Resistance-breaking Isolates Research Articles

Isolates of Citrus tristeza virus that overcome Poncirus trifoliata resistance comprise a novel strain

The economically important rootstock species Poncirus trifoliata is resistant to most isolates of Citrus tristeza virus (CTV), but not to members of the CTV resistance-breaking (RB) strain presently found in New Zealand. In this study, five known and suspected RB isolates were separated from field mixtures, and their genomes were sequenced in full. It was found that the RB isolates are members of a single phylogenetically distinct clade with an average of 90.3% genomic nucleotide sequence identity to the closest extant isolate, T36. These isolates also show evidence of multiple recombination events throughout their evolutionary history, with T36, T30 and VT-like isolates, and with each other. Finally, the genomic sequences of these isolates show that several genes contain unique polymorphisms that may or may not be involved in overcoming resistance. These data will aid in the understanding of host-virus interactions, and the mechanism of resistance in P. trifoliata.

Characterization of Tomato spotted wilt virus isolates that overcome the Sw-5 resistance gene in tomato and fitness assays.

Resistance-breaking (RB) isolates of Tomato spotted wilt virus (TSWV) that overcome the resistance conferred by the Sw-5 gene in tomato have had only a limited spread since they were first detected in north-eastern Spain in 2002. Symptom expression, homogeneity, stability and the transmission capacity of RB and non-resistance breaking (NRB) isolates were biologically compared. The fitness of both types of isolates infecting tomato plants was determined in competition assays. All TSWV isolates induced similar systemic symptoms in a wide range of plant species, except RB isolates in tomato carrying the Sw-5 resistance gene and pepper carrying the Tsw resistance gene . The mechanical transmission of RB isolates to tomato plants with the Sw-5 gene failed in some trials, although NRB isolates did not differ noticeably in transmission efficiency when tested with the thrips Frankliniella occidentalis . Biological clones from individual local lesions obtained by mechanically inoculating Nicotiana glutinosa in some TSWV field samples showed that they were biologically homogeneous. Mixed infections of RB and wilt-type isolates were not found. The RB isolates were relatively stable because no reversion to NRB isolates was seen after serial passages in susceptible tomato plants. In competition assays between RB and NRB isolates, after serial passages in susceptible tomato plants, the prevalence of a particular isolate was not related to its capacity to overcome Sw-5 gene resistance. The low spread of the RB isolates in Spain does not seem to be related to a loss of fitness in tomato plants or to differences in transmission capacity by thrips, but it could be related to the reduction of the selection pressure of RB isolates as consequence of the gradual replacement of susceptible tomato plants by resistant tomato plants by growers.

Identification of Beet necrotic yellow vein virus P25 Pathogenicity Factor–Interacting Sugar Beet Proteins That Represent Putative Virus Targets or Components of Plant Resistance

Beet necrotic yellow vein virus (BNYVV) induces the most important disease threatening sugar beet. The growth of partially resistant hybrids carrying monogenic dominant resistance genes stabilize yield but are unable to entirely prevent virus infection and replication. P25 is responsible for symptom development and previous studies have shown that recently occurring resistance-breaking isolates possess increased P25 variability. To better understand the viral pathogenicity factor's interplay with plant proteins and to possibly unravel the molecular basis of sugar beet antivirus resistance, P25 was applied in a yeast two-hybrid screen of a resistant sugar beet cDNA library. This screen identified candidate proteins recognized as orthologues from other plant species which are known to be expressed following pathogen infection and involved in plant defense response. Most of the candidates potentially related to host-pathogen interactions were involved in the ubiquitylation process and plants response to stress, and were part of cell and metabolism components. The interaction of several candidate genes with P25 was confirmed in Nicotiana benthamiana leaf cells by transient agrobacterium-mediated expression applying bimolecular fluorescence complementation assay. The putative functions of several of the candidates identified support previous findings and present first targets for understanding the BNYVV pathogenicity and antivirus resistance mechanism.

Breakdown of resistance in sweet pepper against Pepper yellow mosaic virus in Brazil

Plants of Capsicum annuum cv. Magali R, resistant to Pepper yellow mosaic virus (PepYMV), which showed severe yellow mosaic, leaf malformation and stunting were observed during the 2003/04 growing season in Lins, São Paulo State, Brazil. Potyvirus-like particles observed in leaf sap from infected plants under the electron microscope reacted with an antiserum against PepYMV in PTA-ELISA. In addition to C. annuum cv. Magali R, this potyvirus also infected systemically the resistant C. annuum cv. Rubia R. The nucleotide sequence of part of the CP gene of this potyvirus shared 96-98% identity with that of other PepYMV isolates. The partial nucleotide sequence of the 3' NTR showed 94-96% identity with that of PepYMV. These data indicate that this potyvirus is a resistance-breaking isolate of PepYMV.

