Formation of sporangia by Phytophthora drechsleri in soil at high matric potentials

J . M . DUNIWAY Department ofplant Pathology, University of California, Davis, California 95616

Received December 10, 1974

DUNIWAY, J. M. 1975. Formation of sporangia by Phytophthora drechsleri in soil at high matric potentials. Can. J. Bot. 53: 1270-1275.

The formation of sporangia by Phytophthora drechsleri was examined in soil at constant matric potentials ($,) between 0 and -0.3 bar, corresponding to soil water contents between saturation and field capacity. At $, = -0.3 bar, P. drechsleri required 1.5 days to form a significant number of sporangia in soil. After 3-1 1 days in soil, when maximum numbers of sporangia were found, all $,values from -0.025 to -0.3 bar were optimum for the formation of sporangia in both sterilized and unsterilized soil. In contrast, few sporangia were formed in soil at $, values of 0 and -0.01 bar. Mycelia on the soil surface at $, = 0 formed many sporangia, indicating that aeration at a depth of 5 mm in saturated soil was inadequate for sporangia to form. Viable sporangia were recovered from soil as long as 35 days after mycelial disks were placed in soil. Evidence is presented for the release of zoospores by sporangia in soil at all $, values tested.

Introduction The association of root and crown rots caused

by Phytophthora spp. with wet soil conditions is frequently attributed to the water requirements for zoospore production and motility in soil (8, 13, 15, 18). Although many factors that influence the formation of sporangia by soil- borne Phytophthora spp. have been examined (5, 17, 19), there has been little quantitative work on the influence of soil moisture, and most of the literature only emphasizes the highly stimulatory influence of liquid water on the formation of sporangia (5, 17, 19). An important exception is a recent study by Sneh and Mclntosh (14) on Phytophthora cactorum. They used three levels of soil moisture and found that numerous sporangia were formed at matric potentials ($,) of -0.1 and -0.3 bar. but that few or no sporangia were formed at $, = -3.0 bars. Because the influence of soil moisture on the formation of sporangia by Phytophthora spp. is largely unknown and is of probable significance in plant disease, the influence of soil moisture on the formation of sporangia by Phytophthora drechsleri Tucker was examined quantitatively. This paper describes the influence of soil $, values between 0 and -0.3 bar, which corre- spond to soil water contents between saturation and field capacity.

Materials and Methods The isolate of P. drechsleri used in all experiments was

the A2 mating type and was originally isolated from safflower (Carthamus tinctorius L.). Mycelium was grown on lima bean agar made by boiling 200 g of frozen lima

beans in distilled water for 20min and blending the cooked beans in the water. The resulting suspension was brought up to 1000 ml with distilled water, and 10 g dextrose, 10 g yeast extract, and 15 g agar were added before autoclaving at 121C for 15 min. Petri-plate cultures were incubated at 25C in the dark, where P. drechsleri produced abundant aerial mycelia with few swellings and no sporangia. When mycelia were to be placed in soil, a 7-mm-diam cork borer was pressed against the agar surface and disks of aerial mycelia were lifted off the agar with forceps (5). Mycelial disks were then submersed in a 400-ppm solution of fluorescent brightener (Calco- fluor @ White M2R New, American Cyanamide Co.) for 3 h. Disks were subsequently rinsed three times for 10 min in water. T o facilitate recovery from soil, each mycelial disk was coated with a thin layer of moist soil and placed in a small envelope of fiber glass window screen. Except where noted, mycelial disks in envelopes were placed at a final depth of 5 mm below the soil surface. Soil was prepared by mixing 2 volumes of sandy loam with 1 volume of sieved (1.4-mm openings) peat. Some of the soil was sterilized by autoclaving at 121C for 1 h on 2 successive days.

Soil moisture was controlled by using Biichner funnels with fritted glass of fine porosity (Kimble 28400-90F) a s tensiometers (6). Continuous columns of water were established between the fritted glass and water reservoirs and the funnels were placed at various heights above the reservoirs to obtain W, values between 0 and -0.3 bar. Soil was packed to a depth of 1 cm on top of the fritted glass, water was added to the soil surface, and tensiometers were allowed to stand overnight. The envelopes con- taining mycelial disks were then buried and 20 ml of water was added to the soil surface. Finally, tops of the Biichner funnels were covered with loosely fitting plastic bags to retard evaporation. The approximate half-time for changes in soil water content in the tensiometers was less than 20 min and soil W, values were essentially constant. Estimates of solute potential (w,) of the soil o n tensiometers were made by withdrawing small volumes of solution from just below the fritted glass after tensio- meters had equilibrated for several weeks. Solution W, was measured in a thermocouple psychrometer (2) and

