THE EFFECT OF AERATION ON COLONY DIAMETER, SPORANGIUM PRODUCTION AND ZOOSPORE GERMINATION OF PHYTOPHTHORA CINNAMOMI

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1 New Phytot. (1986) 103, THE EFFECT OF AERATION ON COLONY DIAMETER, SPORANGIUM PRODUCTION AND ZOOSPORE GERMINATION OF PHYTOPHTHORA CINNAMOMI BY E. M. DAVISONi AND F. C. S. TAY^ ' Department of Conservation and Environment, 1 Mount Street, Perth, Western Australia 6000, and ^ School of Environmental and Life Sciences, Murdoch University, Murdoch, Western Australia 6150 {Accepted 2 April 1986) SUMMARY The reduced aeration which occurs in waterlogged soil was simulated in the laboratory using O /N gas mixtures, and its effects were studied on six isolates of Phytophthora cinnamomi Rands^. Mycelial growth of five isolates grown on agar under a flowing atmosphere, and sporangium production by all of the isolates grown in bubbled liquid culture, were severely reduced under anaerobic conditions. Three of the isolates showed an increase in the number and rate of development of sporangia with increasing O^ concentration of the gas stream. The other isolates showed no increase in the number of sporangia formed at dissolved Oj concentrations above 13 mg 1"'. There was no eftect of Oj concentration, either during growth (13-5 or 21 % O2) or germination, on the proportion of zoospores that germinated on agar or on initial growth of germ tubes. However, after 19 h, the total length of germ tubes including branches increased with increasing Oj concentration during germination. The number of branches in 11 of the 12 combinations examined was also positively correlated with O2 concentration. Key words: Waterlogging, jarrah dieback, anoxia, hypoxia. INTRODUCTION About 14% of the jarrah forest of Western Australia (WA) is infested with the soil-borne, pathogenic fungus Phytophthora cinnamotni Rands (Forests Department WA, 1982). Jarrah {Eucalyptus marginata Donn ex Sm.) dies on infested sites with impeded drainage, following exceptionally heavy rainfall (Podger, Doepel & Zentmyer, 1965; McKinnell, 1981; Shea, Shearer & Tippett, 1982; Davison & Tay, 1985). It is probable therefore that these sites become partly waterlogged for a short time following exceptionally wet weather. In the forest, jarrah normally grows on well-drained sites (Havel, 1975). When glasshouse-grown jarrah seedlings are flooded, the xylem vessels in the tap-root and stem become occluded with tyloses and,' because the plants continue to transpire, they eventually wilt and die (Davison & Tay, 1985). Waterlogging, however, may also affect both Phytophthora and its infection of jarrah trees. To assist in the interpretation of how and why trees die on sites with impeded drainage following exceptionally heavy rainfall, we are investigating the effects of short-term waterlogging on jarrah, on infection of jarrah by P. cinnamomi, and the effects of reduced aeration on P. cinnamomi. This paper examines the latter X/86/ «03.00/0 '^86 The New Phytologist

