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 variability 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 30ЊC and radial growth was slowest at 5 and 35ЊC. 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 30ЊC, 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 susceptible 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 Ϯ 1ЊC (light period, 14 h at 200 ␮Em¹2 s¹1, provided by Osram-Vialox, Molsheim, Conservation of isolates France, NAV-T 600 W lamps) and 17 Ϯ 1ЊC (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 sunflower stem fragment, sterilized at 110ЊC 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 Ϯ 1ЊC under a 12-h cycle Stem fragments showing characteristic symptoms were of illumination (37 ␮Em¹2 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 25ЊC an aluminium foil and kept in the dark at 6ЊC. 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 ␮Em¹2 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 South–west Tour de Faure (46) MP5 Citrine green þþ present 1996 South–west Castanet (31) MP6 Citrine green þ present 1996 South–west Castanet (31) MP7 Citrine þþþ present 1996 South–west Saint Lys (31) MP8 Apricot þ present 1996 South–west Saint Lys (31) MP9 Citrine þþþ present 1996 Ile de France Egreville (77) MP10 Citrine þþþ present 1996 Ile de France Saint Pathus (77) aEach monopycniospore isolate (MP) was obtained from a different lesion and a different plant. Each monoascospore isolate (MA) was from a different perithecium overwintering under field conditions. bCZ, concentric zonation associated with the aerial development of the mycelium and the production of pycnidia. Growth and morphology Aggressiveness The effects of temperature (5, 10, 15, 20, 25, 30 and An inoculation technique was developed to assess 35ЊC) at pH 6 and of pH (4, 5, 6 and 7) at 25ЊC on the aggressiveness of isolates under controlled conditions. radial growth of the isolates on the PDA medium were The pathogen was cultured on PDA for 10 days at 25ЊC investigated. McIlvaine buffers (Anonymous, 1963) under continuous light. A pycniospore suspension was were used to control pH. The growth medium was prepared by flooding the plates with sterile distilled selected following preliminary experiments showing water and stirring mechanically. The concentration was the superiority of PDA over 5 and 10% V8 and adjusted to 106 pycniospores per mL and 20 ␮L of the Czapek-Dox for the growth and sporulation of four spore suspension were deposited with a micropipette polypycniospore isolates (data not published). The into the pit formed by the cotyledon petiole and the effects of temperature and pH were studied under a epicotyl of 12-day-old sunflower seedlings grown to 12-h photoperiod. Colony diameter was measured the first pair of developed leaves, stages 1–2 (Lancashire after 8 days and the isolates were also scored for et al., 1991). A drop of sterile distilled water was used presence or absence of concentric zonation and for pig- on the control plants. During the first 72 h after inocu- mentation of the mycelium. lation, the containers in which the plants were grown were covered with a polyethylene bag to promote pathogen development. Isolate aggressiveness was assessed Asexual morphogenesis (pycnidiogenesis) 10 days after inoculation, on a 1–9 scale, based on For each isolate, pycnidia were counted on three the percentage of necrotic petiole area: 1 (0–5%), 2 different sectors (each sector area 1 cm2) under a bino- (5–10%), 3 (10–20%), 4 (20–30%), 5 (30–40%), 6 cular microscope (Wild Heerbrugg) and 50 pycnidia (40–60%), 7 (60–80%), 8 (80–100%) and 9 (100% were measured on 10-day-old cultures grown as des- and necrosis extending to the epicotyl). cribed above. Diameters were measured under the A completely randomized block design was used with microscope (Biomed Leitz, Rueil-Malmaison, France). three replications (three containers per isolate tested All measurements were made on three replicate plates and 20 sites of contamination per replication). per isolate. Data analysis Pycniospore germination In each case, data were subjected to variance analysis, A Van Tieghem cell was used for assessing the possible and means were compared with the test of Newman– effect on germination of temperature (5, 10, 15, 20, 25, Keuls (P ¼ 0·05). Intensity measurements relative to 30 and 35ЊC), pH (4, 5, 6 and 7, using McIlvaine the aggressiveness of the isolates did not require any buffers), pycniospore concentration (10, 102,103,104, transformation for the standardization of distributions. 105,106 and 107 spores per mL), light (continuous light, continuous darkness and photoperiods of 12 h light at ¹ ¹ Results 37␮Em 2 s 1), and relative humidity (100, 98, 95 and 90%), according to the method used by Barrault (1989). Morphology The pycniospore suspension was spread onto a glass slide, then dried under sterile conditions, at a tempera- Concentric zonation was frequently associated with the ture below 30ЊC. The slide was then put on a glass stand aerial development of mycelium and the production inside a microchamber. The addition of sterile distilled of pycnidia, except for isolates MA1, MA2 and MA5. water at the bottom of the microchamber provided The colonies usually displayed a citrine to citrine green saturated humidity. Sterile water was replaced with colour, according to the nomenclature of Rayner (1970). saturated saline solutions at 20ЊC to obtain relative However, under all conditions tested, the mycelium humidities of 98%; (K2Cr2O7), 95% (Na2SO4,7H2O) of isolate MP8 produced apricot-coloured crystals and 90% (ZnSO4,7H2 O). Humidity was controlled (Table 1), which were released in the medium after with a wet chromel and a dry alumel thermocouple, mycelium autolysis. calibrated with standard solutions (K2SO4, 97% R.H.; LiCl, 13% R.H.). The voltmeter readings were used for Growth and temperature determination of relative humidity, using calibration curves. Variance analysis of growth was carried out separately In each experiment, the percentage germination was for each temperature in all isolates and for each assessed from the examination of 50 spores after 16 h isolate at different temperatures. Significant differences of incubation (after 36 h for the effect of R.H.) at 25ЊC between isolates were found for each temperature and in the dark. A pycniospore was considered germinated between temperatures for each isolate (Table 2). when the germ tube was longer than the spore itself. The data showed significant effects of temperature Three replicate slides per experimental condition were (F ¼ 843·24; P < 0·001) and isolate (F ¼ 9·5; P < 0·001) observed. on growth, as well as a significant isolate–temperature Table 2 Colony diameter (mm) of 13 Phoma macdonaldii isolates grown for eight days on PDA at pH 6 and under a 12-h photoperiod (37 ␮Em¹2 s¹1) in growth cabinets kept at different temperatures. The values represent the mean diameters of three replicate cultures per isolate and temperature