Breaking of Beet necrotic yellow vein virus resistance in sugar beet is independent of virus and vector inoculum densities

Beet necrotic yellow vein virus (BNYVV) is transmitted by Polymyxa betae to sugar beet, causing rhizomania disease. Resistance-breaking strains of BNYVV, overcoming single (Rz1) or double (e.g. Rz1 + Rz2) major resistance genes in sugar beet have been observed in France and recently in the USA and Spain. To demonstrate if resistance-breaking is dependent on inoculum density, the inoculum concentration of BNYVV and P. betae in soil samples where resistance-breaking had been observed was estimated using the most probable number (MPN) method. The MPN-values obtained displayed highly significant differences with respect to the virus concentration in various soils and did not correlate with the ability to overcome resistance. Virus quantification in susceptible plants demonstrated that soils containing resistance-breaking isolates of BNYVV did not produce higher virus concentrations. The MPN assay was repeated with Rz1 + Rz2 partially-resistant sugar beets to see if the resistance-breaking is concentration-dependent. There was no correlation between soil dilution and increased virus concentration in Rz1 + Rz2 plants produced by BNYVV resistance-breaking strains. Determination of the absolute P. betae concentration by ELISA demonstrated that all resistance-breaking soil samples contained elevated concentrations. However, the calculation of the proportion of viruliferous P. betae did not show a positive correlation with the resistance-breaking ability. Finally resistance-breaking was studied with susceptible, Rz1 and Rz1 + Rz2 genotypes and standardised rhizomania inoculum added to sterilised soil. Results from these experiments supported the conclusion that resistance-breaking did not correlate with virus concentration or level of viruliferous P. betae in the soil.

Identification of rhizomania-infected soil in Europe able to overcome Rz1 resistance in sugar beet and comparison with other resistance-breaking soils from different geographic origins

Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV), is vectored by Polymyxa betae. The disease can only be controlled by growing partially resistant sugar beets, which quantitatively reduce virus replication and spread. None of the known major resistance genes (Rz1, Rz2, Rz3), alone or in combination, are able to prevent BNYVV infection entirely. Here we report for the first time the identification of a Spanish soil, containing an A-type BNYVV with RNA 1-4, displaying Rz1 resistance-breaking abilities comparable to soils from the USA and to those from France containing the French (Pithiviers) P-type BNYVV with RNA 5. A resistance test with several soil samples vs. different sugar beet cultivars was conducted under standardised conditions. Sugar beets were analysed after 12 weeks of greenhouse cultivation for taproot weight, BNYVV and relative P. betae content. The soil samples from Spain, France and the USA produced high virus contents and strong rhizomania symptoms in Rz1 plants, indicative of resistance-breaking abilities. In addition, all resistance-breaking soil samples produced detectable virus concentrations in plant lateral roots of the Rz1 + Rz2 cultivar, and plants grown in the Spanish soil sample also had reduced taproot weight and displayed severe rhizomania disease symptoms. Additionally, the main pathogenicity factor P25, responsible for the formation of BNYVV symptoms, showed high sequence variability in the amino acid tetrad at position 67–70. The results suggest the geographically independent selection of BNYVV resistance-breaking isolates following the uniform cultivation of Rz1-containing sugar beet cultivars.

Identification and molecular characterization of two naturally occurring Soybean mosaic virus isolates that are closely related but differ in their ability to overcome Rsv4 resistance

A naturally occurring Rsv4 resistance-breaking isolate (L-RB) and a closely related non-resistance-breaking isolate (L) of Soybean mosaic virus (SMV) were identified in soybean fields in London, Ontario, Canada. The viral genomes of L and L-RB were completely sequenced. Each isolate has a 9585-nucleotide genome with a single open reading frame encoding a polyprotein of approximately 350 kDa. L-RB and L have a very high sequence similarity (99.6%) at both the nucleotide and amino acid levels. Phylogenetic analysis showed that the two isolates belong to the G2 pathotype. Pathogenicity predictions of all virus/soybean combinations, based on the phylogenetic profile, were confirmed by pathogenicity tests using L and L-RB isolates and soybeans carrying different resistance genes, with an exception that L-RB infected a soybean cultivar carrying Rsv4 resistance. The temporal and spatial proximity of L and L-RB and their high sequence similarity suggest L-RB was likely derived from the SMV-L quasispecies. Recombination analysis did not reveal the evidence of genetic recombination for the emergence of L-RB. Mutations introduced by virus-encoded RNA-dependent RNA polymerase during viral genome replication and selection pressure probably contributed to the occurrence of L-RB.