2 736 E. M. DAVISON AND F. C. S. TAY In waterlogged soils the O^ concentration of the soil solution drops rapidly (Ponnamperuma, 1984); when lateritic jarrah forest soil was flooded at 20 C in the laboratory, the Oj concentration of the soil solution dropped to less than 1 mg 1"! after 2 d (Davison & Tay, 1985). Even at matric potentials (^^) close to zero there may be reduced availability of O^ to plant roots and soil micro-organisms (Griffin, 1968; Noordwijk & Willigen, 1984; Willigen & Noordwijk, 1984). Phytophthora cinnamomi requires ^^ close to zero for sporangial production in soil (Gisi, Zentmyer & Klure, 1980; Benson, 1984). In waterlogged soil either numbers of sporangia produced are very low, or none are formed (Nesbitt, Malajczuk & Glenn, 1979; Gisi et al., 1980). Mitchell & Zentmyer (1971b) showed that sporangium production by several Phytophthora spp. was reduced at Oj concentrations lower than in air, thus, in waterlogged soil sporulation of P. cinnamomi is considered to be restricted by reduced aeration (Nesbitt et al., 1979; Gxsietal, 1980). Zoospores, which are liberated from sporangia, are believed to be the main dispersal and infective propagule of P. cinnamomi in the jarrah forest during wet weather (Shea, Gillen & Leppard, 1980). During the hot, dry, mediterranean summer P. cinnamomi survives in infected plant roots (Shea, 1979; D. E. Schild, Murdoch University, pers. commun.). Chlamydospores do not persist in dry forest soil (D. E. Schild, Murdoch University, pers. commun.). Therefore, in WA most work has concentrated on soil conditions which favour sporangium production and zoospore dispersal (Shea, 1975; Shea et al., 1980). The reduced availability of O2 in waterlogged soil can be simulated in the laboratory by using O2/N2 mixtures. We have used this method to study, under sterile conditions, the effect of reduced aeration on mycelial growth, sporangium production, zoospore germination and germ tube growth of six isolates of P. cinnamomi. MATERIALS AND METHODS Isolates of Phytophthora cinnamomi Table 1 shows the mating types and origins of the P. cinnamomi isolates used in this study. Two of the A2 isolates (60 and 230) have been used experimentally in WA the two Al isolates were included for comparison. Effect of aeration on mycelial growth Four O2 concentrations: 0, 6-2, 13-5 and 21 % were used in all the experiments. Industrial dry N2 and industrial dry compressed air were used respectively for the 0% O2 and 21 % O2 concentrations; 6-2 and 13-5% O2 were specially prepared gas mixtures in which the balance was made up with N2 (Commonwealth Industrial Gases Ltd, Perth, WA). All experiments were carried out in a flowing system, at a flow rate of approximately 35 ml min~\ at 20 C. All gases were filtered through Sartorius filter membranes (0-2 /im pore size). A 5-5 mm plug of the test fungus growing on 20% V8 juice agar (200 ml V-8 juice (Campbell's Soups (Aust.) Pty Ltd), 3 g CaCO3, 15 g Gibco bacteriological agar per litre) was placed centrally onto half-strength potato dextrose agar {\ PDA) (19-5 g Gibco PDA, 7-5 g Gibco bacteriological agar per litre) in a 9 cm plastic Petri dish. The agar had been equilibrated for at least 18 h at 20 C in a flowing gas mixture of the appropriate O2 concentration. After 126 h incubation at the same O2 concentration, fungal growth was terminated by adding 10% formalin

3 Aeration and Phytophthora cinnamomi 737 Table 1. Mating types and origins of Phytophthora cinnamomi isolates Isolate number Mating type Culture collection number* Sourcef Isolated from Date Isolated Al Al A2 A2 A2 A2 IMI DAR53101 WAR I CSIRO CSIRO MU MU MU Jarrah forest soil, WA Hibhertia subvaginata Steudel (F. Muell.) WA Eucalyptus marginata WA E. marginata WA. marginata WA * IMI, Commonwealth Mycological Institute, UK: DAR, Department of Agriculture, Rydalmere, New South Wales. t WARI, Waite Agricultural Research Institute, South Australia: CSIRO, CSIRO Division of Forest Research, Kelmscott, WA: MU, Murdoch University, WA. to the Petri dishes. Two colony diameters, at right angles, were measured on each plate. There were nine replicate plates per treatment. It was assumed that the Oj concentration affecting the fungus was that of the gas passing over the colony. Effect of aeration on production of sporangia A modification of the method of Byrt & Grant (1979) was used to study the effect of O.J concentration on sporangium production under sterile conditions. Before use all solutions were bubbled with gas of the appropriate Oj concentration for at least 12 h at 20 C. The Oj concentration in samples of all the solutions was measured with an O.^ electrode (Triton Instruments) immediately before the solutions were used, and just before they were replaced. All measurements were made under a Nj atmosphere. A 3-d-old mycelial mat of the test fungus growing on Miracloth (Chicopee Mills, New York) was incubated in 5 % V8 broth, shaken at 110 orbits per minute and bubbled with the appropriate gas. After 24 h the broth was replaced by mineral solution (Chen & Zentmyer, 1970) and the culture shaken and bubbled as before. After a further 24 h the mat was suspended in sterile deionized water, chilled at 4 C for 5 min and then kept at 25 C for 2 h until zoospore release had occurred. The mycelial mat was flooded with 10 % formalin, and the fungus was stained with ammoniacal congo red [1 g congo red, eight drops NH4OH(s.g.0-88), 100 ml deionized water]. The number and development of sporangia were assessed using a X 40 objective in two random flelds of view (one fleld of view has an area of mm^) for each colony of the test fungus. There were flve colonies on each mycelial mat and three mycelial mats for each treatment. Stages of sporangium development were recognized as follows: a sporangium initial had a definite swelling at the hyphal apex but no cross wall had formed, a delimited sporangium had a septum at the base, an aborted sporangium had a cross wall at the base but shrivelled contents, and discharged sporangia were those from which zoospore release had occurred. Effect of aeration on zoospore germination and germ tube growth Zoospores produced from sporangia grown in liquid culture bubbled with either 13-5 % O.^ or 21 % O2 were used in these experiments. A drop of a freshly