Isolates 5ЊC10ЊC15ЊC20ЊC25ЊC30ЊC35ЊC

MP3 e11ؒ25bd16ؒ42cd c26ؒ83cd b30ؒ58ea44ؒ00ba44ؒ92cd f7ؒ42a MP4 e9ؒ58bde17ؒ08cd cd28ؒ17cd bc36ؒ42cde a57ؒ75bab49ؒ83cd e7ؒ00a MP5 f7ؒ83ce13ؒ83dd23ؒ00dc37ؒ67cde b44ؒ83ba52ؒ00cd f6ؒ83a MP6 f13ؒ50ae23ؒ00bd28ؒ92cd c31ؒ58de a47ؒ33bb44ؒ25cd g6ؒ75a MP7 d7ؒ33cb17ؒ58cd b27ؒ92cd b34ؒ75cde a47ؒ83bb29ؒ00ed6ؒ50a MP8 e9ؒ83bd14ؒ58dc30ؒ59cc33ؒ25cde a44ؒ68bb38ؒ33de f6ؒ00a MP9 d9ؒ75bc21ؒ83bb36ؒ83bb41ؒ83bcde a57ؒ75ba60ؒ83bc d7ؒ25a MP10 d10ؒ08bc16ؒ58cd b24ؒ25da51ؒ42ba55ؒ92ba51ؒ92cd d7ؒ33a MA3 e6ؒ83cd15ؒ00dc27ؒ83cd b44ؒ58bcd b41ؒ17ba57ؒ00ce7ؒ58a MA4 d10ؒ50bc27ؒ42ab57ؒ75aa75ؒ25aa76ؒ92aa77ؒ42ad7ؒ75a MA5 f7ؒ08ce16ؒ33cd d30ؒ83cc38ؒ33cde b51ؒ75ba55ؒ17cf6ؒ83a MA6 d9ؒ92bc20ؒ00bc b31ؒ83ca46ؒ25bc a53ؒ00ba55ؒ83cd7ؒ00a MA7 e7ؒ08ce13ؒ58dd24ؒ42dc35ؒ67cde b51ؒ58ba70ؒ75ab e7ؒ00a

