Carlos Bayon

Mutation discovery for crop improvement

Increasing crop yields to ensure food security is a major challenge. Mutagenesis is an important tool in crop improvement and is free of the regulatory restrictions imposed on genetically modified organisms. The forward genetic approach enables the identification of improved or novel phenotypes that can be exploited in conventional breeding programmes. Powerful reverse genetic strategies that allow the detection of induced point mutations in individuals of the mutagenized populations can address the major challenge of linking sequence information to the biological function of genes and can also identify novel variation for plant breeding. This review briefly discusses recent advances in the detection of mutants and the potential of mutagenesis for crop improvement.

Risk assessment studies on succinate dehydrogenase inhibitors, the new weapons in the battle to control Septoria leaf blotch in wheat

Chemical control of Septoria leaf blotch, caused by Mycosphaerella graminicola, is essential to ensure wheat yield and food security in most European countries. Mycosphaerella graminicola has developed resistance to several classes of fungicide and, with the efficacy of azoles gradually declining over time, new modes of action and/or improvements in host varietal resistance are urgently needed to ensure future sustainable disease control. Several new-generation carboxamide fungicides with broad-spectrum activity have recently been introduced into the cereal market. Carboxamides inhibit succinate dehydrogenase (Sdh) of the mitochondrial respiratory chain (complex II) but, because of their single-site specificity, these fungicides may be prone to resistance development. The objective of this study was to assess the risk of resistance development to different Sdh inhibitor (SDHI) fungicides in M. graminicola. UV mutagenesis was conducted to obtain a library of carboxin-resistant mutants. A range of SDHI resistance-conferring mutations was found in Sdh subunits B, C and D. Pathogenicity studies with a range of Sdh variants did not detect any fitness costs associated with these mutations. Most of the amino acid residues identified (e.g. B-S221P/T, B-H267F/L/N/Y, B-I269V and D-D129E/G/T) are directly involved in forming the cavity in which SDHI fungicides bind. Docking studies of SDHI fungicides in structural models of wild-type and mutated Sdh complexes also indicated which residues were important for the binding of different SDHI fungicides and showed a different binding for fluopyram. The predictive power of the model was also shown. Further diagnostic development, enabling the detection of resistant alleles at low frequencies, and cross-resistance studies will aid the implementation of anti-resistance strategies to prolong the cost-effectiveness and lifetime of SDHI fungicides.

Overexpression of the sterol 14α-demethylase gene (MgCYP51) in Mycosphaerella graminicola isolates confers a novel azole fungicide sensitivity phenotype

BACKGROUND The recent evolution towards resistance to azole fungicides in European populations of the wheat pathogen Mycosphaerella graminicola has been caused by the progressive accumulation of mutations in MgCYP51 gene, encoding the azole target sterol 14α-demethylase. Particular combinations of mutations have been shown specifically to affect the interaction of the MgCYP51 protein with different members of the azole class. Although additional mechanisms, including increased MgCYP51 expression and enhanced active efflux, have been proposed, the genetic changes underlying these mechanisms are unknown. RESULTS Analysis of the azole sensitivities of recent M. graminicola isolates identified a novel phenotype, seemingly independent of changes in MgCYP51 coding sequence. Characterised by a 7-16-fold reduction in in vitro sensitivity to all azoles tested and by growth on seedlings at higher doses of azoles in glasshouse tests compared with isolates carrying the same MgCYP51 variant (L50S, S188N, I381V, ΔY459/G460, N513K), isolates with this phenotype constitutively overexpress MgCYP51 by between 10- and 40-fold compared with the wild type. Analysis of sequences upstream of the predicted MgCYP51 translation start codon identified a novel 120 bp indel, considered to be an insertion, in isolates overexpressing MgCYP51. CONCLUSIONS The identification of an insertion in the predicted MgCYP51 promoter in azole-resistant isolates overexpressing MgCYP51 is the first report of a genetic mechanism, other than changes in target-site coding sequence, affecting sensitivity to multiple azoles in field isolates of M. graminicola. The identification of recent isolates overexpressing MgCYP51 confirms the ongoing evolution and diversification of resistance mechanisms in European populations of M. graminicola.

DArT markers: diversity analyses, genomes comparison, mapping and integration with SSR markers in Triticum monococcum

