Ultrastructural and physiological changes in ...

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lA, B. Electron micrographs of sporangia of Ps. crlbelrsis after exposure to WDR (3 11 at 35Y0 RH;. 20°C) (A), or control (3 11 at 10OL); RH, 20'C) (B) treatments.
Ultrastructural and physiological changes in sporangia of Pseirdoperonospora czrbensis and Phytophthora infestans exposed to water stress YIGALCOIIEN,~ M. PERL,J. ROTEM,HELENA EYAL,AND J. COHEN

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Departinenf of Life Sciences, Bar Ilatz Utziversity, Rat~iaf-Gatr,Israel Departmet11 of Platzf Parlrology, Volcatri Cetzter of Agriciilf~rralResearcll, Bet-Dagatr, Ist,ael Received May 8 , 1973 COHEN, Y., M. PERL,J. ROTEM,H. EYAL,and J. COHEN.1974. Ultrastr~~ctural and physiological changes in sporangia of Pseiirloperotrospo,.a c~~betzsis and P/2yfop/1fl1oraitfesfrrtr.~exposed to water stress. Can. J. Bot. 52: 447-450. crrbet~sisand P I I ~ ! o ~ I I ~ Ii~festatrs I o I . ~ I was greatly reduced Viability of sporangia of Pserrdo~~erot~ospora when sporangia were wetted for a period too short to ensure germination and then allowed to dry out (WDR treatment). No morpl~ologicalchanges were detected in WDR-treated sporangia by ~nicroscopic examinations, but when examined under electron microscope they exhibited partial or complete alterations of the mitochondrial inner membrane integrity, some deterioration of the plasmalemma, and extensive vacuolization. Water uptake was not affected but oxygen uptake was totally inhibited in WDR-treated sporangia and previously labeled sporangia exhibited increased leakage. No incorporation of 14C-leuc~ne(into proteins) and 3zP-ortl~opl~ospl~ate (into nucleic acids) occurred as a result of the treatment. Adding various sugars, alcol~ols,and adenosine triphosphate (ATP) to the treated sporangia did not restore their viability. It was suggested that the water stress exerted by W D R treatment primarily ca~lseda deterioration of the mitochondria1 membrane integrity, which in turn probably led to the other phenomena listed above. COHEN,Y., M. PERL,J. ROTEM,H. EYAL,et J. COHEN.1974. Ultrastructural and physiological changes in sporangia of Pse~idoperot~ospom cubetrsis and Phytopl~fl~ora itfestatrs exposed to water stress. Can. J. Bot. 52: 447450. La viabilitC des sporanges de Pse~~dopelatiospora cr~betrsiset de Pl2yfopl1tI1omit2frstat1sa CtC fortement rCduite quand les sporanges ont CtC humectCs pendant une pCriode trop courte pour assurer la germination, puis mis i sCcher (traitement WDR). Aucu~lcllangement morpholog~quen'a CtC dkelC, en microscopic optique, dans les sporanges trait& au WDR, mais en m~croscopieClectronique, ils ant montrC des alterations partielles ou complktes dans l'intCgritC de la membrane interne des mitochondl~ies, ainsi ue des dCtCriorations dans le plasmalemme et une vacuolisatio~limportante. ~ ' a t s o r ~ t i oden l'eau n'a pas CtC affect& mais I'absorption de I'oxygkne a CtC complktement inhibee chez les sporanges traitis au WDR, et des sporanges prialablement marquis ont montrC des pertes plus grandes. Aucune i~lcorporatio~l de 1 4 C - l e u c ~(dans ~ ~ e les protCines) et de 3ZP-ortl~opl~ospl~ate (dans les acides nuclCiques) n'a eu lieu h la suite du traitement. L'addition de divers sucres, d'alcools et d'ATP aux sporanges trait& ne leur a pas rendu leur viabiliti. On a suggCrC ue la tension en eau exercCe par le traitement WDR a caust d'abord une dttirioration de 1'intCgritC de?a n ~ e n ~ b r a ndes e mitocl~ondries,ce qui par la suite a probablement entrain6 Ies autres CvC~lementsCnumCrCs plus haut. [Traduit par le journal]