Means with the same letter do not differ signiﬁcantly, according to a Newman–Keuls test (P ¼ 0ؒ05). Letters to the right of each value refer to differences between isolates (read vertically); letters to the left of each value refer to differences between temperatures (read horizontally).

interaction (F ¼ 4·92; P < 0·001). Isolate MP3 grew at optimum concentrations, germination was close to the lowest rate, MA4 at the highest. 100% for some isolates (e.g. MP8), but much lower Radial growth was slowest at 5 and 35ЊC in all (70–75%) for others, such as MA7. The optimum isolates. MA3, MA5, MA7 and MP5 grew best at temperatures for pycniospore germination (at pH 6, 30ЊC, isolates MP4, MP6, MP7 and MP8 at 25ЊC, MA4, 106 pycniospores per mL) were 15–25ЊC (MP6) or MA6 and MP10 between 20 and 30ЊC, and MP3 and 15–30ЊC (MA7, MP8, MP9 and MP10). Germination MP9 between 25 and 30ЊC. was very low (1·3–5%) at 5ЊC for all isolates. The maximum temperature is likely to be above 35ЊC, since germination rates ranging between 32% for MA7 and Growth and pH 65% for MP10 were observed at that temperature The variance analysis of growth data showed a significant effect of pH (F ¼ 2104·84; P < 0·001), with the occurrence of an isolate *pH interaction (F ¼ 4·87; < Table 3 Colony diameter (mm) of 13 Phoma macdonaldii isolates P 0·001). For all isolates tested, the optimum pH grown on PDA at different pH values for eight days at 25ЊC and for growth was between 4 and 5 (Table 3). under a 12-h photoperiod (37 ␮Em¹2 s¹1). The values represent the mean diameters of three replicate cultures per isolate and pH value Production and size of pycnidia Significant differences in the number (Fig. 1) and size pH (Fig. 2) of pycnidia produced by the various isolates Isolates 4 5 6 7 were observed. The least-sporulating isolates (MA3, abcde ؒ aabؒ aaؒ baؒ c MP3 and MP6) produced 15–20 pycnidia per mm2 vs. MP3 42 67 43 00 19 67 11 33 MP4 abcd44ؒ33aab42ؒ67aab18ؒ67ba13ؒ00c 45–50 in isolates MP5, MP9, MP10 and MA6. The MP5 bcde41ؒ00aab42ؒ67aa20ؒ00ba10ؒ67c number and size of pycnidia were significantly and MP6 ef35ؒ67acd35ؒ00abc15ؒ67ba13ؒ33b negatively correlated (r ¼¹0·52; P ¼ 0·05). MP7 def38ؒ00abc38ؒ00acd11ؒ67ba7ؒ33c MP8 fg33ؒ33ad31ؒ33ad10ؒ00ba6ؒ33c ab ؒ aaؒ abcؒ baؒ b Pycniospore germination MP9 47 00 48 00 14 67 13 67 MP10 g28ؒ67ad30ؒ67acd12ؒ00ba6ؒ00c Five isolates (MP6, MP8, MP9, MP10 and MA7) differ- MA3 fg32ؒ67ad31ؒ00ad9ؒ33ba6ؒ00b ing in morphological characteristics, radial growth MA4 a49ؒ00aa48ؒ67aab19ؒ00ba12ؒ33b abc aabaabbac rate and pycnidial production were retained for this MA5 45ؒ33 43ؒ33 19ؒ00 8ؒ00 bcde ؒ abcؒ abcؒ baؒ c experiment. MA6 40 33 39 67 15 00 6 67 MA7 cdef38ؒ67abc39ؒ33abc15ؒ33ba13ؒ00b Pycniospore germination was maximum at con- 6 centrations of 10–10 pycniospores per mL for all Means with the same letter do not differ significantly, according to a 7 isolates at pH 6. Increasing the concentration to 10 Newman–Keuls test (P ¼ 0ؒ05). Letters to the right of each value refer pycniospores per mL decreased the germination rate to differences between pH values (read horizontally); letters to the left by 20–30%, depending on the isolate (Table 4). At of each value refer to differences between isolates (read vertically). Figure 1 Pycnidia per mm2 of Phoma macdonaldii after 10 days of culture on PDA medium, temperature 25ЊC, day length 12 h (37 ␮Em¹2 s¹1).

