Short-term retrievability of Pythium propagules in simulated soil environments

Mycot. Res. 93 (2): 199-207 (1989)

199

Printed in Great Britain

Short-term retrievability of Pythium propagules in simulated soil environments

J. M. HARDMAN, D. J. PIKE AND M. W. DICK Departments of Botany and Applied Statistics. University of Reading, 2 Earley Gate. Whiteknights Rd, p.o. Box 239, Reading RG6 2AU, UK

Short-tenn retrievability of Pythium propagules in simulated soil environments. Mycological Research 93 (2): 199-207 (l989). The short term retrievability (3 d) of Pythium propagules was tested in sand. and partially-sterilized-soil plates. Assessment of propagule populations was by dilution plates. Retrievability was tested at two moisture contents, two incubation temperatures and with or without particulate bait amendments of Agrostis leaf blades or Hebe leaves. Sand moisture content, at the non-limiting levels tested, had no significant effect upon Pythium propagule retrievability. In the absence of bait amendments, a temperature of 20°C gave lower retrievability than 10° when the inoculum consisted of sporangia or zoospores but not when it was of hyphal swellings. At 20° recovery rates rarely exceeded the level of the initial inoculum. Retrievability from sporangia of P. intermedium and zoospore cysts of P. aqualile was favoured or even enhanced by a temperature of 10°. Retrievability from propagules in sand plates, soil plates and bait-amended sand plates showed a characteristic decline for the first 6-28 h followed by a recovery which, in some systems, considerably exceeded the levels of the initial inoculum. These characteristic retrievability curves are named 'J' curves. Percentage colonization of Agrostis bait, either at 10° or 20°. was generally greater than on Hebe bait at 20°. This preference was most marked for P. intermedium, but P. aquatile and Pythium H.S. group showed moderate colonization of Hebe and appeared to be able to use this bait to boost the spore population within the test time-scale. The propagule populations in sand plates did not correlate closely with the percentages of bait colonization, a trend most noticeable at 10° with Hebe baits. Key words: Pythium, Ecology, Population fluctuations, Colonization.

Hardman & Dick (1987) have shown that short-term fluctuations in Pyfhium populations are of the same order of magnitude as those found by Ali-Shtayeh, Lim-Ho & Dick (1986 b) for seasonal change. The fluctuations described by Hardman & Dick (1987) could occur in a matter of a day or two, suggesting that both production of propagules (or their 'availability' to sampling procedures) and the mortality of these propagules (or their' unavailability') could be rapid. The assessment of vegetative activity of Pyfhium mycelia in soil has to be indirect, and thus dependent on propagule production and demise. It has often been assumed that while the mycelial phase is ephemeral (Stanghellini, 1974), propagule survival would be of longer duration. Therefore, estimates of activity derived from propagule populations have been considered unreliable. However, there may be substantial reliability in such estimations, depending on the extent to which the fluctuating mycelial biomass and activity stems primarily from short-lived or long-lived propagule types. A balance must be achieved, in the experimental design, between the calibration of the time-scale of the fluctuations under observation and the population densities and probable halflives of the propagule types involved. Conventionally, a short-lived propagule type would be the zoospore cyst, while 14

a long-lived propagule type could be the oospore, but such a concept may be too simplistic and not relate to the biology of individual species. Long-term survival in the habitat may usually depend on long-lived propagule types, such as the oospore, while maintenance of mycelial population levels over both short and medium terms may be much more closely related to more ephemeral propagules (which may include oospores). For many species the oospore population may never represent more than a very small proportion of the total propagule population of the species, except in more or less extended periods of almost total mycelial inactivity. In such periods the oospore population (and the total propagule population) is still likely to be small in absolute, i.e. gm dry wt- 1, terms (Dick, 1966). The life-histories of different species of Pyfhium differ Widely in the relative proportions of different kinds of propagules produced (Ali-Shtayeh ef aI., 1986b), so that the propagule populations of different species of Pythium may not enable inter-specific comparisons of mycelial activity to be made on the basis of dilution plate estimates of available propagules. Autecological studies need not be subject to this constraint if the probable survival and mortality of propagules in simulated soil environments can be elucidated. MYC 93

Short-term retrievability of Pythium

MATERIAL AND METHODS Four taxa of Pythium were used in these experiments: Pythium H.S. group (APCC 204), P. intermedium de Bary (APCC 407e = CBS 221.68), P. torulosum Coker & Patterson (APCC 403a) and P. aquatile Hohnk (APCC HCL 7). They were selected on the basis of the diversity of their asexual sporulation. Asexual propagules and mycelium were produced from young colonies grown on com meal agar.

