Common Bacterial Blight Of Bean Research Articles

DTALE approach demonstrates that induction of common bean OVATE Family Protein 7 (PvOFP7) promotes resistance to common bacterial blight

Abstract Common bacterial blight of bean (CBB) is a devastating seed-transmitted disease caused by Xanthomonas phaseoli pv. phaseoli and Xanthomonas citri pv. fuscans on common bean (Phaseolus vulgaris L.). The genes responsible for CBB resistance are largely unknown. Moreover, the lack of a reproducible and universal transformation protocol limits the study of genetic traits in common bean. We produced X. phaseoli pv. phaseoli strains expressing artificially-designed Transcription-Activator Like Effectors (dTALEs) to target 14 candidate genes for resistance to CBB based on previous transcriptomic data. In planta assays in a susceptible common bean genotype showed that induction of PvOFP7, PvAP2-ERF71 or PvExpansinA17 expression by dTALEs resulted in CBB symptom reduction. After PvOFP7 induction, in planta bacterial growth was reduced at early colonisation stages and RNA-Seq analysis revealed up-regulation of cell wall formation and primary metabolism, together with major down-regulation of Heat Shock Proteins. Our results demonstrate that PvOFP7 contributes to CBB resistance, and underline the usefulness of dTALEs for functional validation of genes whose induction impacts Xanthomonas-plant interaction.

Burkholderia gladioli BNM349 as a promising source of bacterial metabolites for biocontrol of common bacterial blight of bean

Burkholderia gladioli BNM349 as a promising source of bacterial metabolites for biocontrol of common bacterial blight of bean

Whole Genome Sequences of Nine Xanthomonas Strains Responsible for Common Bacterial Blight of Bean.

We report the complete and circularized genome sequences of nine strains of Xanthomonas phaseoli pv. phaseoli and Xanthomonas citri pv. fuscans, which cause common bacterial blight of bean. These assemblies provide high-quality material for functional and evolutionary studies of these legume pathogens.

Complete and Circularized Genome Sequences of 17 Xanthomonas Strains Responsible for Common Bacterial Blight of Bean.

ABSTRACTWe report the complete and circularized genome sequences of 17 strains of Xanthomonas citri pv. fuscans and Xanthomonas phaseoli pv. phaseoli, which cause common bacterial blight of bean. These new assemblies combining PacBio and short-read sequencing methods provide high-quality material for studying the evolution of these plant pathogens.

Common bacterial blight of bean: a model of seed transmission and pathological convergence.

Background Xanthomonas citri pv. fuscans (Xcf) and Xanthomonas phaseoli pv. phaseoli (Xpp) are the causal agents of common bacterial blight of bean (CBB), an important disease worldwide that remains difficult to control. These pathogens belong to distinct species within the Xanthomonas genus and have undergone a dynamic evolutionary history including the horizontal transfer of genes encoding factors probably involved in adaptation to and pathogenicity on common bean. Seed transmission is a key point of the CBB disease cycle, favouring both vertical transmission of the pathogen and worldwide distribution of the disease through global seed trade.TaxonomyKingdom: Bacteria; phylum: Proteobacteria; class: Gammaproteobacteria; order: Lysobacterales (also known as Xanthomonadales); family: Lysobacteraceae (also known as Xanthomonadaceae); genus: Xanthomonas; species: X. citri pv. fuscans and X. phaseoli pv. phaseoli (Xcf‐Xpp).Host rangeThe main host of Xcf‐Xpp is the common bean (Phaseolus vulgaris). Lima bean (Phaseolus lunatus) and members of the Vigna genus (Vigna aconitifolia, Vigna angularis, Vigna mungo, Vigna radiata, and Vigna umbellata) are also natural hosts of Xcf‐Xpp. Natural occurrence of Xcf‐Xpp has been reported for a handful of other legumes such as Calopogonium sp., Pueraria sp., pea (Pisum sativum), Lablab purpureus, Macroptilium lathyroides, and Strophostyles helvola. There are conflicting reports concerning the natural occurrence of CBB agents on tepary bean (Phaseolus acutifolius) and cowpea (Vigna unguiculata subsp. unguiculata).SymptomsCBB symptoms occur on all aerial parts of beans, that is, seedlings, leaves, stems, pods, and seeds. Symptoms initially appear as water‐soaked spots evolving into necrosis on leaves, pustules on pods, and cankers on twigs. In severe infections, defoliation and wilting may occur.DistributionCBB is distributed worldwide, meaning that it is frequently encountered in most places where bean is cultivated in the Americas, Asia, Africa, and Oceania, except for arid tropical areas. Xcf‐Xpp are regulated nonquarantine pathogens in Europe and are listed in the A2 list by the European and Mediterranean Plant Protection Organization (EPPO).GenomeThe genome consists of a single circular chromosome plus one to four extrachromosomal plasmids of various sizes, for a total mean size of 5.27 Mb with 64.7% GC content and an average predicted number of 4,181 coding sequences.Disease controlManagement of CBB is based on integrated approaches that comprise measures aimed at avoiding Xcf‐Xpp introduction through infected seeds, cultural practices to limit Xcf‐Xpp survival between host crops, whenever possible the use of tolerant or resistant bean genotypes, and chemical treatments, mainly restricted to copper compounds. The use of pathogen‐free seeds is essential in an effective management strategy and requires appropriate sampling, detection, and identification methods.Useful websites https://gd.eppo.int/taxon/XANTPH, https://gd.eppo.int/taxon/XANTFF, and http://www.cost.eu/COST_Actions/ca/CA16107.

