analysis of soil water matric potential requirement for infection of turnip with plasmodiophora...
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Analysis of soil water matric potential requirement forinfection of turnip with Plasmodiophora brassicae usingnegative pressure water circulation techniqueHidenori Iwama a , Seiko Osozawa a , Tuneo Ushiroda b & Toru Kubota aa National Institute of Agro-Environmental Sciences , Tsukuba , 305 , Japanb Nagasaki Prefectural Fruit Tree Experimental Station , Omura , 856-01 , Japanc Shikoku National Agricultural Experiment Station , Zentuji , 765 , JapanPublished online: 04 Jan 2012.
To cite this article: Hidenori Iwama , Seiko Osozawa , Tuneo Ushiroda & Toru Kubota (1994) Analysis of soil water matricpotential requirement for infection of turnip with Plasmodiophora brassicae using negative pressure water circulationtechnique, Soil Science and Plant Nutrition, 40:2, 293-299, DOI: 10.1080/00380768.1994.10413303
To link to this article: http://dx.doi.org/10.1080/00380768.1994.10413303
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Soil Sci. Plant Nutr., 40 (2), 283-291, 1994 283
Nitrogen Fixation in Peanut Determined by Acetylene Reduction Method and 15N -Isotope Dilution Technique
Monowar Karim Khan and Tomio Yoshida
Faculty of Horticulture, Chiba University, Matsuda, 271 Japan
Received May 31, 1993; accepted in revised form September 8, 1993
Seasonal pattern of nitrogenase activity of peanut (Arachis hypogaea L.) was studied under concrete plot conditions using samples of six different soils collected from Chiba Prefecture. Nitrogen fixation was determined using the 15N -isotope dilution, N -difference, and acetylene reduction methods. Peanut derived 41-63% of its total nitrogen from fixation depending on the soil types, based on measurements using the 15N -isotope dilution technique. The largest amount of nitrogen was fixed in Humic Andosols followed by Light-coloured Andosols and Sand-dune Regosols and the smallest in Dark Red soils. Amount of nitrogen fixed that was estimated by the N -difference method was 20-30% larger compared to the use of the isotope dilution technique. Calculation of nitrogen fixation based on the determination of acetylene reduction activity using a theoretical conversion factor of one-third N 2 reduced per C2 H 2 reduced markedly underestimated the amount compared to the isotope dilution method. The highest acetylene reduction activity was observed during the period from 95 to 120 d after sowing irrespective of soils.
Key Words: acetylene reduction activity, Arachis hypogaea L., isotope dilution, nitrogen fixation.
The fixation of nitrogen by peanut is largely dependent on various soil factors. For example, the Bradyrhizobium strain which was effective at a particular pH was less effective or not at all, when the pH value changed (Vincent 1965). To optimise the amount of nitrogen fixed in any system it is, therefore, necessary to estimate accurately the magnitude of fixation under different conditions. Only accurate measurements of nitrogen fixation will allow realistic evaluation of the contribution made by peanut to the N balance in a plant-soil system.
Methodology for the estimation of nitrogen fixation by legumes has evolved over the past three decades. Considerable effort has been directed to the development of methods for measuring nitrogen fixation (Hardy et al. 1973; Fried and Broeshart 1975; Rennie et al. 1978; Bergersen et al. 1989; N ambiar 1990; Hardarson et al. 1991). It is very important to identify a suitable method for measuring nitrogen fixation in leguminous crops. Each of the available methods has distinct advantages and disadvantages in terms of complexity and cost of analysis (Herridge 1982; Rennie and Rennie 1983).
Many studies have already been carried out on symbiotic nitrogen fixation by soybean or other legumes unlike for peanut. Peanut derives its nitrogen mainly from the atmosphere through symbiosis with root nodule bacteria and partially from soil and fertilizer N (Giller
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284 M.K. KHAN and T. YOSHIDA
et al. 1987). Hence, the experiment described here was carried out to determine the amount of nitrogen fixed by peanut in different soils over the growing period. Fixation was estimated based on various methods to obtain accurate values and to establish a relationship among the methods for easy, rapid, and accurate estimation of nitrogen fixation by peanut.
