Ultrastructural studies of the mode of penetration byPhoma macdonaldiiin sunflower seedlings

An ultrastructural investigation of the artificial inoculation of sunflower with Phoma macdonaldii conidia was undertaken using light, scanning, and transmission electron microscopy to elucidate the host-parasite relationship. The behavior of the conidia deposited on the cotyledon petiole was investigated at various time intervals after inoculation. Conidia adhesion and germination were observed first. The cotyledon petiole was invaded by the fungus directly through the cuticle and via stomata. Externally, the spore and germ tube were covered with a mucilaginous polysaccharide sheath of a cotton-like appearance and of variable thickness. At the time of penetration, the host cuticle was perforated mechanically. The cuticle was slightly depressed and no enzymatic alteration could be observed. The fungus did not form appressoria on the surface of the host tissues but developed an infection peg. As soon as the cuticle barrier was crossed, the fungus rapidly colonized the host parietal layer. In a first step, the plasmalemma of the host cell appeared to be stuck against the cell wall. As soon as the fungus passed through the epidermal cell wall to reach the host cytoplasm, the plasmalemma was disrupted, and the subsequent rapid breakdown of cell integrity favored the colonization of the tissues by the pathogen

Phenotypic variability of Leptosphaeria lindquistii (anamorph: Phoma macdonaldii), a fungal pathogen of sunflower

Growth of 17 isolates of Phoma macdonaldii , the causal agent of sunflower black stem, was investigated for response to pH and temperature, and for morphology and asexual morphogenesis (pycnidiogenesis and pycnidium size). For all isolates, the optimum pH for growth was between 4 and 5, and the optimum temperature varied between 20 and 30°C and radial growth was slowest at 5 and 35°C. Significant differences in the number and size of pycnidia were observed between isolates. Pycniospore germination was investigated under various conditions in five isolates chosen for their geographical origins, pigmentation, optimum growth temperature and pycnidiogenesis. Increasing the concentration from 106 to 107 pycniospores per mL decreased the germination rate. The optimum temperature for pycniospore germination varied between 15 and 30°C, depending on the isolate, and the optimum and maximum pH values were 5 and 7, respectively. The optimum and minimum relative humidities allowing pycniospore germination were 100 and 95%, respectively. Pycniospore germination was photo‐independent. An artificial inoculation method was developed and the aggressiveness of the pathogen was assessed on a susceptible sunflower cultivar, using a 1–9 scale that integrated the percentage of necrotic area on the cotyledon petiole at the stage when the first pair of leaves was fully developed. Significant differences in aggressiveness were observed among the 17 isolates. The parameters investigated clearly suggest the occurrence of a wide phenotypic variability in Phoma macdonaldii

A QTL analysis of sunflower partial resistance to downy mildew (Plasmopara halstedii) and black stem (Phoma macdonaldii) by the use of recombinant inbred lines (RILs)

Partial resistance to downy mildew (Plasmopara halstedii) and to black stem (Phoma macdonaldii) in sunflower were investigated under natural field infection and a controlled growth chamber respectively. Genetic control for resistance to the diseases was determined in recombinant inbred lines (RILs) and their two parents, ’PAC-2’ and ’RHA-266.’ The experiments were undertaken in a randomized complete block design with two replications, in a field severely infected by downy mildew and in a controlled growth chamber with plants inoculated with an agressive French isolate of P. macdonaldii. Each replication consisted of three rows, 4.6-m long, giving 48 plants per RIL or parent in the field and 15 plants in the growth chamber. Genetic variability was observed among the RILs for resistance to both diseases. When 10% of the selected RILs were compared with the mean of the two parents genetic gain was significant for partial resistance to the diseases. Four putative QTLs for resistance to downy mildew on linkage groups 1, 9 and 17 were detected using composite interval mapping. The QTLs explained 54.9% of the total phenotypic variance. Major QTLs (dmr1–1 and dmr1–2) for resistance were found on linkage group 1 with up to 31% of the phenotypic variability explained by two peaks. QTL analysis of resistance to black stem showed seven QTLs on linkage groups 3, 6, 8, 9, 11, 15 and 17. The detected QTLs together explain 92% of the phenotypic variation of the trait. Crosses between RILs contrasted for their resistance to downy mildew and black stem, and exhibiting molecular polymorphism in detected QTLs, will be made in order to focus more-precisely on the genomic region of interest

Electrophoretic Analysis of Total and Membrane Proteins of Xanthomonas campestris Pathovars, the Causal Agents of the Leaf Streak of Cereals and Grasses in Iran

