Mapping of quantitative trait loci for resistance to <Emphasis Type="Italic">Mycosphaerella pinodes</Emphasis> in <Emphasis Type="Italic">Pisum sativum</Emphasis> subsp. <Emphasis Type="Italic">syriacum</Emphasis>

Aschochyta blight, caused by Mycosphaerella pinodes, is one of the most economically serious pea pathogens, particularly in winter sowings. The wild Pisum sativum subsp. syriacum accession P665 shows good levels of resistance to this pathogen. Knowledge of the genetic factors controlling resistance to M. pinodes in this wild accession would facilitate gene transfer to pea cultivars; however, previous studies mapping resistance to M. pinodes in pea have never included this wild species. The objective of this study was to identify quantitative trait loci (QTL) controlling resistance to M. pinodes in P. sativum subsp. syriacum and to compare these with QTLs previously described for the same trait in P. sativum. A population formed by 111 F_6:7 recombinant inbred lines derived from a cross between accession P665 and a susceptible pea cultivar (Messire) was analysed using morphological, isozyme, RAPD, STS and EST markers. The map developed covered 1214 cM and contained 246 markers distributed in nine linkage groups, of which seven could be assigned to pea chromosomes. Six QTLs associated with resistance to M. pinodes were detected in linkage groups II, III, IV and V, which collectively explained between 31 and 75% of the phenotypic variation depending of the trait. While QTLs MpIII.1 and MpIII.2 were detected both for seedlings and field resistance, MpV.1 and MpII.1 were specific for growth chamber conditions and MpIII.3 and MpIV.1 for field resistance. Quantitative trait loci MpIII.1, MpII.1, MpIII.2 and MpIII.3 may coincide with other QTLs associated with resistance to M. pinodes previously described in P. sativum. Four QTLs associated with earliness of flowering were also identified. While dfIII.2 and dfVI.1, may correspond with other genes and QTLs controlling earliness in P. sativum, dfIII.1 and dfII.1 may be specific to P. sativum subsp. syriacum. Flowering date and growth habit were strongly associated with resistance to M. pinodes in the field evaluations. The relation observed between earliness, growth habit and resistance to M. pinodes is discussed.     

Detection of partial resistance quantitative trait loci against Didymella pinodes in Medicago truncatula

Ascochyta blight caused by Didymella pinodes (formerly Mycosphaerella pinodes) is one of the most important fungal diseases of pea (Pisum sativum) worldwide that can also infect the model legume Medicago truncatula. The objective of this study was to identify quantitative trait loci (QTLs) controlling resistance to D. pinodes in M. truncatula. Response to D. pinodes was studied under controlled conditions in seedlings of a population derived from the cross J6 × F83005.5, two M. truncatula lines that are, respectively, resistant and susceptible to D. pinodes. A combined map using two different recombinant inbred line populations was then used to identify the genomic regions bearing putative QTLs and to improve the position of the QTLs. A single QTL associated with resistance to D. pinodes was detected on linkage group 2, explaining up to 13 % of the total phenotypic variation for relative disease severity against the pathogen. Two simple sequence repeat markers, MTE80 and mtic890 (3 cM apart) were the ones most significantly associated with the QTL. These markers are located in bacterial artifical chromosomes AC119409 and AC125474, respectively, both of them overlapping on M. truncatula chromosome 2. The integration of QTL analysis and genomics in M. truncatula will contribute to the development of new markers and facilitate the identification of candidate genes for Ascochyta blight resistance.     

Identification of quantitative trait loci and candidate genes for specific cellular resistance responses against Didymella pinodes in pea

Key message

Phenotyping of specific cellular resistance responses and improvement of previous genetic map allowed the identification of novel genomic regions controlling cellular mechanisms involved in pea resistance to ascochyta blight and provided candidate genes suitable for MAS.