BackgroundTriticum monococcum (2n = 2x = 14) is an ancient diploid wheat with many useful traits and is used as a model for wheat gene discovery. DArT (D iversity Ar rays T echnology) employs a hybridisation-based approach to type thousands of genomic loci in parallel. DArT markers were developed for T. monococcum to assess genetic diversity, compare relationships with hexaploid genomes, and construct a genetic linkage map integrating DArT and microsatellite markers.ResultsA DArT array, consisting of 2304 hexaploid wheat, 1536 tetraploid wheat, 1536 T. monococcum as well as 1536 T. boeoticum representative genomic clones, was used to fingerprint 16 T. monococcum accessions of diverse geographical origins. In total, 846 polymorphic DArT markers were identified, of which 317 were of T. monococcum origin, 246 of hexaploid, 157 of tetraploid, and 126 of T. boeoticum genomes. The fingerprinting data indicated that the geographic origin of T. monococcum accessions was partially correlated with their genetic variation. DArT markers could also well distinguish the genetic differences amongst a panel of 23 hexaploid wheat and nine T. monococcum genomes. For the first time, 274 DArT markers were integrated with 82 simple sequence repeat (SSR) and two morphological trait loci in a genetic map spanning 1062.72 cM in T. monococcum. Six chromosomes were represented by single linkage groups, and chromosome 4Am was formed by three linkage groups. The DArT and SSR genetic loci tended to form independent clusters along the chromosomes. Segregation distortion was observed for one third of the DArT loci. The Ba (black awn) locus was refined to a 23.2 cM region between the DArT marker locus wPt-2584 and the microsatellite locus Xgwmd33 on 1Am; and the Hl (hairy leaf) locus to a 4.0 cM region between DArT loci 376589 and 469591 on 5Am.ConclusionDArT is a rapid and efficient approach to develop many new molecular markers for genetic studies in T. monococcum. The constructed genetic linkage map will facilitate localisation and map-based cloning of genes of interest, comparative mapping as well as genome organisation and evolution studies between this ancient diploid species and other crops.

Apoplastic recognition of multiple candidate effectors from the wheat pathogen Zymoseptoria tritici in the nonhost plant Nicotiana benthamiana

Summary The fungus Zymoseptoria tritici is a strictly apoplastic, host‐specific pathogen of wheat leaves and causal agent of septoria tritici blotch (STB) disease. All other plants are considered nonhosts, but the mechanism of nonhost resistance (NHR) to Z. tritici has not been addressed previously. We sought to develop Nicotiana benthamiana as a system to study NHR against Z. tritici. Fluorescence microscopy and quantitative reverse transcription polymerase chain reactions were used to establish the interaction between Z. tritici and N. benthamiana. Agrobacterium‐mediated transient expression was used to screen putative Z. tritici effector genes for recognition in N. benthamiana, and virus‐induced gene silencing (VIGS) was employed to determine the role of two receptor‐like kinases (RLKs), NbBAK1 and NbSOBIR1, in Z. tritici effector recognition. Numerous Z. tritici putative effectors (14 of 63 tested) induced cell death or chlorosis in N. benthamiana. For most, phenotypes were light‐dependent and required effector secretion to the leaf apoplastic space. Moreover, effector‐induced host cell death was dependent on NbBAK1 and NbSOBIR1. Our results indicate widespread recognition of apoplastic effectors from a wheat‐infecting fungal pathogen in a taxonomically distant nonhost plant species presumably by cell surface immune receptors. This suggests that apoplastic recognition of multiple nonadapted pathogen effectors may contribute to NHR.

Genetic structure and population dynamics of a heteroecious plant pathogen Melampsora larici‐epitea in short‐rotation coppice willow plantations

Complex life strategies are common among plant pathogens belonging to rust fungi (Uredinales). The heteroecious willow rust Melampsora larici‐epitea produces five spore stages and alternates on larch (Larix). To shed light on the epidemiology of this pathogen, amplified fragment length polymorphisms (AFLPs) were used to determine the genetic diversity and genetic structure of rust samples collected from coppice willow (Salix) plantations at three UK sites (LA, CA and MC) over three sampling dates (September 2000, July 2001 and September 2001). Of the total of 819 isolates, 465 were unique AFLP phenotypes and there was a shift in genotype diversity between the two seasons (0.67 in 2000 and 0.87–0.89 in 2001). No phenotypes were common between the two seasons within a site, suggesting that the rust did not overwinter as an asexual stage within plantations. A temporal analysis detected large amounts of genetic drift (FS = 0.15–0.26) between the two seasons and very small effective population sizes (Ne = 2–3) within sites. These results all point to a new colonization of the plantations by the rust in the second season (2001). The FST‐analogue values were ΦCT = 0.121, Weir and Cockerham’s θ = 0.086 and the Bayesian estimate θB = 0.087–0.096. The results suggest that the sources of inoculum were somewhat localized and the same sources were mainly responsible for disease epidemics in LA and CA over the two seasons. The relatively low FST‐values among sites (0.055–0.13) suggest the existence of significant gene flow among the three sampled sites.