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CULTURE AGE (days)

FIG. 1. Effect of culture age on the fresh weight of mycelial disks and number of sporangia formed by them in soil. Fresh weights were measured immediately after disks were lifted from agar cultures, and sporangia were counted 7 days after the same disks were placed in unsterilized soil at v, = -0.15 bar.

vs ranged from -0.3 to -0.8 bar, with no consistent variation in v, with v,. The water content of soil from tensiometers was determined by drying soil to a constant weight at 105C. Soil temperature in the tensiometers ranged between 23 and 27C, but variation between tensiometers was less than 1C a t any one time.

Sporangia formed in soil were observed with the aid of fluorescence microscopy (16). Mycelial disks were re- covered from the soil in most treatments and all the soil within a screen envelope was sampled in the few treat- ments where mycelia were not found. Each mycelial disk or soil sample was blended in 5 ml water for 10 s (Waring Blender with a microcontainer at medium speed). The suspension was centrifuged at 1600g for 10 min and the pellet was resuspended in water on a vortex mixer in a final volume of 0.5 ml. A drop of the final suspension was placed on a slide under a cover glass supported by small pieces of glass to give a sample of constant depth. Either 100 sporangia or the sporangia in 50 fields (0.4 mm diameter) were counted per slide, depending on which number was reached first, and the total number of sporangia was calculated for each mycelial disk. Treat- ments included four to five disks per tensiometer and a Duncan's test was used to judge significance at the 5% level. A preliminary experiment showed that the blending and centrifugation procedure did not alter the condition or viability of sporangia. It was, however, necessary to complete the counting procedure within 20 min because keeping sporangia in water for longer periods frequently induced indirect germination.

The viability of sporangia was examined by spreading the suspension used for counting sporangia, or some dilution thereof, on a selective medium. Sporangia with new germ tubes were counted after 16 h incubation on the medium. The selective medium contained 17 g corn- meal agar (Difco), 10 mg pimaricin (Delvocid @, Gist- Brocades nv. Delft, Holland), 300 mg vancomycin hydro- chloride (Vancocin @, Eli Lilly & Co.), 100 mg penta- chloronitrobenzine (Aldrich), 50 mg penicillin G potas- sium salt (1585 units/mg, Calbiochem), and 50 mg strep- tomycin sulfate (Sigma) per 1000 ml of distilled water.

The antibiotics were added to autoclaved medium after it had cooled to 50C.

Results

The age of the cultures from which mycelia were taken did not have a marked influence on the number of sporangia formed by mycelial disks in soil (Fig. 1). Because of the relative ease with which mycelial disks could be lifted from older cultures, 14- to 21-day-old cultures were used for the remaining experiments.

Mycelia of P. drechsleri required 1.5 days t o form a significant number of sporangia in soil (Fig. 2A). The maximum number of sporangia was found at 7 days, after which time the number declined significantly. During the first 4 days in soil most of the sporangia were full of cytoplasm and did not have germ tubes (Fig. 2B). Small but significant percentages of sporangia had germ tubes at 9 and 11 days and an occasional germ tube gave rise to a new sporangium. More important were the large and significant per- centages of sporangia at 7, 9, and 11 days which

TIME (days)

TIME (days)

FIG. 2. Effect of time that mycelial disks were in soil and the number (A) and condition (B) of sporangia. Mycelial disks were placed in sterilized soil at v, = -0.3 bar on day 0.

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0 ' I 0 1 2 3 4 5 a 6

NUMBER OF EMPTY SPORAMGIA PER DISK X 10'

FIG. 3. Percentage of mycelial disks from soil that had spores resembling zoospore cysts plotted as a function of the number of empty sporangia per disk. Data from several tensiometer experiments at v, values between 0 and -0 .3 bar are included. Disks were grouped for the calculation of percentages by rounding off numbers of empty sporangia to the nearest multiple of lo4 and each point represents 10-18 disks.

had no germ tubes and were empty of cytoplasm. The empty sporangia having no germ tubes were open at their distal ends and resembled sporangia which had released zoospores in pure culture. Samples from soil with many empty sporangia also contained many spores resembling zoospore cysts. The spores fluoresced brightly and were within the size range of zoospore cysts. Data from several experiments are used to show the positive correlation between the presence of the spores and the number of empty sporangia in each sample (Fig. 3). There was no correlation between $, value and the presence of the spores.