Abstract

Didymella pinodes, causing ascochyta blight, is a major pathogen of the pea crop and is responsible for serious damage and yield losses. Resistance is inherited polygenically and several quantitative trait loci (QTLs) have been already identified. However, the position of these QTLs should be further refined to identify molecular markers more closely linked to the resistance genes. In previous works, resistance was scored visually estimating the final disease symptoms; in this study, we have conducted a more precise phenotyping of resistance evaluating specific cellular resistance responses at the histological level to perform a more accurate QTL analysis. In addition, P665 × Messire genetic map used to identify the QTLs was improved by adding 117 SNP markers located in genes. This combined approach has allowed the identification, for the first time, of genomic regions controlling cellular mechanisms directly involved in pea resistance to ascochyta blight. Furthermore, the inclusion of the gene-based SNP markers has allowed the identification of candidate genes co-located with QTLs and has provided robust markers for marker-assisted selection.     

A Single,Plastic Population of Mycosphaerella pinodes Causes Ascochyta Blight on Winter and Spring Peas (Pisum sativum) in France

Plant diseases are caused by pathogen populations continuously subjected to evolutionary forces (genetic flow, selection, and recombination). Ascochyta blight, caused by Mycosphaerella pinodes, is one of the most damaging necrotrophic pathogens of field peas worldwide. In France, both winter and spring peas are cultivated. Although these crops overlap by about 4 months (March to June), primary Ascochyta blight infections are not synchronous on the two crops. This suggests that the disease could be due to two different M. pinodes populations, specialized on either winter or spring pea. To test this hypothesis, 144 pathogen isolates were collected in the field during the winter and spring growing seasons in Rennes (western France), and all the isolates were genotyped using amplified fragment length polymorphism (AFLP) markers. Furthermore, the pathogenicities of 33 isolates randomly chosen within the collection were tested on four pea genotypes (2 winter and 2 spring types) grown under three climatic regimes, simulating winter, late winter, and spring conditions. M. pinodes isolates from winter and spring peas were genetically polymorphic but not differentiated according to the type of cultivars. Isolates from winter pea were more pathogenic than isolates from spring pea on hosts raised under winter conditions, while isolates from spring pea were more pathogenic than those from winter pea on plants raised under spring conditions. These results show that disease developed on winter and spring peas was initiated by a single population of M. pinodes whose pathogenicity is a plastic trait modulated by the physiological status of the host plant.     

An Endogenous Suppressor of the Defense Response in Pisum sativum

Healthy pea plants contain a substance, tentatively called "endogenoussuppressor", which specifically suppresses the accumulationof pisatin in pea plants that is induced by treatment with CuCl₂or an elicitor from Mycosphaerella pinodes. This suppressorelicits the accumulation of phytoalexins in other legumes, suchas kidney bean, soybean and cowpea. The endogenous suppressorfunctions to delay the accumulation of pisatin, the activationof phenylalanine ammonialyase (PAL) and the accumulation ofmRNAs for PAL and chalcone synthase induced by the elicitorfrom M. pinodes. The substance specifically induces susceptibilityto nonpathogens, such as Mycosphaerella ligulicola and M. melonis,in pea out of four species of legume tested, but the effectis not cultivar-specific. Thus, the endogenous suppressor inhealthy pea plants suppresses a series of self-defense reactionsand induces susceptibility in pea plants in a species-specificmanner, being similar to the exogenous fungal suppressor fromthe pea pathogen, M.pinodes. (Received February 19, 1992; Accepted May 11, 1992)     

Non-Specific Induction of Pisatin and Local Resistance in Pea Leaves by Elicitors from Mycosphaerella pinodes, M. melonis and M. ligulicola and the Effect of Suppressor from M. pinodes

The accumulation of pisatin was induced non-specifically by elicitors prepared from the high molecular weight fraction (molecular weight: more than 10,000 daltons) of the spore germination fluid of three species of Mycosphaerella-plant pathogens in pea leaves with epidermis removed, regardless of the pathogenicity of the fungi to pea. Before the elicitation of pisatin synthesis, local resistance to infection by Mycosphaerella pinodes was induced by elicitors again non-specifically inpea leaves in which wax had been removed from the leaf surface. The substance responsible for local resistance could be extracted with ethylacetate from the elicitor-containing drop diffusate which was placed on pea leaves. The substance prevented the penetration of M. pinodes through heat-killed pea epidermis, but did not affect spore germination. The suppressor prepared from the low molecular weight fraction (molecular weight: less than 10,000 daltons) of the spore germination fluid of M.pinodes counteracted the ability of elicitors to induce both phases of resistance mechanisms.     

