controlling soil water matric potential in root disease studies
TRANSCRIPT
Controlling soil water matric potential in root disease studies1
Department of Plant Science, University of British CoBmbia, Vancouver, B .C. , Crinndn V6T 1 WS
Deportment of Soil Science, University of British Col~rmbia, Vnrzcoin,er, B .C. , Cnrzndn V6T1 W5
AND
Depnrrmerzr of Plnrit Scierlce, Universiry of Brirish Colrrr7lbiri, Vorlcoii~~er, B . C . , Cnrlrirlri V6T 1W5
Received July 28, 1976
WISBEY, B. D., T. A. BLACK, and R. J . COPEMAN. 1977. Controlling soil water matric potentialin root disease studies. Can. J. Bot. 55: 825-830.
Soil contained in 5.6 x 2.0 x 10.5 cm Plexiglas soil containers was separated from solutions of polyethylene glycol (PEG) 6000 by a Pellicon ultrafiltration membrane glued over the inside of 3.8-cm-diameter holes in two sides. Flow rate through the membrane was 4.4 ml d a y - l ~ m - ~ for a 1.0-bar potential difference across the membrane. At the end of 2 weeks, the average matric potential in the soil adjacent to the membranes for soil cells immersed in -0.2-, - 1.0-, and -2.0-bar PEG solutions was -0.3, -1.1, and -2.3 bars respectively. The relatively constant potential and vigorous plant growth during the period indicated that sufficient water was passing through the membrane to meet the plant's water requirements. The Pellicon membrane's toler- ance to microbial deterioration will permit the use of this technique in longer term studies than previously has been possible.
WISBEY, B. D., T. A. BLACK et R. J . COPEMAN. 1977. Controlling soil water matric potential in root disease studies. Can. J . Bot. 55: 825-830.
Les auteurs ont reussi i separer du sol, contenu dans des recipients de Plexiglass mesurant 5.6 x 2.0 x 10.5 cm, de solutions de polyethylene glycol (PEG) 6000 au moyen d'une membrane k ultrafiltration Pellicon collee sur la face interne d'ouvertures de 3.8 cm des deux cbtes. Les taux d'ecoulement B travers la membrane est de 4.4 ml jour-I cm-2 lorsqu'il existe und difference de potentiel de 1.0 bar i travers la membrane. Apres 2 semaines, le potentiel matriciel dans le sol adjacent aux membranes pour des cellules de sol immeugees dans des solutions de -0.2, - 1.0 et -2.0 bars de PEG sont respectivement de -0.3. - 1.1 et -2.3 bars respectivement. La Constance relative du potentiel et lacroissance vigoureuse des plantes pendant cette periode indiquent qu'une quantite suffisante d'eau passe a travers la membrane pour rencontrer les besoins en eau de la plante. La membrane de Pellicon resiste B I'attaque des microorganisnies et permettre d'utiliser cette technique dans des experiences h plus long terme qu'avant.
[Traduit par le journal]
Introd~~ction Soil water is an important environmental
factor in many soil-borne diseases (Cook and Papendick 1972). In our research on the effect of soil moisture on the incidence and develop- ment of Pytlliutn root dieback of carrot, it became necessary to devise a system which would maintain the soil water content for a growing seedling within a narrow range for a t least 3 weeks. An osmotic system developed by Zur (1966, 1967) appeared to offer the greatest potential. In this method soil water content is
'Portion of a thesis submitted by the senior author in partial fulfillment of the requirements for the M.Sc. degree.
ZPresent address: British Columbia Department of Agriculture, Abbotsford, B.C., Canada V2S 1x4.
controlled by using an osmotic solution sepa- rated from the soil by a semipermeable mem- brane. As the soil water content decreases, so does the potential energy of the water in the soil matrix (matric potential). When the matric potential decreases below the osmotic potential of the solution, water flows from the solution through the membrane to the soil until the matric potential is equal to the osmotic potential of the solution. In practice the matric potential is always slightly less than the osmotic potential because of the membrane's resistance to water flow. A solute that has proven to be satisfactory is polyethylene glycol (PEG) 6000, a large inert molecule, which does not pass through semi- permeable membranes (Williams and Shayke- wich 1969). By keeping the PEG solution at a
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y M
cMas
ter
Uni
vers
ity o
n 11
/11/
14Fo
r pe
rson
al u
se o
nly.