MATERIALS AND METHODS
Soils. The experiment was conducted in concrete frame plots in the field. There were 18 plots (I m X I m X I m) with 6 different soils in 6 plots replicated three times. The soils consisted of Sand-dune Regosols (SDR), collected from Nosai Town; Brown Forest soils (BFS), collected from Tateyama City; Dark Red soils (DRS), collected from Kamogawa City; Humic Andosols (Ando. I), collected from Chiba City; Light-coloured Andosols (Ando. 2), collected from Matsudo City; and sub-soil of Light-coloured Andosols (Ando. 3), which was also collected from Matsudo City. All these soil sampling locations are distributed in Chiba Prefecture. Before sowing the seeds the physico-chemical properties of the soils were analyzed as indicated in Table 1. Soil texture was determined by the pipette method, pH was measured potentiometrically in the supernatant of a 1 : 2.5 soil: liquid mixture. The liquid was either water (pH - H 20) or a I M KCI solution (pH - KCl). CEC was estimated following the procedure described by Keeney and Bremner (1969), total-C content by Tyurin (1931) and total-N content by the semimicro-Kjeldahl method (Bremner 1965).
Crop. The crop was peanut (Arachis hypogaea L.) var. Tarapoto and a nonnodulating mutant of the same plant as a reference crop, which was kindly provided by Shoichiro Akao, National Institute of Agrobiological Resources, Tsukuba, Japan. There were three rows of plants in each plot as follows: two rows with nodulating plants and one row with non-nodulating plants and in each row there were seven plants. The row to row and plant to plant distance were 35 and 15 cm, respecti vel y.
The seeds were sown on May 7, 1992. The seeds were germinated at 12-15 d after sowing (DAS) and flowering occured at 45-50 DAS. Irrigation, weeding, and insecticides were applied when necessary.
Data on nodule number and dry weight, and plant dry weight were recorded at 40, 70, 95, 120, and 155 DAS.
Acetylene reduction activity (ARA). At each growth stage plants were uprooted . very carefully from the soil so that nodules did not remain in the soil and the ARA was measured between 10:00 to 12:00 h, according to the procedure described by Yoshida et aJ. (1983). Ethylene production per plant per hour was measured. Nitrogen fixation (mg
Table l. Physico-chemical properties of the soils.
Particle size dist.(%) Textural b pH CEC T-C T-N Soils'
Sand class (cmol( +) kg-I) (g kg-l) (g kg-l) Silt Clay H2O KCl
SDR 98.93 1.07 trace S 5.30 4.37 3.4 3.3 0.3
BFS 60.17 23.68 16.15 CL 5.59 4.20 21.1 8.5 0.7 DRS 37.74 23.39 38.87 LC 6.27 5.24 41.0 40.3 3.6
Ando. I 45.84 45.81 8.30 SL 5.75 5.23 35.3 64.5 5.1 Ando.2 58.00 35.15 6.85 L 6.02 5.57 23.4 20.9 2.5 Ando.3 55.11 37.97 6.92 L 5.97 5.52 23.5 20.7 2.5
a Details on soils are given in MATERIALS AND METHODS. b S, Sand; CL, Clay Loam; LC, Light Clay; SL, Silt Loam; L, Loam.
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Estimation of Nitrogen Fixation in Peanut 285
plane l) was calculated based on the cumulative ethylene production at different sampling times and by adopting the theoretical conversion factor of one-third N 2-reduced per C2 H 2
red uced (Hardy et al. 1968). Nitrogen and 15N analysis. 15N-urea was applied at the rate of 3 g per plot (5.456%
15N excess) in all the plots before sowing of seeds and mixed up thoroughly with the soil. Except for nitrogen no other fertilizer was applied. The whole plant was dried at 70"C for 3 d and ground to pass through a 0.5 mm mesh sieve and was mixed thoroughly. When the pods were formed they were also taken with the whole plant for total Nand 15N determination. Total N content of the whole plant samples was determined following the semimicroKjeldahl procedure (Bremner 1965) using a digestion block chamber (Dry block bath, AL-1000, Sci nics corporation). The 15N abundance was determined for the Kjeldahl distillates according to the Rittenberg method (Rittenberg 1948) by emission spectrography using a JASCO N-150 analyzer.