4 738 E. M. DAVISON AND F. C. S. TAY ) y J - \ r 1 Isolate / - / 1 " i Oxygen in flowing atmosphere (%) Fig. 1. Colony diameter of six isolates of Phytophthora cinnamomi after 126 h growing on \ PDA, at 20 "C, in fiowing gas mixtures containing different O^ concentrations. There were nine plates per treatment. Error bar.s are SD. 20 prepared zoospore suspension was placed on a J PDA plate which had previously been equilibrated for at least 18 h in a fiowing gas mixture of the appropriate Og concentration. The plate was incubated for either 6 or 19 h in a flowing gas mixture. It was assumed tbat the O^ concentration affecting the fungus was that of the gas passing over the colony. Per cent germination was assessed using a x 40 objective in four random fields of view in each drop of zoospore suspension. There were five drops per plate and three plates per treatment. The total germ tube length, including branches, of 20 germinated spores was measured using a x 10 objective; means and standard deviations were calculated using log transformed data. RESULTS Effect of aeration on mycelial growth After 126 h the colony diameter of all isolates except isolate 60 was significantly smaller at 0% Oj than at other O._, concentrations (Fig. 1). The effect of Og concentration on hyphal branching was not quantified, but it was noted that branching was extremely sparse in the absence of O2. Effect of aeration on production of sporangia Under anaerobic conditions sporangia were either not produced (isolate 230) or only produced in very low numbers (Fig. 2). At dissolved O^ concentrations above 13 mg 1"' isolates 25, 50 and 229 showed no further increase in tbe number of sporangia with increasing aeration, while the remaining isolates (60, 210 and 230) showed an increase in sporangium production with increasing O2 concentration. In these three isolates the proportion of discharged sporangia increased as the O.^ concentration increased, indicating a more rapid rate of sporangium development at the higher Oj levels. This pattern was not seen in isolates 25, 50 and 229 (Fig. 2). The proportions of delimited and aborted sporangia were low in all isolates and showed no consistent trends.

5 Aeration and Phytophthora cinnamomi 739 Isolate a..2 0 I concentration (mg I -h Fig. 2. Production and development of sporangia by six isolates of Phytophthora cinnamomi. at 20 C in solutions of different measured O.^ concentrations. Each column is the mean of 30 observations. Error bars are.se. ^, sporangium initials;, delimited sporangia; Q. discharged sporangia;, aborted sporangia ^ Isolote Fig 3 Germination of zoospores of six isolates of Phytophthora cinnamomi on J PDA at 20 C. Zoospores were produced from sporangu, grown in solutions bubbled with 21 >' i\. Zoospores germinated in flowing gas mixtures containing difierent O, concentratu.ns and were assessed after 19 h Each column is the mean of 60 observations. lcrror bars are.so. 0, 0' O,; D. ('2 O,; B 13 5",, ()..;E). 21 " O... Effect of aeration on zoospore germination and germ tube groivth The O., concentration of the gas mixture during growth had no consistent effect on the percentage of zoospores which germinated. Consequently, data are presented only for zoospores produced by sporangia grown at the highest ()., concentration, and germinated for 19 h (Fig. 3). By 19 h at least SO",, of the

