Septoria and Stagonospora Diseases of Cereals - CIMMYT ...

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According to the previous observations, this paper describes physiologic specialization of bread and durum wheat isolates collected from Morocco and Tunisia, respectively, and the sequence of their ITS rDNA. Specific primers determined by Beck and Ligon (1995) for PCR detection of S. tritici and S. nodorum were used to differentiate those pathogens from other fungal pathogens of wheat and to detect their presence in asymptomatic wheat plants. Time course PCR quantification of S. tritici in infected wheat was assessed to analyze the evolution of mycelia in resistant and susceptible cultivars. Materials and Methods Plant material and experimental design Thirty-six durum wheat and eight bread wheat cultivars (Table 1) were employed to study genetic variation for virulence. Seeds of the cultivars were sown in trays with 228 alveolus with 3 cm x 3 cm surface and 5 cm height. Each alveolus contained four seeds. The experiment was conducted in an arbitrary complete block design with three replicates for each isolate. Replicates were blocked in the same tray, separated by two rows of alveolus. Characterization of Septoria tritici Variants and PCR Assay for Detecting Stagonospora nodorum and Septoria tritici in Wheat 27 Experimental procedure Seven isolates of S. tritici taken from durum wheat and seven isolates from bread wheat collected in different regions of Tunisia and Morocco, respectively, were used to inoculate durum and bread wheat cultivars (Table 2). Eleven-day-old seedlings with emerging second leaves were inoculated with a monosporal suspension till run-off. The inoculum was prepared from monosporal culture cultivated on potato dextrose agar (PDA) medium. The spores were scraped from the agar, re-suspended in distilled water, filtered, and adjusted to 106-107 spores/ml. After inoculation the trays were incubated in a humid chamber for 72 hours and returned to a growth chamber at temperature 20-25°C during the day, 17°C during the night, and 12 hours photoperiod. Table 1. List of durum and bread wheat cultivars used to study genetic variation for virulence in Mycosphaerella graminicola. Code Cultivars Code Cultivars 01 Florence aurore* 23 Medea AC3 02 Florence Aurore x PUSA* 24 Medea AC4 03 Derbessi x Biskri 25 Medea AP1 04 Guelma* 26 Medea AP2 05 EAPC6326127* 27 Allorea* 06 Tunis9* 28 Richelle* 07 Tunis23* 29 Sebei glabre 08 Abdelkader 30 Sebei pubescent 09 Agili pubescent AC1 31 Souri AC8 10 Bidi AP4 32 Souri AC9 11 Bidi AP1 33 Medea AC1 12 Bidi AP3 34 Medea AC2 13 Bidi AP 35 Medea AP6 14 Biskri glabre 36 Medea AP10 15 Biskri glabre AP3 37 Medea RP1 16 Derbessi AC1 38 Biancullida ICM27 17 Derbessi AP1 39 Mahmoudi ICM28 18 Derbessi AP2 40 Mahmoudi ICM67 19 Hamira AC2 41 Khotifa x ICM75 20 Hamira AC3 42 ICM313 21 Hamira AC4 43 ICM314 22 Hamira AC5 44 Hamira * Bread wheat cultivars. Disease evaluation and statistical analysis Disease severity was evaluated at 21 days after inoculation on the second leaf of the plant and using the presence of pycnidia as the disease parameter (Kema et al., 1996a). The collected data were treated with SAS software (1993 version). The presence of pycnidia was subjected to analysis of variance. The tables of means of the 14 isolates were subjected to hierarchical clustering using the statistical analysis software CSTAT. DNA extraction from fungal spores and infected plants Spores of S. tritici and S. nodorum cultivated on PDA medium were subcultured in 100 ml flasks of yeast extract and glucose liquid medium. Five-day spore cultures were centrifuged and DNA extraction of fungal spores was performed using CTAB extraction buffer according to the protocol described by Morjane et al. (1995). DNA extraction from three infected leaves was performed according to the protocol described by Möller et al. (1992). DNA pellet after extraction was re-suspended in 50 µl Tris-HCl; 10 mM EDTA; 1 mM (TE) buffer. PCR amplification and sequencing The sequence of ITS rDNA concerned 5 isolates (MAR 1, 2, 3, 4, 5) collected from Morocco (Table 2) and 5 isolates (TUN 1, 2, 3, 4, 5) collected from Tunisia (Table 2). ITS amplification was performed with ITS1 (5’- TCCGTAGGTGAACCTGCGG-3’) and ITS 4 (5’- TCCTCCGCTTATTGATATGC-3’) universal primers. PCR reactions
