The sunflower crop is faced to several diseases caused by fungi and oomycetes in all the regions where it is cultivated. The use of resistant sunflower varieties is an efficient way to control the diseases, and resistance to these diseases re- mains a major target for sunflower breeding . During the last two decades, the premature death  or premature rip- ening (PR) induced by Phomamacdonaldii Boerema (teleo- morph: Leptosphaeria lindquistii) became the most severe and widespread sunflower disease in France, and could be partly responsible for the yield stagnation around 2.5 t.ha −1 . P. macdonaldii is also responsible for black stem disease (BS). However, the damages of PR on seed yield appeared greater than those of black stem . As the chemical con- trol of the disease remains difficult, potentially dangerous for the environment and becomes less socially acceptable, the development of even partially resistant varieties is an important breeding objective. Some genetic variability has been described for sunflower resistance to BS disease [4, 5] and PR . Several studies dealing with the genetic control of sunflower resistance to Phoma attacks on petiole stem base and roots of seedlings in growth chamber have demonstrated the quantitative character of this resistance [6–11]. Cytological observations of a susceptible and of a more resistant inbred line showed that the development of fungal hyphae within the stele was affected in the more re- sistant genotype, suggesting the involvement of a plant compound in the defense . In addition, candidate genes could be identified through transcriptomic studies of the sunflower * P. macdonaldii interaction (e.g. sunflower-like- lipase, MYB-related transcription factor regulating PAL2, a key enzyme involved in the phenylpropanoid pathway) . However, in these studies, the pathosystem was developed on two-leaf-stage plantlets that is not fully representative of natural attacks. In field or in greenhouse, P. macdonaldii has been found to cause higher damages when contamin- ation occurs at the star bud phenological stage (E1) than at earlier phenological stages . Recently, we demonstrated the clear role of aerial Phoma infection in PR compared with soilborne inoculums . In addition, we showed that artificial inoculation at the stem base with pycniospores or mycelium of P. macdonaldii could be used for screening genotypes showing a substantial level of resistance to PR. Using this phenotyping protocol in field, we investigated in this study the phenotypical variability and the genetic archi- tecture of premature ripening and black stem resistance along plant development in Helianthus.
phora teres, was adapted for P. macdonaldii. A sun-
flower stem fragment, sterilized at 110 8C for 25 min, was placed on a culture of a monospore isolate on PDA. Pycnidia were visible on the stem fragment after incubation for 15 days at 25 6 18C under a 12-h cycle of illumination (37 mE m ¹2 s ¹1 ) and darkness. When these fructifications developed, the stem fragment was placed in a sterile haemolysis tube containing CaCl 2 crystals (as a dessiccant) at the bottom. Tubes were closed with an absorbent cotton plug, covered with an aluminium foil and kept in the dark at 6 8C. This method, used since 1996, has proved to be efficient for long-term storage of Phomamacdonaldii isolates (data not published).
