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Dynamics of the survival and infectivity to potato tubers of sporangia of Phytophthora infestans in three different soils
Soil Biol.Biochem.Vol. 26, No. 8, pp. 945-952, 1994
00384717(94)EOO32-U
Copyright 0 1994 Elsevier science Ltd
Printed in Great Britain. All rights reserved 0038-0717/94$7.00+ 0.00
DYNAMICS OF THE SURVIVAL AND INFECTIVITY TO POTATO TUBERS OF SPORANGIA OF PHYTOPHTHORA INFESTANS IN THREE DIFFERENT SOILS D. ANDRIVON INRA, Station de Pathologie V&g&ale, Domaine de la Motte, BP29, F-35650 Le Rheu, France (Accepted 9 February 1994) Sumntary--Changes affecting sporangia of Phytophthora infestans in three soils from Brittany were monitored at nine dates over a 60 day period, using buried filter membranes on which sporangia had been deposited. Germination was poor on all dates (< 30%), but significant differences between soils were found both in germination rates and in the proportion of germlings producing secondary sporangia. Lysis was active in all three soils (up to 4060% of sporangia affected), and was associated with colonization of the pathogen organs by bacteria or fungi. Infectivity to potato tubers persisted for 15-45 days depending on soil. No single index describing inoculum state could be correlated with infectivity to tubers. One of the three soils was suppressive to P. infestans, and was characterized by very low germination rates and intense lysis of the pathogen by fungal hyphae.
INTRODUCTION
The ecology and epidemiology in soil of the potato late blight fungus Phytophthoru infestuns (Mont.) de Bary has been relatively little studied in comparison with the aerial phases of the life cycle of this destructive pathogen. It is, however, an important stage to consider, since it conditions tuber contamination and infection (Murphy, 1921). It may also be of importance for the survival and perennation of the fungus by means of oospores, which might be formed in the field under European conditions (Frinking et al., 1987) since the introduction of the A2 mating type in the late 1970s (Fry et al., 1992). To be able to infect the host, these oospores have to germinate, either through germ tubes (hyphae) or secondary sporangia, which in turn should be capable of some saprophytic life until they reach susceptible host tissue and become parasitic. It is commonly admitted that P. infestans, like many other members of the genus, has poor saprophytic capacities in soil (Malajczuk, 1983). Under optimal conditions, infectivity to potato tuber slices of soils infested with sporangia of P. infestuns persisted for 26-77 days, depending on soil type and experimental procedures (Murphy, 1922; Zan, 1962; Lacey, 1965; Bogulavskaya and Filippov, 1977). This limited persistence in soil is generally attributed to the inactivation and rapid degradation of sporangia and mycelium by soil microorganisms. Lacey (1965) observed that mycelial growth of the pathogen was good in sterilized soil, but was severely impaired in unsterilized soil. He also showed that, in vitro, Cladosporium herbarum, Monosporium sylvaticum, Penicillium sp. and an unidentified actinomycete species limited the
growth of P. infestans but did not affect cytoplasm organization, whereas Mucor spinosus, Rhizoctonia solani and Trichoderma viride had a lytic effect on mycelium and sporangia. Gregory (1983) once described soil infectivity as a ‘poltergeist’, or disembodied ghost, in the case of P. infestam. In fact, although Murphy (1922) and Zan (1962) reported some microscopic observations of the evolution of P. infestans inoculum in contact with natural soil, quantitative data concerning temporal changes in the state and composition of this inoculum, as well as the relationships between inoculum composition and infectivity to potato tubers are missing. Several authors (Murphy, 1922; Zan, 1962; Lacey, 1965; Bogulavskaya and Filippov, 1977) noted that infectivity varies noticeably with soils, as does its persistence over time; however, the reasons for such differences among soils handled with the same experimental procedures remain largely unexplored. This study was therefore undertaken with the aim of (i) describing in a quantitative manner the changes affecting sporangia exposed in different soils and (ii) relating these changes with those of inoculum infectivity to potato tubers. For these purposes, the buried filter membrane method of Adams (1967) was used, as modified by Tivoli et al. (1983, 1990).
MATERIALSAND METHODS Soils Three soils from Brittany were compared. Two (P and R) were obtained from cultivated fields, while the third (L) was from a non-cultivated moor. The P soil is a sandy loam, with 4.6% organic matter and a pH
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of 5.6, often cropped with potatoes. The R soil is a sandy loam, with 2.9% organic matter and a pH of 5.4, with no history of potato cropping. Finally, the L soil is sandy, with extreme pH (3.8) and organic matter content (7.6%). These soils, used and described by Tivoli et al. (l987), had shown different levels of receptivity to Fusarium spp causing potato dry rot. They were air-dried for several months, sieved (0.5 mm) and distributed in 55 mm plastic Petri dishes (10 g of dry soil per dish) before use. Pathogen
An isolate of P. infestans (race 1.3.4.7.10.1 l), recovered in 1991 from a potato field located in the island of Noirmoutier (France), was used. 2-3 week old cultures on clarified V8 agar were flooded with sterile distilled water, and sporangia were harvested by gentle scrapping. Sporangia were then separated from mycelium by filtration through a 20pm mesh nylon cloth. The final concentration was adjusted to 2.104 sporangia ml-’ using a haemacytometer. Preparation of soils and membranes
Soils distributed in Petri dishes were moistened with 2 ml sterile distilled water per dish and homogenized using a spatula. Sporangia were deposited by vacuum filtration of 1 ml of the freshly-prepared sporangial suspension onto cellulose nitrate membranes (Sartorius, 5 cm diam, 0.45 pm pore size). One membrane was then buried, upside down, in each Petri dish.