Estimation of N 2-fixed by the N-difference method followed the classical subtraction procedure:
N 2-fixed (mg plant-l)=yield of N: nodulating peanut (mg plant-I)-yield of N: nonnodulating peanut (mg plant-I).
Estimation of N 2 -fixation by isotope dilution technique (ID). The amount or proportion of N derived from the atmosphere (% Ndfa) was calculated following the procedure described by Fried and Middleboe (1977) and Rennie et al. (1978).
Percent N (plant) derived from fertilizer (% Ndff)
% Ndff atom% 15N excess in plant X 100 atom% 15N excess in fert. .
Percent N (plant) derived from atmosphere (% Ndfa)
% Ndfa= (1- atom% 15N excess (fs) ) X 100 atom% 15N excess (nfs) ,
N -fixed = (1- atom% 15N excess (fs) ) X Nield (fs), 2 atom% 15N excess (nfs) y
where fs, fixing system (nodulating peanut); nfs, non-fixing system (non-nodulating peanut). Percent N (plant) derived from soil (% Ndfs)
% Ndfs= 100-(% Ndff+% Ndfa).
Statistical analysis. Analysis of variance of various crop characteristics was performed and means were compared using Duncan's multiple range test. Correlation of different crop characters was also determined (Gomez and Gomez 1984).
RESULTS AND DISCUSSION
At all the sampling times peanut grown in Ando. I displayed the largest nodule number and weight followed by BFS (Table 2). The increase in nodulation in these soils may be due to the low soil pH and other physico-chemical properties like soil texture, total-C content etc. of the soils. The lowest values for the nodule number and weight were observed in plants grown in DRS at all the sampling dates except for the nodule number at 155 DAS, presumably due to the fact that the soil was not cultivated. The physico-chemical properties
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286 M.K. KHAN and T. YOSHIDA
of Ando. 2 and Ando. 3 were somewhat similar but the nodule number and weight were smaller in Ando. 3, presumably because the bradyrhizobial population in Ando. 3 was low. The largest nodule number and weight were observed at 120 DAS in all the soils except for DRS and Ando. 3. In DRS and Ando. 3 the largest nodule number and weight were observed at 155 DAS.
The seasonal changes of ARA in peanut in various soils are presented in Fig.I. The largest ARA value was observed in Ando. I followed by Ando. 2 and the lowest in DRS. The highest nitrogenase activity in Ando. I can therefore, be ascribed to the fact that the nodule number and weight in this soil were larger than those in other soils and the lowest nitrogenase activity in DRS may be due a decrease in nodulation. These findings also corroborate the results obtained last year (unpublished data).
Further more, irrespective of soils the highest ARA value was recorded between 95 and 120 DAS. In Israel, Ratner et al. (1979) observed the maximum ARA of peanut plant at 90 to 100 DAS, while Dutta and Reddy (1988) in India; observed the highest nitrogenase activity at 76 or 86 DAS. In soybean the maximum ARA was observed during the reproductive stage (Rennie and Kemp 1984), suggesting that the characteristics of the nitrogenase activity of peanut are similar to those of other legumes such as soybean, although the mechanism of nodule formation is different (Nambiar 1990).
The amount of nitrogen accumulated in nodulated peanut was the largest in Ando. 1 followed by BFS and Ando. 2, and the smallest in DRS and Ando. 3. In the case of
Table 2. Nodule number and nodule weight of peanut grown in various soils at different days after sowing.