Laboratoire de Biotechnologie et Amélioration des Plantes (BAP), IFR 40, INP-ENSAT, 18 Chemin de Borde Rouge, BP 32607, 31326 Castanet Tolosan, France
Black stem, caused by Phomamacdonaldii, is one of the most important diseases of sunflower in the world. Quantitative trait loci (QTLs) implicated in partial resistance to two single pycnidiospore isolates of P. macdonaldii (MP8 and MP10) were investigated using 99 recombinant inbred lines (RILs) from the cross between sunflower parental lines PAC2 and RHA266. The experimental design was a randomized complete block with three replications. High genetic variability and transgressive segregation were observed among RILs for partial resistance to P. macdonaldii isolates. QTL-mapping was performed using a recently developed high-density SSR/AFLP sunflower linkage map. A total of 10 QTLs were detected for black stem resistance. The phenotypic variance explained by each QTL (R 2
girdling symptoms appearance were found in this study whereas N supply was shown to enhance disease severity in previous studies (Debaeke and Pérès, 2003; Seassau et al., 2010). Leaf node infections were enhanced by nitrogen fertilisation as observed by Debaeke and Pérès (2003). High nitrogen fertilisation could lead to greater petiole trough size. Petiole trough appeared when sunflower plants have between ten and twelve leaves and it acts as a receptacle for free water. The water-holding capacity of these leaf structures is greater and appears earlier at low plant densities especially when N nutrition is plentiful. Phoma ascospores polluting petiole trough and groove encounter favourable conditions for germination and penetration in the plant even when contaminating rain was followed by dry period. Leaf pathogen such as Phomopsis helianthi is more dependent on microclimatic conditions due to infection point at leaf margin (hydathodes) that makes the fungus more sensitive to relative humidity: humidity saturation is needed for 36 h to result in plant contamination (Desanlis et al., 2013). Petiole susceptibility increases with growth stage as petiole trough deepens with plant growth being thus more prone to water storage (Délos et al., 1997b). In addition, high nitrogen availability favours the bursting of very turgid petiole tissues that facilitates fungus penetration in the plant. Indeed, Phomamacdonaldii penetrates into plants either directly by mechanical pressure or via enzymatic degradation of the cell wall, or indirectly through wounds and natural openings such as lenticels and stomata (Roustaee et al., 2000c).
mean of the parent values was significant for partial resistance to Phomamacdonaldii 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
Une variabilité génétique de la résistance partielle du tournesol au phoma a été décrite aussi bien en champs qu’en conditions contrôlées (Pérès et al., 1994 ; Roustaee, 2000a; Rachid Al-Chaarani et al., 2002; Abou Al Fadil et al., 2004; Bert et al., 2004)). En conditions contrôlées, cette variabilité génétique a été mise en évidence grâce à des familles F3 issues d’un croisement entre un mutant, partiellement résistant et la lignée originelle correspondante, sensible (Abou al Fadil et al., 2004). Roustae et al., (2000a), par utilisation de génotypes parentaux et leurs hybrides F1, ont montré que la variation observée entre génotypes était due à l’aptitude générale à la combinaison (AGC). De ce fait, la majeure partie de la variabilité est attribuée à des effets additifs des gènes. Des lignées recombinantes (Recombinant Inbred Lines, RILs) dérivée d’un croisement entre les génotypes PAC-2 et RHA-266 ont été inoculées par un isolat français agressif de Phomamacdonaldii et 7 QTLs (Quantitative Trait Loci) furent identifiés (Rachid Al-Chaarani, et al. 2002). Ces QTLs permettaient expliquer 92%de la variation phénotypique de la résistance partielle du tournesol. Bert et al. (2004) utilisèrent des familles F2-F3 dérivées d’un croisement entre les génotypes PAZ2 et FU, et mirent en évidence 4 QTLs contrôlant la résistance au phoma.
Abstract Phomamacdonaldii is one of the most important pathogens of sunflower (Heliantus annuus) in France. In order to determine the inheritance of resistance to the disease, five sunflower genotypes with wide genetic variability for resistance to two ‘collar’ and two ‘root’ Phoma isolates were crossed in a diallel programme. Four separate experiments were undertaken under controlled conditions. In each one, the response of parental genotypes and their F1 hybrids were evaluated with one of the four Phoma isolates. Analysis of variance was performed to determine the effects of genotype on disease severity score when inoculated with ‘collar’ or ‘root’ Phoma isolates and showed significant variability among parents and F1 hybrids for disease severity score. Diallel analysis showed that general combining ability (GCA) and specific combining ability (SCA) effects for resistance to ‘collar’ and ‘root’ Phoma isolates were highly significant for each of the four isolates indicating that both kinds of gene effects were important in controlling the resis- tance. The GCA/SCA ratios were more than one for three out of four isolates showing that additive genetic effects were more important than non-
To date, sunflower genotypes with partial resis- tance to P. macdonaldii have been described, but no fully resistant genotypes are available. Previous work showed that partial resistance to black stem disease is conferred by more than one gene with additive effects (Roustaee et al. 2000a ). A more recent study with nine French isolates of P. macdonaldii and four sunflower genotypes suggested the presence of tissue-specific resistance genes (Abou Al Fadil 2006 ) Phomamacdonaldii penetrates into the plants either directly via enzymatic degradation of the plant cell wall or by mechanical pressure, or indirectly through wounds and natural openings such as lenticels and stomata (Isaac 1992 , Roustaee et al. 2000b ). On the same host plants, different tissues may be penetrated differently by the same fungus. A first small-scale transcriptomic study has shown differential expression of defence-related genes in petioles of susceptible and resistant lines (Alignan et al. 2006 ).
