Chapter 5: Resistance to Rice yellow mottle virus (RYMV) in a rice germplasm collection from
5.3 Material and Methods
5.3.1. Plant materials and inoculations
The plant material consisted of 175 accessions from Niger, 52 accessions from Mali, 2 resistant checks (Gigante and TOG5681) and 2 susceptible checks (IR64 and Bouaké189). The 175 accessions from Niger were randomly selected among the collections made in the country early in 2008. This collection included 139 traditional landraces, as well as modern O. sativa varieties, 32 traditional O. glaberrima landraces, and 4 wild O. barthii accessions.
RYMV isolates were multiplied by mechanical inoculation onto the susceptible variety IR64 for wild types of the virus, or varieties Gigante and TOG5681 for RB-isolates, as described by Ndjiondjop et al. (1999). Infected frozen leaves sourced from the Pathology Unit of AfricaRice were ground in a phosphate buffer (0.1 M KH2PO4, 0.1 M Na2HPO4, pH 7.2) using a ratio of 1:10 (w:v). The mixture, with carborundum dust (600 mesh) added as an abrasive, was used directly to inoculate two week old seedlings of the susceptible varieties by finger-rubbing young leaves with the inoculum. Two weeks later, infected leaves were harvested and used to prepare the inoculum needed for the different trials.
Four disease evaluation trials were conducted under screenhouse conditions of which 3 trials were conducted at AfricaRice in Cotonou, Benin and one trial at IRD, Montpellier, France. The first trial involved 227 accessions (175 accessions from Niger and 52 accessions from Mali), plus a susceptible (IR64) and resistant (Gigante) check. Three plants per accession were inoculated, with the two checks grown between every 20 accessions. The RYMV isolate B27 that belongs to the S1 group (Thiémélé et al., 2010) was used to inoculate the plants in Trial 1. Trial 2 had three
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experiments and was conducted on 155 accessions from Niger (the worst 20 accessions from Trial 1 were discarded), plus the 2 susceptible and 2 resistant checks described above. However, one accessions did not germinate, thus data were recorded and analysed for 154 accessions.
Accessions were randomly assigned to five blocks, arranged into an Augmented Design, with three pots per accession per experiment and three plants per pots. In Experiment One, all plants were inoculated with the RYMV isolate Ng122; in Experiment Two plants were inoculated with Isolate Ng144; Experiment Three was run as a negative control and all plants were inoculated with distilled water and carborundum dust. The two virus strains have been reported to induce different symptoms on the susceptible IR64 and resistant TOG5681 and Gigante checks.
In Trial 3, a subset of 24 resistant accessions (eight were O. sativa) that were the most resistant in the previous trials were screened against three RYMV isolates (Ng117b, Ng122 and Ng144) from Niger using a split plot design, with virus isolates as the main plots and the genotypes as subplots. Three replications, of three pot each, each containing three plants, were used for each virus strain. A fourth replication was only inoculated with distilled water and carborundum.
In Trial 4, a subset of 25 accessions (of which only one was O. sativa) that were also the most resistant in the previous trials were also evaluated at IRD, Montpellier,France using the RYMV isolate BF1, sourced from Burkina Faso. Only 8 accessions were common between Trial 3 and Trial 4. They were selected because they were resistant to Isolate B27 in Trial 1, and Isolates Ng122 and Ng144 in Trial 2. For each accession ten plants were inoculated. Varieties IR64 and Gigante were grown between every seven accessions, as a susceptible and resistant checks, respectively.
5.3.2. Data collection and statistical analysis
For all the experiments, a visual evaluation of RYMV symptoms was made using the Standard Evaluation System of IRRI (2002) with a 1-9 visual scale: 1 for no symptoms and plants considered as highly resistant (HR); 3 for sparse dots or streaks, considered as partially resistant (PR); 5 for visible mottling on green to light-green leaves, considered as Intermediate (I); 7 for generalised yellowing and stunting, considered as susceptible (S); 9 for necrosis, and plant death, considered as highly susceptible (HS) (Konate et al., 1997). Depending on the trial, the visual rating was recorded every week from 14 to 42 or 49 days after inoculation (DAI).
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In Trials 1,2 and 3 the chlorophyll content of leaves (ChcL) was measured, with 10 points per accession, either at 42 DAI (Trial 1 and 2), or every week from 14 to 42 DAI (Trial 3), with a SPAD 502 chlorophyll Meter (Minolta C. Co. Osaka, Japan) (Esfahani et al., 2008). Likewise, Plant height (PHgt) was also recorded on 3 plants per accession at 42 DAI in Trials 1, 2 and 3 (Onasanya et al., 2006). Accessions that displayed a high level of resistance (disease score equal to 1) during the first trial were further evaluated for the virus content using an enzyme-linked immunosorbent assay (ELISA), as described by Séré et al. (2007). The last fully expanded leaf was harvested, ground in a coating buffer (Na2CO3, NaHCO3, pH 9.6) at 1:10, w:v) and the homogenized sap was directly adsorbed to microtitre plates overnight, followed by blocking with 1% bovine serum albumin. Double serial dilutions of antisera were made and bound antibody was detected with goat anti-rabbit serum, conjugated to alkaline phosphatase (Sigma-Aldrich Co, MO, USA). The bound conjugate was detected using a p-nitrophenyl phosphate solution (1 mg ml-1) and the optical density was read with a spectrophotometer at 405 nm. A sample was considered to be negative when the absorbance value was lower than the average absorbance of the negative control.
The software package SAS version 9.1 (SAS Institute Inc., Cary, NC) was used to analyse the data sets. First, an analysis of variance (ANOVA) was performed to compare differences in virulence among virus isolates. In addition, a generalized linear model (GLM) for multinomial data procedure was used to analyse discrete data, such as the disease symptom scores. The odds ratio estimates the strength of association between the response of interest (in this case disease scores), which stipulated that resistant accessions should have low disease symptoms and thus low scores). Microsoft Office Excel 2007 was also used to draw charts and compute the area under a symptoms progression curve (AUSPC) estimated using the formula:
AUSPC = ∑ [(Si+S(i+1)-2)(t(i+1)– ti)]/2, (Si is the symptom score at date ti)
Finally, the AUSPC, rPHgt, and rChcL were used to perform a principal component analysis using the software GenStat version 14.
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From Trials 4 and 2, 11 resistant accessions were investigated using primers allel-specific primers that were designed to amplify the three resistant alleles of RYMV1 gene. DNA was extracted from infected plant leaves as described by Edwards et al. (1991). All the 11 accessions selected for genotyping were O. glaberrima; therefore only alleles for rymv1-3, rymv1-4 and rymv1-5 were targeted in this study. PCR was performed as described by Thiémelé et al. (2010), using eight primer pairs, of which two primers were specific for each allele (F18 and R16 for rymv1-3; F17 and R15b for rymv1-4; R18 and F6 for rymv1-5), while the remaining two primers (F5 and R3) were common for all three alleles. Amplified products were separated on 2.5%
stained with 5% ethidium bromide (BET).