Soils' Nodule number plant-I Nodule weight (mg plant-I)
70DAS 95 DAS 120 DAS 155 DAS 70DAS 95 DAS 120DAS 155 DAS
SDR 65 ab 72c 149 b 133b 99 abc 149 bc 329 b 314 ab BFS 97 a 105 b 160 b 139 b 136 a 179 b 358 b 329 ab DRS 50 b 76 c 146 b 161 b 62 c 127 c 225 c 295 ab Ando. 92 ab 152 a 287 a 258 a 123 ab 276 a 446 a 365 a Ando.2 69 ab 147 a 167 b 156 b 85 bc 263 a 341 b 289 b Ando.3 62 b 86 bc 108 c 144 b 69 c 116 c 194 c 279 b LSD (0.05) 31 19 26 48 41 39 69 60
a Details on soils are given in MATERIALS AND METHODS. In a column, means followed by the same letter are not significantly different at 5% level of significance.
25
"7 ... 20 ~.r::
'i:: '" 15
"is.. OJ
"0 E 10 :::t.
~ N 5 U
0
Pod filling MaJ\.. nowcring ~
I
40 70 95 120
Days after sowing
155
Fig. 1. Seasonal changes of nitrogenase activity of peanut in various soils. Nitrogenase activity in SDR (0), BFS (fl.), DRS (0), Ando. I (0), Ando. 2 (.&.), Ando. 3 (II).
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Estimation of Nitrogen Fixation in Peanut 287
non-nodulated peanut a large amount of nitrogen was accumulated in BFS followed by Ando. I and the smallest was recorded in SDR (Table 3). Hence, it appeared that the nitrogen assimilation by nodulating and non-nodulating peanuts did not follow a similar trend in various soils.
Quantities of N 2-fixed by peanut calculated by the isotope dilution, N-difference, and acetylene reduction methods are presented in Table 4. Irrespective of the method of estimation, peanut grown in Ando. I fixed the largest amount of nitrogen followed by Ando. 2 and SDR and the smallest in DRS. Fixation of nitrogen calculated by the N-difference method was about 20- 30% higher and that by the acetylene reduction method was about one-third of the amount estimated by the isotope dilution technique.
The root weight of nodulating plants was larger than that of non-nodulating plants
Table 3. Nitrogen accumulation in nodulating and non-nodulating peanut grown in various soils at different days after sowing.
Nitrogen content (mg plant-I)
Soils" Nodulating Non-nod ulati ng
40 70 95 120 155 40 70 95 120 155 DAS DAS DAS DAS DAS DAS DAS DAS DAS DAS
SDR 66 397 ab 688 bc 993 ab 1,115 bc 56 181 201 d 226 c 271 d BFS 67 436 a 788 b 1,130 ab 1,273 b 56 211 331 ab 522 a 599 a DRS 61 307 b 525 d 862 b 984 c 53 205 287 bc 317 bc 390 bc Ando. I 79 474 a 997 a 1,439 a 1,551 a 61 217 386 a 471 ab 501 ab Ando.2 62 337 b 768 b 1,159 ab 1,249 b 54 195 280 bc 363 abc 382 cd Ando.3 66 328 b 535 c 884 b 954 c 55 189 256 cd 315 bc 344 cd LSD (0.05) 16 90 145 430 225 18 69 66 145 108
• Details on soils are given in MATERIALS AND METHODS. In a column, means followed by the same letter are not significantly different at 5% level of significance.
Table 4. Nitrogen fixation in peanut grown in different soils estimated by various methods.
DAS Methods N2-fixed (mg plant-I) LSD
SDR" BFS DRS Ando. I Ando.2 Ando.3 (0.05)
40 ID 2 3 ns N-diff. 10 II 8 17 8 II ns ARA
70 ID 123 abc 158 ab 65 d 168 a 110 bcd 103 cd 48 N-diff. 215 a 225 a 101 b 257 a 142 b 139 b 46 ARA 20 ab 26 ab 8c 27 a 18 b 18 b 8
95 ID 415 b 396 b 196 c 547 a 455 ab 235 c 87 N-diff. 487 a 457 a 238 b 611 a 488 a 279 b 143 ARA 72 cd 90 b 40 e 104 a 84 bc 61 d 12
120 ID 655 abc 542 bcd 361 d 851 a 717 ab 469 cd 224 N-diff. 767 ab 608 ab 545 b 967 a 796 ab 569 ab 366 ARA 144 c 161 bc 76 e 218 a 181 b 115 d 22
155 ID 699 bc 615 cd 402 e 955 a 775 b 512 cd 144 N-diff. 848 b 673 bc 594 c 1,050 a 867 ab 610c 179 ARA 227 c 225 c 103 e 329 a 281 b 177 d 38
a Details on soils are given in MATERIALS AND METHODS. DAS, days after sowing; ID, isotope dilution; N-diff., nitrogen difference; ARA, acetylene reduction activity (a theoretical value of one-third N 2-reduced per C2H2-reduced was used to calculate N2-fixed). -, not detected; ns, not significant. In a row, means followed by the same letter are not significantly different at 5% level of significance.