Abstract Partial resistance to downy mildew (Plasmo-
para halstedii) and to black stem (Phomamacdonaldii) in sunflower were investigated under natural field infec- tion and a controlled growth chamber respectively. Ge- netic control for resistance to the diseases was deter- mined 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. mac- donaldii. 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 pheno- typic variability explained by two peaks. QTL analysis of resistance to black stem showed seven QTLs on link- age 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 resis- tance 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.
After the exclusion of four ESTs showing significant interac- tion effects, only seven cDNA clones were identified as showing
treatment-specific expression patterns across all experiments (Fig. 2b). Five of these genes were induced in the inoculated plants (located in the upper right square of Fig. 2b) while two clones were found to be repressed (located in the upper left square). These regulated genes belonged to various functional categories and had P-values < 0.001 (summarized in Table 1). It is important to mention that two out of the five clones that were found to be up-regulated in inoculated plants are potentially involved in amino acid metabolism. These cDNA clones are thought to be involved in the biosynthesis of glutamate, arginine and proline (CD855351; EC:220.127.116.11), and tyrosine and phenylalanine (CD849150; EC:18.104.22.168). These results indicate that the metabolism of these amino acids as well as gluconeogenesis (glyceraldehyde 3-phosphate dehydrogenase; CD852523; EC:22.214.171.124) might be associated with plant responses to P. macdonaldii infection.
on two cultivars with artiﬁcial inoculation (AI) and natural infection (NI). Disease assessment was recorded weekly to calculate the area under disease progress curve (AUDPC) and the ﬁnal percentage of PR plants. Data showed that high levels of N fertilization led to signiﬁcantly (P < 0.05) more PR than non fertilization. Water deﬁcit conditions were signiﬁcantly (P < 0.05) involved in disease severity, and AUDPC and PR were increased when dry conditions were associated with high N supply. This was true for two cultivars which differed in their susceptibility to the disease but cv. Heliasol RM was signiﬁcantly (P < 0.05) more affected than cv. Melody, partially resistant to PR. Despite contrasting weather patterns, these results demonstrated a clear role of crop management and environmental conditions on the incidence and severity of stem base attacks responsible for the PR syndrome. These ﬁndings suggest that sunﬂower crop husbandry should be adapted to minimize premature ripening induced by P. macdonaldii.
This study was intended to identify the effect of sunﬂower crop management on PR induced by P. macdonaldii. The effect of nitro- gen and water supply on PR, previously observed, was conﬁrmed. The effect of plant density on disease expression was studied in interaction with these two factors. Field experiments demon- strated that the combination of high plant density and nitrogen fertilization in rainfed conditions after anthesis resulted in high disease pressure in two contrasting growing seasons. From the characterization of the canopy by agronomic indicators related to N shoot status, plant growth and architecture, stem base mor- phology, plant water satisfaction rate and microclimate, we can propose a conceptual framework of the effect of plant density on the expression of the disease ( Fig. 4 ). The microclimate apparently has a moderate effect on disease epidemiology and PR, unlike the other variables that directly affect PR. High leaf area index (due to high N fertilization and high plant density) results more rapidly in soil water exhaustion and a drop in transpiration after anthe- sis. Avoiding excessive N fertilization by using the soil N balance method could signiﬁcantly reduce disease severity. Also, manip- ulating the stem diameter, mainly through planting density and N supply, could be exploited more, instead of resorting to fungi- cide use. This is probably a morphological trait that breeders could exploit in the future. Indeed genetic tolerance to PR should be eval- uated in order to achieve complete non-chemical control ( Bordat et al., 2011 ). Promising cultivars, with thick stems, should there- fore be tested at high density and N supply under water-limited conditions, a procedure which could be used in resistance tests during breeding programmes for an effective control of sunﬂower PR.