826 CAN. J. BOT. VOL. 55, 1977
constant volume, water lost by soil surface evaporation and transpiration is constantly re- placed. Owing to microbial deterioration of the semipermeable membrane, this technique pre- viously has been used only for short duration studies (Babalola et a/. 1968) unless sterilized soil could be used (Painter 1966; Zur 1966, 1967).
This paper describes the design and testing of simple apparatuses which employ the osmotic principle. A preliminary report has been pub- lished (Wisbey et a/. 1974).
Methods and Materials Preliminary experiments employed cellulose dialysis
tubing of different types and ultrafiltration membranes in soil containers of varying design. Thin, rectangular, soil containers were constructed by fitting 9.2-cm-diameter (wall thickness 0.011 cm) dialysis tubing (Arthur H. Thomas Co., Philadelphia, PA) over a U-shaped 12.5 x 1.0 x 30.0 cm polyvinylchloride (PVC) frame. The tubing was sealed at the bottom with a plastic clamp. Soil tubes were constructed by knotting one end of a 15 cm length of 2.22-cm-diameter (wall thickness 0.0025 cm) dialysis tubing (Arthur H. Thomas Co., Philadelphia, PA) and fitting the open end over a single-holed No. 3$ cork. The tubing was held in place by a rubber band. These containers were carefully filled with sieved (10 mesh), pasteurized, greenhouse mix (loam) and sus- pcnded In solutions containing varying concentrations of PEG 6000 (J . T . Raker Chemical Co.. Philliosbur~. N.J.). . -, ,
In some experiments carrot seedlings (Darrcris cnrotcr L. cv. Goldpak Elite) were transplanted into the soil con- tainers. In an attempt to extend membrane longevity 1000 ppm active ingredient (a.i.) benomyl [methyl-l- (butylcarbamoyl)-2-benzimidazole carbamate, Benlate 50 WP, duPont], 1000 ppm a.i. quintozene (pentachloro- nitrobenzene, Terrachlor 7 5 z WP, Olin), and 125 ppm a.i. streptomycin sulfate (Sigma, St. Louis, MO) were added to the PEG solution. Because the ultrafiltration membranes [Pellicon, 500 nominal molecular weight limit (NMWL), Millipore Filter Corp., Bedford, MA] were available only as discs, cylindrical soil containers were tried first. Acrylonitrile butadiene styrene (ABS) plumbing adapters (4.0 cm inside diameter (ID)) were adapted for use (Fig. 1). The membrane was placed between the nylon ring and the screw cap which were both lightly coated with stopcock grease. Soil was added to a height of 3 cm and the plumbing adapters suspended in PEG 6000 solutions. Water content of the soils from the above containers was determined by oven drying at 105°C. Water content was converted into matric potential using a soil water retention curve determined by standard methods (Richards 1965).
Two additional containers both employing the Pellicon membrane were evaluated. Cubical soil containers (5.6 x 5.6 x 10 cm, outside measurements) were made of 0.635-cm-thick, clear Plexiglas (Fig. 2). Five 3.8-cm- diameter holes were centered one in each side and one in the bottom. Thin, rectangular soil containers (5.0 x 2.5 x 10 cm, outside measurements) were similarly made and two 3.8-cm-diameter openings centered 3.5 cm from the bottom of each large side (Fig. 3). Pellicon ultrafiltration
membranes were glued to the inside surface of the con- tainer with Black Plastic Rubber (Duro Plastic, Woodhill Chemical Sales Corp., Cleveland, OH). In these pre liminary studies involving high matric potentials (> - 0.8 bar) we used ceramic-cup tensiometers (2.0 x 0.6 cm outside diameter (OD), Soil Moisture Eqpt. Corp., Santa Barbara, CA) and inserted them into the cells from the open top to the point of interest as shown in Figs. 2 and 3. However, in the system finally selected for intensive testing, we were largely concerned with matric potentials below tensiometer range and deter- mined all matric potentials using the soil water retention curve method described above. The containers were carefully filled with soil and a radish seed (Rapharzus sativlrs L. cv. Cherry belle) was planted in each. The con- tainers were suspended in -0.2-bar PEG solutions and the plants grown at 20 + 3°C under a 14-h photoperiod (2800 Ix) provided by 'cool white' fluorescent lamps and incandescent bulbs. Matric potentials were monitored over a 25-day period.