Forty‐five Iranian isolates of Xanthomonas campestris obtained from wheat, barley and grasses were compared with reference strains using polyacrylamide gel electrophoresis (PAGE) of the whole‐cell and membrane proteins. The PAGE profiles of the whole‐cell and membrane proteins of the Iranian isolates obtained from barley, with the exception of IBLS 11 and IBLS 12, were identical and clearly distinguishable from those of the other isolates. The barley group isolates, which were pathogenic only to barley, were similar to UPB 458 (NCPPB 2389), the reference strain of pathovar hordei. The isolates obtained from wheat and grasses, as well as IBLS 11 and IBLS 12, which can infect wheat, barley and some wild grasses, had similar banding patterns: only IBLS 40 isolated from Hordeum sp. displayed the same profile as the barley group. Reference strains UPB 443 (NCPPB 2821) and UPB 513, which correspond to pathovars undulosa and translucens , respectively, were related to the wheat group. IBLS45, isolated from Bromus sp., had a banding pattern that differed from those observed for strains of the barley and wheat groups. The results suggest that this method can be useful for discriminating different pathovars of X. campestris attacking cereals and grasses, and sodium dodecyl sulphate (SDS)‐PAGE of membrane proteins was not more sensitive than SDS‐PAGE of total proteins for differentiating the isolates

Inheritance of partial resistance to black stem (Phoma macdonaldii) in sunflower

Two programmes to investigate the inheritance of resistance to black stem in sunflowers were undertaken in a controlled-environment growth chamber. In the first, an experiment was performed using a randomized complete block design with 24 lines, six male-sterile (A lines), six maintainers (B lines), six restorers (R lines) and their six F-1 hybrids in six replications. Each treatment consisted of 12 seedlings. Twelve-day-old seedlings were inoculated with a suspension of pycniospores, and 7 days later the two cotyledon petioles of each seedling were scored on a 1-9 scale for the percentage of necrotic area. Some alloplasmic lines (which have the same nucleus and different cytoplasms) gave a significant cytoplasmic effect for improved partial resistance to the disease. Deviation of F-1 hybrids from the mean of the parent values was significant for partial resistance to Phoma macdonaldii in three of the six F-1 hybrids. Two further experiments with eight lines (resistant and susceptible) at the fifth leaf-pair and flowering stages were carried out under the same conditions. At both growth stages the previous classification of lines at the seedling stage was confirmed. In the second programme, five male-sterile sunflower lines were crossed with five fertility-restorers in a factorial mating design. The 10 inbred lines and their 25 F-1 hybrids were studied in two successive experiments under the same conditions and with the same experimental design and isolate of Phoma as in the seedling-stage experiment in the first programme. Analysis of variance showed that male-sterile and restorer lines possessed general combining abilities, and also that specific combining abilities of F-1 hybrids were significant. The estimates of general combining ability for partial resistance were significant in AS617A, AS618A and AS614R inbred lines. These lines are available for developing F-1 hybrids with improved resistance to Phoma in sunflower-breeding programmes

Genetic analysis of partial resistance to bacterial leaf streak ( Xanthomonas campestris pv. cerealis ) in wheat

Genetic variability of partial resistance to bacterial leaf streak was investigated in hexaploid winter wheat (Triticum aestivum. ), using 16 parental genotypes and 48 pure lines (F10) derived from a composite cross programme. Two experiments were undertaken in a controlled growth chamber. Seeds of all genotypes were grown under controlled conditions using a randomized block design with three replications. Each replication consisted of a row of 20 seedlings of each parent and pure line. An Iranian strain of bacterial leaf streak was used for the inoculation of 12‐day‐old seedlings. In a third experiment, eight genotypes from parents and F10 pure lines representing a large variability for partial resistance were inoculated with four other Iranian strains of bacterial leaf streak. A large genetic variability was observed amongst the 64 genotypes for partial resistance to the disease. Partial resistance heritability estimates were rather high (70%), indicating that the resistance factors may be transmitted by crossing. Amongst all genotypes investigated, ‘DC2‐30‐N2’ and ‘IBPT‐66’ displayed the highest partial resistance to the disease. Significant correlations between strains in the third experiment show that a genotype resistant or susceptible to one strain will have similar reactions with other strains. No significant genetic gain was observed for partial resistance in the best pure line of the 48 lines studied, when compared with the best parental line. Increasing the number of pure lines is likely to result in the identification of genotypes that might prove to be more resistant

Potential of doubled-haploid lines and localization of quantitative trait loci (QTL) for partial resistance to bacterial leaf streak (Xanthomonas campestris pv. hordei) in barley

Genetic variability for partial resistance to bacterial leaf streak in barley, caused by Xanthomonas campestris pv. hordei, was investigated in 119 doubled-haploid lines (DH) developed by the Hordeum bulbosum method from the F1 progeny of the cross between two cultivars, ‘Morex’ (resistant) and ‘Steptoe’ (susceptible). Two experiments were undertaken in a randomized complete block design with three replicates, in a controlled growth chamber. Twenty seeds per replicate were planted in plastic containers (60×40×8 cm) containing moistened vermiculite. At the two-leaf stage seedlings were inoculated with an Iranian strain of the pathogen. Genetic variability was observed among the 119 DH lines for partial resistance to the disease. Some DH lines were significantly more resistant than ‘Morex’ (resistant parent) to bacterial leaf streak. Genetic gain in percentage of resistant parent for 5% of the selected DH lines was significant (47.70% and 33.72% in the first and the second experiment, respectively). A QTL analysis of bacterial leaf streak resistance showed that three QTLs were detected on chromosomes 3 and 7. Multilocus allelic effects of the three QTLs account for almost 54% of the mean difference between the parents and nearly 30% of the phenotypic variation of the trait in the mean experiment. The resistance locus on chromosome 3, near ABG377, apprears to be a major gene