200 The concentration of each suspension was calculated using a haemocytometer and adjusted using a series of dilutions to 10 or 20 propagules ml- 1 . A simple viability test was then carried out by using 5 replicate O' 5 ml aliquots of the zoospore cyst suspension spread over nutrient agar. The haemocytometer count was thereby corrected to an estimate of the density of viable inoculum. Only a rough assessment could be made for the hyphal fragments.

Preparation of dishes Zoospores Blocks of agar, ca 15 mm square, were cut from the colony margin from cultures of P. iniermedium, P. forulosum or P. aqua file, and placed in double glass distilled water (DGDW) in a Petri dish. Lengths (40 mm) of Agrosfis leaf blade autoclaved in water, were placed against the agar block and incubated at 20°C (15° for P. iniermedium) for 48 h (72 h for P. interml!dium). If zoospores were not produced by then, cultures were chilled for 30 min at 4°. To isolate the zoospores, the agar block and Agrostis baits were removed and the DGDW containing the motile zoospores was poured through a layer of moist sterile Whatman no. 541 filter paper into a sterile 50 cm 3 beaker. If all hyphal fragments or sporangia were not removed, the filtration was repeated. By the time the suspension dilution was adjusted and incorporated into the experimental system, it was presumed that the zoospores had encysted.

Sporangia or hyphaI swellings of Pythium H.S. group or P. intermedium The cultures were prepared as above except that the incubation temperature was 20° and the incubation period was up to 5 d to ensure sufficient sporangia or hyphal swellings. Then, using a sterile scalpel blade (Swann-Morton # 22A) and forceps, the mycelium and sporangial mass was cut away from the bait and block, and removed by Pasteur pipette into a sterile 50 cm 3 beaker. Two Petri dishes containing a total of 6-8 colonized leaf blades were needed to produce sufficient propagules. The sporangial-mycelium mass so removed was gently chopped in DGDW using a rocking movement of the scalpel blade, and then filtered through a single layer of sterile damp muslin, which retained the tangled hyphae but allowed sufficient sporangia or hyphal swellings to filter through. If necessary the filtration was repeated to remove all hyphal fragments.

Mycelium of Pythium H.S. group or P. intermedium These were also cultured in the same way except that the incubation temperature was 25° and the incubation period was 24 h to ensure that no sporangia or hyphal swellings were formed. The mycelium was cut from the bait and block and pipetted into a sterile 50 cm 3 beaker containing DGDW. The mycelial mass was chopped, using a rocking movement of the scalpel to produce short hyphal fragments.

The simulated soil environments consisted of either sand or partially sterilized soil in 90 mm Petri dishes with or without bait particles. The baits were either Agrosiis leaf blades or Hebe (H. pinguiculifolia Hooker f. cv. Pagei) leaves which were wetsterilized in 'Universal Bottles' immediately before use. The leaves were then chopped into 2-4 mm lengths and about 50 pieces were added to each Petri dish.

Sand plates Sterile fine grade sand (50 g d. wt, pH 7'0) was added to a 90 mm glass Petri dish and moistened with the inoculum suspension and sealed with 'Cling film' before replacing the lid. The sand had a field capacity of 15'5 % and saturation occurred at 22 % moisture content.

Soil plates Soil was collected from the meadow of the Botanic Gardens, adjacent to site 3 (Hardman & Dick 1987), blended and divided amongst the glass Petri dishes, 30 g wet weight per dish. The difference in weights between soil and sand in the plates was necessary because of the volumes involved. The soil was then' partially sterilized' by dry heat (50° for 30 min) in order to remove the natural Pythium population present (Hardman, 1985). Preliminary tests showed that this treatment reduced the soil moisture content from about 16% to about 14%. In order to maintain moisture contents roughly equivalent to those of the sand plates and the known mean annual water content of the natural soil, only 4 ml of inoculum suspension could be added. This was just sufficient to ensure a reasonable distribution across the plate.