Lysk A sporangium was considered lysed either when its membrane (or that of its germ tube) was partly destroyed (Fig. 1) or when its physiological state was bad (severe cell plasmolysis, cell contents not visible or destroyed). The percentage of lysis was probably overestimated, since it included at least some of the sporangia having released zoospores. Pathogenicity test
Potato tubers (cv. Bintje) were surface-disinfected by dipping for 30 s in 96% ethanol, and allowed to dry. A well (5 mm dia, 5 mm depth) was bored in each tuber using a cork borer. A piece of membrane, cut with the same cork borer, was placed inoculum downwards in each well, and covered with the tuber piece removed from the well. Five tubers per treatment (soil-exposure time) were inoculated in this way, and kept at room temperature. After 6 days, tubers were cut through the well and the extension of rotted tissue was measured. The symptoms produced being typical of late blight, no attempt was made to reisolate the pathogen from diseased tubers. Statistical treatment of data
The whole experiment was carried out in duplicate, at several months interval. Data from both experiments were analysed simultaneously by variance analysis (ANOVA), and the Newman-Keuls test was performed when appropriate. RESULTS
Observation of inoculum evolution in soil
Germination rates and modes
After 1, 3, 6, 9, 15, 21, 30, 45 or 60 days exposure at 15°C membranes were taken out of the dishes and gently brushed to remove soil particles. One quarter of each membrane was kept for the pathogenicity test (see later). The remainder was stained with cotton blue and examined microscopically (100 or 250 times magnification). For each treatment, 100-300 sporangia were scored individually for the following. Germination. A sporangium was considered germinated when it had developed either a germ tube at least as long as the sporangium itself (Fig. 1) or a secondary sporangium. Only direct germination was evaluated, as zoospores and cysts could not be unambiguously distinguished from other small organisms or organic debris stained by cotton blue. Bacterial and fungal colonization. Bacteria were observed close to sporangia (Fig. 1) in all three soils at varying proportions. They were intensely stained by cotton blue and seemed to adhere closely to sporangial or germ tube membranes. Fungal colonization of sporangia and germ tubes was also observed (Fig. 2). The hyphae of the colonizing fungus were thin and usually twisted around P. infestans organs. The identity of the colonizing fungi and bacteria were not established, although the growth habit of the fungal hyphae was reminiscent of Trichoderma sp.
In all three soils, germination was always <30%, irrespective of the observation date [Fig. 3(A)]. ANOVA revealed a significant effect of soil type and exposure duration on germination rates (Table 1). Average germination rates were significantly lower in L than in either R or P soils (P = 0.05), whereas no significant difference was found between these two soils. In all soils, germination was maximal after 3-6 days. Its subsequent evolution was cyclical in both R and P (secondary modes at 15 and 45 days, minima at 9, 21-30 and 60 days), but not in L where it remained at a very low level from the ninth day on [Fig. 3(A)]. Modes of germination also differed markedly between soils. Germination was observed as either germ tube production or buildup of secondary sporangia. ANOVA and the Newman-Keuls test performed on the percentages of germinated sporangia having emitted only a germ tube. revealed significant differences between all three soils. The highest average (95.6%) was observed in the L soil and the lowest (48.4%) in the R soil. These patterns were stable over time, since no significant effect of exposure duration was found. Colonization of sporangia by soil microorganisms
Bacteria1 colonization of sporangia fluctuated markedly in all three soils over the 60 day period, but
Evolution of Phytophthora infestam in three soils
Fig. 1. Some aspects of P. infestuns sporangia after incubation in soil. NG: non-germinated; GT: germ tube; L: lysis of sporangium; B: bacterial colonization. Picture from a membrane exposed in contact with the R soil for 3 days.
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Fig. 2. Sporangium of P. infestam colonized by fimgal hyphae. Picture from membrane exposed inI contact with the L soil for 15 days.
Evolution of Phyrophfhora infesruns in three soils A
I 13 6 9
15
21
30
45
60
15
21
30
45
60
B 3oc
1.
I3
6 9
949
no clear pattern of change was discerned [Fig. 3(B)]. This is reflected in the lack of significant effects of either soil pH or exposure duration as an outcome of ANOVA (Table 1). However, bacterial colonization of sporangia tended to be lower in the L than in the other two soils, especially after the second week of exposure. ANOVA showed a highly significant difference between replicates (Table 1), a consequence of the consistently much higher colonization rates in the first experiment, irrespective of the soil considered. On the other hand, fungal colonization of sporangia and germlings was significantly higher in the L soil than in R and P soils [Fig. 3(C); Table 11. In the L soil, it increased markedly in the course of the second week and continued to do so up to day 45, after which it declined. More than half of the propagules scored were colonized by fungi in the L soil at day 45; this proportion was always < 10% in both R and P soils, whatever the exposure duration. Simultaneous colonization of the same propagule by bacteria and fungi was exceptional, irrespective of the soil and date. Lysis of sporangia
13
6 9
15
21
30
45
60
D 60
ANOVA revealed no significant differences in mean lysis percentages between soils (Table 1). However, highly significant effects (P < 0.001) were found for exposure duration and between replicates. This latter effect is due to a much lower incidence of lysis in the second experiment, which was consistently observed throughout the 60 days and for all three soils. This generated high standard deviations, and possibly prevented the recognition of statistical differences between soils. The gradual increase of lysis with time is reflected by the highly significant effect of exposure duration. The kinetics of lysis in the three soils showed some interesting differences [Fig. 3(D)], although these failed to reach statistical significance. In both R and P soils, lysis tended to start very early and to increase steadily until the ninth day, then to reach a plateau before increasing again at the two later dates (45 and 60 days). On the other hand, lysis started slowly in the L soil, but increased rapidly after the ninth day. From that date on, lysis tended to be more active in the L than in any of the other two soils [Fig. 3(D)]. Colonized sporangia, whether by fungi or bacteria, were lysed more often than non-colonized organs (Fig. 4). Infectivity to potato tubers
Infectivity to Bintje tubers was maximal after 1 (L) or 6 days (R, P), and declined rapidly after these dates (Fig. 5). It was always significantly lowest in L, but
13
6 9
15
21
30
Days
45
60
Fig. 3. Kinetics of germination (A), bacterial colonization (B), fungal colonization (C) and lysis (D) of P. infestans sporangia deposited on membranes exposed in contact with one of three soils for up to 60 days. L soil (-); R soil (---); P soil (-. -). Each point is the mean of two replicates (164 k 41 sporangia scored per replicate).