Suppression of Resistance-Breaking Beet necrotic yellow vein virus Isolates by Beet oak-leaf virus in Sugar Beet.

Rhizomania, a serious disease of sugar beet (Beta vulgaris), is caused by Beet necrotic yellow vein virus (BNYVV). Resistance allele Rz1 has been widely incorporated into commercial cultivars. Recently, resistance-breaking isolates of BNYVV (RB-BNYVV) were identified and characterized. When the occurrence of RB-BNYVV was surveyed throughout the sugar-beet-growing areas in the United States, most soil samples contained Beet oak-leaf virus (BOLV) as well. BNYVV and BOLV often occurred in the same field and sometimes in the same sugar beet plant. The possibility of interactions between these two Polymyxa betae-transmitted sugar beet viruses was tested. Plants grown in soils infested with aviruliferous P. betae or carrying RB-BNYVV and BOLV, alone and in combination, were compared with plants grown in noninfested soil for differences in plant fresh weight and virus content as measured by enzyme-linked immunosorbent assay (ELISA). Rz1 and Rz2 resistance genes that condition resistance to BNYVV did not confer resistance to BOLV. BNYVV ELISA values were significantly higher in single infections than in mixed infections with BOLV in both the rhizomania-resistant and -susceptible cultivars. In contrast, ELISA values of BOLV were not significantly different between single and mixed infections in both the rhizomania-resistant and -susceptible cultivars. Results indicate that BOLV may suppress BNYVV in mixed infections. Soils infested with P. betae significantly reduced fresh weight of sugar beet seedlings regardless of whether they were with or without one or both viruses or resistance genes.

A New Pathotype of Pepper mild mottle virus (PMMoV) Overcomes the L4 Resistance Genotype of Pepper Cultivars.

The biological, serological, and molecular characteristics of a newly isolated L4 resistance-breaking isolate of Pepper mild mottle virus (PMMoV) were studied. The new pathotype of PMMoV is closely related to the Israeli pathotypes P1,2 and P1,2,3 of the virus; however, the mosaic symptoms caused by this new pathotype on pepper plants with an L4 genotype were more severe than the mild mosaic symptoms caused by other common pathotypes of the virus in susceptible plants. The predicted amino acid sequence of the putative coat protein (CP) of the newly described pathotype has two amino acid mismatches when compared with the CP of pathotype P1,2, leucine to glutamine at position 47, and alanine to glycine at position 87. The CP of the new pathotype has one amino acid mismatch when compared with P1,2,3, having alanine instead of glycine at position 87. Based on its biological characteristics, the new pathotype was designated P1,2,3,4 of PMMoV-Is. A method is described for the differentiation among the three PMMoV pathotypes using restriction cleavage analysis of reverse-transcription polymerase chain reaction products made from virus-infected plants. An additional unique MnlI site in the CP gene of the newly isolated P1,2,3,4 allows its distinction from the other two isolates, while BglI cleaved only products of the P1,2 pathotype.

Molecular analysis of the coat protein and minor coat protein genes of New ZealandCitrus tristeza virusisolates that overcome the resistance ofPoncirus trifoliata(L.) Raf.

Poncirus trifoliata (L.) Raf. and trifoliate orange hybrid species comprise 90% of the citrus rootstock used in NewZealand. The resistance expressed by P. trifoliata to Citrus tristeza virus is in part responsible for the NewZealand citrus industry not suffering severe losses to this disease. However, isolates of Citrus tristeza virus have been identified in NewZealand that can overcome this resistance and establish a systemic infection of P. trifoliata. This study examined the coat protein (CP) and minor coat protein (CPm) genes of endemic resistance-breaking (RB) and non-RB isolates at the molecular level. Both the major and minor coat genes of RB isolates were paraphyletic, with average nucleotide sequence identities of 95.2% and 92.1%, respectively. The RB isolates were phylogenetically distinct from non-RB isolates in both genes, with nucleotide identities ranging from 91.8 to 98.2% for the CP and from 91.0 to 96.9% for the CPm. However, no correlation was found between specific nucleotide or amino acid polymorphisms in the CP or CPm genes of these isolates and expression of the RB phenotype. These data suggest that the RB isolates comprise at least one or more distinct genotypes, distinct from known non-RB isolates.

