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Phenotypic Variability of Leptosphaeria Lindquistii Phenotypic variability of Leptosphaeria lindquistii (anamorph: Phoma macdonaldii), a fungal pathogen of sunflower Ali Mohammad Roustaee, S Costes, Grégory Dechamp-Guillaume, Gérard Barrault To cite this version: Ali Mohammad Roustaee, S Costes, Grégory Dechamp-Guillaume, Gérard Barrault. Phenotypic vari- ability of Leptosphaeria lindquistii (anamorph: Phoma macdonaldii), a fungal pathogen of sunflower. Plant Pathology, Wiley, 2000, 49 (2), pp.227-234. 10.1046/j.1365-3059.2000.00451.x. hal-02875262 HAL Id: hal-02875262 https://hal.archives-ouvertes.fr/hal-02875262 Submitted on 19 Jun 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Open Archive Toulouse Archive Ouverte OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible This is an author’s version published in: https://oatao.univ-toulouse.fr/26443 Official URL : https://doi.org/10.1046/j.1365-3059.2000.00451.x To cite this version: Roustaee, Ali Mohammad and Costes, S and Dechamp-Guillaume, Grégory and Barrault, Gérard Phenotypic variability of Leptosphaeria lindquistii (anamorph: Phoma macdonaldii), a fungal pathogen of sunflower. (2000) Plant Pathology, 49 (2). 227- 234. ISSN 0032-0862 Any correspondence concerning this service should be sent to the repository administrator: [email protected] Phenotypic variability of Leptosphaeria lindquistii (anamorph: Phoma macdonaldii), a fungal pathogen of sunflower A. Roustaee, S. Costes, G. Dechamp-Guillaume and G. Barrault* Laboratoire de Biotechnologie et Ame´lioration des Plantes, Ecole Nationale Supe´rieure Agronomique de Toulouse Avenue de l’Agrobiopole, Auzeville-Tolosane, BP 107, F-31326 Castanet-Tolosan Cedex, France 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 308C and radial growth was slowest at 5 and 358C. 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 308C, 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 c ultivar, u sing a 1 –9 s cale 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. Keywords: aggressiveness, ascospores, black stem, Helianthus annuus, pycniospores Introduction maculae surround the back of the flower head. Early plant senescence results in yield losses of 10–30% The black stem disease of sunflower (Helianthus annuus) (Penaud, 1996), and reductions in oil content of the is caused by Phoma macdonaldii (Boerema, 1970), seeds (Maric et al., 1988) and thousand seed weight teleomorph Leptosphaeria lindquistii (Frezzi, 1968). (Carson, 1991). Although chemical control can be The pathogen has been reported in various European considered (Penaud, 1996), its efficiency and the opti- countries (Yugoslavia, Bulgaria, Hungary, Romania: mum time of application of the active ingredients Maric et al., 1988), Asia (Iran: Madjidieh-Ghassemi, remain to be ascertained. Within such a context, 1988; Pakistan: Siddique-Mirza et al., 1988; China: genetic variability for resistance of sunflower should Hua & Ma 1996), the Americas (USA: Acimovic (1984); be investigated, as it may to lead to the identification South America: Maric et al., 1988) and Australia of tolerant or resistant cultivars. However, such an (Acimovic, 1984). In France, the disease has been approach requires accurate knowledge of the variabil- spreading steadily since 1990, and the fungus is now ity of the pathogen, which has remained insufficient a major component of the pathogenic complex on so far. sunflower (Peres & Lefol, 1996). This paper reports on the phenotypic variability The disease is characterized mainly by the appearance present among Phoma macdonaldii isolates, as affected of black spots on the stem, around the petiole insertion by abiotic factors. In particular, the effects of culture point. Coalescing spots at the base of the stems develop medium, pH and temperature on growth, sporulation into a wide black sleeve (Peres & Lefol, 1996), and black and pycniospore