The soil containers found most suitable for our carrot disease research were modified from the two-membrane, thin, rectangular design. By using 0.318-cm-thick Plexi- glas for the large sides, a slightly increased soil volume could be obtained even with the reduction in container width (5.6 x 2.0 x 10.5 cm, outside measurements). Openings for the membranes were centered 3.0 cm from the bottom. Membranes were glued on (Devcon Rubber, Devcon Canada Ltd., Scarborough, Ont.) and soil care- fully added. Reservoirs for the PEG solutions were made from 15-cm lengths of 10-cm-ID ABS drainage pipe to which had been glued a 0.65-cm-thick clear Plexiglas bottom. A 0.65-cm-ID Plexiglas tube near the base of the container was connected to an air manifold system enabling agitation of the solution. The change in volume was determined by measuring the change in height of the solution in a 1-nil disposable pipette that had been bent and connected at the base of the container to function as a level gauge. A pair of soil containers was suspended in each reservoir from openings in the wooden lids (Fig. 4).
A test of this system was conducted as follows. Two uniform, 15-day-old carrot seedlings (Dauctr~ carota L. cv. Goldpak Elite) were transplanted into each soil container and the soil moistened. Reservoirs were filled with PEG 6000 solutions having the following osmotic potentials: three at -0.2 bars (30 g/l); three at - 1.0 bars (88 g/l). and three at -2.0 bars (125 g/l). The concentrations of PEG 6000 at the three potentials were approximated from the graphs of Zur (1966). Levels of the PEG solutions were returned daily to the initial volumes and PEG solutions replaced weekly. Because water loss in the system was due primarily to transpiration, the daily volume of water required to return the PEG solution to the initial volume was considered equivalent to the tran- spiration rate. Growing conditions were the same as described above.
At the end of the 1st week a pair of soil containers was removed from one reservoir at each of the three osmotic potentials. The soil within each cell was carefully removed in layers corresponding to the top 2.5 cm, a 2.0-cm zone just above the membrane, a 3.0-cm zone between the membranes, and the bottom 3.0 cm. Three samples were taken from each layer and oven-dried at 105°C to obtain the soil water content. This procedure was repeated at the end of the 2nd and 3rd weeks. Water content was con- verted into matric potential as previously described.
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y M
cMas
ter
Uni
vers
ity o
n 11
/11/
14Fo
r pe
rson
al u
se o
nly.
WISBEY ET AL.
FIG. 1. Acrylonitrile butadiene styrene (ABS) plumbing adapters as modified for use as soil cells. FIG. 2. Cubical, five-membrane, soil containers constructed of Plexiglas with a ceramic-cup tensio- meter (+) inserted equidistant from all membranes. FIG. 3. Thin, rectangular, two-membrane soil cell with a ceramic-cup tensiometer (+) inserted equidistant between the membranes. FIG. 4. Apparatus used for controlling matric potential consisting of osmotic solution reservoir with level gauge and air inlet enabling agitation of the solution, light-tight lid, and a pair of two-membrane soil containers.
Results Dialysis membranes lost their selective per-
meability within 5 to 10 days. Trichoderma spp., several mucors, and bacteria were associated with the breakdown. The inclusion of benomyl, quintozene, and streptomycin sulfate in the PEG solution extended membrane longevity to 14 to 17 days, but phytotoxicity owing to the strepto- mycin was observed. Under these conditions membranes became very brittle and extreme caution had to be exercised when osmotic solu- tions were being changed. Carrot rootlets were also observed growing through the fragile membranes. By contrast the Pellicon membranes maintained selective permeability for 4 to 6 weeks before leaks developed. In addition they had a permeability of 4.4 ml day-' cm-2 bar-' compared with 0.7 ml day-' cm-* bar-' for the narrow, thinner walled dialysis membrane.
These permeability values were obtained by measuring the rate of water flow through the respective membranes from a reservoir of dis- tilled water to a reservoir of - 1 .O-bar PEG solution.
In containers of all designs, an increase in the ratio of soil volume to membrane area increased the gradient of soil water content (and matric potential) in the soil and decreased the ability of the system to keep up with rapidly transpiring plants. The plumbing adapter type was capable of maintaining the water potential of only a small volume of soil. Even the increased mem- brane area of the five-membrane cubical con- tainers did not overcome this problem because the matric potential decreased at a rate of 0.02 bar day-' once the seedlings made transpira- tional demands upon this system.
In the 3-week test of the narrow, two-mem-
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y M
cMas
ter
Uni
vers
ity o
n 11
/11/
14Fo
r pe
rson
al u
se o
nly.