Proteome changes in sugar beet in response to Beet necrotic yellow vein virus

Beet necrotic yellow vein virus (BNYVV) is a devastating sugar beet pathogen. Resistance is limited and resistance-breaking isolates are becoming problematic. Effective disease control strategies will come from a better understanding of disease and resistance. BNYVV-induced differential sugar beet protein expression was evaluated with multidimensional liquid chromatography. Of more than 1000 protein peaks detected in root extracts, 7.4 and 11% were affected by BNYVV in the resistant and susceptible genotypes, respectively. Using tandem MALDI-TOF-MS, 65 proteins were identified; differential expression of some was verified using RT-PCR. Proteomic data suggest involvement of systemic resistance components in Rz1-mediated resistance and phytohormones in symptom development.

Etiology of the sugar beet rhizomania

Beet necrotic yellow vein virus is responsible for sugar beet rhizomania. Root proliferation is characteristic of the viral infection and lead to sugar losses. Pathogenicity is particularly linked to the expression of RNA-3-encoded p25. The extensive use of viral tolerant crops allows maintenance of sugar yields but also permits viruliferous vector to be maintained and therefore the appearance of resistance breaking isolates. The resistance breaking isolates present some amino acid variations within the p25 protein sequence, a key determinant in BNYVV pathogenicity. Here, we will review the molecular biology of BNYVV, of its vector and the antiviral strategies that may be used against rhizomania.

Distribution and Molecular Characterization of Resistance-Breaking Isolates of Beet necrotic yellow vein virus in the United States.

Beet necrotic yellow vein virus (BNYVV) is the causal agent of rhizomania in sugar beet (Beta vulgaris). The virus is transmitted by the plasmodiophorid Polymyxa betae. The disease is controlled primarily by the use of partially resistant cultivars. During 2003 and 2004 in the Imperial Valley of California, partially resistant sugar beet cultivars with Rz1 allele seemed to be compromised. Field trials at Salinas, CA have confirmed that Rz1 has been defeated by resistance-breaking isolates. Distinct BNYVV isolates have been identified from these plants. Rhizomania-infested sugar beet fields throughout the United States were surveyed in 2004-05. Soil surveys indicated that the resistance-breaking isolates not only existed in the Imperial Valley and San Joaquin Valley of California but also in Colorado, Idaho, Minnesota, Nebraska, and Oregon. Of the soil samples tested by baited plant technique, 92.5% produced infection with BNYVV in 'Beta 6600' (rz1rz1rz1), 77.5% in 'Beta 4430R' (Rz1rz1), 45.0% in 'Beta G017R' (Rz2rz2), and 15.0% in 'KWS Angelina' (Rz1rz1+Rz2rz2). Analyses of the deduced amino acid sequence of coat protein and P-25 protein of resistance-breaking BNYVV isolates revealed the high percentage of identity with non-resistance-breaking BNYVV isolates (99.9 and >98.0%, respectively). The variable amino acids in P-25 proteins were located at the residues of 67 and 68. In the United States, the two amino acids found in the non-resistance-breaking isolates were conserved (AC). The resistance-breaking isolates were variable including, AF, AL, SY, VC, VL, and AC. The change of these two amino acids cannot be depended upon to differentiate resistance-breaking and non-resistance-breaking isolates of BNYVV.