Inoculation Plates were inoculated with a standard volume of a suspension of a known concentration of propagules. The inoculum and sand or soil were mixed gently to produce a fairly even distribution of propagules throughout the plate before covering with 'Cling film'.

Sampling To measure the level of the Pyfhium population present after incubation, a sample of known wet weight of the inoculated sand or soil was extracted from several points around the plate, mixed, and divided between ten nutrient agar plates

J. M. Hardman, D. J. Pike and M. W. Dick

201

using 1'0-1'5 ml of cool molten 0'08% water agar per plate to aid dispersal of the sand or soil over the agar surface. Plates were only sampled once, so for experiments involving sampling on eight occasions sixteen sand or soil plates were prepared initially.

Fig. 1. Effect of sand moisture level and size of sample withdrawn upon % retrievability from different kinds of propagule of different species of Pythium. The joining of the sets of four points is merely a vehicle to aid appreciation of the differences between species and conditions. Initial inoculum of: mycelium (my c.) , sporangia or hyphal swellings (s.p.) , zoospores (cysts) (zoo.}----. Species symbols: Pythium aquati/e (P.a.) e, Pythium intermedium (Pj.)

Media

A, Pythium H.S. group (P. H.5.) _, Pythium torulosum (P.t.) O.

The VP 3 medium of Ali-Shtayeh et al. (1986 a) was used for soil plates, but it was modified by the omission of the antibiotics, vancomycin, pimaricin, penicillin and PCNB when sampling sand plates, since axenic systems were used. Rose Bengal was used to restrict colony spread in all cases. Within this experimental framework, incubation temperatures and time of incubation, proportions of inoculum, and proportions of samples withdrawn could be varied.

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Preliminary experiments Two preliminary experiments were necessary to justify a standard procedure. These were to determine a suitable moisture level and sampling system using the medium recommended by Ali-Shtayeh et ai. (1986a), and to check whether this medium was in fact adequate for the kinds of propagules under investigation. In the first such experiment two moisture contents were investigated: 16 % (approximating to field capacity) and 23 % (approximating to saturation). Two wet weights of samples were removed: 10 g and 20 g. Sampling was carried out immediately after the plates had been prepared (time 0). A 16 % moisture content and plate containing 50 g dry weight of sand and 10 ml propagule suspension at 20 propagules ml- 1 should theoretically yield 4 propagules for each gm dry weight. The dry weight equivalents of the wet weight samples removed were 8'4 g and 16'8 g respectively. Similarly a 23 % moisture content sand plate was produced using 50 g dry weight of sand and 15 ml of propagule suspension at 13'3 propagules ml- 1 to give the same theoretical yield. Dry weight equivalents were 7'7 g and 15'4 g respectively. For each of the four treatments and for each kind of propagule suspension used there were two replicate plates. Seven propagule suspensions were used: Pythium H.5. group, hyphal swellings, mycelium; P. intermedium, sporangia, zoospores, mycelium; P. toruiosum and P. aquatile, zoospores. There were thus two replicates of a factorial experiment of seven kinds of propagule x 2 weight samples x 2 moisture contents. The total number of colonies recorded from ten agar plates was used to calculate the '% recovery' for each treatment for each kind of propagule. There was evidence of greater variation between replicate values for larger values of % recovery, with variance being approximately proportional to the mean, so a square root transformation was used (Steel & Torrie, 1980). Frequently % values which lie between 0 and 100 are transformed by use of the arc sine transformation. This is not appropriate here since the % recovery is not bounded above by 100 % and indeed the data frequently gave values in excess of 100 %. After

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transformation the data were analysed using analysis of variance as a 7 x 2 x 2 factorial by means of the GENSTAT computer package. Although % recovery varied widely (Fig. 1), similar relative % recoveries can be seen from the zoospore cysts of Pythium aquatiie and P. toruio5um; from the sporangia of P. intermedium and the hyphal swellings of Pythium H.5. group, and from the mycelia of P. intermedium and Pythium H.5. group. The zoospore cysts of P. intermedium do not fit any of these three patterns. The low % recoveries of mycelium were to be expected because of the ephemeral nature of this life-form (Agnihotri & Vaartaja, 1967; Vaartaja & Agnihotri, 1969; Hendrix & Campbell, 1973) and its sensitivity to mechanical damage (Warcup, 1967). Pythium intermedium zoospore cysts appear similarly vulnerable to handling techniques and in this respect differ from the zoospore cysts of the other two species tested. 14-2

Short-term retrievability of Pythium

202

Fig. 2. Retrievability (by dilution plating) from Pythium H.5. group hyphal swelling inoculum over time, expressed as a percentage of initial inoculum density (haemocytometer count).

