CHAPTER 6: PHENOTYPIC AND GENOTYPIC CHARACTERISATION OF AN O. SATIVA
6.2 M ATERIAL AND METHODS
6.2.1 Virus multiplication and inoculation:
Four RYMV isolates were used in this study: from Burkina Faso (BF1) and Niger (Ng117b, Ng122 and Ng144). The three isolates from Niger were from a collection of isolates maintained at the Plant Pathology Unit of AfricaRice, selected for their interaction with different resistance genes and alleles (Séré Yacouba, Personal Communication). BF1 is an aggressive RYMV S2 strain previously employed in the characterisation of the QTL7 and QTL12 in Azucena (Albar et al., 1998; Pressoir et al., 1998; Ahmadi et al., 2001). The isolates Ng117b, Ng122 and Ng144 were multiplied on the standard susceptible variety IR64 during a two week period. The BF1 isolate provided by the ―Institut de Recherche Développement‖ (IRD) - France had already been multiplied in variety IR64 in 2006 and stored at -80°C in liquid nitrogen. Mechanical inoculation was performed with infected leaf samples ground in phosphate buffer, pH 7.2 (10 ml g-1 of leaf sample). Carborundum (600 mesh) was added to the extracts as an abrasive agent. Mechanical inoculation was carried out by rubbing the extracted sap on the upper and lower leaf surfaces of two week-old plantsby finger-dipping in the inoculum.
6.2.2 Resistance evaluations with the isolates Ng117b, Ng122 and Ng144 (Experiment One)
Experiment One was performed under greenhouse conditions at the AfricaRice Research Station (Cotonou, Benin) in 2009. Resistance of the accession BM24 was evaluated using three isolates (Ng117b, Ng122 and Ng144). The experiment was conducted in the presence of four checks: Gigante and Tog5681 carrying the Rymv1-2 and the Rymv1-3 recessive resistant alleles, respectively, the highly susceptible variety IR64, and the partially resistant variety Azucena. The experimental design was a split-plot with three replicates. The main plots were the four treatments (the three isolates Ng117b, Ng122 and Ng144, and a non-inoculated control) and the sub-plots were the different accessions. The elementary plot was an individual plant in a plastic pot of five litres. The disease scores were monitored every week from 14 days post-inoculation (dpi) until 49 dpi and the plant height measured at 49 dpi to estimate the variation in height. The percentage of plant height reduction was calculated using the formula:
122 The disease notation used was as described in the International Rice Research Institute (IRRI) Standard Evaluation System (IRRI, 2002) for RYMV symptom severity scale from 1 to 9.
Accordingly, accessions were allotted in five classes: Score 1: no symptom observed (Highly Resistant or HR); score 3: green leaves with sparse dots or streaks (Moderately Resistant or MR); score 5: general mottling on the leaves and 6% to 25% of reduction of plant height (Moderately Susceptible or MS); score 7: yellowing and stunting (Susceptible or S) and score 9 for necrosis to plant death (Highly Susceptible or HS).
Analysis of variance was performed on plant heights of the four treatments. The model was defined as yijk = µ + ai + tj + rk + a.tij + Ɛ1 + Ɛ2, where yijk is the plant height for accession i of treatment j in replication k, µ is the mean effect, ai is the effect of the accession i, tj the effect of the treatment j, rk the effect of the replication k, a.tij the interaction between accession i and treatment j, Ɛ1 is the main plot error term, and Ɛ2 the subplot error term. As disease scores were measured at different times, a repeated measure model was adopted for the analysis of variance. The model used was as follows: yijkn = µ + ai + tj + rk + Tn + a.tij + a.Tin + t.Tjn + a.t.Tijn + Ɛ1 + Ɛ2, where yijkn is the disease reaction (disease score) for accession i of treatment j in replication k at time n, µ is the mean effect, ai is the effect of the accession i, tj
the effect of the treatment j, rk the effect of the replication k, Tn the effect of the time n, a.tij
the interaction between accession i and treatment j, a.Tin the interaction between accession i and time n, t.Tjn the interaction between treatment j and time n, a.t.Tijn the interaction between accession i, and treatment j, and time n, Ɛ1 is the main plot error term, and Ɛ2 the subplot error term.