Role of Inhibitors of Fungal Growth in the Limitation of Leaf Spots Caused by Ascochyta pisi and Mycosphaerella pinodes

The phytoalexin, pisatin, was detected in host tissues 24 hafter inoculation of pea leaflets with spores of the leaf-spottingpathogens Ascochyta pisi and Mycosphaerella pinodes. Pisatincontinued to accumulate in infected tissue as A. pisi lesionsdeveloped and was present in inhibitory concentrations in thebrown tissue beyond the region colonized by the pathogen. During the formation of limited M. pinodes lesions, concentrationsof pisatin were highest 2 days after inoculation. Levels weremore variable and lower in older lesions which appeared to containno other inhibitors of germ-tube growth. Spreading lesions causedby M. pinodes on leaflets floating on water contained littleor no pisatin although little was released to the water below.These lesions did, however, contain other highly active inhibitorsof germ-tube growth. The significance of these results in terms of limitation oflesions are discussed. The ease with which M. pinodes lesionscan become progressive may reflect the greater ability of thispathogen to grow in high concentrations of pisatin under certainconditions.     

Specific Inhibition of Cell Wall-Bound ATPases by Fungal Suppressor from Mycosphaerella pinodes

Activities of phosphatases were found in the fractions whichwere solubilized from cell walls of both pea and cowpea seedlingswith 0.5 M NaCl. These phosphatases hydrolyzed triphosphonucleotidesin the order: UTP=CTP>GTP>ATP; and UTP=GTP>CTP=ATP,respectively. The activities of a pyrophosphatase and a p-nitrophenylphosphatasewere also detected in these fractions. The suppressor in thespore germination fluid of a pea pathogen,Mycosphaerella pinodes,inhibited all of these phosphatase activities in the fractionsolubilized from pea cell walls, but it rather enhanced onlythe activity of the ATPase among those phosphatases from thecowpea cell wall. Hydrolysis of ATP by a cell wall fractionof pea was also markedly inhibited by the suppressor, whilehydrolysis of ATP by similar fractions from cowpea, kidney beanand soybean were rather enhanced by the suppressor, as wellas by the elicitor. Thus, the cell wall-bound ATPases respondedto the suppressor species-specifically. These cell wall-boundATPases seemed to be different from the plasma membrane ATPasesin several respects. The results suggest that plants recognizethe fungal signals not only on their plasma membranes but alsoon their cell walls and, moreover that putative receptors forthe fungal signals might be located close to cell wall-boundATPases or might even be these ATPases themselves. (Received November 16, 1994; Accepted April 20, 1995)     

Transcriptomic and Proteomic Analyses of Resistant Host Responses in Arachis diogoi Challenged with Late Leaf Spot Pathogen,Phaeoisariopsis personata

Late leaf spot is a serious disease of peanut caused by the imperfect fungus, Phaeoisariopsis personata. Wild diploid species, Arachis diogoi. is reported to be highly resistant to this disease and asymptomatic. The objective of this study is to investigate the molecular responses of the wild peanut challenged with the late leaf spot pathogen using cDNA-AFLP and 2D proteomic study. A total of 233 reliable, differentially expressed genes were identified in Arachis diogoi. About one third of the TDFs exhibit no significant similarity with the known sequences in the data bases. Expressed sequence tag data showed that the characterized genes are involved in conferring resistance in the wild peanut to the pathogen challenge. Several genes for proteins involved in cell wall strengthening, hypersensitive cell death and resistance related proteins have been identified. Genes identified for other proteins appear to function in metabolism, signal transduction and defence. Nineteen TDFs based on the homology analysis of genes associated with defence, signal transduction and metabolism were further validated by quantitative real time PCR (qRT-PCR) analyses in resistant wild species in comparison with a susceptible peanut genotype in time course experiments. The proteins corresponding to six TDFs were differentially expressed at protein level also. Differentially expressed TDFs and proteins in wild peanut indicate its defence mechanism upon pathogen challenge and provide initial breakthrough of genes possibly involved in recognition events and early signalling responses to combat the pathogen through subsequent development of resistivity. This is the first attempt to elucidate the molecular basis of the response of the resistant genotype to the late leaf spot pathogen, and its defence mechanism.     