germination of the pathogen were investigated. Aggressiveness of the isolates on a sus- ceptible cultivar was evaluated by a test on cotyledon *E-mail: [email protected] petioles. The objectives of this work were to provide information to permit selection of isolates to be included in a screening programme for sunflower resistance, TLD 15 W 33 lamps) and darkness to induce sporu- and to devise a test suitable for such screening. lation. A pycniospore suspension was obtained by placing one cm2 explant from theses cultures in a Petri Materials and methods dish containing 10 mL sterile distilled water. Mono- pycniospore cultures were prepared by serial dilutions (Barrault, 1989) and maintained on PDA for further Plant material subculture (Table 1). Seeds of a susceptible sunflower hybrid (Santiago) were disinfected for 5 min in a sodium hypochlorite solu- Monoascospore isolates tion (6 chlorometric degrees), rinsed three times in Monoascospore isolates (Table 1), obtained by the sterile distilled water and then sown uniformly at a dilution method (Barrault, 1989) from individual, depth of 2 cm in 40 × 30 × 25 cm plastic containers mature perithecia that had developed on overwintering filled with vermiculite. Plants were raised in a growth sunflower stems, were maintained on PDA. chamber regulated at 24 6 18C (light period, 14 h at 200 mEm¹2 s¹1, provided by Osram-Vialox, Molsheim, Conservation of isolates France, NAV-T 600 W lamps) and 17 6 18C (dark period, It is essential that the pathological and physiological 10 h), and at 75–85% relative humidity. Each container characteristics of isolates remain constant during was watered every third day with 500 mL of water, storage. The method described by Barrault (1989), containing 1 mL of a nutrient solution (NPK 6–3-6 and which proved adequate for the conservation of Pyreno- micronutrients; Substral, Boulogne-Billancourt, France). phora teres, was adapted for P. macdonaldii. A sun- flower stem fragment, sterilized at 1108C for 25 min, was placed on a culture of a monospore isolate on Fungal isolates PDA. Pycnidia were visible on the stem fragment after Monopycniospore isolates incubation for 15 days at 25 6 18C under a 12-h cycle Stem fragments showing characteristic symptoms were of illumination (37 mEm¹2 s¹1) and darkness. When cut into pieces (5 × 5 mm), surface-sterilized for 5 min in these fructifications developed, the stem fragment was a sodium hypochlorite solution (6 chlorometric degrees), placed in a sterile haemolysis tube containing CaCl2 washed three times (5 min) in sterile distilled water, crystals (as a dessiccant) at the bottom. Tubes were transferred to Petri dishes containing potato dextrose closed with an absorbent cotton plug, covered with agar (PDA, 39 g L¹1) and incubated for 8 days at 258C an aluminium foil and kept in the dark at 68C. This in the dark to allow mycelial growth. The dishes were method, used since 1996, has proved to be efficient for incubated for a further 10 days under alternating long-term storage of Phoma macdonaldii isolates (data periods of illumination (12 h, 37 mEm¹2 s¹1; Philips not published). Table 1 Isolates of Leptosphaeria lindquistii collected in different regions of France Isolatesa Pigmentation Pycnidiogenesis CZb Year Region Locality (De´partement) MA1 Citrine þþ absent 1997 Ile de France Egreville (77) MA2 Citrine þþ absent 1997 South–west Tour de Faure (46) MA3 Citrine þþþþþ present 1997 South–west Castanet (31) MA4 Citrine þ present 1997 South–west Saint Lys (31) MA5 Citrine green þþþ absent 1997 Ile de France Egreville (77) MA6 Citrine þþ present 1997 South–west Saint Lys (31) MA7 Citrine þþ present 1997 South–west Castanet (31) MP1 Citrine þþ present 1996 Ile de France Egreville (77) MP2 Citrine þþ present 1996 Ile de France Egreville (77) MP3 Citrine þþ present 1996 South–west Tour de Faure (46) MP4 Citrine þþþ present 1996
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