(Table 4). For all isolates at 106 pycniospores per mL, of a Phoma macdonaldii isolate was characterized by the optimum and maximum pH values were 5 and 7, minimum, optimum and maximum temperatures of respectively. The optimum and minimum relative 5ЊC, 22·5ЊC and 32·5ЊC, respectively. As observed humidities allowing pycniospore germination were 100 for mycelial growth, pycniospore germination was and 95%, respectively. Pycniospore germination was often maximum over a wide range of temperatures photo-independent (Table 4). (15–30ЊC). In Phoma lingam, the optimum temperature for pycniospore germination was shown to be between 20 and 25ЊC (Vanniasingham & Gilligan, 1988). Aggressiveness Phoma macdonaldii thrived at pH values in the range Significant differences in symptom production were of 4–5. Such a peculiarity has already been reported apparent between isolates: some (e.g. MA4, MA7 and for other species of fungi, although most phytopatho- MP10) were weakly aggressive, whereas others (MP1, genic fungi grow best at a pH of between 5 and 6·5 MP2, MP3, MP4, MP5 and MP7) were highly patho- (Cochrane, 1958). It should be noted that this acid- genic (Fig. 3). Aggressiveness was not significantly ophilic behaviour was also evident for pycniospore correlated (P > 0·05) with radial growth (r ¼¹0·39), germination, which was maximum at pH 4–5. pycnidia number (r ¼¹0·05) or pycnidia diameter The minimum relative humidity for spore germina- (r ¼ 0·20). tion is 95% and constitutes a major climatic parameter in the knowledge of disease epidemiology, because Discussion germination is essential for field infection. An increase in spore concentration to 107 pycniospores per mL The Leptosphaeria lindquistii isolates investigated decreased markedly the germination rate for each of displayed phenotypic variability relative to growth the isolates. This effect could be expected since Fournet and pycnidia production. Such variability has already et al. (1970) showed that the presence of carbohy- been observed in related species, such as Phoma exigua drates, proteins and a water-soluble compound in the (Decognet, 1994), Phoma lingam (Hassan et al., 1991) sporiferous gels of some Sphaeropsidales and Melanco- or Phoma medicaginis (Angevain, 1984; Gray et al., niales at high concentrations inhibited spore germi- 1990). nation, possibly preventing spore germination within The temperature optimum for pathogen growth the pycnidium itself (Corbaz, 1990). Inhibition could varied with the isolate: some isolates can be called be decreased by dilution, as was the case with other stenothermic (optimum at 25ЊC), whereas others are fungi (Fournet et al., 1970). Boudart (1981) suggested rather eurythermic (optimum between 20 and 30ЊC). that inhibition by sporiferous gels explains why These results are in agreement with those of Maric & pycniospore germination is slower than ascospore Schneider (1979), who showed that the in vitro growth germination in Leptosphaeria maculans.