Management of the Common Bacterial Blight of the Bean by Rhodotorula glutinis and Sporidiobolus johnsonii

Bean common bacterial blight reduces crop productivity and is difficult to control. However, biological control by yeast can be an efficient complementary measure in management. The objective was to evaluate the ability of Rhodotorula glutinis and Sporidiobolus johnsonii to reduce the severity of bean common bacterial blight. The cultivar used was IAPAR Tuiuiú. The first experiment was sown in March and repeated in October, in a 4 × 3 factorial scheme (zero, one, two and three applications and three treatments R. glutinis, S. johnsonii and Acibenzolar-S-Methyl (ASM)). For this purpose were evaluated the area under the disease progress curve (AACPD), number of pods per plant (NVP), number of grains per pod (NGV), thousand grain mass (MMG) and productivity. For the results of the March cultivation, due to the low temperature, the maximum severity of bean common bacterial blight was 8% and the applications of yeasts were not significant for AACPD. The isolate R. glutinis showed the highest average of productivity with two applications, being 1006.44 kg ha-1. For October cultivation, R. glutinis and S. johnsonii isolates reduced AACPD by 66.84 and 58.42%, respectively with three applications. For productivity, R. glutinis and S. johnsonii showed no difference between the number of applications. The ASM showed a productivity of 4418.56 kg ha-1 with three applications. The results indicate that the yeasts R. glutinis and S. johnsonii reduce the severity of bean common bacterial blight and the most appropriate number of applications are two for both isolates.

Genome sequence of Xanthomonas fuscans subsp. fuscans strain Xff49: a new isolate obtained from common beans in Southern Brazil.

The genus Xanthomonas comprises Gram-negative bacteria, many of which are phytopathogens. Xanthomonas fuscans subsp. fuscans is one of the most devastating pathogens affecting the bean plant, resulting in the common bacterial blight of bean (CBB). The disease is mainly foliar and affects a wide variety of bean species, thus acting as the yield-limiting factor for the bean crop. Here, we report the whole-genome sequencing of a new strain of X. fuscans subsp. fuscans, named Xff49, isolated from the infected and symptomatic beans from Capão do Leão, Southern Brazil. The genetic analysis demonstrated the presence of single-nucleotide variants (SNVs) in this strain, potentially affecting the mobilome, cell mobility, and inorganic ion metabolism. In addition, the analysis resulted in the identification of a new plasmid similar to the pAX22 derived from Achromobacter denitrificans, which was named plX, along with plA and plC, previously reported in other strains of X. fuscans subsp. fuscans. Xff49 represents the first Brazilian genome of X. fuscans subsp. fuscans and might provide useful information applicable to the studies of phylogenetics, evolution, and pathogenomics, thereby allowing a better understanding of the genomic features present in the Brazilian strains.