28 Session 1 — S. Hamza, M. Medini, T. Sassi, S. Abdennour, M. Rouassi, A.B. Salah, M. Cherif, R. Strange, and M. Harrabi Table 2. Experimental code and origin of Mycosphaerella graminicola collected from Morocco and Tunisia to study genetic variation for virulence. Isolates collected from Tunisia Isolates collected from Morocco Origin Origin Code Location Wheat species Code Location Wheat species TUN1 Beja DW MAR1 Jemaa Sham BT TUN2 Mateur DW MAR2 Douyet BT TUN3 Mornag DW MAR3 Agadir BT TUN4 El Agba DW MAR4 Ain Orma BT TUN5 Zaghouane DW MAR5 Azrou BT TUN6 Siliana DW MAR6 Essaouira BT TUN7 Mjez El Bab DW MAR7 Tetouan BT were performed in a volume of 50 µl containing 50 mM KCl, 10 mM Tris-HCl, pH8.3, 1.5 mM MgCl2, 200 µM of dNTP, 50 pmole of primer, 2.5 units of Taq polymerase (Boerhringer- Manheim) and 25 ng of genomic DNA. Reactions were run in a Thermolyne, Temptronic model thermal cycler for 30 cycles, each consisting of 15s at 94°C, 15 s at 50°C, and 45 s at 72°C. An additional 5 min polymerization step at 72°C ended the amplification. The products were analyzed by electrophoresis of 20 µl aliquot of each PCR sample on 0.8% agarose gel. Specific DNA amplifications were performed as determined by Beck and Ligon (1995). ITS1 and JB446 (5’- CGAGGCTGGAGTGGTGT-3’) primers were used for specific amplification of S. tritici DNA and JB433 (5’- ACACTCAGTAGTTTACTACT-3’) and JB434 (5’- TGTGCTGCGTTCAATA-3’) were used to amplify S. nodorum DNA. PCR was performed as described above, except the annealing temperature was 57°C. ITS sequencing was performed using 90 ng of 500 bp amplified product with ITS1 and ITS4. The sequence was determined by the dideoxynucleotide chain termination method using an automated sequencer (Perkin Elmer) at the Darwin Building of University College London (London, United Kingdom). Fungal DNA amplification in resistant and susceptible cultivars To evaluate the competition of plant DNA with fungal DNA using ITS1 and ITS4 primers, amplification was performed using 1 µl and 5 µl of plant DNA extract mixed with several amounts ( 0.001, 0.01; 0.1; 1; 10; 100 and 1000 ng) of fungal DNA. The PCR conditions were the same as described before with a 57°C annealing temperature. The time course amplification using ITS1 and ITS4 primers was realized using 2 µl of DNA extract from one infected leaf at 3, 6, 9, 14, 18, and 22 days after inoculation. The PCR conditions were the same as described before, with a 57°C annealing temperature. Results and Discussion Bread wheat derived isolates almost exclusively produced pycnidia in the bread cultivars, whereas pycnidial production by durum wheat derived isolates was almost entirely restricted to durum wheat isolates. The analysis of variance (Table 3) shows highly significant differences (p
Page 1 and 2: Septoria and Stagonospora Diseases
Page 3 and 4: ii The Organizing Committee express
Page 5 and 6: iv 87 Genetic Variability in a Coll
Page 7 and 8: vi Foreword In the mid-1970s the id
Page 9 and 10: 2 Opening Remarks — S. Rajaram ar
Page 11 and 12: 4 Opening Remarks — S. Rajaram Ta
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Page 15 and 16: 8 Opening Remarks — S. Rajaram br
Page 17 and 18: 10 Opening Remarks — S. Rajaram T
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Page 21 and 22: 14 Opening Remarks — S. Rajaram C
Page 23 and 24: 16 Opening Remarks — S. Rajaram r
Page 26 and 27: Session 1: Pathogen Biology Biology
Page 28 and 29: Table 2. The Septoria tritici/Stago
Page 30 and 31: Molecular Analysis of a DNA Fingerp
Page 32 and 33: histidine kinase in yeast, although
Page 36 and 37: cultivars. The presence of pycnidia
Page 38 and 39: Provided that S. nodorum fungal gen
Page 40 and 41: ;; yy ;; yy 6 28% 1 32% ;y; ; y y ;
Page 42 and 43: Table 1. Sources of isolates or DNA
Page 44 and 45: Septoria/Stagonospora Leaf Spot Dis
Page 46: Interrelations among Septoria triti
Page 49 and 50: 42 Session 2 — B.M. Cunfer disper
Page 51 and 52: 44 Session 2 — B.M. Cunfer beyond
Page 53 and 54: 46 Aggressiveness of Phaeosphaeria
Page 55 and 56: 48 Session 2 — P. Halama, F. Lala
Page 57 and 58: 50 Growth of Stagonospora nodorum L
Page 59 and 60: 52 Session 3A — G.H.J. Kema and E