Role of Cell-wall-degrading Enzymes in the Development of Leaf Spots Caused by Ascochyta pisi and Mycosphaerella pinodes

Extracts of limited and spreading lesions caused by Mycosphaerellapinodes on detached pea leaflets contained proteolytic, cellulolytic,and pectolytic enzymes although only in spreading lesions wasthere much degradation of cell walls. The brown tissue fromlimited M. pinodes lesions was resistant to maceration by enzymesfrom spreading lesions. Limited lesions contained water-soluble,95 per cent ethanol insoluble, partially dialysable, inhibitorsof pectin transeliminase which is probably the macerating enzyme. Green, spreading M. pinodes lesions developed only on leafletsfloating on water. Growth of these lesions was accompanied bycontinous loss of phenolic substances to the water while thephenol content in infected tissue remained similar to that inuninoculated controls. In contrast, the phenol content in mature,limited M. pinodes lesions on leaflets suspended just abovethe water level was about four times that in healthy tissue.It is suggested that loss of phenolics from floating leafletsprevents tissue browning and the development of resistance ofthe cell walls to maceration. But this type of resistance doesnot appear to be a major factor in the limitation of lesionson suspended tissue. Extracts of limited Ascochyta pisi lesions on leaflets floatingon water contained pectolytic and hemicellulolytic enzymes.Some cellulase (Cx) activity was detected although there waslittle evidence of cellulose degradation in cell walls in infectedtissue. The nature of the macerating factor remains uncertainbut it was found that extracts from lesions contained inhibitorsof pectic enzymes and that tissue just beyond that colonizedby the fungus was resistant to maceration; this resistance isprobably important in restricting the growth of the pathogenin the leaf.     

A complex genetic network involving a broad-spectrum locus and strain-specific loci controls resistance to different pathotypes of Aphanomyces euteiches in Medicago truncatula

A higher understanding of genetic and genomic bases of partial resistance in plants and their diversity regarding pathogen variability is required for a more durable management of resistance genetic factors in sustainable cropping systems. In this study, we investigated the diversity of genetic factors involved in partial resistance to Aphanomyces euteiches, a very damaging pathogen on pea and alfalfa, in Medicago truncatula. A mapping population of 178 recombinant inbred lines, from the cross F83005.5 (susceptible) and DZA045.5 (resistant), was used to identify quantitative trait loci for resistance to four A. euteiches reference strains belonging to the four main pathotypes currently known on pea and alfalfa. A major broad-spectrum genomic region, previously named AER1, was localized to a reduced 440 kb interval on chromosome 3 and was involved in complete or partial resistance, depending on the A. euteiches strain. We also identified 21 additive and/or epistatic genomic regions specific to one or two strains, several of them being anchored to the M. truncatula physical map. These results show that, in M. truncatula, a complex network of genetic loci controls partial resistance to different pea and alfalfa pathotypes of A. euteiches, suggesting a diversity of molecular mechanisms underlying partial resistance.     

Origin of pisatin demethylase (PDA) in the genus Fusarium

Host specificity of plant pathogens can be dictated by genes that enable pathogens to circumvent host defenses. Upon recognition of a pathogen, plants initiate defense responses that can include the production of antimicrobial compounds such as phytoalexins. The pea pathogen Nectria haematococca mating population VI (MPVI) is a filamentous ascomycete that contains a cluster of genes known as the pea pathogenicity (PEP) cluster in which the pisatin demethylase (PDA) gene resides. The PDA gene product is responsible for the detoxification of the phytoalexin pisatin, which is produced by the pea plant (Pisum sativum L.). This detoxification activity allows the pathogen to evade the phytoalexin defense mechanism. It has been proposed that the evolution of PDA and the PEP cluster reflects horizontal gene transfer (HGT). Previous observations consistent with this hypothesis include the location of the PEP cluster and PDA gene on a dispensable portion of the genome (a supernumerary chromosome), a phylogenetically discontinuous distribution of the cluster among closely related species, and a bias in G + C content and codon usage compared to other regions of the genome. In this study we compared the phylogenetic history of PDA, beta-tubulin, and translation elongation factor 1-alpha in three closely related fungi (Nectria haematococca, Fusarium oxysporum, and Neocosmospora species) to formally evaluate hypotheses regarding the origin and evolution of PDA. Our results, coupled with previous work, robustly demonstrate discordance between the gene genealogy of PDA and the organismal phylogeny of these species, and illustrate how HGT of pathogenicity genes can contribute to the expansion of host specificity in plant-pathogenic fungi.     