Foliar spraying with bacterial biocontrol agents for the control of common bacterial blight of bean

Abstract: The objective of this work was to evaluate the effect of foliar spraying with bacterial biocontrol agents (BBAs) on the control of common bacterial blight (CBB) of bean, and on the induction of systemic resistance in bean plants. CBB control by BBAs was evaluated by spraying bean leaves 48 and 24 hours before and after pathogen inoculation (BPI and API, respectively), with: DFs93, Bacillus cereus; DFs513, Pseudomonas veronii; DFs769, B. cereus; the C01 combination, DFs93 + DFs769 + DFs831 (Pseudomonas fluorescens); the C03 combination, DFs348 (Bacillus sp.) + DFs769 + DFs831; and water (control). Systemic effects were analyzed after spraying DFs513, DFs769, C03, and water 72 and 48 hours BPI. Phaseolin production induced by DFs348, DFs513, DFs769, DFs831, and water was also assessed. DFs513, DFs769, and C03 significantly reduced disease incidence (area under disease progress curve), regardless of spraying time and disease severity when sprayed 72 and 48 hours BPI. The DFs769 and DFs831 isolates induced the accumulation of phytoalexin (phaseolin). Therefore, DFs513, DFs769, and C03 show potential for the biocontrol of CBB when applied preventively on bean leaves, besides inducing systemic resistance.

Horizontal gene transfer plays a major role in the pathological convergence of Xanthomonas lineages on common bean

BackgroundHost specialization is a hallmark of numerous plant pathogens including bacteria, fungi, oomycetes and viruses. Yet, the molecular and evolutionary bases of host specificity are poorly understood. In some cases, pathological convergence is observed for individuals belonging to distant phylogenetic clades. This is the case for Xanthomonas strains responsible for common bacterial blight of bean, spread across four genetic lineages. All the strains from these four lineages converged for pathogenicity on common bean, implying possible gene convergences and/or sharing of a common arsenal of genes conferring the ability to infect common bean.ResultsTo search for genes involved in common bean specificity, we used a combination of whole-genome analyses without a priori, including a genome scan based on k-mer search. Analysis of 72 genomes from a collection of Xanthomonas pathovars unveiled 115 genes bearing DNA sequences specific to strains responsible for common bacterial blight, including 20 genes located on a plasmid. Of these 115 genes, 88 were involved in successive events of horizontal gene transfers among the four genetic lineages, and 44 contained nonsynonymous polymorphisms unique to the causal agents of common bacterial blight.ConclusionsOur study revealed that host specificity of common bacterial blight agents is associated with a combination of horizontal transfers of genes, and highlights the role of plasmids in these horizontal transfers.

Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors

BackgroundCommon bacterial blight is a devastating bacterial disease of common bean (Phaseolus vulgaris) caused by Xanthomonas citri pv. fuscans and Xanthomonas phaseoli pv. phaseoli. These phylogenetically distant strains are able to cause similar symptoms on common bean, suggesting that they have acquired common genetic determinants of adaptation to common bean. Transcription Activator-Like (TAL) effectors are bacterial type III effectors that are able to induce the expression of host genes to promote infection or resistance. Their capacity to bind to a specific host DNA sequence suggests that they are potential candidates for host adaption.ResultsTo study the diversity of tal genes from Xanthomonas strains responsible for common bacterial blight of bean, whole genome sequences of 17 strains representing the diversity of X. citri pv. fuscans and X. phaseoli pv. phaseoli were obtained by single molecule real time sequencing. Analysis of these genomes revealed the existence of four tal genes named tal23A, tal20F, tal18G and tal18H, respectively. While tal20F and tal18G were chromosomic, tal23A and tal18H were carried on plasmids and shared between phylogenetically distant strains, therefore suggesting recent horizontal transfers of these genes between X. citri pv. fuscans and X. phaseoli pv. phaseoli strains. Strikingly, tal23A was present in all strains studied, suggesting that it played an important role in adaptation to common bean. In silico predictions of TAL effectors targets in the common bean genome suggested that TAL effectors shared by X. citri pv. fuscans and X. phaseoli pv. phaseoli strains target the promoters of genes of similar functions. This could be a trace of convergent evolution among TAL effectors from different phylogenetic groups, and comforts the hypothesis that TAL effectors have been implied in the adaptation to common bean.ConclusionsAltogether, our results favour a model where plasmidic TAL effectors are able to contribute to host adaptation by being horizontally transferred between distant lineages.