Introduction In contrast to the extensively studied effects of water stress on longevity of fungal spores (13), the physiological alterations associated with desiccation are less known. We studied some of these changes using sporangia of P/~ytophthora iifestans Mont. DBy. (race 0), which survive better under conditions of high relative humidity (RH) (2, ll), and sporangia of Pseudoperonospora cubensis (Berk. & Curt.) Rost., which 'Present address: Department of Plant Pathology, Macdonald Campus of McGill University, Ste. Anne d e Bellevue, Quebec.

survive better under low RH but are sensitive to alternating wetting and drying (1). Materials and Methods Unless otherwise stated, sporangia of Ps. crrbetrsis and

P. itlfesrarls were collected from infected leaves of cucumbers (Cucnmis safivus L. var. "Bet-Alpha") and potatoes (Solannnz fuberosrrm L. var. "Up-to-date") respectively, in distilled water. The infected plants were grown in cabinets kept a t 20°C 0.5"C and with light intensity of 1560 ft-c for 12 h a day. In all cases sporangial suspensions (50-100 X lo3 sporangia/ml) were filtered through 5-micron ( P ) millipore membranes (10 ml suspension per membrane) within 10-15 min after preparation and were washed with 20 X 25 ml of sterile distilled

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able to infect 4 out of 100 inoculated cotyledonary leaves, but when exposed to 35y0 RH, none of the cotyledons becaine infected. Control sporangia (100% RH) infected 777'0 of the inoculated leaves. Three, 0, and 50% of sporangia released zoospores in the 65y0, 35y0, and 100% RH treatments respectively. Naturally dispersed sporangia lost their infectivity after 3 days of storage at 65% RH (20°C) (1). Inoculation of potato leaflets (100 per treatment) with sporangia of P, i/$estmz.s exposed to 657; and 35% RH for 30min resulted with average lesion development of 2.3 (in 0-4 scale) and 0 respectively; percentages of sporangia which released zoospores were 30 and 0 respectively. The control sporangia gave rise to nearly maximal lesion development, viz. 3.7, and 80% released zoospores. Light microscopic observation of sporangia of both species showed no morphological deformation with the WDR treatment. Electron microscopic observations, however, showed partial alteration or total deterioration of the mitochondria1 eristae in the WDR-treated sporangia. Some mitochondria also exhibited deforined shape. In addition, the WDR treatment seemed to cause extensive vaeuolization of the cytoplasm and a partial disintegration of the plasmalemma (Figs. I and 2). Some treated sporangia of P. infestans showed total destruction of the inner cell integrity. Control and WDR-treated sporangia of both fungi were able to absorb similar quantities of water as determined by weighing the respective samples. Leakage of metabolites was tested with control and WDR-treated sporangia of Ps. cube~zsis previously labeled with 14C02. It was found that the amount of radioactive materials leaked from WDR-treated sporangia was 2.3 times higher than that leaked from the untreated controls. As measured in sporangia of P. itgestnns the WDR treatment resulted in the complete inhibiResults tion of oxygen uptake, whereas the control When exposed to an atmosphere of 65% RH showed a respiratory rate of 0 . 1 6 6 ~ 1O2/min for 3 h (20°C), sporangia of Ps. cuben.ris were per loGsporangia.