Mutations Associated with Resistance-Breaking Isolates of Beet necrotic yellow vein virus and Their Allelic Discrimination Using TaqMan Technology

ABSTRACT Genetic resistance in sugar beet (Beta vulgaris) to Beet necrotic yellow vein virus (BNYVV), which causes the disease rhizomania, is conferred by the single dominant gene Rz1. However, since 2002, Rz1 cultivars grown in the Imperial Valley of California have been increasingly damaged by a new strain of BNYVV. Viral RNA 3 was extracted from asymptomatic and symptomatic sugar beets and, after amplification and sequencing of a region including the p25 cistron, two polymorphic sites, A67V and D135E, associated with the capability of the virus to overcome resistance were identified. Using the real-time reverse transcription-polymerase chain reaction allelic discrimination technique, TaqMan probes designed to detect the responsible nucleotide substitutions permitted the differentiation between wild type (WT) and resistance-breaking (RB) isolates. This method also allowed easy detection of mixed infections by giving a heterozygous call, which was verified by DNA sequencing of individual clones. The capability of this technology to typify numerous isolates facilitated the analysis of the spatial distribution of virus haplotypes in the field. Thus, RB variants were mostly baited from yellow strips with high incidence of rhizomania, whereas WT variants predominated in the surrounding green areas. Mixed infections were found mainly in green areas and transitional zones. The predominance of the RB isolates in yellow strips suggests that they have gained fitness in Rz1 cultivars and will eventually become the dominant haplotype.

Cooperative effect of two amino acid mutations in the coat protein of Pepper mild mottle virus overcomes L 3 -mediated resistance in Capsicum plants

We found that an L3 resistance-breaking field isolate of Pepper mild mottle virus (PMMoV), designated PMMoV-Is, had two amino acid changes in its coat protein (CP), namely leucine to phenylalanine at position 13 (L13F) and glycine to valine at position 66 (G66V), as compared with PMMoV-J, which induces a resistance response in L3-harboring Capsicum plants. The mutations were located to a CP domain corresponding to the outer surface of PMMoV particles in computational molecular modeling. Analyses of PMMoV CP mutants containing either or both of these amino acid changes revealed that both changes were required to efficiently overcome L3-mediated resistance with systemic necrosis induction. Although CP mutants containing either L13F or G66V could not efficiently overcome L3-mediated resistance, these amino acid changes had different effects on the elicitor activity of PMMoV CP. L13F caused a slight reduction in the elicitor activity, resulting in virus restriction to necrotic local lesions that were apparently larger than those induced by wild-type PMMoV, while G66V rendered wild-type PMMoV the ability to overcome L3-mediated resistance, albeit with a lower efficiency than PMMoV with both changes. These results suggest that a cooperative effect of the L13F and G66V mutations on the elicitor activity of CP is responsible for overcoming the L3-mediated resistance.

Identification and Sequence Analysis of RNA3 of a Resistance-Breaking Cucumber mosaic virus Isolate on Capsicum annuum

Cultivated hot pepper crops showing severe mosaic symptom were found in Korea in 2004 and their causal agent was identified as Cucumber mosaic virus (CMV). These pepper crops was resistant to the virus in the filled, and they belonged to pathotype 0 (P0) resistant pepper. Resistance screening of selected pepper plants showed that a pepper isolate of CMV was the P0 resistance-breaking virus. This P0 resistance-breaking isolate of CMV, named as Ca-P1, was isolated from leaves of the virus-infected Capsicum annuum cv. Manidda that showed systemic severe mosaic symptom. Ca-P1-CMV could induce systemic mosaic symptoms on P0-susceptible (P0-S) and P0-resistant (P0-R) cultivars whereas an ordinary strain (Fny-CMV) could not infect P0-R. This result suggests that Ca-P1-CMV can overcome P0 resistant pepper cultivars. To analyze its genome sequence, the complete nucleotide sequence of RNA3 of Ca-P1-CMV was determined from the infectious full-length cDNA clone of the virus. RNA3 of Ca-P1-CMV consisted of 2,219 nucleotides. Overall sequence homology of RNA3-encoded two viral proteins (movement protein and coat protein) revealed high similarity (75.2-97.2%) with the known CMV strains. By sequence analysis with known representative strains of CMV, Ca-P1-CMV belongs to a typical member of CMV subgroup IB. The resistance and resistance-breaking mechanisms of pepper and counterpart CMV, respectively, remain to be investigated, which will enrich the genetic resources and accelerate CMV-resistant pepper breeding programs.

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

The recessive gene rymv-1, responsible for the high resistance of Oryza sativa 'Gigante' to Rice yellow mottle virus (genus Sobemovirus), was overcome by the variant CI4*, which emerged after serial inoculations of the non-resistance-breaking (nRB) isolate CI4. By comparison of the full-length sequences of CI4 and CI4*, a non-synonymous mutation was identified at position 1729, localized in the putative VPg domain, and an assay was developed based on this single-nucleotide polymorphism. The mutation G1729T was detected as early as the first passage in resistant plants and was found in all subsequent passages. Neither reversion nor any additional mutation was observed. The substitution G1729T, introduced by mutagenesis into the VPg of an nRB infectious clone, was sufficient to induce symptoms in uninoculated leaves of O. sativa 'Gigante'. This is the first evidence that VPg is a virulence factor in plants with recessive resistance against viruses outside the family Potyviridae.