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D.
ANDRWON
Table 1.ANOVA analysis of variablea describing the evolution of sporangia of P. in/esransexposed in contact with
three different soils and scored at nine dates between 1and 60 days Variable factor d.f. F P Germination
Soil Exposure duration Interaction Replicates
2 8 16 1
22.29
3.51 0.76 2.29
0.007 0.716 0.136
2 8 16
3.41 0.79 0.71 16.73
0.048 0.619 0.760 0.001
2 8 16
16.87 2.14 1.74 0.95
0.000 0.068 0.100 0.340
2 8 16
0.30 8.05 0.69 30.33
0.747 0.000 0.782 0.000
0.000
Bacterial colonization
Soil Exposure duration Interaction Replicates Fungal colonization Soil Exposure duration Interaction Replicates
1
1
report of Murphy (1922) that germination rates of P. infestmu sporangia in unsterilized soil are generally low. Murphy (1922) reported as well that he was unable to find mycelium in natural soil, although mycelial development is possible in sterilized soil (de Bruyn, 1922; Lacey, 1965). Similarly I found very few hyphae on the membranes, irrespective of the soil and date of observation. Two mechanisms shaped the survival of P. infestuns sporangia in soils: fungistasis (i.e. inhibition of sporangial germination; Lockwood, 1964) and lysis of fungal organs. Part of the differences observed between soils reflects the varying intensity of these two phenomena. In particular, both fungistasis and inoculum lysis were more intense in the L soil than either the R or P soils, which behaved in a comparable way.
Lysis
Soil Exposure duration Interaction Replicates
1
.
100
A
Soil Exposure duration Interaction _ . Kepkates
.
.
Infectivity to tubers
2 8 16 1
4.42 2.18 0.69 13.95
0.022 0.063 0.775 0.001
.
/
/
‘A.”
/
A/ /
/
/
/
I ‘* ‘A
/ /
.
/ /
no significant differences were found between infectivity of inoculum exposed in contact with either the R or P soils. Infectivity was completely lost from L after day 15, from R and P at day 60. A highly significant difference was found between replicates (Table l), since infectivity of inoculum was always much lower in the second experiment than in the first for all three soils. There was no correlation between infectivity and the percentages of germinated sporangia, of lysed sporangia, of sporangia colonized with fungi or of germinated but unlysed sporangia in any of the three soils. A significant, positive correlation was recorded between infectivity and the percentage of sporangia colonized with bacteria in both the R and P soils (r 2 = 0.584, P < 0.001 and r 2 = 0.375, P = 0.009), but not in the L soil.
/A / /
20
.
100
40
60
80
100
.
A A
/
/
/
. 80
DISCUSSION
This study confirmed previous observations concerning the duration of infectivity to potato tubers of soils infected with sporangia of P. infestam, although the methodology for the appraisal of soil infectivity differed. The persistence of infectivity during 45 days in both R and P soils is comparable to the 26-77 days recorded in several British soils (Murphy, 1922; Zan, 1962; Lacey, 1965). However, none of the soils investigated previously was as inhibitory to disease development as the L soil examined here. This soil can be considered as suppressive to the late blight pathogen, according to the definition of Baker and Cook (1974). My observations also confirmed and extended the
.a
3
/ //
ms*
/
1
I
I
20
40
60
I 80
I 100
Lysis among all sporangia (9%) Fig. 4. Comparison of lysis rates of P. infestanssporangia colonized by bacteria (A) or fungi (B) with those of all sporangia. Each point represents one observation (i.e. one membrane). Points on the dashed line represent cases where colonized organs are lysed in the same proportion as non-colonized ones. Membranes exposed in contact with the L (A), R (0) or P (m) soils.
951
Evolution of P&toph~hora infesruns in three soils
1369
15
21
30
45
60
Days Fig. 5. Kinetics of infectivity to potato tubers of P. i~~stu~ssporangia deposited on membranes exposed in contact with one of three soils for up to 60 days. L soil (-); R soil (- - -); P soil (- 1 -). Each point is the mean rot extension measured in two replicate experiments (five tubers per replicate).
