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Influence of temperature and water potential interactions on the germinability of Cylindrocladium crotalariae microsclerotia in naturally infested soil
Copyright r
o(338-07I7 88 53.00 + 0.00 1988 Pcrgamon Press plc
INFLUENCE OF TEMPERATURE AND WATER POTENTIAL INTERACTIONS ON THE GERMINABILITY OF CYLINDROCLADIUM CROTALARIAE MICROSCLEROTIA IN NATURALLY INFESTED SOIL P. GRAHAM and G. J. GRIFFIN Department
of Plant Pathology.
Physiology and Weed Science, Virginia Polytechnic University, Blacksburg. VA 24061. U.S.A. (/(cceprrd
5 /anuar,r
Institute
and State
1988)
Summary-Microsclerotia of C. crotaluriar in naturally-infested soils were placed in the field during late fall at depths of 5, 13 or 25 cm and exposed for I-yr. Germinable populations of microsclerotia declined rapidly at all soil depths during the winter, leveled off during April. May and June, and declined gradually from July to November. Soil at the S-cm depth was frozen in the winter and exposed to 0°C or lower for numerous days, whereas soil at the 25-cm depth was saturated and exposed extensively to chilling (65 C) temperatures. In laboratory trials. chilling plus saturation of soil with water was highly detrimental to microsclerotium germinability. and similar in its effect to soil freezing. Chilling alone had a more moderate effect on microsclerotium germinability. High soil temperatures (2 3O’C) were common in the field in the upper soil horizons during the late summer months. In laboratory tests. high soil temperatures (30, 35 and 4OC) greatly decreased microsclerotium germinability compared to moderate soil temperature (25‘C). and the effects of high temperatures were much greater at low (-22.4 to -23.5 MPa) water potentials than at high (-0.03 to - 1.5 MPa) water potentials.
MATERIALS
INTRODL’CTION
Soils und assay
Survival and germinability of Cvlindrocludium microsclcrotia are affected by low temperatures and low water potentials (Griffin er al., 1978: Phipps and Brute, 1979; Roth er al., 1979; Taylor ef al., 1981), and both factors are thought to play important roles in nature. Virginia soils containing C_vlindrocladium crotaluriue (Loos) Bell and Sobers, causal agent of Cylindrocladium black rot of peanut, are always exposed to chilling (,< 5-C) conditions and sometimes are frozen. The unusually severe winter of 19761977. when the soil was frozen for several weeks. was associated with a 95% decline in the C. crofaluriae microsclerotium population in the plow layer (O-25cm deep) (Taylor et al., 1981). A 1975 summer drought, which resulted in very dry soil conditions, was associated with a large decline in the C. crofuiariae microsclerotium population in the upper one-half (&12.7cm depth) of the pi’ow layer (Taylor et al., 1931). While low soil temperature and water potentials may have appreciable and independent effects on C. croruluriar microsclerotia, low temperature in midwinter is commonly associated with saturated soil conditions, and dry soil conditions in the summer months are often associated with high soil temperatures. The effects of high soil temperatures (2 30-C) on C. crotaluriae microsclerotia populations have not been investigated. Also, the effects of low temperatures at soil saturation have not been examined. We examined the influence of these factors on C. crotuluriue microsclerotium germinability in naturally-infested soil and estimated their relative importance in the survival of microsclerotia in nature.
AND METHODS
of germinuhle
microsclerotiu
Naturally-infested sandy loams with large C. crofuluriue microsclerotium populations, were taken from the root zones of symptomatic peanut plants in three peanut fields (A, B and C) in the Tidewater area of Virginia, and were used throughout the study. Soils were sieved (< I .9 mm mesh), placed in separate plastic bags with pin holes for air exchange, kept moist by periodic additions of distilled water, and by stored at 2YC. Soils were mixed thoroughly shaking in a plastic bag for 30min before use. Microsclerotium populations in all experiments were assayed quantitatively by wet sieving and a I :7 soil-dilution plating on sucrose-QT medium (Griffin, 1977). Colonies of C. croruluriue on 10 Petri plates for each sample were counted after 7-10 days growth at room temperature (2%28C). The recovery of microsclerotium populations from soils was used as an indicator of germinable microsclerotia in the soil. Eflects ofjeld nubility
of C.