Occurrence and Importance of Xanthomonas axonopodis pv. Phaseoli in Common Bean (Phaseolus vulgaris L) Seed Produced under Different Seed Production System in Central Rift Valley of Ethiopia

Common bacterial blight of bean caused by the seed-borne bacteria Xanthomonas axonopodis pv. phaseoli (Xap) (Smith) Vauterin and X. axonopodis pv. phaseoli var. fuscans (Burkholder) Starr and Burkholder is one of the most constraint of common bean production all over the world. The pathogen is seed-borne and survives as long as the seed remains viable. Use of pathogen-free seeds has been the main method used to control the disease in most bean production areas, and detection of this pathogen in seeds is essential for effective disease control. This study was carried out to detect and characterize Xap in seed lots collected from different seed dealers and local markets in Central Rift Valley of Ethiopia. A semi-selective medium Xanthomonas campestris pv. phaseoli (XCP1) and yeast extract-dextrose-calcium carbonate agar (YDCA) were used to recover the bacterium from whole bean seed extract and direct seed plating respectively. The pathogenicity test was done on Mexcan-142 bean cultivar to confirm pathogen identification. Colonies of the bacterium were yellow, mucoid and convex on XCP1 media and zone of hydrolysis formed around them. Further biochemical test results also confirm that the colonies were gram negative, rod shape and hydrolyze starch, casein and Tween80. The results confirmed the presence of seed borne Xap in all seed dealers and local market seed lots in the study area. The result reveled Xap was prevalent in 79.27% of the total seed samples collected. Lower prevalence (21.43%), seed infection percentage (1.643%) and bacterial population were resulted in seed lots from Melkassa Agricultural Research Center seed lots; while the higher prevalence, seed infection percentage and bacterial population were observed in cooperative union, local market, and seed producer’s cooperative seed lots. From the result, it can be concluded that Xap was potentially recovered from naturally infected seeds using XCP1 media and the pathogen is highly distributed in seed lots in the study area with high prevalence in farmer’s produced seed lots. Therefore, seed plating on semi selective medium XCP1 can be used as standard method for routine analysis of Xap from bean seeds. Seed dealers in the study area should follow strict disease free seed production programs and farmers in the study area should be encouraged not to use local market and/or their own saved seeds for planting purpose.

Identificación de razas de Xanthomonas campestris pv. phaseoli en hojas de phaseolus vulgaris.

Bean Common Bacterial Blight is a limiting factor on seed yield production of Phaseolus vulgaris in many countries of Central America and the Caribbean. In order to clarify the plant-pathogen interaction, a collection of Xanthomonas campestris pv. phaseoli was made in Costa Rica, Cuba, Dominican Republic and Puerto Rico. The Xcp collections were inoculated on leaves of different P. vulgaris genotypes. The response to foliage inoculation identified genotypes useful for the differentiation of the pathogenicity FXcp. differences at the level of physiological races were found. On the other and, genotypes with resistance to multiple Xcp races, were also found therefore, based on foliage response, the detection of physiological races of Xcp within the collection of Costa Rica, Cuba, Dominican Republic and Puerto Rico.

Genome sequence of Xanthomonas fuscans subsp. fuscans strain 4834-R reveals that flagellar motility is not a general feature of xanthomonads.

BackgroundXanthomonads are plant-associated bacteria responsible for diseases on economically important crops. Xanthomonas fuscans subsp. fuscans (Xff) is one of the causal agents of common bacterial blight of bean. In this study, the complete genome sequence of strain Xff 4834-R was determined and compared to other Xanthomonas genome sequences.ResultsComparative genomics analyses revealed core characteristics shared between Xff 4834-R and other xanthomonads including chemotaxis elements, two-component systems, TonB-dependent transporters, secretion systems (from T1SS to T6SS) and multiple effectors. For instance a repertoire of 29 Type 3 Effectors (T3Es) with two Transcription Activator-Like Effectors was predicted. Mobile elements were associated with major modifications in the genome structure and gene content in comparison to other Xanthomonas genomes. Notably, a deletion of 33 kbp affects flagellum biosynthesis in Xff 4834-R. The presence of a complete flagellar cluster was assessed in a collection of more than 300 strains representing different species and pathovars of Xanthomonas. Five percent of the tested strains presented a deletion in the flagellar cluster and were non-motile. Moreover, half of the Xff strains isolated from the same epidemic than 4834-R was non-motile and this ratio was conserved in the strains colonizing the next bean seed generations.ConclusionsThis work describes the first genome of a Xanthomonas strain pathogenic on bean and reports the existence of non-motile xanthomonads belonging to different species and pathovars. Isolation of such Xff variants from a natural epidemic may suggest that flagellar motility is not a key function for in planta fitness.