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288 M.K. KHAN and T. YOSHIDA
(data not presented). As a result, the nodulating plants had the advantage of taking up more nutrients from the soil and growing better than the non-nodulating plants. Hence, the difference method overestimated the fixation. The overestimation of nitrogen fixation by the N-difference method may be due to the difference in root weight and distribution, and nutrient assimilation by nodulating and non-nodulating peanuts (Peoples and Herridge 1990). Calculation of nitrogen fixation from ARA using a theoretical conversion factor of one-third N2 reduced per C2H2 reduced greatly underestimated the amount of N2-fixed compared to 10. When the ARA method was compared with other measurement techniques under field conditions, the ARA method greatly underestimated the nitrogen fixation activity (Boddey et al. 1984; Rennie and Kemp 1984; Kumar Rao and Dart 1987). This phenomenon may be due to the use of the theoretical conversion factor of one-third N2-reduced per C2H2 reduced (Hardy et al. 1968) or to the decline of the nitrogenase activity in the presence of acetylene during the assay (Minchin et al. 1983). Bergersen (1970), Burris (1974), and Bremner (1975) have reported that it is not suitable to convert C2H2-reduction values to N2 using a theoretical factor of 3. Our findings also confirmed these reports.
A positive correlation between the nodule number and ARA at 70 and 95 DAS, and between the nodule number and N2-fixation determined by 10 only at 70 DAS was observed (Table 5). However a positive correlation between the nodule weight and ARA and between the nodule weight and N 2-fixation determined by 10 was also observed at all the sampling dates except for 155 DAS. At 155 DAS, there was no correlation between the nodule number or weight and ARA and also between the nodule number or weight and N 2-fixation estimated by 10, presumably because, after 120 DAS the nodules started to become senescent although a few nodules were still being formed.
The amount of nitrogen in peanut derived from air, fertilizer and soils is shown in Fig 2. The amount of N derived from the atmosphere ranged from 41-63% at 155 DAS. The percentage of N derived from the atmosphere was the highest in SDR (62.6%) followed by Ando. 2 (61.9%) and Ando.1 (61.6%), and the lowest in DRS (41.0%). By measuring the
Table 5. Relationship of nodule number and weight to ARA and N,-fixation estimated by ID at different growth stages of peanut.
Parameters DAS Correlation coefficient (r) p-value
Nodule number vs ARA 70 95
120
0.943 0.919 0.769
0.005 0.009 0.074
155 0.071 0.894 --------.----------------------------- ------------------------. -------------------------- -----------------.---------. -------------------
Nodule weight vs ARA 70 0.914 0.01 I 95
120 0.968 0.831
155 0.153
0.001 0.041 0.772
.-------------------------------------------------------------------------------------------------------------- -----------------------------Nodule number vs N,-fixation by ID 70 0.842
Nodule weight vs N,-fixation by ID
95 120 155
70 95
120 155
p>0.05: not significant. psO.05: significant. psO.OI: highly significant.