encironmental conditions on the germicroluluriue microsclerotium populutions
Twenty-seven glass cylinders (2.5 x 3 cm), open at both ends, were filled with 27 g of naturally-infested soil for each of soils A, B and C. A series of nine cylinders of each soil were buried at S-. 13- and 25-cm depths on a 2.4 x 1.5 m plot at the Tidewater Agricultural Experiment Station, Holland, Va. The initial population for each soil was assayed the day after samples were buried (I2 November 1977). One soil cylinder of each soil from each depth was sampled on each of eight dates for the year 1978. At the time of collection. the appearance of the soil (frozen. satur731
732
P. Gttt~u~
and G. J. Gtums
ated, etc.) was recorded and the soil water content determined gravimetrically. The water content was compared to a soil moisture retention curve to estimate the water potential. The retention curve was prepared with a soil pressure plate at - 0.0 1, - 0.033, -0.51 and - 1.52 MPa. Soil temperature was monitored at each depth by temperature probes (model 15606, with No. 44019 thermilinear sensor, A. H. Thomas Co., Philadelphia, Penn.). Soil temperature data from the plot site at Holland, Va were obtained from I January to 22 Februav 1978 and 1 August to 30 September 1978. Effects of chilling ut soil saturation, and high temperatures at high and low water potentials on the germinubility of C. crotalariae microsclerotium populations under laboratory conditions Soil samples were maintained in incubators at 5 or 25YZ for I and 5 weeks. Soil moisture of the sample was maintained at saturation or moist levels (-0.03 to -0.22 MPa) by partially immersing a 50 ml beaker, with about 40 g of soil, in a 94-ml canning jar containing distilled water. Two such systems were made for each temperature-time intecval. Similar samples of soil were used for studying effects of high temperature (3@-40-C) under conditions of either 100, 85 or 75% relative humidity. Water potential of soils was calculated using the following formula: ‘Y=TinRH where Y = water potential, R = universal gas constant, T = absolute temperature. Y = partial modal volume of water, and RH = relative humidity (Brown, 1970). Saturated solutions of NaCl and KCI were used to maintain the humidity of a 94-ml closed container to 75 and 85% relative humidity, respectively (Winston and Bates, 1961). Approximately
I .3-2.0 cm of saturated salt solution or distilled water was dispensed into a 94-ml canning jar. A glass ring (2.5 x 3 cm) was placed at the bottom of the jar to support an aluminum pan containing 4Og of soil above the solution. A wire screen shelf was placed over the pan to support another pan containing a soil sample. The jar lid was coated with Vaseline and tightly sealed for the test period; or. in other instances, the lids were opened periodically. Two jar systems of either distilled water or saturated solution of KCI or NaCl were placed in incubators at 25, 30, 35 or 40X. Populations of microsclerotia were assayed after 2 and 3 weeks. After the specified periods, water potential of soils incubated with distilled water was determined by comparing percentage water content to a soil moisture reten;ion curve. RESULTS
Germinabiiit_v of C. crotalariae microsclerotium lutions under field conditions
Overall, there was a progressive decline at all soil depths in the number of germinable microsclerotia through the winter and spring months until about the end of March (Fig. 1). During April, May and June the germinable populations remained relatively constant or increased slightly. From July to November. however, there was a gradual decline in the germinable populations at all soil depths. With the exception of one data point (23 August for the 13 cm depth), germinable populations during this period at the 25-cm depth were largest. populations at the 5 cm depth were least, and the populations at the 13-cm depth were intermediate. Soil samples were frozen at the time of collection only at the j-cm soil depth in January and February (Fig. 1). At these times. the samples collected at the other depths were either saturated or nearly satur-
-.----
Cl
’
I
12
IO
NW
Jan
I
I
I
I
23
I
5
2
Apr
May
Jutv
Feb
popu -
5 cm l3Crn 25cm
I
&
I
I
CL
tEv
Fig. I. Survival of microsclerotium populations of C~lindrocludium crotnluriae in naturally infested soil at three depths in the soil profile during I yr at Holland, Virginia. Soil was buried in glass cylinders with open tops and bottoms on I2 November. 1977. F = frozen soil (3 -0.01 MPa). S = saturated soil. NS = nearly saturated soil (> -0.01 MPa). and M =moist soil (-0.01 to -0.1 MPa) indicate the appearance and water potential (by gravimetric and standard cume assay) of soil samples at the time of collection. Data are the means of three soils at each collection date and for each depth.
Temperature
and
water interactions
conditions. only 1.5% of the initial microsclerotium population was germinable. whereas 30% of the initial population was germinable in moisr soil at 5’C after 5 weeks. When the soil was maintained at 25’C. populations of germinable microsclerotia decreased under saturated conditions by 26% after I week and by 56% after 5 weeks. When soil was maintained at moist conditions and 25’C. the germinable population did not decline significantly (P = 0.05), even after 5 weeks. In a separate test with this soil, 26 and 0.396 of the initial germinable population was recovered after I and 5 weeks. respectively, when moist soil was held at O’C. Numbers of germinable microsclerotia in soil decreased significantly with increasing temperature at all water potentials examined (Table 3). Effects were greatest when soils were maintained at very low water potentials (-39.6 to 41.6 IMPa). Even at the high water potentials (-0.03 MPa). significant decreases in germinable microsclerotia were found after 3 and 3 weeks at 3O’C; no germinable microsclerotia were recovered after exposure to 4O’C. However, at 25’C in moist soil (-0.07 and -0.08 MPa) germinable microsclerotium populations were not significantly different from initial populations after 2 and 3 weeks. After 3 weeks at 35’C and -0.52 MPa, germinable populations were almost as low and not significantly different (P = 0.05) from populations recovered after 4O’C and about - I.5 MPa incubation. In soil maintained at low water potentials (-22.4 to - 23.5 MPa). germinable microsclerotium populations declined significantly even at 25’C, after 2 and 3 weeks exposure. Similar trends were noted for 25’C very low water potentials (-39.6 MPa); no germinable microsclerotia were recovered after 3 weeks at 25’C and - 39.6 .CIPa. Results were similar whether jars were periodically opened and resealed or not.