Bean common bacterial blight: pathogen epiphytic life and effect of irrigation practices

In recent years, bean common bacterial blight (CBB) caused by Xanthomonas axonopodis pv. phaseoli (Xap) has caused serious yield losses in several countries. CBB is considered mainly a foliar disease in which symptoms initially appear as small water-soaked spots that then enlarge and become necrotic and usually bordered by a chlorotic zone. Xap epiphytic population community has a critical role in the development of the disease and subsequent epidemics. The epiphytic population of Xap in the field has two major parts; solitary cells (potentially planktonic) and biofilms which are sources for providing and refreshing the solitary cell components. Irrigation type has a significant effect on epiphytic population of Xap. The mean epiphytic population size in the field with an overhead sprinkler irrigation system is significantly higher than populations under furrow irrigation. A significant positive correlation between the epiphytic population size of Xap and disease severity has been reported in both the overhead irrigated (r=0.64) and the furrow irrigated (r= 0.44) fields.

Comparison of Seed Transmission and Survival of Xanthomonas axonopodis pv. phaseoli and Xanthomonas fuscans subsp. fuscans in Common Bean Seeds

Xanthomonas axonopodis pv. phaseoli (Smith) Vauterin (Xap) and Xanthomonas fuscans subsp. fuscans Schaad et al. (Xff) cause indistinguishable symptoms known as common bacterial blight of bean (Phaseolus vulgaris L.). These results confirm a higher disease incidence and seed transmission frequency of Xff compared to Xap and reinforce the need for seed health tests that can differentiate the two species. Accepted for publication 21 August 2013. Published 23 September 2013.

Genetic diversity of the common bacterial blight pathogen of bean, Xanthomonas axonopodis pv. phaseoli, in Iran revealed by rep-PCR and PCR–RFLP analyses

Xanthomonad-like bacteria that are associated with common bacterial blight of bean in Iran were identified on the basis of their colonial morphology, biochemical and serological properties, presence of a specific DNA fragment using PCR primers and pathogenicity on bean. Xanthomonas axonopodis pv. phaseoli (Xap) strains were further characterized using rep-PCR and restriction fragment length polymorphism (RFLP). RFLP profiles generated by the restriction endonucleases RsaI, TaqI, HaeIII and Sau96I and rep-PCR analysis revealed that Iranian strains were relatively genetically homogenous. The similarity coefficients among the strains ranged from 0.87 to 1. The genetic diversity coefficients among strains from three infected provinces, Isfahan, Markazi and Lorestan, were 0.019, 0.072 and 0.033, respectively. The low overall level of polymorphism within Xap isolates collected from the three Iranian infected regions could suggest that few initial inoculum introductions might have distributed among these different bean-growing areas in Iran.

Detecção de Xanthomonas axonopodis pv. phaseoli em sementes de feijoeiro provenientes do estado do Paraná, Brasil

O crestamento bacteriano comum do feijoeiro causado por sobrevivência e disseminação da Xap, a semente representa o mais Xanthomonas axonopodis pv. phaseoli (Xap) é a principal doença eficiente. A qualidade sanitária de 34 amostras de sementes de feijoeiro do feijoeiro comum no Brasil. O patógeno encontra-se disseminado produzidas no estado do Paraná, nas safras 1998/99 e 1999, foram em todas as regiões produtoras do país, porém com maior importância avaliadas quanto à presença de Xap em macerados de sementes nos estados do Paraná, Rio de Janeiro, São Paulo e na região do Brasil plaqueados em meio semi-seletivo. Cinqüenta por cento dos lotes de Central, sobretudo na safra das águas. Dentre os vários meios de sementes foram portadores de Xap com incidência de 0,1% a 1,7%.