CAN. J. BOT. VOL. 55 , 1977
TABLE 1. Soil water content (%) and matric potential (bars; in parentheses) after 2 weeks in soil containers supporting carrot seedlings while suspended in solutions of three osmotic potentials
Osmotic potential, bars Soil layer,
cm -0.2 -1.0 -2.0
*Each value, expressed o n a dry weight basis, is the average of three deter- minations in each of two soil containers.
tWater content less than that a t the lower limit (-5 bar) of the soil water retention curve.
TABLE 2. Soil water content (%) and matric potential (bars; in parentheses) at membrane level in soil containers supporting carrot seedlings while suspended in solutions of three osmotic
potentials
Osmotic potential, bars
Days -2.0 - 1 .O -2.0
*Each value, expressed o n a dry weight basis, is the average of three deter- minations in each of two soil containers.
tBo th cells dcveloped leaks.
brane containers, soil water measurements in- dicated that the gradient in matric potential was kept small in the lower half of the container but was substantial in the upper half (Table 1). Soil at the level of the membranes generally had the highest water content and a matric potential closest to that of the PEG solution. In the con- tainers suspended in the - 0.2-bar solution, the matric potential of soil at the level of the mem- brane was essentially constant at about -0.4 bar for the duration of the test (Table 2). There was good regulation of the matric potential in con- tainers in the - I-bar solutions for the first 2 weeks. Leakage occurring in both containers after 2 weeks was caused by a faulty seal between the membrane and container wall, not membrane deterioration. The lag in reaching equilibrium in the -2-bar soil containers was caused by 'watering-in' the carrot seedlings during trans- planting. Toward the end of the 3-week period, the matric potential at membrane level in con- containers suspended in the -2-bar PEG solu- tion was about -2.5 bars.
Transpiration of the two carrot seedlings de- creased as the osmotic potential of the PEG
TABLE 3. Average transpiration (mllday) of two carrot seedlings grown in soil containers exposed
to different osmotic solutions
Osmotic potential, bars Time interval --
days -0.2 -1.0 -2.0
3 t o 7 0.8 0.6 0.G 8 to 10 1.6 1 .O 1.9
11 to 15 5.7 1.7 2.8 16 to 18 7.8 4.0 2.4
solutions decreased (Table 3). Plants grown at - 2.0 bar transpired about half as much as plants at -0.2 bar. After 3 weeks, plants at -2.0 bar had only one or two leaves with an average leaf area of 3 cm2, whereas those at -0.2 bar had three leaves and a leaf area of 8.5 cm2. These transpiration and leaf area measurements indi- cate that the Pellicon membrane system was able effectively to control matric potential of a soil system supporting carrot seedlings.
Discussion The design and use of soil containers employ-
ing the Zur (1966) osmotic principle for control-
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y M
cMas
ter
Uni
vers
ity o
n 11
/11/
14Fo
r pe
rson
al u
se o
nly.
WlSBEY ET AL. 829
ling soil moisture are determined largely by the characteristics of the semipermeable membrane employed. Soil containers using the Pellicon ultrafiltration membrane were capable of main- taining selective permeability for much longer periods than previously had been possible. The longevity of these membranes was about three times that of dialysis tubing exposed to identical conditions. Since membrane flow rates are im- portant in determining the volume of soil which can be used, the higher flow rates characteristic of the Pellicon membrane permitted a reduction in membrane area. This enabled evaluation of containers of varying geometry. The elimination of some membrane surface in favor of imper- meable container wall permitted easier handling but it also resulted in the formation of gradients within the containers. The design providing the fewest gradient problems was one in which the center of the soil mass was only 0.7 cm from the membrane. Even at this distance. with our soil mix, hydraulic conductivity was a limiting factor. As the potential of the soil decreased, hydraulic conductivity rapidly decreased. For example, Gardner (1965) has estimated that the con- ductivity of a sandy loam at saturation, - 1.0 and - 15 bar, is in the order of 10, lop3, and
cm day-', respectively. If the Pellicon membrane becomes available in sheet form, the vertical gradient observed in this study could likely be eliminated by having the membranes extend the full length of the soil container. However, horizontal gradients would not be corrected, so if this technique is to be success- fully used at low potentials,?he distance between membranes should probably not exceed the 1.4 cm used in this study.