Table 1. Percentage of da)s the minimtun soil temperature was at or belo* 0 and 5-C during tw mid-winter months and the maximum soil temperature was a: or stwe 30 and 3?‘C durrng two summer months for various soil depths at Holland. Virginia’ SolI depth (cm) Temperature (V g5 SO
g30 332
7.6
15.2
% days *. dqs 9.? 96 3 58
30.5 9% da)s w 0
5.0
IO.?
50.8
81 62
72 55
31 0
‘Data are for January and February 1973 (mmus 6 days each in January and Fcbrusry) and August and September 1978.
ated. and environmental data (Table I) indicated they were exposed most of the time to chilling soil temperatures. In addition to January and February, freezing air temperatures were observed in March (14 days) but not in April, while chilling air temperatures occurred through April (IO days). Rainfall was fairly uniform from November to July (III-16.8cm). except for February (3.7cm). Very high air temperatures ( 2 30-C) occurred in May (2 days). June (I3 days), July (I9 days). August (21 days) and September (I? days), but not in October. Maximum soil temperatures were equal to or greater than 32’C most days monitored in August and September to at least a soil depth of IOcm (Table I). and for only a small number of days did they not reach 3O’C. Rainfall was adequate in August (I 6.6 cm), but low in September (3.4 cm) and October (4.8 cm). At the time of collection. from May until November, soil samples were never dry, but were moist or near saturation (Fig. I). Water potentials of these soil samples were never below -0.4 MPa (Fig. I).
DISCUSSION
Efltcrs of chilling in saturated soil, and high temperatures at high and low wafer potentials on the grrminahilit~ o/ C. croralariue microsclerotium populurions under laboratory conditions
There was a great decline in germinable microsclerotia in soil maintained under saturation conditions at 5~C, at I week and at 5 weeks (Table 2). Germinable populations declined under moist (-0.22 MPa) conditions at 5-C also, but not as much. After 5 weeks at S’C and saturated soil
i33
on Cylindmc!~dium
The results of the field study indicated that the population of germinable C. crotalariae microsclerotia declined rapidly during the winter, regardless of soil depth. However, the causes of the declines are not likely to be the same for the three soil depths, as soil freezing was observed only in the upper stratum. Roth er al. (1979) found freezing of soil was much more detrimental to microsclerotium germinability than chilling soil temperatures in trials conducted at water potentials near field capacity. In our study, soil at the ‘j-cm depth was not frozen at collections and probably was not exposed to O’C.
Table 2. Influence of chilling under saturated and unsawrated solI conditwns on the germ,nabihtr of C~irn&xludium crorolariur microw-lcrotium popuiat~ons xn naturally infested soil’ Microsclerotia g Saturated soil (0 MPa) Temperature ( C) lninal population 25 5
I week 394 h’.b 292 B 139c
5 Necks 394 A’,b 173 B 6C
’ sod
Unsa[urarcd soil (--0.22 MPa) I week 394A“’ A04 A 310 B
5 weeks 394 A’.b 388 A l1OB
‘Daw are based on averaged C. crorularrae colony counts from IO Pcrn plates of sucrose-QT medium for each of two samples of soil B. “For each populat:on. column means labeled with the same letter are not significantly different (P = 0 05) xwxdmg to Duncan’s multlplc range test.
P. GIUHM and G. J. Gatmz Table
3. Influence
srur&rbc
of high
microsclcrorium
temperatures (ms)
on the prmmab:hty
populations
and very low
of C~iindrocladium
tn naturalI)-Xested
water
soil
_- wcki
Intttal
xl~~~‘~Oll ?JY A
25
35: A
-0.07
336 .A
-0.08
30
X8
-0.03
IO9B
-0.03 -0.52
B
35
72 c
-O..tY
5C
40
OD
- I.5
oc
349 x
-0.03
i M Pa,’ - 0.03
-I
5
-0.03
349 A
25
UB
- 12.4
-X.4
IZC
-27
28
30
oc
- 22.7
35
OD
-23.1
OC
-23.1
10
OD
-23.5
OC
- 23.5
population
4O?A
25
55
-0.03
B
-
JO? A
39.6
-0.03
08
- 39.6
30
5C
-40.2
OB
-JO.2
35
oc
-40.9
0
B OB
-409 -41 6
water
oc
potentials
water content low water
ptrntials
relative
significantly
rela~c
humidity
were maintained
retention with
humidity
relative
for each of two
potential
regime. column
ddkrcnt
(P = 0.05)
sampler
Nevertheless, the decline in microsclerotium germinability was about as rapid as at the 5cm depth where the soil was frozen and was exposed to long periods at 0-C or lower. The results from the laboratory trials provide evidence that saturated soil plus a chilling temperature was highly detrimental to microsclerotium germinability, was similar in its effect to soil freezing, and may be a principal cause of the germinability decline observed at the 2%cm depth. At this depth. soil samples were saturated or nearly saturated at all winter and spring collections and were exposed to chilling temperatures on about 90% of the days during January and February. Thus, the combined effects of chilling and soil saturation apparently exert a critical interacting influence onmicrosclerotium germinability. Whether the influence of soil saturation on microsclerotia is due to decreased partial pressure partial pressure of O?, increased both. or other factors, requires further of co:. studv The apparent stability, or even increase, of microsclerotium germinability at all soil depths over April. May and June may be due to favorable or moderate temperatures during that time. Soil data are not available but air temperatures during these months did not reach the chilling or high temperature regimes common in earlier and later months, respecttvely. April had only IO days of chilling temperatures and June had I3 days of high (2 30%) temperatures. Partial reversal of low temperature injury to microsclerotium germinability by exposure to moderate temperature (26C) has been demonstrated by Roth er al. (1979). and this reversal effect may be a factor in the good or high germinability of microsclerotia found in April. May and early July. In fact, two of the three soils used (A and B) showed significantly (P = 0.05) greater germinable microsclerotium popu-
to
NaCl
solution.