<Emphasis Type="Italic">STAYGREEN</Emphasis> (<Emphasis Type="Italic">CsSGR</Emphasis>) is a candidate for the anthracnose (<Emphasis Type="Italic">Colletotrichum orbiculare</Emphasis>) resistance locus <Emphasis Type="Italic">cla</Emphasis> in Gy14 cucumber

Key message

Map-based cloning identified a candidate gene for resistance to the anthracnose fungal pathogen Colletotrichum orbiculare in cucumber, which reveals a novel function for the highly conserved STAYGREEN family genes for host disease resistance in plants.

Abstract

Colletotrichum orbiculare is a hemibiotrophic fungal pathogen that causes anthracnose disease in cucumber and other cucurbit crops. No host resistance genes against the anthracnose pathogens have been cloned in crop plants. Here, we reported fine mapping and cloning of a resistance gene to the race 1 anthracnose pathogen in cucumber inbred lines Gy14 and WI 2757. Phenotypic and QTL analysis in multiple populations revealed that a single recessive gene, cla, was underlying anthracnose resistance in both lines, but WI2757 carried an additional minor-effect QTL. Fine mapping using 150 Gy14?×?9930 recombinant inbred lines and 1043 F₂ individuals delimited the cla locus into a 32 kb region in cucumber Chromosome 5 with three predicted genes. Multiple lines of evidence suggested that the cucumber STAYGREEN (CsSGR) gene is a candidate for the anthracnose resistance locus. A single nucleotide mutation in the third exon of CsSGR resulted in the substitution of Glutamine in 9930 to Arginine in Gy14 in CsSGR protein which seems responsible for the differential anthracnose inoculation responses between Gy14 and 9930. Quantitative real-time PCR analysis indicated that CsSGR was significantly upregulated upon anthracnose pathogen inoculation in the susceptible 9930, while its expression was much lower in the resistant Gy14. Investigation of allelic diversities in natural cucumber populations revealed that the resistance allele in almost all improved cultivars or breeding lines of the U.S. origin was derived from PI 197087. This work reveals an unknown function for the highly conserved STAYGREEN (SGR) family genes for host disease resistance in plants.

     

Bidirectional backcrosses between wild and cultivated lettuce identify loci involved in nonhost resistance to downy mildew

Key message

The nonhost resistance of wild lettuce to lettuce downy mildew seems explained by four components of a putative set of epistatic genes.

Abstract

The commonplace observation that plants are immune to most potential pathogens is known as nonhost resistance (NHR). The genetic basis of NHR is poorly understood. Inheritance studies of NHR require crosses of nonhost species with a host, but these crosses are usually unsuccessful. The plant-pathosystem of lettuce and downy mildew, Bremia lactucae, provides a rare opportunity to study the inheritance of NHR, because the nonhost wild lettuce species Lactuca saligna is sufficiently cross-compatible with the cultivated host Lactuca sativa. Our previous studies on NHR in one L. saligna accession led to the hypothesis that multi-locus epistatic interactions might explain NHR. Here, we studied NHR at the species level in nine accessions. Besides the commonly used approach of studying a target trait from a wild donor species in a cultivar genetic background, we also explored the opposite, complementary approach of cultivar introgression in a wild species background. This bidirectional approach encompassed (1) nonhost into host introgression: identification of L. saligna derived chromosome regions that were overrepresented in highly resistant BC1 plants (F1?×?L. sativa), (2) host into nonhost introgression: identification of L. sativa derived chromosome regions that were overrepresented in BC1 inbred lines (F1?×?L. saligna) with relatively high infection levels. We demonstrated that NHR is based on resistance factors from L. saligna and the genetic dose for NHR differs between accessions. NHR seemed explained by combinations of epistatic genes on three or four chromosome segments, of which one chromosome segment was validated by the host into nonhost approach.

