RÉSUMÉ
Sugarcane streak mosaic virus (SCSMV; Poaceavirus; Potiviridae) is the causal agent of streak mosaic disease of sugarcane (Saccharum interspecific hybrids), a major industrial crop that is widely cultivated in tropical and subtropical regions for sugar and ethanol production. was first reported by Hall et al. (1998) from quarantined germplasm material exhibiting mosaic symptoms imported from Pakistan into the USA. Subsequently, the virus was also reported to occur in most of the Asian countries like Bangladesh, India, Indonesia, Iran, Sri Lanka, Thailand, Vietnam and China (Chatenet et al. 2005; Hema et al. 2008, Kasemsin et al. 2016, Putra et al. 2014, Xu et al. 2010, Moradi et al. 2015; Moradi et al. 2018, Zhang et al. 2018). Until now, there is no report of SCSMV outside the Asian continent. From February to October 2018, sugarcane plants exhibiting symptoms such as irregular yellow and green mosaic, interveinal chlorotic specks, and streaks were observed in Bafing (Borotou-Koro), Marahoué (Zuénoula) and Tchologo (Ferkéssédougou) regions of Côte d'Ivoire (Fig. 1a). Varieties under large-scale commercial cultivation such as R570, R579, SP711406, Co997, Co449, M1176/77, M2593/92, M2580/95, and M1400/86 were all symptomatic. A total of 94 sugarcane leaf samples were collected from these regions and, among those, 82 showed disease symptoms and 12 were symptomless. Samples were first tested for the presence of sugarcane mosaic virus (SCMV), which causes mosaic a disease that is already present in Africa. Serological tests with infected sap using a Double Antibody Sandwich (DAS)- Enzyme Linked Immuno Sorbent Assay (ELISA) kit (DSMZ, RT-0166, Braunschweig, Germany) were negative for SCMV and no amplification product was obtained by RT-PCR using primers specific to the coat protein (CP) gene of SCMV (Putra et al. 2003). The 82 symptomatic leaves tested positive by DAS-ELISA with SCSMV antiserum (polyclonal antibodies were graciously provided by Prof. Hema M. of the Sri Venkateswara University, Tirupati, AP, India), whereas the 12 symptomless samples tested negative. To confirm these results, virus free greenhouse-grown sugarcane varieties Co997 and M1176/77, were mechanically inoculated with 10 sap extracts from 10 SCSMV-infected sugarcane leaf samples. Sap was also extracted from DAS-ELISA negative sugarcane leaves and used as negative control. For sap preparation, leaves were homogenized with a mortar in 2 mL of phosphate buffer 0.01 M pH 7.2 (ratio 1:10). Fifteen 4-week-old plants per variety were inoculated separately with each sap. All inoculated plants exhibited streak mosaic symptoms 13 days post-inoculation (fig. 1b), and the presence of SCSMV in the inoculated plants was confirmed by DAS-ELISA. Total RNA was extracted from four symptomatic leaf samples, one symptomless and one DAS-ELISA positive sample using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized using M-MLV Reverse Transcriptase (Promega, Cat.No.M1705, Madison, WI, USA) following the manufacturer's instructions. A 690-nucleotide fragment of the CP gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) with primers SCSMV-F690 and SCSMV-R690 (Viswanathan et al. 2008). All primers were synthesized by Eurogentec (Seraing, Belgium). Aliquots of RT-PCR products (5 µl) were analyzed by electrophoresis on 1.2 % (w/v) agarose gel, stained with ethidium bromide and visualized on a UV transilluminator (Fig. 2). An amplification product of the expected size was obtained for all five symptomatic or DAS ELISA positive but not for the symptomless sample. Two RT-PCR products were sequenced and deposited in GenBank under accession Nos. LR594547 and LR594582. These partial CP gene sequences shared highest nucleotide identity with two isolates of SCSMV from India in GenBank: 91% with JN315855 and 90% with EF655859, thus confirming that SCSMV was occurring in sugarcane in Côte d'Ivoire. To our knowledge, this is the first report of natural infection of sugarcane by SCSMV in Africa. Streak mosaic is a serious threat to the entire sugar industry in West Africa and needs further investigations as it may affect sugarcane yields and impact local economies. Our findings further illustrate the need to develop virus-free germplasm for local, national, and international distribution of sugarcane.
RÉSUMÉ
The diversity of a highly variable RNA plant virus was considered to determine the range of virulence substitutions, the evolutionary pathways to virulence, and whether intraspecific diversity modulates virulence pathways and propensity. In all, 114 isolates representative of the genetic and geographic diversity of Rice yellow mottle virus (RYMV) in Africa were inoculated to several cultivars with eIF(iso)4G-mediated Rymv1-2 resistance. Altogether, 41 virulent variants generated from ten wild isolates were analyzed. Nonconservative amino acid replacements at five positions located within a stretch of 15 codons in the central region of the 79-aa-long protein VPg were associated with virulence. Virulence substitutions were fixed predominantly at codon 48 in most strains, whatever the host genetic background or the experimental conditions. There were one major and two isolate-specific mutational pathways conferring virulence at codon 48. In the prevalent mutational pathway I, arginine (AGA) was successively displaced by glycine (GGA) and glutamic acid (GAA). Substitutions in the other virulence codons were displaced when E48 was fixed. In the isolate-specific mutational pathway II, isoleucine (ATA) emerged and often later coexisted with valine (GTA). In mutational pathway III, arginine, with the specific S2/S3 strain codon usage AGG, was displaced by tryptophane (TGG). Mutational pathway I never arose in the widely spread West African S2/S3 strain because G48 was not infectious in the S2/S3 genetic context. Strain S2/S3 least frequently overcame resistance, whereas two geographically localized variants of the strain S4 had a high propensity to virulence. Codons 49 and 26 of the VPg, under diversifying selection, are candidate positions in modulating the genetic barriers to virulence. The theme and variations in the evolutionary pathways to virulence of RYMV illustrates the extent of parallel evolution within a highly variable RNA plant virus species.