可以新鲜使用,在-20°C快速冷冻,或在植物标本中干燥。
    用组织裂解器(QIAGEN)进行样品制备时,将25毫克新鲜材料(或10毫克干燥材料)放入带有金属珠的2毫升试管中。将管放入液氮中5分钟。以30Hz的振荡频率在组织裂解器中研磨30秒。加入500μl1x PBST。剧烈旋涡。或者,用500μl1x PBST研磨相同数量的样品,用研杵和研钵研磨,然后转移至2 ml管中。在5,200×gg下离心5分钟。在将样品用于ELISA测试之前,将样品保持在4℃下24小时或在-20℃下冷冻。
    1. 涂料
      将包被缓冲液(配方2)中纯化的RYMV病毒粒子的抗体稀释至浓度为1μg/ ml IgG或按供应商推荐的稀释倍数。每孔加入100μl抗体溶液,除了应填充的板边缘的孔中以避免边缘效应。将盖板盖上盖子,在潮湿的环境(湿纸巾盒)中37°C孵育2小时。
    2. 洗涤
      丢弃孔中的涂层溶液。用1x PBST用洗瓶填充孔板并立即倒出洗涤缓冲液(快速洗涤)或3分钟后洗涤平板。继续进行快速洗涤和3次3分钟洗涤。清除纸巾上残留的缓冲液(视频3)。

      视频3
    3. 阻挡
      每孔加入1μlPBST中的200μl3%(3×100ml)脱脂牛奶。盖上培养板并在37°C孵育1小时。
    4. 洗涤
      弃去溶液,用1x PBST洗3分钟。清除纸巾上的残留缓冲液。
    5. 抗原的保存
      向孔中加入100μl样品上清液。盖上平板并在37℃孵育2小时。在样品中始终包括阳性对照(通常是RYMV接种物)和阴性对照(单独的PBST缓冲液和未接种的植物)。
      两个对照和样本的技术重复
    6. 洗涤
      弃去孔中的样品溶液,并在纸巾上除去残留的样品溶液以避免井间污染。
      按照上述步骤C2所述继续用1x PBST清洗并去除纸巾上残留的缓冲液。
    7. 添加酶结合抗体
      根据提供者的推荐或根据Hill等人(1981)实验确定的在1x PBST中稀释与碱性磷酸酶缀合的RYMV的抗体。每孔加入100μl结合抗体溶液。覆盖平板并在37°C孵育2小时。
    8. 洗涤
      继续按上述步骤C2所述进行清洗。
    9. 添加基材
      将pNPP片剂溶解在底物缓冲液中(1mg / ml;方案3)。由于pNPP是对光敏感的,所以要尽可能小心保持在黑暗中。每孔加入100μl底物溶液。在黑暗中和室温下孵育1小时。
      在酶标仪上读取405 nm处的光密度(OD)。

数据分析

  1. 症状严重程度
    1. 易感品种IR64一般产生10〜15 DAI的第一症状,严重性指数在新出现的叶片上迅速达到7或9。部分抗性品种Azucena在IR64发生后大约一周出现症状,并且通常达到5的严重性指数。这两个品种通常显示清晰的斑点。相反,其他行,尤其是 O。 glaberrima 品种可以显示较少的特异性症状,这些症状可能难以与由非生物胁迫引起的萎黄病区分开来。高抗品种,如Gigante,Tog5681,Tog7291或Tog5307(Ndjiondjop等人,1999;Thiémélé等人,2010; Orjuela等人, / ,2013; Pidon et al。,<2017>),除非使用抗性破坏的分离株作为接种源或在抗性突变株出现实验。然而,在短期实验中,电阻分解的出现通常仅限于少数植物。因此,建议停止对WAI症状的观察以减少耐药性分解病例的发生。图3显示了整株植物的RYMV症状及其对植物发育的影响。


      图3.整个稻株上RYMV的症状A.接种后一个月,接种IR64植株(左)与未接种IR64对照(右)比较。 B-d。具有不同抗性水平的植物:具有症状严重性指数为1(B)的高抗性植物;症状严重程度指数为5(C)的易感植物;症状严重程度指数为7(D)的易感植物。

    2. 与其他植物/病毒病理系统可观察到的情况相反(Ouibrahim等人,2014; Poque等人,2015),RYMV的机械接种是高效的 - 预计所有易感或部分抗性植物都会受到感染,预计症状严重程度在具有相同基因型的植物中是同质的。
    3. 可以根据相同样本的算术平均值或症状进展曲线下面积(AUSPC)计算相应样本上不同观察日期的症状严重程度指数,计算公式如下:

    4. AUSPC =Σ[(S 1 + S 1 + 1))×(t i + 1 -t i) )/ 2]


      其中,S 是在日期t i 处观察到的症状严重性指数。

  2. DAS-ELISA
    1. OD读数后,从所有值中减去阴性对照(PBST)的平均值。计算技术复制的手段。当样品优于0.1时,样品被认为是阳性的,两倍于未接种对照的平均值。表1提供了ELISA数据分析的一个例子。

      表1.每个样品两次重复和PBST对照重复四次的ELISA数据分析实例

      * OD_cor对应于样品的OD 405nm减去PBST对照的OD 405nm的平均值。
      **如果平均值(OD )优于0.1和两倍于未接种的对照(此处为0.002),则认为样品为阳性。

    2. 高度抵抗的控制是负面的,除了阻力分解的情况下,而Azucena和IR64预计是积极的。使用半定量分析可以区分敏感和部分抗性种质,但需要进行初步测试以确定最大化样品间差异的稀释范围。

笔记

  1. RYMV是感染水稻和野生草的病毒病原体,其操作可能受到生物安全问题的限制。
    根据你所在国家的法律来操纵病毒
  2. 抗性或易感性取决于植物和病毒基因型之间的特定相互作用,抗性或易感对照的选择也可能取决于您的RYMV分离株和实验目标。如果高度耐药的对照在15日龄之前显示出RYMV症状,那么您的接种物可能含有抗性突变基因型(Hébrard et al。2006;Traoré et al。,2010; Pinel-Galzi等人,2017; Pinel-Galzi等人,2016;Hébrard等人,<2018年>),并且将是不适当的用于评估您材料的抗性表型。此外,为了避免错误解释,强烈建议使用与抗性等位基因相关的分子标记来检查每批种子中的抗性等位基因的存在(Thiémélé et al。,2010; Orjuela et al。,2013; Pidon et al。,2017)。
  3. 被感染的叶片可用作接种源,可保存在-20°C数月或数年。但是,当接种源是在田间采集的叶子样品或保持在干燥条件下时,推荐在敏感对照或初步DAS-ELISA测试中增殖病毒。
  4. 在DAS-ELISA试验过程中,在37°C孵育1或2小时,可以在4°C温育过夜,前提是在必须进行比较的实验之间使用相同的程序。
  5. 如果需要,可以通过结合以下步骤来减少DAS-ELISA中的背景反应:(i)将2%聚乙烯吡咯烷酮(PVP-40)加入到1x PBST(样品制备缓冲液)中; (ii)将2%PVP-40和1%脱脂牛奶加入共轭稀释溶液(1×PBST)中。

食谱

  1. 磷酸盐接种缓冲液(0.1 M pH 7.2)
    准备0.1M KH 2 PO 4:1.36g在100ml MilliQ水中的溶液。
    准备0.1M Na 2 HPO 4:1.41g在100ml MilliQ水中的溶液。
    将32ml KH 2 PO 4 0.1M与68ml Na 2 HPO 4 0.1M混合, 用pH计控制pH值。它应该是pH值为7.2
    在4°C储存
  2. 用于ELISA测试的缓冲液

    1. 磷酸盐缓冲盐水吐温(PBST)浓缩10倍(1升) 79.5克NaCl
      1.9克KH 2 PO 4 4克/升 11.36克Na 2 HPO 4无水
      1.93克KCl
      称量并溶解900毫升MilliQ水中的上述化学物质,pH值应为6.8
      加入5毫升吐温20,调节体积至1升。在室温下保存
      使用时,用MilliQ水稀释10倍。
    2. 涂层缓冲液pH 9.6(500毫升)
      0.79克Na 2 CO 3 3 1.425g NaHCO 3 /
      称量并溶解450毫升MilliQ水中的上述化学物质,pH应为9.6
      用MilliQ水将终体积调整至500 ml
      在4°C储存
  3. 底物缓冲液(二乙醇胺)
    取49毫升二乙醇胺,加入400毫升MilliQ水,拌匀。

    用10 N HCl调pH至9.8 用MilliQ水完成高达500毫升
    在4°C储存

致谢

该协议是从先前在不同实验室中使用的程序开发的(Fauquet和Thouvenel,1977; Yassi等人,1994;Konaté等人,1997; N'Guessan等人,等人,2001; Albar等人,1998)。这项工作由全球水稻科学伙伴关系(GRiSP)资助,作为新前沿研究计划(MENERGEP项目)。作者感谢Ajayi博士在手稿更正方面的帮助。作者宣称没有利益冲突。

参考

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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Pinel-Galzi, A., Hébrard, E., Traoré, O., Silué, D. and Albar, L. (2018). Protocol for RYMV Inoculation and Resistance Evaluation in Rice Seedlings. Bio-protocol 8(11): e2863. DOI: 10.21769/BioProtoc.2863.
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