Fig. 3. Retrievability (by dilution plating) from Pythium intermedium sporangial inoculum over time, expressed as a percentage of initial inoculum density (haemocytometer count).

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There was no obvious overall trend to justify the use of 23 % as opposed to 16 % moisture content: nor the use of 20 g compared with 10 g sample weight. But 16% moisture and 20 g samples were selected for a second preliminary

experiment on different isolation media, because these conditions gave workable recovery levels of all propagule types. The results of this second experiment (not presented) showed again that recoveries from mycelial propagules were almost negligible. The presence or absence of Rose Bengal from the VP 3 medium (without antibiotics) had no significant effect upon propagule recovery except with P. intermedium zoospore cysts, where recovery was significantly less in the absence of Rose Bengal. Corn meal agar, used for comparison, was significantly the poorest medium for the recovery of Pythium H.5. group hyphal swelling propagules and zoospore cysts of P. aquatile and P. torulo5um. These results justified the use of VP 3 medium (minus antibiotics) with Rose Bengal for all subsequent work. Main series of experiments Substantial short-term fluctuations in Pythium populations

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occur within a period of a day or two. The following experiments were therefore continued for 72 h and were designed to investigate the survival of propagules under representative environmental conditions. Two plates were withdrawn for sampling at 0, 6. 18, 28, 41, 52, 60 and 72 h. As with the preliminary experiments, logistic factors curtailed the degree of replication. Each experiment, using a pair of replicate plates. was repeated twice. The pair of measurements at each of the eight time points was used to provide a measure of replicate to replicate variability for that data set (with 8 degrees of freedom). This approach is justified here since there was no evidence from the data that this variability changed systematically with the values of % recovery. and without more genuine replicates such a hypothesis cannot be tested. From these values the average standard error of any individual mean value was calculated. It must be stressed that this study was designed to be exploratory and comparative amongst taxa. For subsequent. stringently designed. autecological studies more replications would be made. It might then be sensible to make a square-root transformation of the data if it was apparent that the variance was larger for the higher values of % recovery.

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Fig. 4. Retrievability (by dilution plating) from Pythium aquatile zoospores (cysts) inoculum over time, expressed as a percentage of initial inoculum density (haemocytometer count).

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In the absence of any baits, the Pythium population never fully recovered beyond the level of the initial inoculum. Even the addition of a bait at 10° had no effect upon the population. In contrast, at 20° the addition of either Agrostis or Hebe baits produced a significantly better recovery at 72 h, such that the final population much exceeded the initial inoculum (9 x with Agrostis, 6'5 x with Hebe).

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The experiments compared retrievability with and without Agrostis bait amendment at 10° and 20°, and at 20° only, in soil plates, and in sand plates amended with Hebe baits. The results are given in Figs 2-4. The families of curves of these three figures show some general similarities. The most notable observation is that there was an initial decline in percentage recovery, irrespective of species of Pythium or kind of propagule, during the first 28 h. Thereafter the % recovery depended upon the environmental conditions, the species and the kind of propagule. Since Figs 2-4 are organized by species and propagule a short discussion of the results will be given under each species heading.

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With all treatments the inoculum population followed the same initial decline to a trough at 18-28 h. At 20° (without baits) the population again never recovered from the trough and remained fairly constant around a 35 % recovery level until 72 h. Only a marginally better recovery was obtained in the presence of Hebe at 20°. However, in contrast to Pythium H.5. group, P. intermedium showed a higher % recovery at the lower temperature in the absence of any bait. Nevertheless,

Fig. 5. Pythium H.S. group: percentage of baits of Agrostis or Hebe colonized over time. following inoculation of sand plates with

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Short-term retrievability of Pythium Fig. 6. Pythil.lm intennedil.lm: percentage of baits of Agroslis or Hebe colonized over time, following inoculation of sand plates with sporangial inoculum. 100

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Fig. 4. Pythium aquatile: zoospore cysts

The % recovery data for Pythium intermedium zoospore cysts were so low and erratic that no conclusions could be drawn.