     

The genetic organization and evolution of the broad host range mercury resistance plasmid pSB102 isolated from a microbial population residing in the rhizosphere of alfalfa

Employing the biparental exogenous plasmid isolation method, conjugative plasmids conferring mercury resistance were isolated from the microbial community of the rhizosphere of field grown alfalfa plants. Five different plasmids were identified, designated pSB101–pSB105. One of the plasmids, pSB102, displayed broad host range (bhr) properties for plasmid replication and transfer unrelated to the known incompatibility (Inc) groups of bhr plasmids IncP-1, IncW, IncN and IncA/C. Nucleotide sequence analysis of plasmid pSB102 revealed a size of 55 578 bp. The transfer region of pSB102 was predicted on the basis of sequence similarity to those of other plasmids and included a putative mating pair formation apparatus most closely related to the type IV secretion system encoded on the chromosome of the mammalian pathogen Brucella sp. The region encoding replication and maintenance functions comprised genes exhibiting different degrees of similarity to RepA, KorA, IncC and KorB of bhr plasmids pSa (IncW), pM3 (IncP-9), R751 (IncP-1β) and RK2 (IncP-1α), respectively. The mercury resistance determinants were located on a transposable element of the Tn5053 family designated Tn5718. No putative functions could be assigned to a quarter of the coding capacity of pSB102 on the basis of comparisons with database entries. The genetic organization of the pSB102 transfer region revealed striking similarities to plasmid pXF51 of the plant pathogen Xylella fastidiosa.     

Wild peas vary in their cross-compatibility with cultivated pea (Pisum sativum subsp. sativum L.) depending on alleles of a nuclear–cytoplasmic incompatibility locus

Key message

Divergent wild and endemic peas differ in hybrid sterility in reciprocal crosses with cultivated pea depending on alleles of a nuclear ‘speciation gene’ involved in nuclear–cytoplasmic compatibility.

Background

In hybrids between cultivated and wild peas, nuclear–cytoplasmic conflict frequently occurs. One of the nuclear genes involved, Scs1, was earlier mapped on Linkage Group III.

Results

In reciprocal crosses of seven divergent pea accessions with cultivated P. sativum, some alleles of Scs1 manifested incompatibility with an alien cytoplasm as a decrease in pollen fertility to about 50 % in the heterozygotes and lack of some genotypic classes among F2 segregants. Earlier, we defined monophyletic evolutionary lineages A, B, C and D of pea according to allelic state of three markers, from nuclear, plastid and mitochondrial genomes. All tested representatives of wild peas from the lineages A and C exhibited incompatibility due to Scs1 deleterious effects in crosses with testerlines of P. sativum subsp. sativum (the common cultivated pea) at least in one direction. A wild pea from the lineage B and a cultivated pea from the lineage D were compatible with the testerline in both directions. The tested accession of cultivated P. abyssinicum (lineage A) was partially compatible in both directions. The Scs1 alleles of some pea accessions even originating from the same geographic area were remarkably different in their compatibility with cultivated Pisum sativum cytoplasm.

Conclusion

Variability of a gene involved in reproductive isolation is of important evolutionary role and nominate Scs1 as a speciation gene.     

Fine genetic mapping of RXopJ4, a bacterial spot disease resistance locus from Solanum pennellii LA716

The RXopJ4 resistance locus from the wild accession Solanum pennellii (Sp) LA716 confers resistance to bacterial spot disease of tomato (S. lycopersicum, Sl) caused by Xanthomonas perforans (Xp). RXopJ4 resistance depends on recognition of the pathogen type III effector protein XopJ4. We used a collection of Sp introgression lines (ILs) to narrow the RXopJ4 locus to a 4.2-Mb segment on the long arm of chromosome 6, encompassed by the ILs 6-2 and 6-2-2. We then adapted or developed a collection of 14 molecular markers to map on a segregating F₂ population from a cross between the susceptible parent Sl FL8000 and the resistant parent RXopJ4 8000 OC₇. In the F₂ population, a 190-kb segment between the markers J350 and J352 cosegregated with resistance. This fine mapping will enable both the identification of candidate genes and the detection of resistant plants using cosegregating markers. The RXopJ4 resistance gene(s), in combination with other recently characterized genes and a quantitative trait locus (QTL) for bacterial spot disease resistance, will likely be an effective tool for the development of durable resistance in cultivated tomato.