It may be supposed that specific factors act on fungistasis and inoculum lysis. Concerning fungistasis, the low ge~ination rate of sporangia observed in the three soils may be due to the limited supply of water. Both free water and matric potential are of utmost importance for sporangial germination in a number of Phytophthoru species (see reviews by Duniway, 1983 and Gisi, 1983). In our experiments, the water applied to the soil samples was calculated as a percentage of dry soil weight, and not as the quantities necessary to reach equai manic potentials in the three soils. Therefore, it is possible that differences in germination rates between the soils arose from differences in water matric potential. However, the extremely low germination rate of sporangia in the L soil might also depend on characteristics peculiar to this soil, such as low pH and high concentrations of exchangeable A13+and Mn*+ . It has been shown that this soil was suppressive to F. solani var. coeruleum due to inhibition of spore germination and altered inocuium evolution caused by A13+ ions (Ridao et al., 1990). A similar report by Meyer and Shew (1992) attributes soil suppressiveness to Thiefaviopsis basicola to high amounts of exchangeable aluminum. A number of studies, reviewed by Schmitthenner and Canaday (1983), reported an inhibitory effect of soil pH on late blight incidence, but it is not known whether this inhibition is due to a direct effect on pH itself or to an indirect action through the release of toxic soil components, such as Al’+ or Mn2+ ions. Bogulavskaya and Filippov (1977) showed that the infectivity of P. infestans persisted longer in soils at near-neutral pH than in either acid or basic soils. Muchovej et al. (1980) showed that A13+ was inhibitory to P. cap&i growth in vitro, but no data on the influence of that compound on P. infestans are available. Observations of inoculum evolution in the L soil amended with various doses of CaCO, confirmed the influence of soil pH on fungistasis and on fungal colonization (Andrivon, 1994). Work is in progress to
determine the mechanism of pH influence on the pathogen, as well as the sensitivity to soil toxicmetallic ions of P. infesta~s strains. Intense lysis of P. infestans sporangia and mycehum was observed in the three soils after 15-30 days exposure. This lysis was correlated with inoculum colonization by fungi (L soil). Lacey (1965) also mentioned the presence in some of the soils he used of both fungi and bacteria antagonistic in vitro to P. ~~festa~s,but he did not observe them in situ. Differences between soils were noticeable in the kinetics of lysis. The later development of lysis in the L soil was correlated with a delay in the colonization of P. infestans propagules by fungi as compared with bacterial colonization, rapidly occurring in both Rand P soils. A further step now will be to identify the colonizing organisms, their diversity and lytic activity. Although disease was clearly less severe in the soil showing the highest lytic and fungistatic activity (L soil), changes in the state of inoculum and its infectivity to tubers were not correlated when soils were considered separately. It may be noticed that after 60 days, some ungerminated but undamaged sporangia remained in all three soils, although this inoculum was not infective anymore. The presence of such a residual, non-infective inoculum raises several questions, which remain to be answered. The first is to know whether this inoculum is only temporarily inactivated because of unfavourable environmental conditions, or if it lost definitely its ability to infect host tubers. In the first case, it would be important to determine whether such latent sporangia can be reactivated by modifying the conditions of exposure, for instance by increasing the water availability, Another important point is the fate of this residual inoculum: delayed germination, lysis, or persistence as such. Again, further experiments are needed to answer these questions important for a better understanding of the ecology and epidemiology in soil of P. infestans.
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D. ANDRIVON
Acknowledgements-Critical comments on this manuscript by P. Lucas (Plant Pathology Station, Le Rheu) are gratefully acknowledged. I am aGo very grateful to J C. P;quet (Linguistic Service, INRA Versailles) for translating the paper of Bogulavskaya and Filippov.
REFERENCES Adams P. B. (1967) A buried membrane filter method for studying behaviour of soil fungi. Phytopathology 57, 602-603.
Andrivon D. (1994) Fate of Phytophthora infestans in a suppressive soil in relation to pH. Soil Biology & Biochemistry 26, 953-956.
Baker K. F. and Cook R. J. (1974) Biological Control of Plant Pathogens. Freeman, San Francisco. Boaulavskava N. V. and FiliDDov A. V. 0977) lSurviva1 rates of Phytophthora infest&s (Mont.) de Bary in different soils.] Mikologyia i Fitopatologyia 11, 239-241 (in Russian). de Bruyn H. G. L. (1922) The saprophytic life of Phytophthora in the soil. Mededelingen van den Lanbouwhoogeschool te Wageningen 24, 1-37.
Duniway J. M. (1983) Role of physical factors in the development of Phytophthora diseases. In Phytophthora: Its Biology, Taxonomy, Ecology, and Pathology (D. C. Erwin, S. Bartnicki-Garcia and P. H. Tsao, Eds), pp. 175-187. American Phytopathological Society Press, St. Paul. Frinking H. D., Davidse L. C. and Limburg H. (1987) Oospore formation by Phytophthora infestans in host tissue after inoculation with isolates of opposite mating type found in the Netherlands. Netherlands Journal of Plant Pathology 93, 147-149.
Fry W. E., Goodwin S. B., Matuszak J. M., Spielman L. J., Milgroom M. G. and Drenth A. (1992) Population genetics and intercontinental migrations of Phytophthora infestans. Annual Review of Phytopathology 30, 107-129.
Gisi U. (1983) Biophysical aspects of the development of Phytophthora. In Phytophthora: Its Biology, Taxonomy, Ecology, and Pathology (D. C. Erwin, S. Bartnicki-Garcia and P. H. Tsao, Eds), pp. 109-119. American Phytopathological Society Press, St. Paul. Gregory P. H. (1983) Some major epidemics caused by Phytophthora. In Phytophthora: Its Biology, Taxonomy, Ecology and Pathology (D. C. Erwin, S. Bartnicki-Garcia and P. H. Tsao, Eds), pp. 271-278. American Phytopathological Society Press, St. Paul. Lacey J. (1965) The infectivity of soils containing Phytophthorn infestans. Annals of Applied Biology 56, 363-380.
Lockwood J. L. (1964) Soil fungistasis. Annual Review of Phytopathology 2, 341-362.
Malajczuk N. (1983) Microbial antagonism to Phytophthora. In Phytophthora : Its Biology,lhxonomy, Ecology, and Pathology (D. C. Erwin, S. Bartnicki-Garcia and P. H. Tsao, Ed;), pp. 197-218. American Phytopathological Society Press, St. Paul. Meyer J. R. and Shew H. D. (1992) Soils suppressive to black root rot of burley tobacco, caused by Thielaviopsis basicola. Phytopathology 81, 946-954.
Muchovej J. J., Maffia L. A. and Muchovej R. M. C. (1980) Effect of exchangeable soil aluminum and alkaline calcium salts on the pathogenicity and growth of Phytophthora capsici from green pepper. Phytopathology 70, 1212-1214.
Murphy P. A. (1921) The sources of infection of potato tubers with the blight fungus, Phytophthora infestans. ScientiJc 353-368.
Proceedings of the Royal Dublin Society 16,
Murphy P. A. (1922) The bionomics of the conidia of Phytophthora infestans. Scientific Proceedings of the Royal Dublin Society 16, 442466.
Ridao A., Tivoli B., Robert M. and Lemarchand E. (1990) Role de l’aluminium sur le m&canisme de resistance des sols a Fusarium solani var. coeruleum, agent de la pourriture s&he des tubercules de pomme de terre. In Proceedings of the Eleventh EAPR
Triennal Conference.