Molecular Characterization of Soybean Mosaic Virus NIa Protein and its Processing Event in Bacterial Expression

Soybean mosaic virus (SMV)-CN18 is an Rsv resistance-breaking (RB) isolate to overcome soybean resistance genes Rsv1, Rsv3 and Rsv4. The aim of this study was to characterize nuclear inclusion protein a (NIa protein) of RB isolate at the molecular level and demonstrate its processing into genome-linked protein (VPg) and NIa-Pro domains in Esherichia coli containing a bacterial expression pET vector inserted with NIa gene. The full-length of NIa gene was synthesized by reverse transcription-polymerase chain reaction (RT-PCR) and its 1298 nucleotides (nt) and 432 amino acids (aa) were deduced. The nt and aa sequences of NIa gene of SMV-CN18 shared high identities with the corresponding sequences of the NIa gene of the known SMV isolates, suggesting that the NIa is a highly conserved protein. The NIa-Pro domain contains a highly conserved structural motif for proteolysis, while the VPg domain contains a nuclear localization signal (NLS), a putative NTP-binding site and cellular factor-binding sites. The phylogenetic tree revealed that less divergence of NIa protein exists among twelve SMV isolates, which can be supported by a low bootstrap value between clades. In addition, the full-length of NIa gene, amplified by RT-PCR, was ligated into pET-28b E. coli expression vector with an N-terminal His6-tag. Optimal conditions for expression were at 1mM treatment of IPTG at 25°C for 5 hr. The released protein from bacterial lysates remained soluble and proved the processing form of the NIa polyprotein. E. coli expression system shows the processed product of 29 kDa VPg in SDS-PAGE confirmed by western blot analysis in both crude extracts and purified elution products, using Ni2+-NTA resin. The present study indicates that the N-terminal region of NIa which is processed and expressed in bacteria.

Occurrence of Resistance-Breaking Isolates of Rice yellow mottle virus in West and Central Africa.

Rice yellow mottle virus (RYMV) is the most important rice-infecting virus in Africa. Highly resistant rice (Oryza spp.) cultivars Gigante and Tog5681 were challenged with virus isolates from five countries of the west and central African Sudano-savannah zone in order to investigate the occurrence and prevalence of resistance-breaking (RB) isolates. High resistance was overcome by 38.6% of the isolates. RB isolates could be divided into three main pathogenic groups. Isolates of the first group (17.5%) and of the second group (16.4%) were able to break down the resistance of Gigante only and of Tog5681 only, respectively. Resistance in both cultivars was overcome simultaneously by isolates of the third group (4.7%). In each group, some isolates induced symptoms, whereas plant infection by others was evidenced only by serological tests. RB isolates occurred in all five countries with varying frequencies (19 to 57%). The wide geographical distribution and high frequencies of RB isolates represent a high risk for the durability of resistance to RYMV in the Sudano-savannah zone.

Durability of natural and transgenic resistances in rice to Rice yellow mottle virus

A monogenic recessive resistance to Rice yellow mottle virus (RYMV) found in the Oryza sativa indica cultivar Gigante and in a few Oryza glaberrima cultivars provided a higher level of resistance than either a polygenic partial resistance found in some japonica cultivars which delayed symptom expression or transgenic resistances which were partial and temporary. This high resistance was overcome by several isolates, but the percentage of such virulent isolates in the fields was low. There was no relationship between the virulence of an isolate towards the high resistance and its aggressiveness in other cultivars. Isolates with either of the two components of pathogenicity – virulence and aggressiveness – were found in each strain and in all regions of Africa, in both wild and cultivated grass species. There was no loss of fitness of resistance-breaking (RB) isolates as they were not counter-selected, impaired or outperformed after serial passages in susceptible cultivars, even in mixture with avirulent quasi-isogenic wild type isolates. Resistance breaking was highly dependent on the amount of virus inoculated and on the mode of transmission. Implications of these results for the durability of the resistances to RYMV and for the development of integrated disease management strategies are discussed.