0.758 0.782 0.665
0.913 0.871 0.874 0.695
0.036 0.081 0.066 0.149
0.011 0.024 0.023 0.125
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1000
750
500
250
750 SDR
Estimation of Nitrogen Fixation in Peanut
~
r-
BFS
o ~ N
DRS
0'N
Ando.l
o
~-r-
Ando.2
289
Ando.3 Fig. 2. Contribution of various sources of N to the total-N of peanut grown in various soils at different growth stages. c:::=J, air; liliiii, fertilizer; ~::::::::::::1, soil. • Values indicate days after sowing.
nitrogen fixation using the 15N method, Yoneyama et al. (1990) observed that peanut derived 40-61% of its total nitrogen from the atmosphere. The largest amount of nitrogen taken up from soil was found in the plants grown in BFS and the smallest in the plants grown in SDR. The difference in the utilization of fertilizer N (34-45%) by peanut among the soils was not significant although the utilization of fertilizer N in SDR was the lowest (34%). Amount of nitrogen derived from soil, fertilizer, and also the atmosphere in peanut increased with the progression of plant growth in all the soils.
Acknowledgments. We thank Dr. Shoichiro Akao, Department of Applied Physiology, National Institute of Agrobiological Resources, for his generous gift of seeds and Dr. Kazunori Sakamoto, Faculty of Horticulture, Chiba University for the technical assistance.
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Soil Sci. Plant Nu tr. , 40 (2), 293-299, 1994 293
Analysis of Soil Water Matric Potential Requirement for Infection of Turnip with Plasmodiophora brassicae
Using Negative Pressure Water Circulation Technique
Hidenori Iwama, Seiko Osozawa l , Tuneo Ushiroda*, and Toru Kubota
National Institute of Agro-Environmental Sciences, Tsukuba, 305 Japan; and • Nagasaki Prefectural Fruit
Tree Experimental Station, Omura, 856-01 Japan
Received June 3, 1993; accepted in revised form August 31, 1993
Critical soil water matric potential for infection of turnip with Plasmodiophora brassicae was examined using the negative pressure water circulation system for the regulation of the soil water matric potential of infested soils. A series of 12 gradients of soil water matric potential ranging from -3.2 kPa to -16.5 kPa was developed and maintained with fluctuations less than a few millibars (hectopascals) for 10 d after seeding. The clubroot symptoms were detected in plants in soil pots controlled at a higher soil water matric potential than -11.2 kPa. The disease was not detected in plants in soil pots controlled at a lower soil matric water potential than -12.2 kPa.
Key Words: critical soil water matric potential, Plasmodiophora brassicae, soil moisture control.
Clubroot disease of cruciferous plants is a soil-borne disease which can not be easily controlled. The disease is caused by infection of roots of crucifers with zoospores of Plasmodiophora brassicae. Improvement of the soil moisture conditions by drainage, as well as liming, is an effective measure to conirol clubroot disease of crucifers (Monteith 1924). The relations between the infection with the zoospores of Plasmodiophora brassicae and the soil water conditions are well understood. Based on studies carried out by Dobson et aI. (1982), root-hair infection with primary zoospores occurred at a soil water matric potential higher than - 800 mbar (- 80 kPa), while cortical infection with the secondary zoospores did not occur below -150 mbar (- 15 kPa). Based on the difference in the matric potential requirements of both types of zoospores, Dobson and Gabrielson (1983) determined the time required by each type of zoospore for the infection process. The germination of resting spores and root-hair infection with primary zoospores occurred within 1 d after planting of the crucifer seedlings. The mature zoosporangia in the root-hair appeared 2 d after planting. The release of secondary zoospores was observed on the third day, which coincided with the initiation of cortical infection. They confirmed that secondary zoospores were necessary for cortical infection and clubroot formation. Therefore, appropriate soil water regulation may enable to analyze the influence of several external factors on each stage of the infection
1 Present address: Shikoku National Agricultural Experiment Station, Zentuji, 765 Japan.
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process and also the complex influence of soil factors by differentiating physical from the other factors.
The purpose of this study was to examine accurately the critical soil water matric potential required for infection of turnips with clubroot disease pathogen in a humic volcanic ash soil using a soil water regulation technique recently developed, i.e. negative pressure water circulation system (Lipiec et al. 1989; Iwama et al. 1991).