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Edinburgh. Schmitthenner A. F. and Canaday C. H. (1983) Role of chemical facts in the development of Phytophthora diseases. In Phytophthora: Its Biology, Taxonomy, Ecology, and Pathology (D. C. Erwin, S. Bartnicki-Garcia and P. H. Tsao, Eds), pp. 1899196. American Phytopathological Society Press, St. Paul. Tivoli B., Corbidre R. and Jouan B. (1983) Influence de le temperature et de l’humidite sur le comportement dans le sol de trois espbces ou varietes de Fusarium responsables de la pourriture s&he des tubercules de pomme de terre. Agronomie 3, 1001-1009.
Tivoli B., Corbiere R. and Lemarchand E. (1990) Relation entre le pH des sols et leur niveau de receptivite a Fusarium solani var. coeruleum et Fusarium roseum var sambucinum agents de pourriture &he des tubercules de pomme de terre. Agronomie 10, 6368. Tivoli B., Tika N. and Lemarchand E. (1987) Comparaison de la receptivitt des sols aux agents de la pourriture &he des tubercules de pomme de terre: Fusarium spp. et Phoma sp. Agronomie 7, 531-538. Zan K. (1962) Activity of Phytophthora infestans in soil in relation to tuber infection. Transactions of the British Mycological Society 45, 205-221.
00384717(94)EOO32-U
Copyright 0 1994 Elsevier science Ltd
Printed in Great Britain. All rights reserved 0038-0717/94$7.00+ 0.00
DYNAMICS OF THE SURVIVAL AND INFECTIVITY TO POTATO TUBERS OF SPORANGIA OF PHYTOPHTHORA INFESTANS IN THREE DIFFERENT SOILS D. ANDRIVON INRA, Station de Pathologie V&g&ale, Domaine de la Motte, BP29, F-35650 Le Rheu, France (Accepted 9 February 1994) Sumntary--Changes affecting sporangia of Phytophthora infestans in three soils from Brittany were monitored at nine dates over a 60 day period, using buried filter membranes on which sporangia had been deposited. Germination was poor on all dates (< 30%), but significant differences between soils were found both in germination rates and in the proportion of germlings producing secondary sporangia. Lysis was active in all three soils (up to 4060% of sporangia affected), and was associated with colonization of the pathogen organs by bacteria or fungi. Infectivity to potato tubers persisted for 15-45 days depending on soil. No single index describing inoculum state could be correlated with infectivity to tubers. One of the three soils was suppressive to P. infestans, and was characterized by very low germination rates and intense lysis of the pathogen by fungal hyphae.
INTRODUCTION
The ecology and epidemiology in soil of the potato late blight fungus Phytophthoru infestuns (Mont.) de Bary has been relatively little studied in comparison with the aerial phases of the life cycle of this destructive pathogen. It is, however, an important stage to consider, since it conditions tuber contamination and infection (Murphy, 1921). It may also be of importance for the survival and perennation of the fungus by means of oospores, which might be formed in the field under European conditions (Frinking et al., 1987) since the introduction of the A2 mating type in the late 1970s (Fry et al., 1992). To be able to infect the host, these oospores have to germinate, either through germ tubes (hyphae) or secondary sporangia, which in turn should be capable of some saprophytic life until they reach susceptible host tissue and become parasitic. It is commonly admitted that P. infestans, like many other members of the genus, has poor saprophytic capacities in soil (Malajczuk, 1983). Under optimal conditions, infectivity to potato tuber slices of soils infested with sporangia of P. infestuns persisted for 26-77 days, depending on soil type and experimental procedures (Murphy, 1922; Zan, 1962; Lacey, 1965; Bogulavskaya and Filippov, 1977). This limited persistence in soil is generally attributed to the inactivation and rapid degradation of sporangia and mycelium by soil microorganisms. Lacey (1965) observed that mycelial growth of the pathogen was good in sterilized soil, but was severely impaired in unsterilized soil. He also showed that, in vitro, Cladosporium herbarum, Monosporium sylvaticum, Penicillium sp. and an unidentified actinomycete species limited the
growth of P. infestans but did not affect cytoplasm organization, whereas Mucor spinosus, Rhizoctonia solani and Trichoderma viride had a lytic effect on mycelium and sporangia. Gregory (1983) once described soil infectivity as a ‘poltergeist’, or disembodied ghost, in the case of P. infestam. In fact, although Murphy (1922) and Zan (1962) reported some microscopic observations of the evolution of P. infestans inoculum in contact with natural soil, quantitative data concerning temporal changes in the state and composition of this inoculum, as well as the relationships between inoculum composition and infectivity to potato tubers are missing. Several authors (Murphy, 1922; Zan, 1962; Lacey, 1965; Bogulavskaya and Filippov, 1977) noted that infectivity varies noticeably with soils, as does its persistence over time; however, the reasons for such differences among soils handled with the same experimental procedures remain largely unexplored. This study was therefore undertaken with the aim of (i) describing in a quantitative manner the changes affecting sporangia exposed in different soils and (ii) relating these changes with those of inoculum infectivity to potato tubers. For these purposes, the buried filter membrane method of Adams (1967) was used, as modified by Tivoli et al. (1983, 1990).