Phoma du tournesol (déterminisme de la tolérance de l'hôte à la maladie)

La maladie des taches noires du tournesol, dont l'agent responsable est le champignon Phoma macdonaldii, n'a jusqu'alors été que très peu étudiée. En France, si son apparition remonte au début des années 1980, elle provoque aujourd'hui des dégâts de plus en plus inquiétants. Elle a été classée, en 2004, deuxième maladie plus importante après le Mildiou, par les acteurs de la filière tournesol. Aucun génotype de tournesol n'a pu à ce jour être répertorié comme résistant à la maladie ; nous ne disposons que de génotypes tolérants. Nos travaux, qui se sont orientés selon trois axes, ont permis de mettre en évidence différents points : - Une étude histologique a révélé que le premier frein à la pénétration du champignon dans les cellules de l'hôte semble être des ornementations cuticulaires particulières plus prononcées chez le génotype tolérant par rapport au génotype sensible. Une cinétique de l'infection a également été réalisée. - L'étude des phytoalexines du tournesol, la scopolétine et l'ayapine, a permis de mettre en évidence que ces dérivés coumariniques ont un effet inhibiteur important sur la germination du champignon, et que l'ayapine est également capable d'inhiber la croissance de Phoma macdonaldii. De plus, le dosage de la scopolétine in planta, à différents temps après contamination par le Phoma, a prouvé que la molécule présentait un niveau de synthèse et d'accumulation jusqu'à deux fois plus élevé chez le génotype tolérant étudié par rapport au génotype sensible. - Enfin, grâce à la mise au point d'une puce à ADN composée de 1000 unigènes, dont plus de 100 gènes impliqués dans la résistance des plantes aux maladies, nous avons pu mettre en exergue l'expression différentielle de certains gènes entre un génotype tolérant et génotype sensible, en réponse à l'infection. Ces gènes pourraient être impliqués dans la tolérance du tournesol à Phoma macdonaldii. Les différents résultats obtenus nous laissent envisager une amélioration du tournesol afin d'augmenter son niveau de résistance aux maladies.TOULOUSE-ENSAT-Documentation (315552324) / SudocSudocFranceF

La maladie des tâches noires du tournesol causée par Phoma macdonaldii Boerema (variabilité phénotypique et moléculaire)

Ce travail relatif à l'étude du pathosystème Phoma-Tournesol, s'est orienté selon 3 axes : l'étude de la variabilité phénotypique et moléculaire du champignon, l'évaluation de la sensibilité des génotypes de tournesol et les modalités de l'infection. Les résultats suivants ressortent de cette étude. 1) Le Phoma présente une importante variabilité phénotypique (croissance, pycnidiogénèse, pigmentation, agressivité). La croissance, à 25C, à la lumière, est corrélée négativement avec l'agressivité. La forte agressivité des isolats est associée à une pigmentation atypique, à une origine géographique (Sud-Ouest) et à un type de symptôme (tige). L'importante variabilité moléculaire observée n'est pas corrélée avec les caractéristiques phénotypiques. 2) Le comportement des génotypes est identique quel que soit le stade phénologique ou l'environnement. La maladie évolue rapidement pour les génotypes sensibles. A partir de la floraison, l'intensité des symptômes est accrue. 3) Au cours de la pénétration, aucune structure d'infection spécialiasée de type appressorium n'a été observé. Sur les pétioles de cotylédons, la durée d'incubation est majorée de 25 % pour la variété tolérante. Le champignon, chez le génotype sensible, atteint les vaisseaux conducteurs de l'hôte quarante-huit heures après la contamination alors que chez la variété tolérante, il se cantonne dans la zone corticale. Sur le limbe, le champignon reste localisé dans la zone corticale.TOULOUSE-ENSAT-Documentation (315552324) / SudocSudocFranceF

Assessment of sunflower genotype tolerance to Phoma macdonaldii

Sunflower genotypes with differing susceptibility to Phoma macdonaldii, the causal agent of sunflower black stem disease, were inoculated artificially with an aggressive fungal strain according to various modalities. A first series of experiments carried out in a greenhouse on plants at various phonological stages (2-leaf pairs, 5-6 leaf pairs, budding and early flowering) showed that the behaviour of sunflower cultivars, distributed into different susceptibility groups, remained unchanged irrespective of the phenological stage reached at the time of the inoculation. The development of the symptoms varied with genotype susceptibility to the disease: it was fast with coalescing necrotic spots on the stems for the most susceptible cultivars. The intensity and the development rate of the symptoms were also increased when the inoculation had been carried out at later stages. Two additional series of experiments were carried out in the field. In each series, a first block accounted for natural contaminations and a second block was inoculated artificially. The development of the symptoms and the susceptibility groups within the two blocks were identical to those observed in the greenhouse. These results led to the validation of an early inoculation test, at the cotyledon stage, which accounts satisfactorily for the behaviour of sunflower cultivars under natural conditions and is thus likely to be used in breeding programs