hltmidity
with
COWIF from of soil
means labeled
accordrng
distilled assay of
Low water potentials
curve.
at 85%
with
by gravimetric
a saturated
Data arc based on C. crodwiaecolony
sucrose-QT medium
bl;or each vutrr
at YY-100%
at the end of incubation
and use of a moisture
at 75%
KC1 solution.
-41.6
were maintamed
and were determined
maintained
of
potential
-0.03
populahon
water.
Water
potential (SIPa)’
population
JO ‘High
’ soil
Water
349 A’
msg
( Cl
lnitlal
low
3 uceks
TcIllpQ-JtU??
Initial
at high.
potentials’
wth
IO
were
and very a saturated
Prtn
plates
8. the s.wne lrttcr
Duncan’s multiple
are not
range test.
lations at the two lower soil depths on 2 July than on I April. High soil temperatures greatly influenced the germinability of microsclerotia in laboratory assays and the effects of high temperatures (30 and 35’C) were much greater at low and very low water potentials than in moist soil. While very dry soil conditions have been observed in the field during periods of high temperature in previous field experiments (Taylor er al., 1981), fairly uniform rainfall prevailed during our study. Similarly, soils were moist at all collections for all soil depths from August until November. Thus, only high soil temperature appeared to be an important factor in our field study. Based on the soil temperature data for the three soil depths, and the laboratory results, a greater decline in microscierotium germinability than observed might be expected following the months of July, August and September. Maximum soil temperatures were commonly over 3O~C at the 5 and IOcm soil depths and temperatures of 30 and 35’C had large effects on microsclerotium germinability in laboratory trials. However, the laboratory trials were conducted at constant high temperatures whereas the soil data are maximum daily soil temperatures. When maximum daily soil temperatures are greater than 30°C. minimum soil temperatures are often 26°C or lower at both the 5 and IOcm soil depths (unpublished). These differences may possibly explain why there was only a gradual decline in microsclerotium germinability following the months of July, August and September. .4nother hypothesis is that microsclerotia surviving the winter months may be “hardened” to adverse physical environmental factors. In general. however. the soil horizons that probably had the higher soil temperatures showed the more rapid decline inmisrosclerotium germinability.
Temperature and water interactions on Cylindrocladium Acknowledgement-We thank N. Pow.4 for providing information from the Agroenvironmental Monitoring System on soi! and air temperatures and rainfall at the research plot in Holland, Virginia. REFERENCES
Brown R. W. (1970) Measurement of water potential with thermocouple psychometers: construction and applications. USDA, Forest Service Res. Paper INT-80. 27 pp. Odgen. Utah. Griffin G. J. (1977) Improved selective medium for isolation C,vlindrocludim crotaluriae microsclerotia from naturally infested soils. Canadian Journal of Microbiology 23, 68683.
Griffin G. J., Roth D. A. and Powell N. L. (1978) Physical factors influence the recovery of Cylindrocladium cro-
735
ruluriae microsclerotium populations from naturally infested soils. PhTfoparhology 68, 887-89 I. Phipps P. M. and Reute M. K. (1979) Population dynamics of Cylindrocludium crofalariue microsclerotia in naturally infested soil. Phytopothology 69, 240-243. Roth D. A., Griffin G. J. and Graham P. J. (1979) Low temperature induces decreased germinability of Cyfindrocladium microscierotia. Canadian Journal of Microbiology 25, 157-162.
Taylor J. D.. Griffin G. J. and Garren K. Inoculum pattern. inoculum density disease relationships and population fluctuations of cludium crofalariue microsclerotia in peanut
H. (1981) incidence C_vlindrofield soil.
Phyloparhology 71, 1247-1302.