Several attempts were made to germinate the spores from soil which appeared to be zoospore cysts. Unfortunately, germination of the spores was rarely observed on the selective medium. In one experiment at 14 days after mycelial disks were placed in soil, several of the spores ger- minated when transferred to a medium contain- ing 9 g glucose, 1.6 g L-asparagine monohydrate, 0.2 mg thiamine hydrochloride, and 15 g agar per 1000 ml water, as well as vancomycin, penicillin G, and streptomycin in the same con- centrations as were used in the selective medium. Six of the germinated spores were on uncon-

taminated portions of the Petri dishes and were individually transferred to cornmeal agar. My- celium resulting from these spores was finally identified as P. drechsleri. However, in other experiments the identity and viability of the spores were not successfully determined by culture techniques.

All $, values between -0.025 and -0.3 bar were optimum for the formation of sporangia 3-11 days after mycelial disks were placed in both sterilized and unsterilized soils (Fig. 4). Sterilized soil at $, 2 -0.01 bar and unsteril- ized soil at $, 2 -0.005 bar yielded signifi- cantly fewer sporangia than did lower $, values when mycelial disks were buried 5 mm below the soil surface. When soil was completely saturated at $, = 0, mycelia on the soil surface formed

b 0 '

0 - 0.1 -0.2 -0.3

MATRIC POTENTIAL (bars)

- r 3 Z

d g 12

FIG. 4. Number of sporangia per mycelial disk plotted as a function of decreasing matric potential. Circles represent disks 5 mm below the soil surface, squares represent disks on the soil surface, and open and closed symbols, respectively, represent sterilized and unsterilized soil. Sporangia were counted at 3-11 days and again a t 25-35 days after mycelial disks were placed in soil.

rn LL

0 10

25-35 DAYS --

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DUNIWAY: P. DRECHSLERI SPORANGIA 1273

I 0 -0.1 -0.2 - 0.3

MATRIC POTENTIAL (bars)

FIG. 5. Relationships between water content and matric potential for sterilized and unsterilized soil.

significantly more sporangia than did mycelia at a depth of 5 mm. Large numbers of sporangia were present in most soil treatments 25-35 days after mycelial disks were placed in soil (Fig. 4). However, the only significant increase during the interval between the 3- to 11- and 25- to 35-day samples occurred in unsterilized soil at $, = -0.005 bar. In sterilized soil, with the exception of $, = -0.2 bar, the numbers of sporangia at 3-11 and 25-35 days were not significantly different, whereas in unsterilized soil there were significantly fewer sporangia at 25-35 days than at 3-11 days at all $, values between -0.01 and -0.3 bar. Differences between sterilized and unsterilized soil at 25-35 days and $, 5 -0.01 bar were also significant. Relationships between water content and $, for the soils in which sporangia formed (Fig. 4) are shown in Fig. 5.

Throughout the experiment in Fig. 4 and a second experiment which gave similar results there was no significant influence of $,, soil sterilization, or depth on the condition of sporangia. The average percentage of sporangia which were full of cytoplasm and ungerminated ranged from 50 to 60% at 3-11 days and from 25 to 45% at 25-35 days. An average of 93 and 78% of such sporangia at 3-1 1 and 25-35 days, respectively, germinated directly on selective medium. Furthermore, many ungerminated sporangia from soil released zoospores when left

in water for 2 h. Less than 10x of the sporangia in the soil of any treatment had germinated directly.

Discussion The sporangium was the predominant propa-

gule formed by mycelia of P. drechsleri in soil. No sexual structures were observed on the single mating type used and the chlamydospores recent- ly reported for P. drechsleri (4) were not found. Mycelial disks were recovered from all but the 25- to 35-day samples of unsterilized soil at $, 2 - 0.1 bar (Fig. 4). However, the recovered hyphae rarely contained cytoplasm. Evidently, complete lysis of walls of empty hyphae, as well as sporangia, was not so rapid as that reported for other Phytophthora spp. in soil (14, 16). The presence of large numbers of viable sporangia as long as 35 days after mycelia were placed in soil suggests that the sporangium may have some survival value in soil. Unfortunately, the long- evity of sporangia cannot be deduced from the present study because new sporangia may have been formed throughout the course of the experi- ments. Zoospores are frequently thought to be the infectious propagule of soil-borne Phytoph- tllora spp. (8, 13, 15, 18) and the results presented here (Fig. 3) suggest zoospore cysts may have an additional role as a propagule in soil. Zoospore cysts are reported to persist in soil (10, 1 I), and experiments are now underway to establish more clearly the release and viability of zoo- spores of P. drecluleri in soil.