water t o decrease bacterial contamination. Membranes with sporangial deposits were then kept a t 35-1007, RH for 0.5-3 h. The controls were kept on wet Whatman No. 1 filter papers in closed petri dishes. The samples were then resuspended in sterile distilled water and used for tests. This is referred to as "wetting-drying-rewetting" (WDR) procedure. For examination of ultrastructure, the control and WDR-treated sporangia were first fixed in 5% glutaraldehyde in 0.1 M phosphate buffer a t pH 7 for 2 h (4°C). After repeated washing with the buffer, samples were centrifuged and the sporangia were resuspended in 27, agar. Thin blocks were cut and postfixed in OsOJ for 24 h (4°C). The material was washed again with the buffer, dehydrated in graded series of ethanol followed by propylene oxide, and embedded in Epon 812 epoxy resin. Sections were cut with a glass knife on an LKB Ultratome 111, stained for 15 min with 67, uranyl acetate and with Reynold's lead citrate (12), and examined with a Jem-7A electron nlicroscope. Although thoroughly washed, sporangia collected from leaves still contained bacterial contaminants. Therefore, biochemical tests with control and WDR-treated sporangia were conducted with those of P. i ~ f e s t a t ~ obtained s from axenic cultures (5). Respiration (oxygen uptake) was tested polarographically with an oxygen electrode. Incorporation of 1°C-leucine into protein and of 3ZPorthophosphate into nucleic acids was tested by adding 5 X lo5 and 2 X lo5 disintegrations per minute (dpm) of these materials, respectively, to sporangial suspensions (252 X lo3 sporangialml) kept at 19'C. Samples (0.5 ml) were removed at intervals (&I80 min), mixed with 0.5 ml of 10% trichloroacetic acid (TCA), and kept at P C for 60 min. They were then filtered through 0.45-p millipore membranes and washed with 2 X 5 ml of TCA. Radioactivity of the samples was counted in a Packard TriCarb Scintillation Counter. Leakage of 14C-labeled materials from control and WDR-treated sporangia of Ps. cllbelrsis was studied as follows. Isotope uptake by sporangia resulted from previous labeling by 14C02 of infected cucumber plants (10). Labeled sporangia were removed, washed (20 X 25 ml of distilled water), and resuspended in distilled water. Thereafter, sporangia were filtered, exposed to WDR (or control) treatment, resuspended, and kept in distilled water for 90 min and centrifuged. Radioactivity in the pellet (sporangia) and supernatant was then counted. Samples of sporangia treated for ultrastructural and ~11~ysiological observations and tests were simultaneously tested for their germinability (zoospore release) and infectivity on host plants (1, 10, 11). Tests were repeated a t least three times.

FIG. l A , B. Electron micrographs of sporangia of Ps. crlbelrsis after exposure to WDR (3 11 a t 35Y0 R H ; 20°C) (A), or control (3 11 at 10OL); R H , 20'C) (B) treatments. X 10 000. Note: plasrnalemma in Fig. - 1B remained intact. FIG.2A, B. Electron micrographs of sporangia of P. bfesrans after exposure t o WDR (30 rnin a t 350j,, 20°C) (A), or control (30 min at 10OC,,6RH. 20°C) (B) treatments. X 10 000.

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COHEN ET AL.: WATER STRESS EFFECTS O N SPORANGIA

WDR-treated sporangia of P. infestans also failed to incorporate 14C-leucine into proteins (Fig. 3A) and 33P-orthophosphate into nucleic acids (Fig. 3B). N o restoration of germinability and (or) infectivity of WDR-treated sporangia of either fungi was achieved by adding to the suspensions several sugars, alcohols, "Carb-wax" (10-110-4 M), or ATP (10-2-10-6 M) before and (or) after the treatment. The materials tested were glucose, fructose, sucrose, trehalose, mannitol, sorbitol, glycerol, and inositol. Artificially grown bacteria, isolated from the contaminated sporangia of either species, also failed to restore

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viability when added to WDR-treated sporangia before and (or) after treatment.

Discussion Propagules of plant pathogenic Peronosporales seem to exhibit a higher sensitivity to water stress exerted by wetting-drying-rewetting as compared with other fungi. Botrytis cinerea, Cercos~~ora musea, and Monili~ziafructicoln are ones reported to tolerate considerably repeated cycles of WDR treatments (4). Sten71>11yli~~1?~ botryosum f. sp. Iycopersici was found to benefit from these repeated cycles (Bashi and Rotem, unpublished). Sporangia of Ps. cubensi.~and P. itfestans (mentioned above) as well as those of Plrs~nopara viticola (Berk. & Curt.) Berl. & DeT., Plaslnoparn lzalstedii (Farl.) Berl. et de Toni, and conidia of Perono.l;>orn tnbacina Adam (unpublished data) were found to lose a great extent of their capacity for germination and infection when exposed to WDR treatments. These findings may have some practical importance as young field-grown cucun~bers sprayed with water daily were protected from attack by downy mildew (unpublished data). Viability of detached sporangia of P. irfistans was markedly reduced by a relatively short exposure to dryness (30 min at 35y0 RH) (1 1) as 0 compared with Ps. cubensi.~(3 h at 35-65% RH, 0 30 90 150 180 or if kept not wetted, by 3 days exposure to INCUBATION TIME ( m i n l 657; RH (1)). In all cases, no morphological deformations of the treated sporangia were observed, but ultrastructural and physiological alterations were detected. Partial or con~plete deterioration of the mitochondria1 cristae and of the cell membrane was seen in both species; less advanced deterioration was observed in Ps. cube~lsis. This may explain its higher survivability under dry conditions (I). It seems likely that during the first brief wetting of the sporangium, some ultrastructural changes are taking place (3, 6, 7). These waterdependent events, leading to zoosporogenesis, are abruptly stopped when the sporangiu~nis dried up and irreversible damage occurs to the cell, preventing its later germination. A much INCUBATION TIME (rninl longer dry period is needed to induce the same FIG.3A, B. Incorporation of lJC-leucine into proteins damage if the sporangii~mhas not been wetted (A), and of 3T-ortl~ophosphate into nucleic acids (B) in before (I). sporailgia (252 X 103/ml) of P. itfestatzs exposed to Water stress has been reported to alter selecWDR or control treatments.