Winston P. W. and Bates D. H. (1961) Saturated solutions for the control of humidity in biological research. Ecology 41, 232-236.
o(338-07I7 88 53.00 + 0.00 1988 Pcrgamon Press plc
INFLUENCE OF TEMPERATURE AND WATER POTENTIAL INTERACTIONS ON THE GERMINABILITY OF CYLINDROCLADIUM CROTALARIAE MICROSCLEROTIA IN NATURALLY INFESTED SOIL P. GRAHAM and G. J. GRIFFIN Department
of Plant Pathology.
Physiology and Weed Science, Virginia Polytechnic University, Blacksburg. VA 24061. U.S.A. (/(cceprrd
5 /anuar,r
Institute
and State
1988)
Summary-Microsclerotia of C. crotaluriar in naturally-infested soils were placed in the field during late fall at depths of 5, 13 or 25 cm and exposed for I-yr. Germinable populations of microsclerotia declined rapidly at all soil depths during the winter, leveled off during April. May and June, and declined gradually from July to November. Soil at the S-cm depth was frozen in the winter and exposed to 0°C or lower for numerous days, whereas soil at the 25-cm depth was saturated and exposed extensively to chilling (65 C) temperatures. In laboratory trials. chilling plus saturation of soil with water was highly detrimental to microsclerotium germinability. and similar in its effect to soil freezing. Chilling alone had a more moderate effect on microsclerotium germinability. High soil temperatures (2 3O’C) were common in the field in the upper soil horizons during the late summer months. In laboratory tests. high soil temperatures (30, 35 and 4OC) greatly decreased microsclerotium germinability compared to moderate soil temperature (25‘C). and the effects of high temperatures were much greater at low (-22.4 to -23.5 MPa) water potentials than at high (-0.03 to - 1.5 MPa) water potentials.
MATERIALS
INTRODL’CTION
Soils und assay
Survival and germinability of Cvlindrocludium microsclcrotia are affected by low temperatures and low water potentials (Griffin er al., 1978: Phipps and Brute, 1979; Roth er al., 1979; Taylor ef al., 1981), and both factors are thought to play important roles in nature. Virginia soils containing C_vlindrocladium crotaluriue (Loos) Bell and Sobers, causal agent of Cylindrocladium black rot of peanut, are always exposed to chilling (,< 5-C) conditions and sometimes are frozen. The unusually severe winter of 19761977. when the soil was frozen for several weeks. was associated with a 95% decline in the C. crofaluriae microsclerotium population in the plow layer (O-25cm deep) (Taylor et al., 1981). A 1975 summer drought, which resulted in very dry soil conditions, was associated with a large decline in the C. crofuiariae microsclerotium population in the upper one-half (&12.7cm depth) of the pi’ow layer (Taylor et al., 1931). While low soil temperature and water potentials may have appreciable and independent effects on C. croruluriar microsclerotia, low temperature in midwinter is commonly associated with saturated soil conditions, and dry soil conditions in the summer months are often associated with high soil temperatures. The effects of high soil temperatures (2 30-C) on C. crotaluriae microsclerotia populations have not been investigated. Also, the effects of low temperatures at soil saturation have not been examined. We examined the influence of these factors on C. crotuluriue microsclerotium germinability in naturally-infested soil and estimated their relative importance in the survival of microsclerotia in nature.
AND METHODS
of germinuhle
microsclerotiu
Naturally-infested sandy loams with large C. crofuluriue microsclerotium populations, were taken from the root zones of symptomatic peanut plants in three peanut fields (A, B and C) in the Tidewater area of Virginia, and were used throughout the study. Soils were sieved (< I .9 mm mesh), placed in separate plastic bags with pin holes for air exchange, kept moist by periodic additions of distilled water, and by stored at 2YC. Soils were mixed thoroughly shaking in a plastic bag for 30min before use. Microsclerotium populations in all experiments were assayed quantitatively by wet sieving and a I :7 soil-dilution plating on sucrose-QT medium (Griffin, 1977). Colonies of C. croruluriue on 10 Petri plates for each sample were counted after 7-10 days growth at room temperature (2%28C). The recovery of microsclerotium populations from soils was used as an indicator of germinable microsclerotia in the soil. Eflects ofjeld nubility
of C.