MATERIALSAND METHODS Soils Three soils from Brittany were compared. Two (P and R) were obtained from cultivated fields, while the third (L) was from a non-cultivated moor. The P soil is a sandy loam, with 4.6% organic matter and a pH
945
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D. ANDRNON
of 5.6, often cropped with potatoes. The R soil is a sandy loam, with 2.9% organic matter and a pH of 5.4, with no history of potato cropping. Finally, the L soil is sandy, with extreme pH (3.8) and organic matter content (7.6%). These soils, used and described by Tivoli et al. (l987), had shown different levels of receptivity to Fusarium spp causing potato dry rot. They were air-dried for several months, sieved (0.5 mm) and distributed in 55 mm plastic Petri dishes (10 g of dry soil per dish) before use. Pathogen
An isolate of P. infestans (race 1.3.4.7.10.1 l), recovered in 1991 from a potato field located in the island of Noirmoutier (France), was used. 2-3 week old cultures on clarified V8 agar were flooded with sterile distilled water, and sporangia were harvested by gentle scrapping. Sporangia were then separated from mycelium by filtration through a 20pm mesh nylon cloth. The final concentration was adjusted to 2.104 sporangia ml-’ using a haemacytometer. Preparation of soils and membranes
Soils distributed in Petri dishes were moistened with 2 ml sterile distilled water per dish and homogenized using a spatula. Sporangia were deposited by vacuum filtration of 1 ml of the freshly-prepared sporangial suspension onto cellulose nitrate membranes (Sartorius, 5 cm diam, 0.45 pm pore size). One membrane was then buried, upside down, in each Petri dish.
Lysk A sporangium was considered lysed either when its membrane (or that of its germ tube) was partly destroyed (Fig. 1) or when its physiological state was bad (severe cell plasmolysis, cell contents not visible or destroyed). The percentage of lysis was probably overestimated, since it included at least some of the sporangia having released zoospores. Pathogenicity test
Potato tubers (cv. Bintje) were surface-disinfected by dipping for 30 s in 96% ethanol, and allowed to dry. A well (5 mm dia, 5 mm depth) was bored in each tuber using a cork borer. A piece of membrane, cut with the same cork borer, was placed inoculum downwards in each well, and covered with the tuber piece removed from the well. Five tubers per treatment (soil-exposure time) were inoculated in this way, and kept at room temperature. After 6 days, tubers were cut through the well and the extension of rotted tissue was measured. The symptoms produced being typical of late blight, no attempt was made to reisolate the pathogen from diseased tubers. Statistical treatment of data
The whole experiment was carried out in duplicate, at several months interval. Data from both experiments were analysed simultaneously by variance analysis (ANOVA), and the Newman-Keuls test was performed when appropriate. RESULTS
Observation of inoculum evolution in soil
Germination rates and modes
After 1, 3, 6, 9, 15, 21, 30, 45 or 60 days exposure at 15°C membranes were taken out of the dishes and gently brushed to remove soil particles. One quarter of each membrane was kept for the pathogenicity test (see later). The remainder was stained with cotton blue and examined microscopically (100 or 250 times magnification). For each treatment, 100-300 sporangia were scored individually for the following. Germination. A sporangium was considered germinated when it had developed either a germ tube at least as long as the sporangium itself (Fig. 1) or a secondary sporangium. Only direct germination was evaluated, as zoospores and cysts could not be unambiguously distinguished from other small organisms or organic debris stained by cotton blue. Bacterial and fungal colonization. Bacteria were observed close to sporangia (Fig. 1) in all three soils at varying proportions. They were intensely stained by cotton blue and seemed to adhere closely to sporangial or germ tube membranes. Fungal colonization of sporangia and germ tubes was also observed (Fig. 2). The hyphae of the colonizing fungus were thin and usually twisted around P. infestans organs. The identity of the colonizing fungi and bacteria were not established, although the growth habit of the fungal hyphae was reminiscent of Trichoderma sp.
In all three soils, germination was always <30%, irrespective of the observation date [Fig. 3(A)]. ANOVA revealed a significant effect of soil type and exposure duration on germination rates (Table 1). Average germination rates were significantly lower in L than in either R or P soils (P = 0.05), whereas no significant difference was found between these two soils. In all soils, germination was maximal after 3-6 days. Its subsequent evolution was cyclical in both R and P (secondary modes at 15 and 45 days, minima at 9, 21-30 and 60 days), but not in L where it remained at a very low level from the ninth day on [Fig. 3(A)]. Modes of germination also differed markedly between soils. Germination was observed as either germ tube production or buildup of secondary sporangia. ANOVA and the Newman-Keuls test performed on the percentages of germinated sporangia having emitted only a germ tube. revealed significant differences between all three soils. The highest average (95.6%) was observed in the L soil and the lowest (48.4%) in the R soil. These patterns were stable over time, since no significant effect of exposure duration was found. Colonization of sporangia by soil microorganisms
Bacteria1 colonization of sporangia fluctuated markedly in all three soils over the 60 day period, but
Evolution of Phytophthora infestam in three soils
Fig. 1. Some aspects of P. infestuns sporangia after incubation in soil. NG: non-germinated; GT: germ tube; L: lysis of sporangium; B: bacterial colonization. Picture from a membrane exposed in contact with the R soil for 3 days.
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Fig. 2. Sporangium of P. infestam colonized by fimgal hyphae. Picture from membrane exposed inI contact with the L soil for 15 days.
Evolution of Phyrophfhora infesruns in three soils A
I 13 6 9
15
21
30
45
60
15
21
30
45
60
B 3oc
1.