Figure 2 Pycnidium size after 10 days of culture on PDA medium, temperature 25ЊC, day length 12 h (37␮Em¹2 s¹1). Table 4 Effects of temperature, pH, spore concentration, relative humidity and photoperiod on the percentage of pycniopore germination in ﬁve strains of Phoma macdonaldii, determined on 150 pycniospores (3 replications, each including 50 spores) after 16 h of incubation. Temperature effects were investigated at pH ¼ 6, 106 pycniospores per mL, and in the dark; pH effects were investigated at 25ЊC, 106 pycniospores per mL, and in the dark; concentration effects were studied at 25ЊC, pH ¼ 6, and in the dark; relative humidity was investigated at 25ЊC, pH ¼ 6, 106 pycniospores per mL, and in the dark; light effects were studied at 25ЊC, pH ¼ 6, and 106 pycniospores per mL

Treatments MP6 MP8 MP9 MP10 MA7

Temperature ЊC) 5 a2ؒ33ea4ؒ33da1ؒ67da5ؒ00da1ؒ33c) ab32ؒ00da37ؒ00cb25ؒ33ca35ؒ00cb25ؒ33b 10 a82ؒ33aa83ؒ33aa73ؒ33aa83ؒ00aa72ؒ33a 15 ab83ؒ67aa93ؒ33aab78ؒ67aa92ؒ00ab71ؒ33a 20 b87ؒ67aa97ؒ00ac75ؒ33aab93ؒ00ac74ؒ33a 25 abc74ؒ00ba88ؒ33abc65ؒ00aab82ؒ67ac58ؒ33a 30 a51ؒ67ca63ؒ33bb37ؒ33ba65ؒ33bb32ؒ33b 35 pH 4 a31ؒ67ba28ؒ33ba26ؒ33ba25ؒ00ba18ؒ33b a41ؒ67abc31ؒ67aab35ؒ00aa40ؒ00ac26ؒ67a 5 a16ؒ67cab11ؒ67ca16ؒ67cab13ؒ33cb8ؒ33c 6 a8ؒ33da5ؒ00da8ؒ00da5ؒ00da5ؒ00c 7

Concentration spores per mL) 10 ab85ؒ00aa94ؒ00ab b80ؒ67aa96ؒ67ab80ؒ33a) 102b86ؒ00aa99ؒ33ab80ؒ33aa100ؒ00ab75ؒ33a 103bc84ؒ33aa100ؒ00ac80ؒ00aab93ؒ33ac75ؒ00a 104b84ؒ33aa100ؒ00ac71ؒ67aa100ؒ00ac70ؒ00a 105b85ؒ33ab90ؒ67bc70ؒ67aa98ؒ33ac70ؒ33a 106ab85ؒ33aa98ؒ33aab82ؒ67aa98ؒ33ab70ؒ00a 107a20ؒ00ba30ؒ67ca26ؒ33ba20ؒ00ba26ؒ67b

RH 100% a99ؒ33aa96ؒ00ab83ؒ67aa96ؒ67ab88ؒ33a a88ؒ33ba81ؒ67bb66ؒ00ba88ؒ67bb68ؒ00b 98% a15ؒ00ca13ؒ33cb5ؒ00cb5ؒ00cb5ؒ00c 95% a0ؒ00da0ؒ00da0ؒ00ca0ؒ00ca0ؒ00c 90%

Light Light a87ؒ33aa95ؒ00ab80ؒ00aa95ؒ00ac71ؒ67a Darkness b84ؒ00aa100ؒ00ab83ؒ33aa100ؒ00ac70ؒ00a L–D ab85ؒ00aa96ؒ67ab78ؒ00aa93ؒ33ab71ؒ60a**

D–L c83ؒ33aa99ؒ00ad80ؒ00ab94ؒ67ae68ؒ00a

**L–D: 8 h of light then 8 h of darkness, D–L: 8 h of darkness then 8 h of light. Means with the same letter do not differ signiﬁcantly, according to a Newman–Keuls test (P ¼ 0ؒ05). Letters to the right of each value refer to differences between treatments (read vertically); letters to the left of each value refer to differences between isolates (read horizontally).