encironmental conditions on the germicroluluriue microsclerotium populutions
Twenty-seven glass cylinders (2.5 x 3 cm), open at both ends, were filled with 27 g of naturally-infested soil for each of soils A, B and C. A series of nine cylinders of each soil were buried at S-. 13- and 25-cm depths on a 2.4 x 1.5 m plot at the Tidewater Agricultural Experiment Station, Holland, Va. The initial population for each soil was assayed the day after samples were buried (I2 November 1977). One soil cylinder of each soil from each depth was sampled on each of eight dates for the year 1978. At the time of collection. the appearance of the soil (frozen. satur731
732
P. Gttt~u~
and G. J. Gtums
ated, etc.) was recorded and the soil water content determined gravimetrically. The water content was compared to a soil moisture retention curve to estimate the water potential. The retention curve was prepared with a soil pressure plate at - 0.0 1, - 0.033, -0.51 and - 1.52 MPa. Soil temperature was monitored at each depth by temperature probes (model 15606, with No. 44019 thermilinear sensor, A. H. Thomas Co., Philadelphia, Penn.). Soil temperature data from the plot site at Holland, Va were obtained from I January to 22 Februav 1978 and 1 August to 30 September 1978. Effects of chilling ut soil saturation, and high temperatures at high and low water potentials on the germinubility of C. crotalariae microsclerotium populations under laboratory conditions Soil samples were maintained in incubators at 5 or 25YZ for I and 5 weeks. Soil moisture of the sample was maintained at saturation or moist levels (-0.03 to -0.22 MPa) by partially immersing a 50 ml beaker, with about 40 g of soil, in a 94-ml canning jar containing distilled water. Two such systems were made for each temperature-time intecval. Similar samples of soil were used for studying effects of high temperature (3@-40-C) under conditions of either 100, 85 or 75% relative humidity. Water potential of soils was calculated using the following formula: ‘Y=TinRH where Y = water potential, R = universal gas constant, T = absolute temperature. Y = partial modal volume of water, and RH = relative humidity (Brown, 1970). Saturated solutions of NaCl and KCI were used to maintain the humidity of a 94-ml closed container to 75 and 85% relative humidity, respectively (Winston and Bates, 1961). Approximately
I .3-2.0 cm of saturated salt solution or distilled water was dispensed into a 94-ml canning jar. A glass ring (2.5 x 3 cm) was placed at the bottom of the jar to support an aluminum pan containing 4Og of soil above the solution. A wire screen shelf was placed over the pan to support another pan containing a soil sample. The jar lid was coated with Vaseline and tightly sealed for the test period; or. in other instances, the lids were opened periodically. Two jar systems of either distilled water or saturated solution of KCI or NaCl were placed in incubators at 25, 30, 35 or 40X. Populations of microsclerotia were assayed after 2 and 3 weeks. After the specified periods, water potential of soils incubated with distilled water was determined by comparing percentage water content to a soil moisture reten;ion curve. RESULTS
Germinabiiit_v of C. crotalariae microsclerotium lutions under field conditions
Overall, there was a progressive decline at all soil depths in the number of germinable microsclerotia through the winter and spring months until about the end of March (Fig. 1). During April, May and June the germinable populations remained relatively constant or increased slightly. From July to November. however, there was a gradual decline in the germinable populations at all soil depths. With the exception of one data point (23 August for the 13 cm depth), germinable populations during this period at the 25-cm depth were largest. populations at the 5 cm depth were least, and the populations at the 13-cm depth were intermediate. Soil samples were frozen at the time of collection only at the j-cm soil depth in January and February (Fig. 1). At these times. the samples collected at the other depths were either saturated or nearly satur-
-.----
Cl
’
I
12
IO
NW
Jan
I
I
I
I
23
I
5
2
Apr
May
Jutv
Feb
popu -
5 cm l3Crn 25cm
I
&
I
I
CL
tEv
Fig. I. Survival of microsclerotium populations of C~lindrocludium crotnluriae in naturally infested soil at three depths in the soil profile during I yr at Holland, Virginia. Soil was buried in glass cylinders with open tops and bottoms on I2 November. 1977. F = frozen soil (3 -0.01 MPa). S = saturated soil. NS = nearly saturated soil (> -0.01 MPa). and M =moist soil (-0.01 to -0.1 MPa) indicate the appearance and water potential (by gravimetric and standard cume assay) of soil samples at the time of collection. Data are the means of three soils at each collection date and for each depth.
Temperature
and
water interactions
conditions. only 1.5% of the initial microsclerotium population was germinable. whereas 30% of the initial population was germinable in moisr soil at 5’C after 5 weeks. When the soil was maintained at 25’C. populations of germinable microsclerotia decreased under saturated conditions by 26% after I week and by 56% after 5 weeks. When soil was maintained at moist conditions and 25’C. the germinable population did not decline significantly (P = 0.05), even after 5 weeks. In a separate test with this soil, 26 and 0.396 of the initial germinable population was recovered after I and 5 weeks. respectively, when moist soil was held at O’C. Numbers of germinable microsclerotia in soil decreased significantly with increasing temperature at all water potentials examined (Table 3). Effects were greatest when soils were maintained at very low water potentials (-39.6 to 41.6 IMPa). Even at the high water potentials (-0.03 MPa). significant decreases in germinable microsclerotia were found after 3 and 3 weeks at 3O’C; no germinable microsclerotia were recovered after exposure to 4O’C. However, at 25’C in moist soil (-0.07 and -0.08 MPa) germinable microsclerotium populations were not significantly different from initial populations after 2 and 3 weeks. After 3 weeks at 35’C and -0.52 MPa, germinable populations were almost as low and not significantly different (P = 0.05) from populations recovered after 4O’C and about - I.5 MPa incubation. In soil maintained at low water potentials (-22.4 to - 23.5 MPa). germinable microsclerotium populations declined significantly even at 25’C, after 2 and 3 weeks exposure. Similar trends were noted for 25’C very low water potentials (-39.6 MPa); no germinable microsclerotia were recovered after 3 weeks at 25’C and - 39.6 .CIPa. Results were similar whether jars were periodically opened and resealed or not.