I3
6 9
949
no clear pattern of change was discerned [Fig. 3(B)]. This is reflected in the lack of significant effects of either soil pH or exposure duration as an outcome of ANOVA (Table 1). However, bacterial colonization of sporangia tended to be lower in the L than in the other two soils, especially after the second week of exposure. ANOVA showed a highly significant difference between replicates (Table 1), a consequence of the consistently much higher colonization rates in the first experiment, irrespective of the soil considered. On the other hand, fungal colonization of sporangia and germlings was significantly higher in the L soil than in R and P soils [Fig. 3(C); Table 11. In the L soil, it increased markedly in the course of the second week and continued to do so up to day 45, after which it declined. More than half of the propagules scored were colonized by fungi in the L soil at day 45; this proportion was always < 10% in both R and P soils, whatever the exposure duration. Simultaneous colonization of the same propagule by bacteria and fungi was exceptional, irrespective of the soil and date. Lysis of sporangia
13
6 9
15
21
30
45
60
D 60
ANOVA revealed no significant differences in mean lysis percentages between soils (Table 1). However, highly significant effects (P < 0.001) were found for exposure duration and between replicates. This latter effect is due to a much lower incidence of lysis in the second experiment, which was consistently observed throughout the 60 days and for all three soils. This generated high standard deviations, and possibly prevented the recognition of statistical differences between soils. The gradual increase of lysis with time is reflected by the highly significant effect of exposure duration. The kinetics of lysis in the three soils showed some interesting differences [Fig. 3(D)], although these failed to reach statistical significance. In both R and P soils, lysis tended to start very early and to increase steadily until the ninth day, then to reach a plateau before increasing again at the two later dates (45 and 60 days). On the other hand, lysis started slowly in the L soil, but increased rapidly after the ninth day. From that date on, lysis tended to be more active in the L than in any of the other two soils [Fig. 3(D)]. Colonized sporangia, whether by fungi or bacteria, were lysed more often than non-colonized organs (Fig. 4). Infectivity to potato tubers
Infectivity to Bintje tubers was maximal after 1 (L) or 6 days (R, P), and declined rapidly after these dates (Fig. 5). It was always significantly lowest in L, but
13
6 9
15
21
30
Days
45
60
Fig. 3. Kinetics of germination (A), bacterial colonization (B), fungal colonization (C) and lysis (D) of P. infestans sporangia deposited on membranes exposed in contact with one of three soils for up to 60 days. L soil (-); R soil (---); P soil (-. -). Each point is the mean of two replicates (164 k 41 sporangia scored per replicate).
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ANDRWON
Table 1.ANOVA analysis of variablea describing the evolution of sporangia of P. in/esransexposed in contact with
three different soils and scored at nine dates between 1and 60 days Variable factor d.f. F P Germination
Soil Exposure duration Interaction Replicates
2 8 16 1
22.29
3.51 0.76 2.29
0.007 0.716 0.136
2 8 16
3.41 0.79 0.71 16.73
0.048 0.619 0.760 0.001
2 8 16
16.87 2.14 1.74 0.95
0.000 0.068 0.100 0.340
2 8 16
0.30 8.05 0.69 30.33
0.747 0.000 0.782 0.000
0.000
Bacterial colonization
Soil Exposure duration Interaction Replicates Fungal colonization Soil Exposure duration Interaction Replicates
1
1
report of Murphy (1922) that germination rates of P. infestmu sporangia in unsterilized soil are generally low. Murphy (1922) reported as well that he was unable to find mycelium in natural soil, although mycelial development is possible in sterilized soil (de Bruyn, 1922; Lacey, 1965). Similarly I found very few hyphae on the membranes, irrespective of the soil and date of observation. Two mechanisms shaped the survival of P. infestuns sporangia in soils: fungistasis (i.e. inhibition of sporangial germination; Lockwood, 1964) and lysis of fungal organs. Part of the differences observed between soils reflects the varying intensity of these two phenomena. In particular, both fungistasis and inoculum lysis were more intense in the L soil than either the R or P soils, which behaved in a comparable way.
Lysis
Soil Exposure duration Interaction Replicates
1
.
100
A
Soil Exposure duration Interaction _ . Kepkates
.
.
Infectivity to tubers
2 8 16 1
4.42 2.18 0.69 13.95
0.022 0.063 0.775 0.001
.
/
/
‘A.”
/
A/ /
/
/
/
I ‘* ‘A
/ /
.
/ /
no significant differences were found between infectivity of inoculum exposed in contact with either the R or P soils. Infectivity was completely lost from L after day 15, from R and P at day 60. A highly significant difference was found between replicates (Table l), since infectivity of inoculum was always much lower in the second experiment than in the first for all three soils. There was no correlation between infectivity and the percentages of germinated sporangia, of lysed sporangia, of sporangia colonized with fungi or of germinated but unlysed sporangia in any of the three soils. A significant, positive correlation was recorded between infectivity and the percentage of sporangia colonized with bacteria in both the R and P soils (r 2 = 0.584, P < 0.001 and r 2 = 0.375, P = 0.009), but not in the L soil.
/A / /
20
.
100
40
60
80
100
.
A A
/
/
/
. 80
DISCUSSION
This study confirmed previous observations concerning the duration of infectivity to potato tubers of soils infected with sporangia of P. infestam, although the methodology for the appraisal of soil infectivity differed. The persistence of infectivity during 45 days in both R and P soils is comparable to the 26-77 days recorded in several British soils (Murphy, 1922; Zan, 1962; Lacey, 1965). However, none of the soils investigated previously was as inhibitory to disease development as the L soil examined here. This soil can be considered as suppressive to the late blight pathogen, according to the definition of Baker and Cook (1974). My observations also confirmed and extended the
.a
3
/ //
ms*
/
1
I
I
20
40
60
I 80
I 100
Lysis among all sporangia (9%) Fig. 4. Comparison of lysis rates of P. infestanssporangia colonized by bacteria (A) or fungi (B) with those of all sporangia. Each point represents one observation (i.e. one membrane). Points on the dashed line represent cases where colonized organs are lysed in the same proportion as non-colonized ones. Membranes exposed in contact with the L (A), R (0) or P (m) soils.
951
Evolution of P&toph~hora infesruns in three soils
1369
15
21
30
45
60
Days Fig. 5. Kinetics of infectivity to potato tubers of P. i~~stu~ssporangia deposited on membranes exposed in contact with one of three soils for up to 60 days. L soil (-); R soil (- - -); P soil (- 1 -). Each point is the mean rot extension measured in two replicate experiments (five tubers per replicate).
