AACIMP 2011 Summer School. Neuroscience Stream. Lecture by Khaled Khanchouch.

1. General introduction

2. Phytopathology is the study field of plant diseases caused by pathogens and physiological factors. Plant pathology involves the study of pathogen identification, disease etiology, disease cycles, economic impact, epidemiology, plant disease resistance, effects on humans and animals and disease management. Phytopathology

3. keywords Plant Pathology : The study of plant diseases Plant Disease : Any physiological or morphological change in a plant that results in abnormal appearance or development Pathogen : An organism that causes disease Host: The infected plant Symptom : Abnormal appearance of a plant

4. Components of a Plant Disease - Conducive Environment - Susceptible Host - Virulent Pathogen - Adequate Time

5. WSU Extension San Juan County DISEASE Pathogen Susc. Host Environment Time Components of a Plant Disease Time DISEASE Environment Pathogen Susc. Host Components of a Plant Disease

6. Disease Classes - Abiotic (not transmissible) - Biotic (transmissible)

8. Causes of Biotic Diseases (Plant Pathogens)

9. Viruses Electron micrograph of purified tristeza virus particles negatively stained with uranyl formate. Bar represents 100 nm

10. Bacteria Pseudomonas syringae bacterial cells on the surface of a bean leaf ( Phaseolus vulgaris )

11. Fungi Pea Powdery Mildew Erysiphe pisi

12. Nematode ( Meloidogyne sp . ) captured by a the mycelium of a fungus at the moment of its penetration in the root (Root-knot) Nematodes

13. Meloidogyne sp . Root-knot namatode penetrating a root

14. Parasitic Plants Cuscuta campestris , parasitic on Glechoma hederacea . This is one of the most common species of dodder worldwide.

15. Schematic representation of some Plant Pathogens

16. Plant Disease Groups (By Symptoms) - Leaf Spots - Leaf and Shoot Blights - Mildews - Rusts - Cankers - Root Rots - Wilts - Galls - Mosaics and Ringspots

17. Leaf Spots Cause: Fungi, bacteria, insects, mites, or abiotic factors Symptoms: Circular or “somewhat circular” spots showing chlorosis (yellowing) or necrosis (brown and dead) areas on leaf

18. Photinia fungal leaf Spot Entomosporium mespili

19. Leaf and Shoot Blights Cause: Fungi and bacteria Symptoms: Larger areas of leaves (not circular) and shoots showing localized discoloration and/or flagging

20. Dogwood Anthracnose (Fungal)

21. Wilts Cause: Fungi ( Verticillium, Fusarium ) and Bacteria Symptoms: Vascular Plugging Wilted leaves and shoots

22. Verticillium Wilt Smoke Bush Dark colored xylem shows vascular plugging

23. Galls Cause: Fungi, bacteria, insects, mites, nematodes, and abiotic factors Symptoms: Abnormal proliferation of plant tissue Specific to a certain plant organ (leaf, stem, crown or root)

24. Almond Leaf gall Curl Fungus

25. Rusts Cause: Fungi Symptoms: Fungal spore masses in yellow, orange, white, brown, and black on leaves and stems

26. Rust ribes host Fungus requires two host to complete its life cycle. One part on Pinus and the other on Ribes.

27. Cankers Cause: Fungi or bacterial infection resulting in dead, non-growing areas on stems and branches Symptoms: Dark, sunken areas with distinct margins on stems sometimes have small red or purple fruiting bodies Same fungi also cause fruit rots (e.g. bulls-eye rot)

28. Fungal apple Anthracnose Notice sunken (dead) areas of trunk that have failed to grow and inrease in girth as the rest of the trunk (results in depressed canker)

29. Root Rots Cause: Fungi ( Armillaria, Phytophthora, Sclerotinia ) and bacteria ( Erwinia ) Symptoms: Shoot wilting, chlorosis/necrosis Stem and root discoloration Dead roots often with fungal hyphae growth under bark

30. Tomato (Stem and Fruit Rot)

31. Mildews Cause: Fungi Types: - Powdery (common) “Dry” - Downy “Wet” Symptoms: Chlorosis, yellowing or purple blotches on leaves. White hyphae and fruiting bodies (fuzzy looking) on plant (leaf) surface

32. Powdery Mildew Cucumber Does not like free moisture. Likes high humidity (warm days and cool nights)

33. Abiotic Plant Disease (Not Transmissable)

34. Inability to uptake iron from pH neutral or basic soils. Not related to amount of iron (Fe) in soil.

35. Irregular watering appears as a brown spot at the base of the tomato fruit, this develops into a hard, sunken ring, caused by insufficient calcium flow.

37. Biocontrol

38. Bacterial antagonist primary selection

39. Colletotrichium gloesporoides

40. Botrytis cinerea

41. Fusarium oxysporum

42. Fusarium culmorum

43. Direct confrontation Temperature effect on the antagonistic bacterial activity B31 B8 B29 B12 B42 B31 B12 B29 B42 B8 B8 B29 B42 B12 B31 T 19°C T 30°C T 25°C

44. Percentage of inhibition Strains Bacterial antagonisme °c °c °c

45. Inhibition de la croissance mycélienne par les protéines de la souche B8 A: mycelium growth inhibition ( 70.80% ) by bacterial protein of B8 ( proteins are at 0.356µg /well). B: mycelium development on PDA medium without bacterial extract proteins. A B