Table 1. Percentage of da)s the minimtun soil temperature was at or belo* 0 and 5-C during tw mid-winter months and the maximum soil temperature was a: or stwe 30 and 3?‘C durrng two summer months for various soil depths at Holland. Virginia’ SolI depth (cm) Temperature (V g5 SO
g30 332
7.6
15.2
% days *. dqs 9.? 96 3 58
30.5 9% da)s w 0
5.0
IO.?
50.8
81 62
72 55
31 0
‘Data are for January and February 1973 (mmus 6 days each in January and Fcbrusry) and August and September 1978.
ated. and environmental data (Table I) indicated they were exposed most of the time to chilling soil temperatures. In addition to January and February, freezing air temperatures were observed in March (14 days) but not in April, while chilling air temperatures occurred through April (IO days). Rainfall was fairly uniform from November to July (III-16.8cm). except for February (3.7cm). Very high air temperatures ( 2 30-C) occurred in May (2 days). June (I3 days), July (I9 days). August (21 days) and September (I? days), but not in October. Maximum soil temperatures were equal to or greater than 32’C most days monitored in August and September to at least a soil depth of IOcm (Table I). and for only a small number of days did they not reach 3O’C. Rainfall was adequate in August (I 6.6 cm), but low in September (3.4 cm) and October (4.8 cm). At the time of collection. from May until November, soil samples were never dry, but were moist or near saturation (Fig. I). Water potentials of these soil samples were never below -0.4 MPa (Fig. I).
DISCUSSION
Efltcrs of chilling in saturated soil, and high temperatures at high and low wafer potentials on the grrminahilit~ o/ C. croralariue microsclerotium populurions under laboratory conditions
There was a great decline in germinable microsclerotia in soil maintained under saturation conditions at 5~C, at I week and at 5 weeks (Table 2). Germinable populations declined under moist (-0.22 MPa) conditions at 5-C also, but not as much. After 5 weeks at S’C and saturated soil
i33
on Cylindmc!~dium
The results of the field study indicated that the population of germinable C. crotalariae microsclerotia declined rapidly during the winter, regardless of soil depth. However, the causes of the declines are not likely to be the same for the three soil depths, as soil freezing was observed only in the upper stratum. Roth er al. (1979) found freezing of soil was much more detrimental to microsclerotium germinability than chilling soil temperatures in trials conducted at water potentials near field capacity. In our study, soil at the ‘j-cm depth was not frozen at collections and probably was not exposed to O’C.
Table 2. Influence of chilling under saturated and unsawrated solI conditwns on the germ,nabihtr of C~irn&xludium crorolariur microw-lcrotium popuiat~ons xn naturally infested soil’ Microsclerotia g Saturated soil (0 MPa) Temperature ( C) lninal population 25 5
I week 394 h’.b 292 B 139c
5 Necks 394 A’,b 173 B 6C
’ sod
Unsa[urarcd soil (--0.22 MPa) I week 394A“’ A04 A 310 B
5 weeks 394 A’.b 388 A l1OB
‘Daw are based on averaged C. crorularrae colony counts from IO Pcrn plates of sucrose-QT medium for each of two samples of soil B. “For each populat:on. column means labeled with the same letter are not significantly different (P = 0 05) xwxdmg to Duncan’s multlplc range test.
P. GIUHM and G. J. Gatmz Table
3. Influence
srur&rbc
of high
microsclcrorium
temperatures (ms)
on the prmmab:hty
populations
and very low
of C~iindrocladium
tn naturalI)-Xested
water
soil
_- wcki
Intttal
xl~~~‘~Oll ?JY A
25
35: A
-0.07
336 .A
-0.08
30
X8
-0.03
IO9B
-0.03 -0.52
B
35
72 c
-O..tY
5C
40
OD
- I.5
oc
349 x
-0.03
i M Pa,’ - 0.03
-I
5
-0.03
349 A
25
UB
- 12.4
-X.4
IZC
-27
28
30
oc
- 22.7
35
OD
-23.1
OC
-23.1
10
OD
-23.5
OC
- 23.5
population
4O?A
25
55
-0.03
B
-
JO? A
39.6
-0.03
08
- 39.6
30
5C
-40.2
OB
-JO.2
35
oc
-40.9
0
B OB
-409 -41 6
water
oc
potentials
water content low water
ptrntials
relative
significantly
rela~c
humidity
were maintained
retention with
humidity
relative
for each of two
potential
regime. column
ddkrcnt
(P = 0.05)
sampler
Nevertheless, the decline in microsclerotium germinability was about as rapid as at the 5cm depth where the soil was frozen and was exposed to long periods at 0-C or lower. The results from the laboratory trials provide evidence that saturated soil plus a chilling temperature was highly detrimental to microsclerotium germinability, was similar in its effect to soil freezing, and may be a principal cause of the germinability decline observed at the 2%cm depth. At this depth. soil samples were saturated or nearly saturated at all winter and spring collections and were exposed to chilling temperatures on about 90% of the days during January and February. Thus, the combined effects of chilling and soil saturation apparently exert a critical interacting influence onmicrosclerotium germinability. Whether the influence of soil saturation on microsclerotia is due to decreased partial pressure partial pressure of O?, increased both. or other factors, requires further of co:. studv The apparent stability, or even increase, of microsclerotium germinability at all soil depths over April. May and June may be due to favorable or moderate temperatures during that time. Soil data are not available but air temperatures during these months did not reach the chilling or high temperature regimes common in earlier and later months, respecttvely. April had only IO days of chilling temperatures and June had I3 days of high (2 30%) temperatures. Partial reversal of low temperature injury to microsclerotium germinability by exposure to moderate temperature (26C) has been demonstrated by Roth er al. (1979). and this reversal effect may be a factor in the good or high germinability of microsclerotia found in April. May and early July. In fact, two of the three soils used (A and B) showed significantly (P = 0.05) greater germinable microsclerotium popu-
to
NaCl
solution.