6.2.3 Evaluation of resistance to isolate BF1 (Experiment Two)
The experiment was performed at ―Institut de Recherche Développement‖, Montpellier, France in a glasshouse under controlled conditions (28 to 32°C, 12 h of light per 24 h and 80- 90% relative humidity). The accession BM24 was evaluated with one O. sativa accession (HB18B) from a Burkina Faso collection, seven O. sativa accession from the ―Centre de Coopération International en Recherche Agricole pour le Développement‖ (CIRAD) Montpellier, France rice collection of the Mini Gene Bank (MiniGB) and three check varieties (IR64, CG14, and Azucena) against the isolate BF1. The seven accessions of the MiniGB were composed of three O. s. indica (ASD1, CO18 and PTB9), three O. s. japonica (Pagaiyahan, Jumali and Malapkit-Pirurutong) and one accession belonging to the group V of
123 Glaszmann (ARC13829). In particular, Jumali is an admixture between aromatic and temperate O. japonica, while Pagaiyahan is an admixture between tropical japonica and indica (Garris et al., 2005).
Twenty eight plants of each accession were sown in a tray, with 14 plants in two trays. Each tray included two different accessions allocated randomly. The seed trays were replicated twice in the two treatments (the control non-infected and the infected) and arranged in a completely randomised design. The last leaf of infected plants was mechanically inoculated two weeks after sowing with BF1. Such an aggressive isolate was selected to maximise differences in response to infection between resistant and susceptible cultivars. The following parameters: disease scores (1; 3; 5; 7 and 9), and leaf number were monitored at 4; 7; 11; 14 and 21 dpi while the plant heights were measured at 7, 14, and 21 dpi with aim to compare height differences between accessions at early stages. The Area Under Symptoms Progression Curve (AUSPC) was calculated as:
to measure disease progress. Si and S(i+1) correspond to the symptom scores at time Ti and T(i+1), respectively, and n is the total number of observations (Boisnard et al., 2007). The ANOVAs of plant height, number of leaves, and disease score data were implemented and the interaction effects of accession x treatment were primarily considered. A t test was used for means comparison between inoculated accessions and their respective control.
At 14 dpi the last leaf of each individual plant was collected to evaluate virus content through Enzyme-Linked Immunosorbent Assay (ELISA). ELISA tests were performed as described in Ndjiondjop et al. (1999). An ELISA response of the inoculated and systemically infected leaves was measured by direct Double Antibody Sandwich (DAS)-ELISA. Plates were coated with 1:1,000 dilution of the polyclonal antiserum in a carbonate buffer (0.015 M Na2CO3, 0.034 M NaHCO3, pH 9.6). After incubation for 2 h at 37°C, wells were saturated with 200 μl of 3% skimmed milk in PBS-T buffer (pH 7.4) for 1 h at 37°C. Plates were washed three times with PBS-T buffer after each step. Twenty millimetres of infected leaves were ground in PBS-T buffer at 1:1000 or 1:5,000 dilutions. Samples of 100 μl were incubated for 2 h at 37°C in a microtiter plate, and two replicated wells were used to score Optical Density (OD) values. After 2 h incubation at 4°C, 100 μl of a 1 mg ml-1 solution of p-nitrophenyl phosphate
124 in diethanolamine (pH 9.8) was added to each well, and plates were incubated for 3 h at 37°C.
The ELISA responses were expressed by scoring the optical densities measured at 405 nm. In all experiments, the non-inoculated variety IR64 and the PBS-T were used as a negative control, whereas the leaves of RYMV infected IR64 was used as positive control.
The leaves collected at 14 dpi were also used for DNA extraction in order to compare allelic profile in locus RM101 (Chromosome 12). The DNA was extracted as described by Edwards et al. (1991). The PCR amplification was performed in a 96 well thermocycler (Tgradient, Biometra) on 5 ng of DNA in a 15 µl final volume of buffer (10 mM Tris-HCl pH 8, 100 mM KCl, 0.05% w/v gelatin, and 2.0 mM MgCl2) containing 0.1 µM of reverse primer RM101, 0.08 µM of forward primer RM101, 200 µM of dNTP, and 0.1 U of Taq DNA polymerase.
The PCR program was: initial denaturation at 94°C for 4 min; 35 cycles of 94°C for 60 s, hybridisation temperature 55°C for 60 s, and 72°C for 60 s; and a final elongation step at 72°C for 8 min. The products of amplification were revealed on 2% agarose gel.