At $, values between -0.025 and -0.3 bar, the soil used here was a comparatively favorable medium for the formation of sporangia by P. drechsleri. Water extract of the same soil (200 g moist soil per litre) induced a maximum of only 2 x lo4 sporangia per mycelial disk. Even fewer sporangia were formed in water extracts of two other soils and in 0.01 M KNO,, but differences between soil extracts were not so extreme as those reported by Broadbent and Baker (3). The maximum number of sporangia reported here for soil extract is equivalent to the maximum number of sporangia formed per unit area of mycelial disk by P. parasitica in 0.01 M KNO, (5).

Gas exchange between the atmosphere and a depth of 5 mm in saturated or nearly saturated soil is inadequate for sporangium formation (Fig. 4). Perhaps the strongest evidence for this conclusion is the large number of sporangia

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1274 CAN. J . BOT. VOL. 53, 1975

formed on the surface of saturated soil. The correlation between the decrease in soil water content (Fig. 5) and increase in number of sporangia at 3-1 1 days (Fig. 4) with decreasing $, also suggests that aeration has a major effect on sporangium formation in soil. In fact, at $, values of -0.2 and -0.3 bar, sporangia were probably formed in air-filled rather than water-filled soil pores. Pores 15 pm in diameter, which are the smallest pores able to accommo- date most sporangia, are expected to drain water at $, = -0.2 bar (6). However, in con- trast with the conclusion of Bainbridge (1) for sporangia of Pythium ultimum, it cannot be concluded from the present data that sporangia of P. drechsleri form only in the air-filled pores of soil. The similarity between results for steril- ized and unsterilized soil (3-1 1 days in Fig. 4) indicates that living soil microflora do not bring about the limiting influence of high $, values, or for that matter any $, value tested, on the formation of sporangia.

Inhibition of the formation of sporangia by poor soil aeration is expected from the literature. As long ago as 1933-1934, Toxopeus (cited in 18) found moist but well-aerated soil to be the most suitable for P. nicotianae to form sporangia. Several authors (5,7, 17) reported that sporangia formed by Phytophthora spp. in solution or soil extract are located at or near the air-liquid interface. This was also the case for P. drechsleri in soil extract. Although other factors such as strength of the soil solution may be important, adequate aeration may explain why more spor- angia were formed in soil at optimum $, values than were formed in soil extract. Formation of sporangia by Phytophthora spp. is inhibited by both low oxygen and high carbon dioxide con- centrations (12) and it is not known which of these gases was more limiting in saturated soil. In one experiment, some of the Biichner funnels were ventilated with 6 litres of water-saturated air or nitrogen per minute. Nitrogen over soil at $, = -0.3 bar inhibited the formation of sporangia to the same extent as did a depth of 5 mm in saturated soil under air. The experiment also showed that aeration under the plastic usually used to cover the Biichner funnels was adequate for maximum numbers of sporangia to form.

The absence of significant formation of spor- angia by P. drechsleri and perhaps other Phytoph- thora spp. in saturated soil may affect the

isolation of this genus from soil. The usual iso- lation procedure for Phytophthora spp. involves trapping the fungus in plant tissues placed in the water over flooded soil (e.g. 7). Zoospores are the propagule most likely to infect plant tissues under these conditions. Unless sporangia are already present in the soil, more moderate wetting of the soil before flooding may enhance the trapping of Pl~ytophthora spp.

Much of the literature on the genus Phytoph- thora gives the impression that an abundance of liquid water is necessary for sporangia to form (5, 13, 17, 19). However, the lowest $, used in the present study, -0.3 bar, was opti- mum for the formation of sporangia by P. drech- sleri. Furthermore, the results suggest that spor- angia of P. drechsleri can release zoospores at $, values down to -0.3 bar. In contrast with P. drechsleri, formation of sporangia by P. cac- torurn was somewhat greater in soil a t $, = -0.1 bar than at $, = -0.3 bar, and zoospore cysts were seen only rarely (14). Phytophthora cactorum formed few sporangia in soil a t $, = - 3.0 bars, the only other $, value tested by Sneh and McIntosh (14). The tensiometers used with P. drechsleri are not suitable for obtaining 9, values much lower than -0.3 bar and other techniques are required to examine sporangium formation by P. drechsleri at low soil water potentials. It should be noted, however, that because of the contribution of $,, soil at the lowest $, value obtained in the tensiometers had a total water potential of -0.6 to - 1.1 bars. With the possible exception of zoospore release in Aphanomyces euteiches (9), water potentials which have been found to be limiting to fungi are much lower than - 1.1 bars (6).