These propagules differed from those of Pythium HS. group and P. intermedium in that at 20° in the presence of either kind of bait, the duration of the initial propagule decline was relatively short, after which the population steadily increased to about 8 times the level of the initial inoculum. At 10° with or without the presence of Agrostis baits, the initial decline

In general, the partially sterilized soil plates provided data that were intermediate between the bait-amended and unbaited sand. There was again an initial decline in % recovery up to 28 h followed usually by an increase to a level similar to that of

Fig. 7. Pythium intennedil.lm: percentage of baits of Agwsfis or Hebe colonized over time, following inoculation of sand plate with zoospore cyst inoculum. 100 S.E. bars

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the initial inoculum, or higher in the case of Pythium H.5. group. At the same time as the surface spread plate was sampled for Pythium recovery, 25 particulate bait pieces of Agrostis or Hebe were also plated out. Mean percentages of baits colonized by Pythium are shown in Figs 5-8. A general preference for Agrostis over Hebe was shown for all taxa and propagules tested. Hebe was particularly poorly colonized by P. intermedium, irrespective of the kind of inoculum. As would be predicted from the plating data, colonization of Agrostis at 20 0 was greater than at 10°. The levels of bait colonization did not reflect the population levels recorded by plating either within the propagule comparison for P. intermedium or between the zoospore cyst propagule comparison for P. intermedium and P. aquatile. For P. intermedium colonization was approximately 60% at 52 h for both kinds of propagule whereas the population level from a sporangial inoculum was much higher than from a zoospore cyst inoculum. In the comparison of zoospore cyst inocula the colonization levels of 60-80 % for both species may be contrasted with the propagule population levels of approximately 80 % of the initial inoculum for P. intermedium and 800% for P. aquatile.

DISCUSSION The results relate almost exclusively to spore populations because the technique did not favour the isolation of hyphal fragments (Fig. I). The most distinctive feature of the data is the substantial increase in retrievability within a relatively short time-scale (less than 72 h) from bait-amended systems. By 72 h the differences amongst kinds of bait and temperatures for incubation are statistically significant within each of the three fungal populations tested (Figs 2-4). For Pythium H.S. group (hyphal swellings), P. intermedium (sporangia) and P.

aquatile (zoospore cysts) Agrostis-amended systems at 20° gave the greatest increase in retrievability. However, differences did occur amongst these three species of Pythium. For example, the kind of bait was an important factor for P. intermedium - more so than for the other species. In systems without amendments, an incubation temperature of ICo tended to allow enhanced retrievability for inocula of P. aquatile zoospore cysts and P. intermedium sporangia but not for inocula of Pythium H.5. group hyphal swellings. When retrievability from partially sterilized soil systems was compared to Hebe-amended systems, the results from inocula of P. intermedium sporangia and Pythium H.S. group hyphal swellings were similar, but retrievability of P. aquatile zoospore cysts was substantially poorer from the soil system. All fungal populations in all test systems showed an initial decline in retrievability followed by some recovery or enhancement of the initial population level. Future experiments designed to test this particular observation by using more frequent time intervals and more replicates may show this decline to be statistically significant. From the data presented here the selection of a suitable test organism would be critical: inocula of P. aquatile zoospore cysts would be the least appropriate population to use in an attempt to prove this phenomenon. In general, the pattern of retrievability over time in bait-amended systems can be characterized as a 'J' shaped curve. In systems without amendments the ascending limb of this curve is much less pronounced, especially at the higher incubation temperature. Theoretical explanations (Pearson, 1988) will be published elsewhere. In the regimes under consideration, natural death within a propagule-form population such as a hyphal swelling is unlikely to occur within a few hours, but if this were so, then there would be a rapid and irreversible population decline. For most of the spore forms and regimes tested here this is not