It may be supposed that specific factors act on fungistasis and inoculum lysis. Concerning fungistasis, the low ge~ination rate of sporangia observed in the three soils may be due to the limited supply of water. Both free water and matric potential are of utmost importance for sporangial germination in a number of Phytophthoru species (see reviews by Duniway, 1983 and Gisi, 1983). In our experiments, the water applied to the soil samples was calculated as a percentage of dry soil weight, and not as the quantities necessary to reach equai manic potentials in the three soils. Therefore, it is possible that differences in germination rates between the soils arose from differences in water matric potential. However, the extremely low germination rate of sporangia in the L soil might also depend on characteristics peculiar to this soil, such as low pH and high concentrations of exchangeable A13+and Mn*+ . It has been shown that this soil was suppressive to F. solani var. coeruleum due to inhibition of spore germination and altered inocuium evolution caused by A13+ ions (Ridao et al., 1990). A similar report by Meyer and Shew (1992) attributes soil suppressiveness to Thiefaviopsis basicola to high amounts of exchangeable aluminum. A number of studies, reviewed by Schmitthenner and Canaday (1983), reported an inhibitory effect of soil pH on late blight incidence, but it is not known whether this inhibition is due to a direct effect on pH itself or to an indirect action through the release of toxic soil components, such as Al’+ or Mn2+ ions. Bogulavskaya and Filippov (1977) showed that the infectivity of P. infestans persisted longer in soils at near-neutral pH than in either acid or basic soils. Muchovej et al. (1980) showed that A13+ was inhibitory to P. cap&i growth in vitro, but no data on the influence of that compound on P. infestans are available. Observations of inoculum evolution in the L soil amended with various doses of CaCO, confirmed the influence of soil pH on fungistasis and on fungal colonization (Andrivon, 1994). Work is in progress to
determine the mechanism of pH influence on the pathogen, as well as the sensitivity to soil toxicmetallic ions of P. infesta~s strains. Intense lysis of P. infestans sporangia and mycehum was observed in the three soils after 15-30 days exposure. This lysis was correlated with inoculum colonization by fungi (L soil). Lacey (1965) also mentioned the presence in some of the soils he used of both fungi and bacteria antagonistic in vitro to P. ~~festa~s,but he did not observe them in situ. Differences between soils were noticeable in the kinetics of lysis. The later development of lysis in the L soil was correlated with a delay in the colonization of P. infestans propagules by fungi as compared with bacterial colonization, rapidly occurring in both Rand P soils. A further step now will be to identify the colonizing organisms, their diversity and lytic activity. Although disease was clearly less severe in the soil showing the highest lytic and fungistatic activity (L soil), changes in the state of inoculum and its infectivity to tubers were not correlated when soils were considered separately. It may be noticed that after 60 days, some ungerminated but undamaged sporangia remained in all three soils, although this inoculum was not infective anymore. The presence of such a residual, non-infective inoculum raises several questions, which remain to be answered. The first is to know whether this inoculum is only temporarily inactivated because of unfavourable environmental conditions, or if it lost definitely its ability to infect host tubers. In the first case, it would be important to determine whether such latent sporangia can be reactivated by modifying the conditions of exposure, for instance by increasing the water availability, Another important point is the fate of this residual inoculum: delayed germination, lysis, or persistence as such. Again, further experiments are needed to answer these questions important for a better understanding of the ecology and epidemiology in soil of P. infestans.
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Acknowledgements-Critical comments on this manuscript by P. Lucas (Plant Pathology Station, Le Rheu) are gratefully acknowledged. I am aGo very grateful to J C. P;quet (Linguistic Service, INRA Versailles) for translating the paper of Bogulavskaya and Filippov.
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Baker K. F. and Cook R. J. (1974) Biological Control of Plant Pathogens. Freeman, San Francisco. Boaulavskava N. V. and FiliDDov A. V. 0977) lSurviva1 rates of Phytophthora infest&s (Mont.) de Bary in different soils.] Mikologyia i Fitopatologyia 11, 239-241 (in Russian). de Bruyn H. G. L. (1922) The saprophytic life of Phytophthora in the soil. Mededelingen van den Lanbouwhoogeschool te Wageningen 24, 1-37.
Duniway J. M. (1983) Role of physical factors in the development of Phytophthora diseases. In Phytophthora: Its Biology, Taxonomy, Ecology, and Pathology (D. C. Erwin, S. Bartnicki-Garcia and P. H. Tsao, Eds), pp. 175-187. American Phytopathological Society Press, St. Paul. Frinking H. D., Davidse L. C. and Limburg H. (1987) Oospore formation by Phytophthora infestans in host tissue after inoculation with isolates of opposite mating type found in the Netherlands. Netherlands Journal of Plant Pathology 93, 147-149.
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Gisi U. (1983) Biophysical aspects of the development of Phytophthora. In Phytophthora: Its Biology, Taxonomy, Ecology, and Pathology (D. C. Erwin, S. Bartnicki-Garcia and P. H. Tsao, Eds), pp. 109-119. American Phytopathological Society Press, St. Paul. Gregory P. H. (1983) Some major epidemics caused by Phytophthora. In Phytophthora: Its Biology, Taxonomy, Ecology and Pathology (D. C. Erwin, S. Bartnicki-Garcia and P. H. Tsao, Eds), pp. 271-278. American Phytopathological Society Press, St. Paul. Lacey J. (1965) The infectivity of soils containing Phytophthorn infestans. Annals of Applied Biology 56, 363-380.
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Tivoli B., Corbiere R. and Lemarchand E. (1990) Relation entre le pH des sols et leur niveau de receptivite a Fusarium solani var. coeruleum et Fusarium roseum var sambucinum agents de pourriture &he des tubercules de pomme de terre. Agronomie 10, 6368. Tivoli B., Tika N. and Lemarchand E. (1987) Comparaison de la receptivitt des sols aux agents de la pourriture &he des tubercules de pomme de terre: Fusarium spp. et Phoma sp. Agronomie 7, 531-538. Zan K. (1962) Activity of Phytophthora infestans in soil in relation to tuber infection. Transactions of the British Mycological Society 45, 205-221.