Acknowledgment This work was supported by National Science

Foundation Grant GB 32809. The author thanks Mrs. H. D. Zumwalt for technical assistance.

I. BAINBRIDGE, A. 1970. Sporulation by Pyrhirrm ~rlrimrrm a t various soil moisture tensions. Trans. Br. Mycol. Soc. 55: 485488.

2. BARRS. H. D. 1968. Determination of water deficits in plant tissues. Water deficits and plant growth. Vol. I. Edited by T. T . Kozlowski. Academic Press, New York. pp. 235-368.

3. BROADBENT, P., and K. F. BAKER. 1974. Behavior of Phyrophrhorn cinnatnomi in soils suppressive and conducive to root rot. Aust. J . Agric. Res. 25: 121-137.

Can

. J. B

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4. COTHER, E. J . , and D. M. GRIFFIN. 1973. Formation of chlamydospores by Pl~ytophtl~ora drecl~sleri. Trans. Br. Mycol. Soc. 61: 379402.

5. GOODING, G. V., and G. B. LUCAS. 1959. Factors influencing sporangial formation and zoospore activ- ity in Phytophthora parasilica var. tlicotirrnae. Phy- topathology, 49: 277-281.

6. GRIFFIN, D. M. 1972. Ecology of soil fungi. Syracuse Univ. Press, Syracuse, New York.

7. G R I M M , G . R., and A. F . ALEXANDER. 1973. Citrus leaf pieces as traps for Pl~ytopl~tlzora parnsitica from soil slurries. Phytopathology, 63: 540-541.

8. HICKMAN, C. J . 1970. Biology of Phytophtl~ora zoo- spores. Phytopathology, 60: 1128-1 135.

9. HOCH, H. C. , and J . E. MITCHELL. 1973. The effects of osmotic water potentials on Aplronot~iyces elllei- c11es during zoosporogenesis. Can. J . Bot. 51: 413- 420.

10. MCINTOSH, D. L. 1972. Effects of soil water suction, soil temperature. carbon and nitrogen amendments, and host rootlets on survival in soil of zoospores of Phytopl~tl~orrr cactorrrtn. Can. J. Bot. 50: 269-272.

11. M E H R O ~ R A , R. S. 1972. Behavior of zoospores of Phyfopllthora tnegaspertna var. sojae and P. dre- cl~sleri in soil. Can. J . Bot. 50: 2125-2130.

12. MITCHELL,D. J . , a n d G . A.ZENTMYER. 1971. Effects of oxygen and carbon dioxide tensions on sporangium

and oospore formation by Phytopl~tl~ora spp Phy- topathology, 61: 807-81 1 .

13. PRATT, R. G., and J . E. MITCHELL. 1973. Conditions affecting the detection of Pl~ytophtlrorrr megasperma in soils of Wisconsin alfalfa fields. Phytopathology, 63: 1374-1379.

14. SNEH, B. , and D. L. MCINTOSH. 1974. Studies on the behavior and survival of Pl~ytophtl~orn cactorrrm in soil. Can. J . Bot. 52: 795-802.

15. STOLZY, L . H., J. LETEY. L. J . KLOTZ, and C. K. LABANAUSKAS. 1965. Water and aeration as factors in root decay of Citr~ts sinensis. Phytopathology, 55: 270-275.

16. TSAO, P. H. 1970. Applications of the vital fluorescent labeling technique with brighteners to studies of sap- rophytic behavior of Phytopl~tl~ora in soil. Soil Biol. Biochem. 2: 247-256,

17. WATERHOUSE, G . M. 193 1. The production of conidia in the genus Plrytopl~rl~ora . Trans. Br. Mycol. Soc. 15: 311-321.

18. WESTSTEIJN. G. 1973. Pl~ytophthora nicotiannc. var. tlicotianoe on tomatoes. Neth. J . Plant Pathol. 79: suppl. 1.

19. ZENTMYER, G. A., and D. C. ERWIN. 1970. Develop- ment and reproduction of P l ~ y t ~ p l ~ t h o r a . Phytopath- ology, 60: 1120-1 127.

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