71-4945 HEMMES, Don Evan, 1942- ULTRASTRUCTURE AND PHYSIOLOGY OF GERMINATION 2014. 6....

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Transcript of 71-4945 HEMMES, Don Evan, 1942- ULTRASTRUCTURE AND PHYSIOLOGY OF GERMINATION 2014. 6....

  • 71-4945

    HEMMES, Don Evan, 1942- ULTRASTRUCTURE AND PHYSIOLOGY OF GERMINATION IN PHYTOPHTHOR~ PARASITICA.

    University of Hawaii, Ph.D., 1970 Microbiology

    University Microfilms, Inc., Ann Arbor, Michigan

    THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED

  • ULTRASTRUCTURE AND PHYSIOLOGY OF GERMINATION IN

    PHYTOPHTHORA PARASITICA

    A DISSERTATION SUBNITTED TO THE GRADUATE DIVISION OF THE

    UNIVERSITY OF H~WAII IN PARTIAL FULFILLMENT

    OF THE REQUIREMENTS FOR THE DEGREE OF

    DOCTOR OF PHILOSOPHY

    IN MICROBIOLOGY

    MAY 1970

    by

    Don Evan Hammes

    Dissertation Committee

    Leslie R. Berger, Chairman Richard D. Allen Hilmer Ae Frank Roger D. Goos Barbara Z. Siegel

  • ABSTRACT

    Flagella and cleavage vesicles form during the initial stages

    of direct sporangial germination in Pbyt0phthora parasitica in the

    same manner as they do in indirect germination. Later, however, the

    flagella degenerate and cleavage of the cytoplasm is not complete.

    Instead, a new wall layer is deposited onto the existing sporangial

    wall, and this germination wall extends with the gem tube and

    forms the hyphal wall. Material for the germination wall first

    appears around vesicular aggregates concentrated at the periphery

    of the sporangial cytoplasm. As the wall forms the vesicular

    aggregates become encased in it, and the wall eventually consists of

    irregular deposits of wall material interspersed with the vesicular

    aggregates. These are clearly cytoplasmic in nature and different

    from lomasornes, since they are associated with ribosomes and other

    small elements of the cytoplasmo Likely to contribute to the wall

    formation are the endoplasmic reticulum, microbodies, dictyosome-

    derived vesicles, and possibly polyvesicular bodies and larger

    vacuoles with fibrillar contents. The basal plug of the sporangium

    foms via the same mechanism as the germination wall and also

    contains numerous vesicular inclusions. Fewer vesicular inclusions

    are found in the sporangial wall, and none have been observed in

    the walls of the growing hyphae. This difference in wall structure

    can be e~"Plained on the basis of their different growth patterns"

    Penetration of the sporangiaI plug is concomitant with prominent

    lomasornal activity and the cytoplasm at the hypha1 tip is characterized

  • by the presence of numerous vesicles J which are probab~ derived

    from dictyosomes.

    Ultrastructural studies of cyst germination and inhibition of

    direct cyst germination confinn the role of cellular organelles

    in wall formation and hyphal growth. In direct sporangial germ-

    ination and in elongating hypha1 tips, dictyosome-derived vesicles

    appear to function as a transport system for wall precursor

    material. In indirect gennination, on the other hand, vesicles

    occur which are also most likely produced by dictyosomes, but

    their main function is to form the plasmalemma of the developing

    zoospores in the sporangium. The dictyosome thus probably performs

    a dual role in Phyt0phthora: during direct germination it might

    serve primarily as a transport system for wall material; during

    indirect germination it appears to function as a membrane donor.

    iv

  • TABLE OF CONTENTS

    TITLE PAGE • • • • • • • • • • • • • • • • • • • • • • • • • • • • i ABSTRACT • • • • • • • • • • • • • • • • • • • • • • • • • • • • iii

    . . .. . . . . . . .. . . . . '....INTRODUCTION • LIST OF FIGURES •

    CHmER I

    • • • • • • • • • • • • • • •• • • • • • • •• • vi.

    • 1

    CHAPl'ER II MATERIALS AND METHODS • • • • • • • • • • • • • • • • • 5

    A. Culture Methods • • • • • • • • • • • • • • • • • •••• 5

    B. PQysiological Studies • • • •••• • • • • • • • • • • • .5

    C. Light Microscope Studies • .. • • • • • .. .. • • • • • • • • 6

    D. Electron Microscope Techniques • • • • • • .. .. • • • • • • 6

    Inhibition Studies • • • • • • • • · ... • • • e • .. ... • 8 CHAPTER III RESULTS • • • • • • • • • ••••••• • • • • • • • 9

    • • • • • • • • • • • • • • • •• • • • 9A.