6 74O E. M. DAVISON AND F. C. S. TAY 20 = / / = 217e r : / r--0-5 i = Isolote Fig. 4. Total germ tube length, including branches, of zoospores of six isolates of Phytophthora cinnamomi on \ PDA, at 20 C. Zoospores were produced from sporangia grown in solutions bubbled with 21 % O^. Zoospores germinated in flowing ga.s mixtures containing different Oj concentrations and were assessed after 19 h. Means, calculated from log transformed data, are from 20 measurements. Error bars are SD. All r values are significant, P = ^, 0*X) O._,; E3, f>-2 % O.,, 13-5% % O,. zoospores had germinated in almost all treatments, and in isolates 50, 229 and 230 this proportion had germinated within 6 h. At 6 h there was no effect of O^ concentration on total germ tube length (data not presented) but, after germination for 19 h, total germ tube lengtb, including branches, was positively correlated {P = 0-005) witb the O.^ concentration of the gas mixture (Fig. 4). The O.^ concentration during sporulation had no effect on tbe behaviour of the zoospores, therefore, only results for zoospores produced from sporangia grown at the highest O^ concentration are shown. After 19 b tbe number of branches was positively correlated {P = 0-005) with increasing O^ concentration, i.e. with increasing germ tube length, in 11 of tbe 12 experiments (six isolates, two O.^ concentrations for sporulation). An example is shown in Figure 5. DISCUSSION The results confirm and extend the observations of other workers on the effect of reduced O.^ concentration on Phytophthora spp. Measurements of growth in bubbled and unbubbled culture solutions and on solid media bave shown tbat in

7 Aeration and Phytophthora cinnamomi 741 la) (bl (c) (d) r-- I Fig..S. Zoospores of Phvtophthora cinnamomi isolate 60 germinated for 19 h on PDA, at 20 "C, stained with ammoniacal Congo red, photographed through a green filter. Sporangia, from which the zoospores were liberated, developed at a measured Oj concentration of 56 mg T'. Germination in (a) 0% O^; (b) 6-2% O.,; (c) 13-5% O.,; (d) 21 % Oj. Scale line is 100//m. I f. P. cactorum (Lebert & Cohn) Schroeter, P. capsici Leonian, P. citrophthora (Smith & Smith) Leonian, P. megasperma Dreschler, P. palmivora (Butler) Butler, P. parasitica Dastur and P. parasitica var. nicotianae (Breda de Haan) Tucker growth is reduced at very low Oj concentrations (Klotz, Stolzy & DeWolfe, 1963; Dukes & Apple, 1965; Covey, 1970; Mitchell & Zentmyer, 1971a; Mitchell & Mitchell, 1973). P. cinnamomi behaves in a similar way (Figs 1 and 4). Measurement of colony diameter on agar is a relatively insensitive method for measuring fungal growth because it does not take into account any differences in the density of hyphae. Dry weight production in liquid culture or measurement of individual propagules would give a more accurate measurement of fungal growth. This would possibly explain the greater effect of O.^ concentration on germ tube length (Fig. 4) than on mycelial growth (Fig. 1). Sporangium production by P. cactorum, P. capsici, P. cinnamomi, P. citrophthora, P. palmivora, and P. parasitica in culture solution was less under an atmosphere