Variation in pathogenicity of isolates has been var. linicola (Decognet, 1994), and is now reported in observed in Phoma lingam (Cunningham, 1927; Phoma macdonaldii. No correlation between aggres- Williams, 1985, 1992; Mengistu et al., 1991), Phoma siveness and growth or other phenotypic characteristics medicaginis (Angevain, 1984) and Phoma exigua could be detected, in contrast with the observations

Figure 3 Aggressiveness of Phoma macdonaldii isolates recorded 10 days after inoculation; 25/18ЊC day/night temperature, relative humidity 75–85% and day length 14 h (200 ␮Em¹2 s¹1). on Phoma lingam (Pound, 1947; Delwiche, 1980; Hill sporife`res de quelques Sphaeropsidales et Melanconiales. et al., 1984; Petrie, 1988; Koch et al., 1989). However, Annales de Phytopathologie 2, 31–41. before any decisive conclusion can be drawn, it will Gray FA, Fernandez JA, Horton JL, 1990. Variation among be necessary to increase the isolate pool to include isolates of Phoma medicaginis var. medicaginis in spore nonaggressive isolates characterized for their pheno- production in vitro and symptom expression on excised types including biochemical and/or molecular charac- leaves of alfalfa. Plant Disease 74, 668–70. ters, as already reported for Phoma lingam (Koch et al., Hassan AK, Schulz C, Sacristan MD, Wo¨ stemeyer J, 1991. 1991; Williams, 1992). Biochemical and molecular tools for the differentiation of The investigation of isolate aggressiveness led to aggressive and non-aggressive isolates of the oilseed rape pathogen Phoma lingam. Journal of Phytopathology 131, the development of a method of inoculation under 120–36. controlled conditions that will be a valuable tool, not Hill CB, Xu XH, Williams PH, 1984. Correlations of only for the study of sunflower tolerance or resist- virulence growth rate, pigment production and allozyme ance, but also for more fundamental approaches to banding patterns which differentiate virulent and avirulent the genetics of the interaction. isolates of Leptosphaeria Maculans. Cruciferae Newsletter 9, 79. References Hua Z, Ma G, 1996. A review of sunflower disease research in China. Beijing, China: Proceedings of the Fourteenth Acimovic M, 1984. Sunflower diseases in Europe, the United International Sunflower Conference 2, 754–9. States and Australia. 1981–83. Helia 7, 45–54. Koch E, Badawy HMA, Hoppe HH, 1989. Differences Angevain M, 1984. Mise au point d’une me´thode de se´lection between aggressive and nonaggressive single spore lines of de la luzerne contre. Phoma Medicaginis Malbr. et Roum. Leptosphaeria maculans in cultural characteristics and Montpellier, France: Universite´ des Sciences et Techniques phytotoxin production. Journal of Phytopathology 124, du Languedoc, PhD Thesis. 52–62. Anonymous, 1963. Tables scientifiques 6e`me e´dition. Paris, Koch E, Song K, Osborn TC, Williams PH, 1991. France Documenta Geigy. Relationship between pathogenicity and phylogeny based Barrault G, 1989. L’helminthosporiose de l’orge causeé par. on restriction fragment length polymorphism in Drechslera Teres. Toulouse, France: Institut National Leptosphaeria maculans. Molecular Plant–Microbe Polytechnique, PhD Thesis. Interactions 4, 341–9. Boerema GH, 1970. Additional notes on Phoma herbarum. Lancashire PD, Bleiholder H, Langelu¨ ddecke P, Stauss R, Persoonia 6, 15–48. Van Den Boom T, Weber E, Witzenberger A, 1991. An Boudart G, 1981. Modalite´s de l’attaque parasitaire des uniform decimal code for growth stages of crops and crucife`res par Leptosphaeria maculans (Desm) et de Not. weeds. Annals of Applied Biology 119, 