Pathogenic variation in poplar rust Melampsora larici-populina from England

AbstractUsing a leaf disc method, 19 isolates of the poplar rust, Melampsoralarici-populina , and one isolate of M.␣populnea from England were inoculated on to 25 poplar clones belonging to Populus nigra and P.␣trichocarpa, and hybrids between P. deltoides and P. nigra, P. deltoidesand P. trichocarpa, P.␣tacamahaca and P.␣trichocarpa, and P. alba and P. tremula. Disease was scored based on the pustule area and inoculum density. In terms of whether sporulating uredinia formed, the 19 isolates showed seven different patterns to the tested poplar clones. The majority of the rust isolates infected P. nigra ‘P3090’ and ‘Vereecken’, P.␣nigra × P. deltoides ‘Casale’ and ‘Tasman’, P. tacamahaca × trichocarpa ‘36’ and ‘Balsam Spire’, and P.␣trichocarpa ‘Blom’. Populus trichocarpa × P. deltoides ‘69039/4’ was infected by only three isolates collected from southern England. No visible symptoms appeared on P. alba  × P. tremula‘Tower’ and P.␣trichcarpa × P. deltoides × P. deltoides‘76028/5’ in inoculations with M. larici-populina isolates. Populus alba × P.␣tremula ‘Tower’ was infected only by M. populnea. When M.larici-populina isolates were tested using AFLP, no differences were found either between isolates from different geographical regions or between those having ‘narrow’ spectrum of virulence and those showing ‘wide’ spectrum of virulence on the tested clones. The results suggest that the UK rust populations possess virulences which were found in races E1, E2, E3 and E4 in continental Europe and that rust having virulence patterns similar to race E4 has occurred in UK poplar plantations since 1996.

Quantitative inoculation of willow rust Melampsora larici-epitea with the mycoparasite Sphaerellopsis filum (teleomorph Eudarluca caricis).

The mycoparasite Sphaerellopsis filum (teleomorph Eudarluca caricis) was applied simultaneously with Melampsora larici-epitea on to willow leaf discs using eight concentrations of conidia. Inoculum densities were quantified and the numbers of uredinia of the rust, pycnidia and conidia of S. filum and rust spores produced per leaf disc were measured 13 d after inoculation (first assessment). Higher S. filum inoculum densities resulted in more rust uredinia being infected, but did not reduce the number of uredinia produced. The ratios of infected rust pustules: S. filum conidia applied were in a range of 0.25-0.31 when less than 20 S. filum spores were inoculated on to a leaf disc (0.95 cm2). Suppressive effects of S. filum on rust spore production were more obvious in the second assessment, carried out 23 d after inoculation. Inoculum densities of S. filum were significantly (P < 0.001) correlated with the frequency of uredinia infected (% variance accounted for [VAF] = 85.8), the number of S. filum pycnidia (% VAF = 81.4), S. filum spores produced (% VAF = 72.3) and rust spore production (% VAF = 48.6). Rust spore production was significantly (P < 0.001) negatively correlated with the frequency of uredinia infected (% VAF = 51.1), the number of S. filum pycnidia (% VAF = 42.0) and the number of S. filum spores produced (% VAF = 40.6). The best correlation was found between the number of pycnidia and the number of S. filum spores produced (% VAF = 88.8).

Genetic variation in Melampsora larici-epitea on biomass willows assessed using AFLP

Five pathotypes belonging to formae specialeslarici-epitea typica (LET), larici-retusae (LR) and larici-daphnoides (LD) of Melampsora larici-epitea were examined using amplified fragment length polymorphism (AFLP). Of 213 AFLP markers scored, several were found to be exclusive to different formae speciales. The dendrogram placed the five pathotypes into distinct groups. Within pathotypes, average Nei & Lis similarity coefficients were calculated as 0.71–0.85. The similarities were 0.66–0.72 among the three pathotypes within LET and 0.34–0.44 between pathotypes belonging to different formae speciales. When assessed using the Shannon index, the diversity within locations was estimated as 0.55–0.59, greater than that found within pathotypes (0.24–0.42). The average per-locus diversity was 0.37 among the pathotypes and 0.11 among the locations. When the data from both LET and LR isolates were examined using AMOVA, the majority of the variation (70.85%) was attributed to among pathotypes within location. When only LET types were included, approximately half of the variation was partitioned to among pathotypes within location and the other half to among the isolates within collection. It appears that the degree of differentiation of LET4 on S. × mollissima between Loughgall and Long Ashton sites has decreased markedly since 1992, when it was first detected.

Characterization of an antimicrobial and phytotoxic ribonuclease secreted by the fungal wheat pathogen Zymoseptoria tritici

Summary The fungus Zymoseptoria tritici is the causal agent of Septoria Tritici Blotch (STB) disease of wheat leaves. Zymoseptoria tritici secretes many functionally uncharacterized effector proteins during infection. Here, we characterized a secreted ribonuclease (Zt6) with an unusual biphasic expression pattern. Transient expression systems were used to characterize Zt6, and mutants thereof, in both host and non‐host plants. Cell‐free protein expression systems monitored the impact of Zt6 protein on functional ribosomes, and in vitro assays of cells treated with recombinant Zt6 determined toxicity against bacteria, yeasts and filamentous fungi. We demonstrated that Zt6 is a functional ribonuclease and that phytotoxicity is dependent on both the presence of a 22‐amino‐acid N‐terminal ‘loop’ region and its catalytic activity. Zt6 selectively cleaves both plant and animal rRNA species, and is toxic to wheat, tobacco, bacterial and yeast cells, but not to Z. tritici itself. Zt6 is the first Z. tritici effector demonstrated to have a likely dual functionality. The expression pattern of Zt6 and potent toxicity towards microorganisms suggest that, although it may contribute to the execution of wheat cell death, it is also likely to have an important secondary function in antimicrobial competition and niche protection.