Adhesion and Fitness in the Bean Phyllosphere and Transmission to Seed of Xanthomonas fuscans subsp. fuscans

Deciphering the mechanisms enabling plant-pathogenic bacteria to disperse, colonize, and survive on their hosts provides the necessary basis to set up new control methods. We evaluated the role of bacterial attachment and biofilm formation in host colonization processes for Xanthomonas fuscans subsp. fuscans on its host. This bacterium is responsible for the common bacterial blight of bean (Phaseolus vulgaris), a seedborne disease. The five adhesin genes (pilA, fhab, xadA1, xadA2, and yapH) identified in X. fuscans subsp. fuscans CFBP4834-R strain were mutated. All mutants were altered in their abilities to adhere to polypropylene or seed. PilA was involved in adhesion and transmission to seed, and mutation of pilA led to lower pathogenicity on bean. YapH was required for adhesion to seed, leaves, and abiotic surfaces but not for in planta transmission to seed or aggressiveness on leaves. Transmission to seed through floral structures did not require any of the known adhesins. Conversely, all mutants tested, except in yapH, were altered in their vascular transmission to seed. In conclusion, we showed that adhesins are implicated in the various processes leading to host phyllosphere colonization and transmission to seed by plant-pathogenic bacteria.

The Type III Secretion System of Xanthomonas fuscans subsp. fuscans Is Involved in the Phyllosphere Colonization Process and in Transmission to Seeds of Susceptible Beans

Understanding the survival, multiplication, and transmission to seeds of plant pathogenic bacteria is central to study their pathogenesis. We hypothesized that the type III secretion system (T3SS), encoded by hrp genes, could have a role in host colonization by plant pathogenic bacteria. The seed-borne pathogen Xanthomonas fuscans subsp. fuscans causes common bacterial blight of bean (Phaseolus vulgaris). Directed mutagenesis in strain CFBP4834-R of X. fuscans subsp. fuscans and bacterial population density monitoring on bean leaves showed that strains with mutations in the hrp regulatory genes, hrpG and hrpX, were impaired in their phyllospheric growth, as in the null interaction with Escherichia coli C600 and bean. In the compatible interaction, CFBP4834-R reached high phyllospheric population densities and was transmitted to seeds at high frequencies with high densities. Strains with mutations in structural hrp genes maintained the same constant epiphytic population densities (1 x 10(5) CFU g(-1) of fresh weight) as in the incompatible interaction with Xanthomonas campestris pv. campestris ATCC 33913 and the bean. Low frequencies of transmission to seeds and low bacterial concentrations were recorded for CFBP4834-R hrp mutants and for ATCC 33913, whereas E. coli C600 was not transmitted. Moreover, unlike the wild-type strain, strains with mutations in hrp genes were not transmitted to seeds by vascular pathway. Transmission to seeds by floral structures remained possible for both. This study revealed the involvement of the X. fuscans subsp. fuscans T3SS in phyllospheric multiplication and systemic colonization of bean, leading to transmission to seeds. Our findings suggest a major contribution of hrp regulatory genes in host colonization processes.

Contamination of bean seeds by Xanthomonas axonopodis pv. phaseoli associated with low bacterial densities in the phyllosphere under field and greenhouse conditions

Xanthomonas axonopodis pv. phaseoli and its variant fuscans are the causal agents of common bacterial blight of bean. Production of seeds is recommended in arid climates with the use of pathogen-free seeds. However, contamination of seeds still occurs in these seed production areas. To verify if low contamination levels of sown seeds could explain these field contaminations, we used seeds that were naturally contaminated with CFBP4834-R, a rifamycin-resistant X. axonopodis pv. phaseoli fuscous strain, to contaminate field plots at different rates. We also inoculated seeds to verify some parameters of plant colonization and seed transmission. In growth chambers, seedling contamination was always successful from seeds contaminated with CFBP4834-R having population sizes greater than 1 × 103 CFU seed−1 and were not successful below 1 × 102 CFU seed−1. In the greenhouse, the efficiency of contamination of seeds was not significantly different between contaminated plants that had a low or a high CFBP4834-R population size and reached between 40% and 52% whatever the origin of the inoculum (aerial or seed-borne). In field experiments, under low relative humidity, plots with 0.1–0.003% contamination rates or plots sown with seeds that were inoculated with low CFBP4834-R population sizes (1 × 102 and 1 × 104 CFU seed−1) led to an asymptomatic colonization of bean during the entire growing season with low CFBP4834-R population sizes. Seeds were contaminated both in primary and secondary foci. The contamination of seeds without symptom expression during the growing season represents a risk for eventual disease outbreaks.