    B.

    Germination TYPes

    Physiology and Control • •••• • • • • • .. . . • • • • .10 • .44

    . . . .~ • • • •

    • • • • • • • •

    Electron Microscopy of Sporangial Germination • • • .. • • • • • • •

    RESULTS

    Initial, Intrasporangial Changes.

    B. Perforation of Apical Plug and Sporangial Wall. • • • .. .48

    C. Hyphal Tips ..... • • • • • • .. • • .. • • .. • • .. .. • • .49

    CHAPTER IV

    CHAPl'ER V RESULTS Electron Microscopy of Encystment and Cyst Germination. .. • .. .. • .. .. • II .. • • .. • 125

    • • • • • • • • • • • • • •

    Secondar,y Zoospore Production

    Germ Tube Production

    .... 127 • 127

    .. .. .. • ••

    • ..... 125

    • •

    · ....· .. .. . ..• •• •

    · .. · .... .. .

    • •••EncystationA.

    B.

    CHAPTER VI RESULTS Electron MicroscoP,1 of Inhibition • •••• 169

    DISCUSSION • • • • • • • • • • • • • • • • • • • • • • ••••• 179

    BIBLIOGRAPHY • • • • • .... • • • • .. • .. .. 0 0 .. • • .. .. .. .. . 190

  • FIGURE

    1-1.

    LIST OF FIGURES

    Germination types of Phytophthora parasitica•••• • • •

    PAGE

    • 13

    1-2. Sporangia prior to germination. • • • • • • • • • • • • • • 15

    Cysts of Phytophthora parasitica prior to germination.

    • •••1-3.

    1-4.

    1-5.

    Direct sporangial germination.

    Indirect sporangial germination. • • •

    •••••••••• 15

    • • • • • • • • •• 15

    •• 15

    1-6. Cysts with short germ tubes prior to secondary zoospore release. • • • • • • • • • • • • • • • • • • • • • 17

    1-7. Secondary zoospores being released in. indirect cyst germination. •••••• • • • • • • • • • • • • • • • •• 17

    1-8. Empty cyst cases after secondary zoospore release. • •• • 17 1-9. Direct cyst germination after 3 hrs of gemination••••• 19

    1-10. Direct cyst germination after 5 hra of incubation. • •• • 19 1-11. Appressoria formation. • • • • • • • • • • • • • • • • • • 21

    1-12. High magnification of appressoria formation. o 0 e " e • e 21

    1-13. Sporangia after 3 hrs of incubation in 50% papaya extract. 23

    1-14. Sporangia after 3 hrs of incubation in artificial extract. 23

    1-15. Sporangia after 3 brs of incubation in dialyzed extract. • 23

    1-16. Sporangia after 3 brs of incubation in 12% sucrose. • • • • 23

    1-17. Sporangia after 5 hrs of incubation in 50% papaya extract. 25

    1-18. Sporangia after 5 brs of incubation in artificial extract. 25

    1-19. Sporangia after 5 brs of incubation in dialyzed extract. • 25 1-20. Sporangia after 5 hrs of incubation L~ 12% sucrose. • • • • 25 1-21. Kinetics of cyst germination in 50% papaya extract~ • II .. 0 27

    1-22. Kinetics of cyst germination in distilled water. • • • • • 29

    1-230 Effect of sporangial density on germination. • • • • • • • 31

  • • ••• 33

    FIGURE

    1-24.

    1-25.

    Effect of pH on sporangial gemination. • • • • • •

    Effect of sporangial age on germination. • • • • •

    • •

    vii

    PAGE

    • • 31

    1-26. Effects of modification of papaya extract on direct sporangial germination. • • • • • • • • • • • • • • • • • • 35

    1-21. Effects of artificial extract components on direct sporangial germination. • • • • • • • • • • • • • • • • • • 37

    1-28. Effect of carbohydrate component of artificial extract on direct sporangial gemination. • • • • • • .. 0 0 0 e e • 39

    1-29. Effect of amino acid component of artificial extract on direct sporangial germination. • • • • • • • • • • • • • 41

    • •

    1-30.

    2-1.

    Effect of pH of artificial extract on direct sporangial germination. • • • • • • • • • • • • • • • •

    Resting sporangium of Phytophthora parasitica. ••••

    • • 43

    52

    2-3. ~porangium before formation of germination wall• .....-....

    Section through sporangium and basal attachment.2-2.

    2-4. Sporangium fixed with glutaraldehyde. • •••

    • • • • • 54

    • • • • • 56