hltmidity
with
COWIF from of soil
means labeled
accordrng
distilled assay of
Low water potentials
curve.
at 85%
with
by gravimetric
a saturated
Data arc based on C. crodwiaecolony
sucrose-QT medium
bl;or each vutrr
at YY-100%
at the end of incubation
and use of a moisture
at 75%
KC1 solution.
-41.6
were maintamed
and were determined
maintained
of
potential
-0.03
populahon
water.
Water
potential (SIPa)’
population
JO ‘High
’ soil
Water
349 A’
msg
( Cl
lnitlal
low
3 uceks
TcIllpQ-JtU??
Initial
at high.
potentials’
wth
IO
were
and very a saturated
Prtn
plates
8. the s.wne lrttcr
Duncan’s multiple
are not
range test.
lations at the two lower soil depths on 2 July than on I April. High soil temperatures greatly influenced the germinability of microsclerotia in laboratory assays and the effects of high temperatures (30 and 35’C) were much greater at low and very low water potentials than in moist soil. While very dry soil conditions have been observed in the field during periods of high temperature in previous field experiments (Taylor er al., 1981), fairly uniform rainfall prevailed during our study. Similarly, soils were moist at all collections for all soil depths from August until November. Thus, only high soil temperature appeared to be an important factor in our field study. Based on the soil temperature data for the three soil depths, and the laboratory results, a greater decline in microscierotium germinability than observed might be expected following the months of July, August and September. Maximum soil temperatures were commonly over 3O~C at the 5 and IOcm soil depths and temperatures of 30 and 35’C had large effects on microsclerotium germinability in laboratory trials. However, the laboratory trials were conducted at constant high temperatures whereas the soil data are maximum daily soil temperatures. When maximum daily soil temperatures are greater than 30°C. minimum soil temperatures are often 26°C or lower at both the 5 and IOcm soil depths (unpublished). These differences may possibly explain why there was only a gradual decline in microsclerotium germinability following the months of July, August and September. .4nother hypothesis is that microsclerotia surviving the winter months may be “hardened” to adverse physical environmental factors. In general. however. the soil horizons that probably had the higher soil temperatures showed the more rapid decline inmisrosclerotium germinability.
Temperature and water interactions on Cylindrocladium Acknowledgement-We thank N. Pow.4 for providing information from the Agroenvironmental Monitoring System on soi! and air temperatures and rainfall at the research plot in Holland, Virginia. REFERENCES
Brown R. W. (1970) Measurement of water potential with thermocouple psychometers: construction and applications. USDA, Forest Service Res. Paper INT-80. 27 pp. Odgen. Utah. Griffin G. J. (1977) Improved selective medium for isolation C,vlindrocludim crotaluriae microsclerotia from naturally infested soils. Canadian Journal of Microbiology 23, 68683.
Griffin G. J., Roth D. A. and Powell N. L. (1978) Physical factors influence the recovery of Cylindrocladium cro-
735
ruluriae microsclerotium populations from naturally infested soils. PhTfoparhology 68, 887-89 I. Phipps P. M. and Reute M. K. (1979) Population dynamics of Cylindrocludium crofalariue microsclerotia in naturally infested soil. Phytopothology 69, 240-243. Roth D. A., Griffin G. J. and Graham P. J. (1979) Low temperature induces decreased germinability of Cyfindrocladium microscierotia. Canadian Journal of Microbiology 25, 157-162.
Taylor J. D.. Griffin G. J. and Garren K. Inoculum pattern. inoculum density disease relationships and population fluctuations of cludium crofalariue microsclerotia in peanut
H. (1981) incidence C_vlindrofield soil.
Phyloparhology 71, 1247-1302.
Winston P. W. and Bates D. H. (1961) Saturated solutions for the control of humidity in biological research. Ecology 41, 232-236.