The matric potential of the soil containers in the -0.2-bar solution remained relativelv con- stant, around -0.4 bar, for a 3-week period. Several factors may have contributed to the dis- crepancy between the matric potential observed (-0.4 bar) and that expected (-0.2 bar). Because a different lot of PEG was used in this study, extrapolation of PEG concentrations for desired osmotic potentials using Zur's (1966) Fig. 2 may have been a source of error. Con- version to matric potential using the soil water retention curve and membrane resistance to water flow may also have been partially respon- sible for the difference. The soil containers were calculated to have a volume of 57.5 cm3 which contained 40 g of oven-dry (105°C) soil. If no
water entered the soil cells and the carrot seedlings were transpiring 7.8 ml day-', as was observed, the matric potential would theoretic- ally decrease from - 0.2 to about - 2.0 barwithin 1 day. A further 12 h without water would result in the potential reaching that of the permanent wilting point. Because the carrots were growing well and the soil water content remained vir- tually constant, it is apparent that water was flowing from the PEG solution into the soil. Thus, the Pellicon membranes in the soil con- tainers used had sufficient permeability and area to maintain the water requirements of young, actively transpiring seedlings.
Although the matric potentials observed in the - 1.0- and -2.0-bar solutions were more variable, this technique still permitted better control at these potentials than would be possible using other techniques. The reason for failure of both soil containers in the - 1.0-bar solution by 3 weeks was determined to be due to leakage at the membrane-Plexiglas interface. Owing to a temporary withdrawal of Duro Plastic, Black Plastic Rubber from the market, we were forced to switch to Devcon Plastic Rubber. The Duro Plastic proved to be more durable for attaching membranes to Plexiglas.
The use of the Pellicon ultrafiltration mem- branes has extended the time during which matric potentials down to -2 bars can be maintained using Zur's (1966) osmotic system. It should now be possible to grow seedlings for several weeks at these matric potentials. During this time a non-cellulolytic pathogen may be added and disease development observed under closely defined water potentials. This technique may also find application in studies of spore or sclerotial germination at lower potentials than are currently possible with other techniques.
Acknowledgment Financial aid in the form of an NRCC
scholarship to the senior author and a Faculty of Agricultural Sciences Research and Develop- ment Fund grant to Drs. Copeman and Black is gratefully acknowledged.
BABALOLA. 0.. L. BOERSMA, and C. T. YOUNGBERG. 1968. Photosynthesis and transpiration of Monterey pine seedlings as a function of soil water suction and soil temperature. Plant Physiol. 43: 515-521.
COOK. R. J., and R. I. PAPENDICK. 1972. Influence of water potential of soils and plants on root disease. Annu. Rev. Phytopathol. 10: 349-374.
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y M
cMas
ter
Uni
vers
ity o
n 11
/11/
14Fo
r pe
rson
al u
se o
nly.
830 CAN. J. BOT.
GARDNER. W. R. 1965. Rainfall, run-off and return. 111 Agricultural meteorology. Erlitecl by P. E. Waggoner. Meteorol. Monogr. No. 28. Am. Meteorol. Soc., Bos- ton, MA. pp. 138-148.
PAINTER, L. 1. 1966. Method of subjecting growing plants to a continuous soil moisture stress. Agron. J . 58: 459460.
RICHARDS. L. A. 1965. Physical condition of water in soil. 111 Methods of soil analysis. Agronomy 9. Eclirerl by C. A. Black. Am. Soc. of Agron., Madison, WI. pp. 128-152.
VOL. 5 5 , 1977
WILLIAMS. J. , and C. F. SHAYKEWICH. 1969. An evalua- tion of polyethylene glycol (P.E.G.) 6000 and P.E.G. 20,000 in the osmotic control of Boil water matric poten- tial. Can. J . Soil Sci. 49: 397401.
WISBEY. B. D., R. J . COPEMAN. and T. A. BLACK. 1974. Control of soil water matric potential in studies of root diseases. Proc. Am. Phytopathol. Soc. 1: 59. (Abstr.).
ZUR. B. 1966. Osmotic control of soil water matric poten- tial. I. Soil-water system. Soil. Sci. 102: 394-398.
1967. Osmotic control of soil water matric poten- tial. 11. Soil-plant system. Soil Sci. 103: 30-38.
Can
. J. B
ot. D
ownl
oade
d fr
om w
ww
.nrc
rese
arch
pres
s.co
m b
y M
cMas
ter
Uni
vers
ity o
n 11
/11/
14Fo
r pe
rson
al u
se o
nly.