Page 61 and 62: 54 A Possible Gene-for-Gene Relatio
Page 63 and 64: 56 Diallel Analysis of Septoria Tri
Page 65 and 66: 58 Session 3A — X. Zhang, S.D. Ha
Page 67 and 68: 60 Session 3A — L. Gilchrist, C.
Page 69 and 70: 62 Session 3A — L. Gilchrist, C.
Page 71 and 72: 64 Session 3B — E. Arseniuk and P
Page 75 and 76: 68 Cultivar variances Cultivar vari
Page 79 and 80: 72 Session 3B — N.E.A. Murphy, R.
Page 81 and 82: 74 Inheritance of Septoria Nodorum
Page 83 and 84: 76 Session 3B — N.E.A. Murphy, R.
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78 Session 4 — B.A. McDonald, C.C
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84 Session 4 — E. Arseniuk, H.S.
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86 Session 4 — C. Cowger, C.C. Mu
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88 each isolate. Two of the probes
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90 Mating Type-Specific PCR Primers
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92 Session 4 — Bennett, R.S., S.-
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94 Session 5 — M.W. Shaw and the
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96 Session 5 — M.W. Shaw The path
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98 Spore Dispersal of Leaf Blotch P
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Session 5 — C.A. Cordo, M.R. Sim
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102 Epidemiology of Seedborne Stago
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Session 5 — D.A. Shah and G.C. Be
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106 Session 6A / Session 6B — J.M
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Session 6A / Session 6B — C.C. Mu
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118 Session 6C — M. van Ginkel an
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Session 6C — G. Shaner 128 An exa
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Session 6C — G. Shaner 130 Anothe
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Session 6C — M. Díaz de Ackerman
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134 Septoria tritici Resistance Sou
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Session 6C — L. Gilchrist et al.
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Session 6C — L. Gilchrist et al.
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140 Selecting Wheat for Resistance
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Session 6C — R.M. Gonzalez I., S.
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Session 6C — R.M. Gonzalez I., S.
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Session 6C — R. Loughman, R.E. Wi
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148 Field Resistance of Wheat to Se
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150 Using Precise Genetic Stocks to
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Session 6C — C.M. Ellerbrook, V.
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154 Evaluating Triticum durum x Tri
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156 Sources of Resistance to Septor
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Session 6C — H. Toubia-Rahme and
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160 Partial Resistance to Stagonosp
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Session 6C — C.G. Du, L.R. Nelson
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Session 6C — D.E. Fraser, J.P. Mu
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Session 6C — C.G. Du, L.R. Nelson
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Session 6C — M. Mincu 168 Arcsin
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170 Comparison of Greenhouse and Fi
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Session 6C — S.L. Walker, S. Leat
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Session 6D — L.N. Jørgensen, K.E
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176
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Concluding Remarks — Z. Eyal 178
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184 Dr. Patrice Halama Professor In
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186 Dr. Hala Toubia-Rahme Research
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