561–601. (f.c. Phoma Lingam), agent de la nećrose du collet. Madjidieh-Ghassemi S, 1988. Studies on some important De´terminisme molećulaire du pouvoir pathoge`ne. Lille, fungal diseases of sunflower in Iran. Novi Sad, Yugoslavia: France: Universite´ des Sciences et Techniques de Lille, PhD Proceedings of the Twelfth International Sunflower Thesis. Conference 2, 22–3. Carson ML, 1991. Relationship between Phoma black stem Maric A, Camprag D, Masirevic S, 1988. Bolesti i severity and yield losses in hybrid sunflower. Plant Disease Stetocine suncokreta. Beograd, Yugoslavia: Injihovo 75, 1150–3. suzbijanje Nolit 37–45. Cochrane VW, 1958. Physiology of fungi. Cultivation and Maric A, Schneider R, 1979. Die Schwartzfleckenkrankheit Growth. New York, USA: Wiley & Sons. der Sonnenblume in Jugoslawien und der Erreger Phoma Corbaz R, 1990. Principes de phytopathologie et de lutte macdonaldii Boerema. Phytopathologische Zeitschrift 94, contre les maladies des plantes. Lausanne, Switzerland: 226–33. Presses Polytechniques et Universitaires Romandes. Mengistu A, Rimmer SR, Koch E, Williams PH, 1991. Cunningham GH, 1927. Dry-rot of swedes and turnips: Pathogenicity groupings of isolates of Leptosphaeria its cause and control. Auckland, New Zealand: New maculans on Brassica napus cultivars and their disease Zealand Department of Agriculture, Bulletin 133–51. reaction profiles on rapid cycling brassicas. Plant Disease Decognet V, 1994. Phoma exigua var. Linicola, agent du 75, 1279–82. mort-lin: variabilite´ et mode d’infection du parasite, Penaud A, 1996. Phoma du tournesol: recherche des e´poques expression de la maladie. Rennes, France: Universite´ de de contamination et mise au point de la protection Rennes, PhD Thesis. fongicide. Beijing, China: Proceedings of the Fourteenth Delwiche PA, 1980. Aspects of blackleg (Leptosphaeria International Sunflower Conference 2, 694–9. maculans) resistance to rapeseed Brassica napus. Madison, Peres A, Lefol C, 1996. Phoma macdonaldii Boerema: USA: University of Wisconsin, PhD Thesis. e´le´ments de biologie et mise au point d’une me´thode de Frezzi MJ, 1968. Leptosphaeria lindquistii n. sp., forma contamination artificielle en conditions controleés. Beijing, sexual de Phoma oleracea var. helianthi-tuberosi Sacc., China: Proceedings of the Fourteenth International hongo causal de la ‘mancha negra del tallo’ del girasol Sunflower Conference 2, 687–93. (Helianthus annuus L.), en Argentina. Patologia Vegetal 5, Petrie GA, 1988. The rapid differentiation of virulent and 73–80. weakly virulent strains of Leptosphaeria maculans Fournet J, Pauvert P, Rapilly F, 1970. Proprie´te´s des geleés (blackleg or stem canker) and related pycnidial fungi from Brassica seeds and stems. Canadian Journal of Plant Vanniasingham VM, Gilligan CA, 1988. Effects of biotic and Pathology 10, 188–90. abiotic factors on germination of pycniospores of Pound GS, 1947. Variability of Phoma lingam. Journal of Leptosphaeria maculans in vitro. Transactions of the Agricultural Research 75, 113–33. British Mycological Society 90, 415–20. Rayner RW, 1970. A mycological colour chart. Kew, UK: Williams PH, 1985. Crucifer Genetics Cooperative (CrGC) Commonwealth Mycological Institute. Resource Book. Madison, USA: Department of Plant Siddique-Mirza M, Masood AR, Ayub M, 1988. Sunflower Pathology, University of Wisconsin. diseases in Pakistan in the period 1980–87. Novi Sad, Williams PH, 1992. Biology of Leptosphaeria maculans. Yugoslavia: Proceedings of the Twelfth International Canadian Journal of Plant Pathology 14, 30-5 Sunflower Conference 2, 25.