8 742 E. M. DAVISON AND F. C. S. TAY of 1 % O.^ than of air, but tbere was variation between different isolates of tbe same species (Mitcbell & Zentmyer, 1971 b). A more detailed study of the effect of O^ on sporangium production by P. parasitica (Ioannou & Grogan, 1985) showed that between 21 and 2-5 % O^ the concentration had no effect on the number of sporangia, but below 2-5% O._, sporangium formation decreased; at 0-3% O.^ no sporangia were formed. Our results show that under anaerobic conditions sporulation of P. cinnamomi either does not occur, or occurs only at very low levels. With increasing O^ concentrations three isolates showed an increase in both the number of sporangia and tbeir rate of development, altbough three others showed no increase in sporangium numbers at O^ concentrations above 13 mg T^ (Fig. 2). Klotz et al. (1963) showed that the proportion of germinated zoospores of P. citrophthora increased with increasing O^ concentration, and the rate of germ tube growth of both P. citrophthora and P. parasitica was affected by aeration. In our experiments hypoxic and anoxic conditions did not affect zoospore germination or initial germ tube growth. Zoospores were liberated into deionized water which had been previously bubbled with either 13-5 or 21 % O^. Even though drops of zoospore suspension were placed on agar which had been maintained in the appropriate fiowing gas mixture for at least 18 h, and the plates were subsequently incubated in a fiowing atmosphere, we do not know how rapidly the O^ concentration of the drop equilibrated with the ambient conditions. If zoospore germination is affected by the O^ level of the surrounding medium, equilibration of the O.^ concentrations may have occurred too slowly to demonstrate this in our experiments. However, after 19 h total germ tube lengtb was correlated with O.^ concentration (Fig. 4). The isolates used in these experiments show considerable variation in their growth and sporulation under a range of O2 concentrations. Isolate 229 which was recovered from a recently dead jarrah sapling from a poorly drained site (Davison and Tay, unpublished data), sporulated vigorously at low O^ concentrations, and zoospores showed both high rates of germination and rapid germ tube growth under hypoxic conditions. Unfortunately, we do not know the drainage characteristics of the sites from which the other isolates were obtained. Phytophthora root rots are frequently most severe in poorly drained sites (e.g. Hansen et al., 1979; Bumbieris, Wicks & Windle, 1982; Mircetich & Matheron, 1983; Von Broembsen & Brits, 1985). It has been suggested tbat this could be due to increased growtb or sporulation of the pathogen, increased infection of tbe bost by the pathogen, decreased response of the host to infection, or direct effects of hypoxic and anoxic conditions on the host (Drew & Lynch, 1980). In soil, P. cinnamomi forms sporangia at ^/^ between 1 and 250 kpa (Gisi et al., 1980). In poorly drained sites there is a gradient of both xjf^ and aeration between tbe soil surface and the watertable. At the surface, where aeration is adequate, the soil may be too dry for sporulation wbile, with increasing depth in the profile, moisture may be adequate but hypoxic or anoxic conditions may restrict sporulation. Zoospores produced from sporangia in moist soil may be passively dispersed by water moving through the profile (Newhook et al., 1981; Shea et al., 1983). Our results suggest that the soil O.^ concentration does not affect the proportion of zoospores which germinate, but does affect their subsequent growth. Thus, although sporulation of the pathogen in moist but not saturated soil may partly explain the severity oi Phytophthora root rots in poorly drained sites, other factors.