46. Effet of the proteine treatment on the disease development Indice of the disease Days

47. Development of resistant plant

48. A. in situ initiation of callogenesis from ovules explants by transversal cutted of younger fruits; B. white cals proliferation from ovular explants; C. development of the isolateds cals on MS+NAA; D. green organogenesis cal. in situ induction and development of ovular cals A B D C

49. A : adventive buds formation from ovular cals on MS+BAP; B : young shoot formation from ovular cals on MS+BAP medium . Formation of structural organogene cal from ovulair explants A B

50. Artificial MS medium. Dicotylydonary embryos cal obtained from styl explant.

51. A : Friable cal with embryogenic structures B : Initiation of the rhizogenic structures. D : developed root from friables cals Organogenesis obtained from no zygotic parts of seeds A B C

52. Plant regeneration from friable cals induced from non zygotic parts of the seeds A: shoot regeneration on MS+BAP+2,4-D medium; B: young plant regenerated from friable cals; C: Vitroplant de Citrus eurêka developed in vitro on MS medium. B A C

54. Highly contaminated Zones Lower contaminated Zones Geographic Distribution of the Mal secco disease

55. A: Pycnidia of P. tracheiphila on lemon dying shoot; B: M ucilaginous pycnidia gel (the cyrrhus) containing pycnidiospores. A: Mycelium; B: Pycnidium; C: Phialoconidia; D: Arthroconidia; E: Pycnidiospores Total lemon diedback caused by the fungal infection A D B C E A B

57. Phoma tracheiphila DPR DP R Pycnides Red Pigment Pycnides Absence of red pigment Sterile Red Pigment Types of P. tracheiphila

58. Morphological varaibility of P. tracheiphila isolates

59. Isolation of the parasite

60. Double stranded Eeectrophoretic profil of the Avirulent isolate on agarose gel ( 1 % ) 9 kb 10 kb 3 kb 1 2 3 4 5

61. P. Tracheiphila collection isolates Référence de l’isolat Date de l’isolement Origine géographique Plante hôte Type V29 2001 Bizerte Eureka Chromogène VK 1998 Nabeul lime douce Chromogène V22 2001 Tunis Eureka Chromogène V25 2001 Tunis Eureka Chromogène V23 2001 Nabeul Eureka Chromogene VH2 2001 Bizerte Eureka Chromogène VX 2001 Tunis Eureka Chromogène V4 2001 Tunis Eureka Chromogène V14 2001 Nabeul Eureka Chromogène V30 2001 Nabeul Eureka Chromogène V12 2001 Nabeul Eureka Chromogène V13 2001 Nabeul Eureka Chromogène VD 2001 Tunis Eureka Chromogène VY 2001 Tunis Eureka Chromogène VH3 2001 Nabeul Eureka Chromogène V21 2001 Nabeul Eureka Chromogène V2 2001 Nabeul Eureka Chromogène VPM 2000 Tunis Eureka Chromogène V6 2001 Tunis Eureka Chromogène V7 2001 Nabeul Eureka Chromogène V8 2001 Tunis Eureka Chromogène V5 2001 Tunis Eureka Chromogène V24 2001 Tunis Eureka Chromogène V26 2001 Tunis Eureka Chromogène VT1 2001 Tunis Eureka Chromogène V3 2001 Tunis Eureka Chromogène VI26 - Italie - Chromogène V28 2001 Tunis Eureka Chromogène V66 - Italie - Chromogène V10 2001 Tunis Eureka Chromogène V11 2001 Tunis Eureka Chromogène VI1 - Tunis - Chromogène V48 - Tunis - Chromogène V27 2001 Bizerte Eureka Chromogène VR 2000 Tunis Eureka N. Chromogène VPt 2000 Tunis Eureka N. Chromogène

65. Empirical scale of evaluation 0 : No drying symptoms 1 : Limited drying to the annual shoots 2 : Drying reach the two years branches 3 : Drying exceeded to the main branches 4 : Trunk drying 5 : Total drying and death of the infected plant

66. Greenhouse artificial infection

67. Inoculations Methods Foliar inoculation Stem inoculation Root inoculation Foliar inoculation 0= No symptoms; 1= Chlorosis on the inoculation point; 2= Chlorosis around the inoculation point; 3= Chlorosis extended till the foliar borders; 4= general chlorosis; 5= foliar necrosis. Stem and root Inoculation 0= No symptoms; 1= Chlorosis at principal vein of the upper leaves; 2= General chlorosis of the upper leaves; 3= Apical necrosis of the branches; 4= Necrosis extended to the trunk of the inoculated plant; 5= Total necrosis and death of the inoculated plant.

68. In planta toxin inoculation

70. VR Te VPt V11 V21 VH2 V13 Foliar phytotoxicity toxin Test V13: Isolat de G1 VH2: Isolat de G2 V21: Isolat de G3 V11: Isolat de G4 VR: Isolat de G5 VPt: Isolat de G5 Te: non treated by the toxin

71. Development of new technic of artificial reproduction of the disease Parameters: 1- Micropropagated stem necrosis (Fig. a) 0= No symptomes; 1= Necrosis under the 1/3 of the micropropagated stem; 2= Necrosis between 1/3 et 2/3 of the micropropagated stem; 3= Necrosis exceed 2/3 of the micropropagated stem; 4= Total necrosis of the micropropagated stem. 2- Inhibition of the regeneration (Fig. b) 0= No regeneration; 1= Initiation of regeneration; 2= Young shoot; 3= shoot with foliar formation . 2 0 1 3 b a

72. Classification of P. tracheiphila isolates