- Un matériel d’adhésion permet dans les deux cas considérés, la fixation des spores à la surface de l’hôte. Cette gaine mucilagineuse avait déjà été décrite par Roustaee et al. (2000b) pour ce même couple hôte-parasite. Le mucilage est généralement composé de polyssacharides et parfois d’enzymes. Il permet de protéger la spore de la dessiccation et favorise l’adhésion au végétal. Pascholati et al. (1993), ont démontré que cette matrice protégeait Colletotrichum graminicola des métabolites toxiques et exsudats synthétisés par la plante en réponse à l’infection. Ce mucilage est retrouvé chez d’autres champignons phytopathogènes. Chez Phoma lingam (Abadie et Boudart, 1982), Aschochyta rabiei (Hohl et al., 1990), et Aschochyta fabae (Maurin et al., 1993), il ne présente aucune activité enzymatique. A l’opposé, chez Colletotrichum graminicola (Pascholati et al.,1993) et Erysiphe graminis f. sp. hordei (Pascholati et al.,1992), la matrice mucilagineuse produit des enzymes impliquées dans la dégradation des parois, généralement des cutinases. Deising et al. (1992) ont également mis en exergue le fait qu’une association cutinases et estérases pourrait permettre à l’agent pathogène de mieux adhérer à l’hôte, ce qui donnerait à ces enzymes un rôle nouveau dans le processus d’infection fongique. Les observations de Roustaee semblaient révéler que chez Phomamacdonaldii, ce mucilage ne présente aucune activité enzymatique (Roustaee et al., 2000b) ; il serait cependant judicieux d’effectuer des dosages biochimiques afin de confirmer ces constatations.
Phomamacdonaldii infects different tissues of sunflower and causes reduction in yield and oil content. The aim of present research was to identify genomic regions involved in partial resistance of sunflower to four Phomamacdonaldii basal stem and root necrosis isolates using our improved map constructed with 191 SSR and 304 AFLP markers. The experiment was conducted using F9 recombinant inbred lines (RILs) from a cross between ‘PAC2’ and ‘RHA266’. Results showed that ‘PAC2’ was more resistant than ‘RHA266’ to basal stem necrosis isolate ‘TA6’ and root necrosis isolate ‘TA4’. By contrast ‘RHA266’ was more resistant than ‘PAC2’ to basal stem necrosis isolate ‘TA9’ and root necrosis isolate ‘TA2’. Transgressive segregation was observed for partial resistance to all four isolates. Some recombinant lines presented partial resistance or susceptibility to all isolates. Twenty seven QTL with phenotypic variance ranging from 7 to 29% were detected. Among them 13 were ‘isolate specific’ and others were common for partial resistance to different isolates (isolate non specific). Most of the QTLs in common have major effects for resistance to each isolate. The ‘isolate non specific’ QTLs were located on linkage groups (LG) 5, 6, 8, 12, 13 and 15. The markers ‘HA3555’ on LG12 and ‘E33M48 26’ on LG6 as well as ‘E33M48 20’ on LG13, which are each linked to QTLs of different basal stem and root necrosis isolates, could be used in marker assisted selection to introduce tolerance to four Phomamacdonaldii isolates into elite sunflower breeding lines.
Keywords: Biplot, general combining ability (GCA), Helianthus annuus L., partial resistance, phoma black stem, specific combining ability (SCA).