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1967. 4. GOOD,H. M., and P. G. M. ZATHURECZKY. Effects of drying on the viability of germinated spores of Botrytis cirzeren, C e ~ o s p o r arrilrsae, and Morzilirriafr~icticolr~. Phytopathology, 57: 719-722. 5. HODGSON, W. P., and P. N. GRAINCER. 1964. Culture of Plrytop11rlror.a irrfeestrrrrs on artificial media prepared from rye seeds. Can. J. Plant. Sci. 44: 583. 1967. Ultra6. HOHL, H . R., and S. T. HAMAMOTO. structural changes during zoospore formation in P1rytol)lrthorri parrrsiticci. Am. J . Bot. 54: 1 131-1 139. 7. KING,J. E., J. COL~IOUN, and R. D. BUTLER.1968. Changes in the ~ ~ l t r a s t r u c t ~of~ rsporangia e of P l y toplrtlrora ii!fi's~ri~rs associated with indirect germination and aging. Trans. Br. Mycol. Soc. 51: 269-281. 8. MILLER,R. J., D. T. BELL,and D. E. KOEPPE.1971. The effect of water stress on some membrane characteristics of corn mitochondria. Plant Physiol. 48: 229-23 1. 9. NIR. I., A. POLJAICOFF-MAYBER, and S. KLEIN.1970. The effect of water stress on niitochondria of root cells. Plant Physiol. 45: 173-177. The technical assistance of Mr. Abraham 10. PERL,M., Y. UOHEN,and J. ROTELI.1972. The etfect of darkness on transfer of assimilates from cucunlber Cohen is gratefully acknowledged. to sporangia of Pseirrloperorro.s~~orri clrberlsis. Physiol. Plant Pathol. 2 : 113-122. 1. COHEN,Y., and J. ROTEM.1971. Dispersal and via- 11. ROTEM,J., and Y. COHEN.1973. Epidemiological bility of sporangia of P~e~rdo~~erorzospo~~ri crrberrsis. patterns of Plrq~tophtl~ornirrfestcirrs under semi-arid conditions. Phytopathology. Ln press. Trans. Br. Mycol. Soc. 57: 67-74. 2. CROSIER, E. S. 1963. The use of lead citrate at high W. 1934. Studies in the biology of P I I J ~ o I ~ I I12. - REYNHOLD, p H as an electron opaque stain in electron microsrhorri irrfestrirzs (Mont.) De Bary. Cornell Univ. copy. J . Cell Biol. 17: 208-212. Agric. Exp. Stn. Mem. No. 153. 3. ELSNEII,P. R., G. E. VANDERMOLEN, J. C. HORTEN, 13. SUSSMAN, A. S. 1968. Longevity and survivability of fungi. 111 The fungi. Eclitecl by Ainsworth and Sussand C. C. BOWEN.1970. Fine structure of Plrytoplltlrora iifestairs during sporangial differentiaticn and man. Academic Press, New York and London. germination. Phytopathology, 60: 1765-1772. pp. 447-486.

tive permeability, membrane integrity, and respiration of plant nlitochondria (8,9). It seems, therefore, reasonable to assume that loss of germinability and infectivity of WDR-treated sporangia of Ps. c~rbensis and P. ilzfestans derives primarily from the destruction of cellular and (or) of nlitochondrial membranes. These destructions are probably responsible for the excess leakage of metabolites, lack of respiration, and the decrease in the synthesis of proteins and nucleic acids found in the treated sporangia.