Short-term retrievability of Pythium true. Lifshitz & Hancock (1984) found that increases in the population of P. ultimum hyphal swellings over five days were favoured by temperatures of between 16-27° and a matric potential of - 0'25 to - 0'5 bars - both conditions being similar to those favoured by Pythium H.5. group hyphal swellings in our experiments. (They also found that maximum leaf colonization in sterile soil occurred after 3 d incubation at 27-30° but that the optimum temperature for saprophytic growth was shifted from 27-30° in sterile soil to below 27° in a non-sterile environment.) Similar results were also found by Barton (1960). We suggest that, following inoculation, a percentage of the propagules germinate or start to germinate. In this state, being mycelial, they are not available to the sampling procedure. Within 20-30 h many of these germinated spores will have produced secondary sporulation of the same or another kind of propagule. Such sporulation could be achieved solely through a recycling of endogenous reserves and thus occur in non-nutrient systems. Although there are many observations from other oomycetes of depauperate growths from zoospore cysts to form small sporangia (Willoughby, 1977), this phenomenon has been ignored for Pythium. It does occur (Hallett & Dick, 1986), but its occurrence may be variable amongst species. Peethambaran & Singh (1977) found that, after infesting a non-sterile soil with lobulate sporangia of P. aphanidermatum and P. graminicola, within the first 24 h there was a transient increase in the propagule numbers due to sporangia I germination and production of germlings. However, by 3 d the population began to decline as germlings underwent lysis and no further survival units were produced. This is at variance with the results from this work in which zoospore cysts, spherical sporangia and hyphal swellings were capable of both germination and the production of further propagules in soil systems. However, we did not use lobulate sporangia because of the difficulty in suspending such units uniformly. Results that are available on the longevity of zoospores (or cysts) in vitro are often contradictory. Encysted zoospores which have a low survival capacity have been shown to germinate readily in naturally fungistatic field soil (Burr & Stanghellini, 1973); in sterile and non-sterile lake water (Hallett & Dick, 1981) and in water without an exogenous source of nutrients (Stanghellini, 1974). In contrast, Luna & Hine (1964) found that zoospores of P. aphanidermatum formed resting structures in both sterile and non-sterile soil and only germinated upon the addition of exogenous nutrients to the soil. Estimates of the life expectancy of zoospores range from less than 24 h (Burr & Stanghellini, 1973; Hallett & Dick, 1981) to 7 d in moist soil (Luna & Hine, 1964). The extent of any recycling of endogenous reserves would be limited by the threshold levels of endogenous reserves which control viability. Normally, endogenous reserves will be in excess. Thus, in natural populations very rapid population increases could be envisaged, limited only by the mean duration of mitotic interphase. Such a population increase would be considerably facilitated by short-term utilization of ephemeral exogenous resources. In the natural environment such population changes have been recorded (AIi-Shtayeh et al., 1986b; Hardman & Dick, 1987), but the populations can

206

decline almost as rapidly and stabilize at a level approximating to the seasonal mean level for that site. Causes of this subsequent decline could be microcycling with rapid exhaustion of endogenous capacity in the absence of ephemeral exogenous resources and predation as well as natural death in the population. The fact that such a proposed cycle of sporulation does not correlate closely with colonization (Figs 5-8) may mean that there are two kinds of activity which can coexist in a population: the short-term microcycling of readily accessible, ephemeral exogenous resources into potential inoculum via a minimal mycelial involvement, and the longer-term mycelial colonization of larger, solid substrata accompanied by a more complex pattern of spore production. It is difficult to predict which might be the more important in total biomass recycling and this might differ for different species. It may not be constant from site to site even for a single species. The data presented here support a general principle of propagule regeneration, but also show that individual species and spore life-forms respond, in different degrees, to simulated soil environment conditions. The patterns of population fluctuations over medium and short-term periods (Ali-Shtayeh et al., 19Mb; Hardman & Dick, 1987) that are held in common for the genus, and the different relative population levels of different species in different habitats (Ali-Shtayeh et al., 1986b; Dick & A1i-Shtayeh, 1986) are both consistent with the hypothesis presented here. The definition of the spore life-form is complicated because it must avoid confusion between function and cytomorphogenesis: resting propagules are not always oospores and 'Oospores are not necessarily primarily adapted for longterm survival for all species. In environmental circumstances where the resistant spore life-form is a minor component of the total life-form populations of the species, e.g. P. aquatife in wet soils, or when a single spore life-form fulfils both survival and multiplication functions, e.g. Pythium H.S. group in grassland, dilution plating provides an adequate measure of vegetative activity over the precedi~g few days. We envisage that the occurrence and probable time-scale of any propagule regeneration could be deduced from the 'J' curves. From theoretical models (Pearson, 1988), it will be possible to return to field work and simulated systems, using dilution plating supplemented by direct observation of propagules, to verify some of the suggestions made here.