Phoma black stem caused by Phomamacdonaldii is one of the most important diseases of sunflower in France (Debaeke & Pe´re`s, 2003). The disease is characterized mainly by black spots that appear on the stem at the base of leaf petioles and spread along stems. Black stem can occur at any time during the growing season, but is most common after flowering and the disease is most severe when abundant moisture is available during and after flowering. When phoma girdles the stem base, symptoms of premature ripening may occur (Donald et al., 1987) resulting in small heads and seeds that are empty or not completely filled, reducing seed and oil yields
It is generally considered that L. lindquistii overwinters as pseudothecia, pycnidia and mycelium produced on infected sunflower stubble residues (Gulya et al. 1997). Phoma black stem epidemic onset typically occurs at flowering, with lesions on the stem progressing from bottom to upper stem nodes (Schwanck et al. 2016). Lesions on the petiole or on the stem are associated to leaf senescence (Quiroz et al. 2014). In France, the main source of primary inoculum appears to consist in ascospores released from infected sunflower residues (Délos et al. 1997; Seassau et al. 2010; Bordat et al. 2011).The occurrence of pycnidia on lesions developed on the stems during the growing season were observed in the USA (McDonald 1964), and in the former Yugoslavia (Maric et al. 1988). In South West of France, however, the presence of pycnidia on black stem lesions has not been observed during epidemics (Délos et al. 1997; Bordat et al. 2011). In this area, the disease is therefore considered to be associated to monocyclic epidemics (sensu Van der Plank 1965; Zadoks and Schein 1979), i.e., to not involve secondary infections over the course of the crop cycle. Nevertheless, this appears to be an untested working hypothesis, since no study to our knowledge has formally explored the occurrence of secondary cycles in phoma black stem epidemics.
2.1. Résumé / Abstract
Résumé. À cause des impacts environnementaux de l’agriculture intensive, il est indispensable de concevoir des systèmes de culture innovants permettant le contrôle des bioagresseurs, mais moins consommateurs de pesticides et où l’efficacité des méthodes de lutte est conservée. La production intégrée nécessite de combiner les différentes méthodes de lutte pour répondre à des exigences économiques, écologiques et toxicologiques. Les expérimentations au champ sont difficiles pour tester des stratégies de production intégrée aux échelles régionales et pluriannuelles. SIPPOM-WOSR, Simulator for Integrated Pathogen Population Management for Winter OilSeed Rape, a été développé pour tester et hiérarchiser des stratégies de contrôle du phoma du colza, préservant l’efficacité des résistances spécifiques. Les variables d’entrée sont les pratiques culturales (dont le choix des variétés) et leur répartition spatiale, la quantité initiale, la répartition et la structure génétique initiale de l’inoculum primaire, ainsi que les conditions pédoclimatiques. Le modèle simule la sévérité de la maladie et les dommages associés pour chaque parcelle de la région, de même que les rendements, la marge brute et le coût environnemental des pratiques. Préalablement à l’utilisation du modèle pour aider à la conception de stratégies de production, il est nécessaire d’analyser la sensibilité du modèle aux variations des paramètres et des variables d’entrée. L’objectif de l’analyse de sensibilité présentée était de tester la sensibilité des variables d’état de SIPPOM (variables de sortie de chaque module) aux variations des paramètres. Un plan factoriel complet a été choisi pour analyser l’effet des variations des paramètres : trois valeurs de chaque paramètre ont été définies et testées sous des combinaisons de variables d’entrée contrastées, choisies selon leur impact attendu ou supposé sur le rendement et sur la maladie. L’analyse de sensibilité présente des variations des sorties attendues (à dire d’expert) en fonction des variations de entrées et paramètres. Les paramètres ayant le plus d’impact sur les sorties et qui nécessitent d’être estimé plus précisément ont été identifiés. Des améliorations possibles du modèle sont proposées. Les résultats de l’analyse de sensibilité montrent que SIPPOM peut être utilisé pour hiérarchiser des stratégies de contrôle, malgré l’incertitude de certains paramètres.
AVALIAÇÃO DO ATAQUE DA MANCHA DE PHOMA EM VARIEDADES DE CAFÉ ARÁBICA
C.A. Krohling, Engº Agrº Incaper; J.B. Matiello e S.R. Almeida, Eng o Agr os Mapa-Fundação Procafe, e V.R. Rodrigues, Engº Agrº Consultor autônomo, C.C.K. Krohling, Graduando em Administração