9 Aeration and Phytophthora cinnamomi 743 such as increased number of infections or decreased host response, may be equally important. ACKNOWLEDGEMENTS We thank A. Leith for technical assistance, D. Jones for typing the manuscript and B. Stewart for drawing the diagrams. F.M.D. thanks Murdoch University for laboratory facilities. REFERENCES BENSON, M. (1984). Influence of pine bark, matric potential and ph on sporangium production by Phytophthora cinnamomi. Phytopathology, 74, BuMBiERis, M., WICKS, T. J. & WINDLE, B. E. (1982). Phytophthora species in apple and cherry orchards in South Australia. Australasian Plant Pathology, 11, BYRT, P. & GRANT, B. R. (1979). Some conditions governing zoospore production in axenic cultures of I Phytophthora cinnamomi Rands. Australian Journal of Botany, 27, ' CHEN, D. W. & ZENTYMER, G. A. (1970). Production of sporangia by Phytophthora cinnamomi in axenic culture. Myeologia, 62, 397^02. COVEY, R. P. (1970). Effect of oxygen tension on the growth oi Phytophthora cactorum. Phytopathology, 60, DAVISON, E. M. & TAY, F. C. S. (1985). The effect of waterlogging on seedlings of Eucalyptus margtnata. New Phytologist, 101, DREW M. C. & LYNCH, J. M. (1980). Soil anaerobiosis, microorganisms, and root function. Annual Review of Phytopathology, 18, DUKES P D & APPLE, J. A. (1965). Effect of oxygen and carbon dioxide tensions on growth and inoculum potential of Phytophthora parasitica var. nicotianae. Phytopathology, 55, FORESTS DEPARTMENT, W.A. (1982). Working plan no. 87 Part 1. General Working Plan for State Forests in Western Australia. Perth. Gisi, U., ZENTMYER, G. A. & KLURE, L. J. (1980). Production of sporangia by Phytophthora cinnamomi and P. palmivora in soils at different matric potentials. Phytopathology, 70, GRIFFIN, M. (1968). A theoretical study relating the concentration and diffusion of oxygen to the biology of organisms in soil. New Phytologist, 67, HANSEN, E. M., HAMM, P. B., JULIS, A. J. & ROTH, L. F. (1979). Isolation, incidence and management of Phytophthora in forest tree nurseries in the Pacific northwest. Plant Disease Reporter, 63, HAVEL, J. J. (1975). Site vegetation mapping in the Northern Jarrah Forest (Darling Range). 1. Definition of'site types. Bulletin of the Forests Department, Western Australia, 87. IOANNOU, N. & GROGAN, R. G. (1985). Formation and germination of chlamydospores of Phytophthora parasitica under various oxygen and carbon dioxide tensions. Plant Disea.'^e, 69, 400^03. KLOTZ, L. J., STOLZY, L. H. & DEWOLFE, T. A. (1963). Oxygen requirement of three root rotting fungi in a liquid medium. Phytopathology, 53, McKiNNELL, F. H. (1981). Review of the dieback disease situation Forests Department, Western Australia, Technical Paper 2. MiRCETiCH, S. M. & MATHERON, M. E. (1983). Phytophthora root and crown rot of walnut trees. Phytopathology, 73, u j- ^ a MITCHELL D J & MITCHELL, J. E. (1973). Oxygen and carbon dioxide concentration effects on the growth and reproduction of Aphanomyces euteiches and certain other soil-borne plant pathogens. Phytopathology, 63, MITCHELL, D.J. & ZENTMYER, G. A. (1971a). Effects of oxygen and carbon dioxide tensions on growth of several species of Phytophthora. Phytopathology, 61, MITCHELL, J. & ZENTYMER, G. A. (1971 b). Effects of oxygen and carbon dioxide tensions on sporangium and zoospore formation by Phytophthora spp. Phytopathology, 61, NESBITT, H. J., MALAJCZUK, N. & GLENN, A. R. (1979). Effect of soil moisture and temperature on the survival oi Phytophthora cinnamomi Rands in soil. Soil Biology and Biochemistry, 11, NEWHOOK, F. J.. YOUNG, B. R., ALLEN, S. R. & ALLEN, R. N. (1981). Zoospore motility of Phytophthora cinnamomi in particulate substrates. Phytopathologische Zeitsehrift, 101, NooRDWijK, M. VAN & WiLLiGEN, P. DE (1984). Mathematical models on diffusion of oxygen to and within plant roots, with special emphasis on effects of soil-root contact. II. Applications. Plant and Soil, 77, PoDGER, F. D., DoEPEL, R. F. & ZENTMYER, G. A. (1965). Association of Phytophthora cinnamomi with a disease of Eucalyptus marginata forest in Western Australia. Plant Disease Reporter, 49,

10 744 E. M. DAVISON AND F. C. S. TAY PoNNAMPERUMA, F. N. (1984). Effects of flooding on soil. In: Flooding and Plant Growth (Ed. by T. T. Kozlowski). pp. 9^5, Academic Press, New York. SHEA, S. R. (1975). Environmental factors of the northern jarrah forest in relation to pathogenicity and survival of Phytophthora cinnamomi. Forests Department, Western Australia, Bulletin, 85. SHEA, S. R. (1979). Phytophthora cinnamomi (Rands) A collar rot pathogen of Banksia grandis Willd. Australasian Plant Pathology, 8, SHEA, S. R., GILLEN, K. J. & LEPPARD, W. I. (1980). Seasonal variation in population level oi Phytophthora cinnamomi Rands in soil in diseased, freely drained Eucalyptus marginata Sm. sites in the northern jarrah forest of southern-western Australia. Protection Ecology, 2, SHEA, S. R., SHEARER, B. L. & TIPPETT, J. (1982). Recovery of Phytophthora cinnamomi Rands from vertical roots of jarrah {Eucalyptus marginata Sm.). Australasian Plant Pathology, 11, SHEA, S. R., SHEARER, B. L., TIPPETT, J. T. & DEEGAN, P. M. (1983). Distribution, reproduction and movement of Phytophthora cinnamomi on sites highly conducive to jarrah dieback in south western Australia. Plant Disease, fil, VON BROEMBSEN, S. L. & BRITS, G. J. (1985). Phytophthora root rot of commercially cultivated proteas in South Africa. Plant Disease, 69, WH^LINGEN, P. DE & NoRDWijK, M. VAN (1984). Mathematical models on diffusion of oxygen to and within plant roots, with special emphasis on effects of soil-root contact. I. Derivation of the models. Plant and Soil, 77,

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