J.M.H. gratefully acknowledges support from an SERC studentship.

REFERENCES V. P. & VAARTAJA, 0. (1967). Effects of amendments, soil moisture content and temperature on germination of Pythium sporangia under the influence of soil mycostasis. Phytopathology 57, 1116-1120. ALI-SHTAYEH, M. 5., L1M-HO, C. L. & DICK, M. W. (1986a). An improved method and medium for quantitative estimates of populations of Pythium species from soil. Transactions of the British AGNIHOTRI,

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ALI-SHTAYEH, M. S., LIM-HO, C. L. & DICK, M. w. (1986 b). The phenology of Pythium (Peronosporomycetidae) in soil. journal of Ecology 74, 823-840. BARTON, R. (1960). Antagonism amongst some sugar fungi. In The Ecology of Soil Fungi - an International Symposium (ed. D. Parkinson & J. S. Waid), pp. 160-167. Liverpool, UK: Liverpool University Press. BURR, T. J. & STANGHELLINI. M. E. (1973). Propagule nature and density of Pythium aphanidermatum in field soil. Phytopathology 65, 1499-1501. DICK, M. W. (1966). The Saprolegniaceae of the environs of Blelham Tam: Sampling techniques and the estimation of propagule numbers. journal of General Microbiology 42, 257-282. DICK, M. W. & ALI-SHTAYEH, M. S. (1986). Distribution and frequency of Pythium species in parkland and farmland soils. Transactions of the British Mycological Society 86, 49---62. HALLETT, I. C. & DICK, M. W. (1981). Seasonal and diurnal fluctuations of oornycete propagule numbers in the free water of a freshwater lake. journal of Ecology 69, 671---692. HALLETT, I. C. & DICK, M. W. (1986). Fine structure of zoospore cyst ornamentation in the Saprolegniaceae and Pythiaceae. Transactions of the British Mycological Society 86, 457-463. HARDMAN, J. M. (1985). The ecology of Pythium: propagule survival and retrieval in natural and artificial systems. Ph.D. Thesis, University of Reading. HARDMAN, J. M. & DICK, M. W. (1987). Short-term fluctuations in the availability of Pythium propagules for isolation from soil. Transactions of the British Mycological Society 88, 29-39.

HENDRIX, F. F. & CAMPBELL, W. A. (1973). Pythium as plant pathogens. Annual Review of Phytopathology II, 77-98. LIFSHITZ, R. & HANCOCK, J. G. (1984). Environmental influences on the passive survival of Pythium ultimum in soil. Phytopathology 74, 128-132. LUNA, L. V. & HINE, R. B. (1964). Factors influencing saprophytic growth of Pythium aphanidermatum in soil. Phytopathology 54, 955-959. PEARSON, J. (1988). Empirical modelling of population dynamics in Oomycetes. M.Sc. Thesis (Applied Statistics). University of Reading. PEETHAMBARAN, C. K. & SINGH, R. S. (1977). Survival of different structures of Pythium species in soil. Indian Phytopathology 30, 347-352. STANGHELLINI. M. E. (1974). Spore germination, growth and survival of Pythium in soil. Proceedings of the American Phytopathological Society I, 211-214. STEEL, R. G. D. & TORRIE, J. H. (1980). Principles and Procedures of Statistics, 2nd Edn. New York: McGraw-HilI. V AARTAJA, O. & AGNIHOTRI. V. (1969). Interaction of nutrients and four antifungal antibiotics in their effects on Pythium species in vitro and in soil. Plant and Soil 30, 49---61. WARCUP, J. H. (1967). Fungi in soil. In Soil Biology (ed. A. Burges & F. Raw), pp. 51-110. New York. U.s.A.: Academic Press. WILLOUGHBY, L. G. (1977). An abbreviated life cycle in the salmonid fish Saprolegnia. Transactions of the British Mycological Society 69, 133-135.

(Received for publication 17 August 1988)

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