MATERIALS AND METHODS
Discription of negative pressure water circulation system. In this technique which enables to control soil water conditions, water is supplied and drained through a ceramic tube wall embedded in a soil pot until the suction pressure of soil water is equilibrated with the negative pressure of water in the ceramic tube. The system, shown schematically in Fig. I, consists of a water pump, a water reservoir, and soil pots. The negative pressure of the circulating water is determined by the reduction of the air pressure in the water reservoir. The distribution of the negative pressure in the circulating system is a composite of the negative pressure indicated in the reservoir and the pump pressure which generates a pressure gradient along the circulation system (Iwama et al. 1991). In order to obtain a sequential setting of several soil water potential levels, soil pots are linked in series as shown in Fig.!. On the other hand, uniform soil moisture conditions can be obtained by parallel arrangement of the soil pots.
Critical soil water matric potential for infection of turnip with Plasmodiophora brassicae. Small soil pots ( 400 mL in volume) with a ceramic tube were packed with upland surface soil taken from an experimental field of the National Institute of Agro-Environmental Sciences in Tsukuba consisting of Humic Andosols. The soil was infested with the resting spores of the clubroot pathogen at the concentration of 106 cm- 3 .
The suspension of resting spores was prepared by grinding club-roots of Chinese cabbage (Brassica campestris L.) stored in a freezer (- 20T). Resting spores were refined by washing three times with distilled water in centrifugation tubes (Yoshikawa et al. 1981). Concentration of the resting spores in the suspension was determined using Thomas' hemocytometer. These soil pots were linked in series as shown in Fig. 1. Two sets of negative pressure water circulation systems were prepared and 12 gradients of soil water matric potentials ranging from -3.2 kPa to -16.5 kPa were obtained. The soil water potential values in each pot were
air pressure regulation system '\
water reservoir
water pump
soil pots
Fig. 1. Negative pressure water circulaiton system.
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Soil Water Potential and Infection 295
measured 2 to 3 times per day using mercury tensiometers embedded at a depth of 5 cm in soil. The water consumption rate was measured based on the changes in water level of the reservoir.
Eight turnip seeds per pot were planted on the soil surface and covered with quartz sand (diameter: 1-2 mm) about 5 mm in thickness to minimize the water loss. After germination, the seedlings were thinned to 4 plants per pot. Turnips were cultured for 1 month under natural light conditions near a window with supplementary fluorescent light to maintain a day light length of more than 13 h. The ambient temperature was not controlled and ranged between 15-28T.
After I month of cultivation, the turnip roots were washed and the severity of the symptoms was graded. The severity of the symptoms (club formation on main root) was rated into 4 scores, 0 to 3, where O=no symptoms; I =slight symptoms (deformation of root); 2=clear club formation; 3=severe club formation.
RESULTS
Control of soil water mat ric potential The values of the soil water matric potential of each pot measured during the I-month
period of cultivation are shown in Fig. 2. During the 2 week period after seeding which included the most critical period for the infection process (Katsura et al. 1970; Dobson et al. 1982), the original potential values were maintained with fluctuations of less than a few
....:I <t: H
E-< Z ~ E-< 0 Po.
hPa -600
-500
-400
-300
Fig. 2. Changes in soil water matric potential of soil pots during turnip culture for 1 month. L 1-5: Soil pots linked in a negative pressure circulation system supplied with water at a low negative pressure of -4 kPa. H 1-7: Soil pots linked in a negative pressure circulation system supplied with water at a relatively high negative pressure of -13.0 kPa. Soil water matric
O'---'-....L..~-'--'-................. -'-.L...J.-'--'-L-L....I...~ ......... ....I.-.L...J.-'--'-L...J......L..~....... potential in soil pots was mea-o 1 5 10 15 20 25 days sured at a depth of 5 cm using
DAYS AFTER SEEDING tensiometer.
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millibars (hectopascals), except for one pot (H-4). Also the diurnal fluctuations of the potential values were less than a few millibars (hectopascals). It was considered that the values of the soil water matric potential of the soil pots were precisely maintained in the pot culture experiment.
After 10 d, the soil water potential values of the pots decreased. Especially after 15 d, the water potential values of the soil pots which were regulated at less than -10.0 kPa decreased drastically due to the shortage of water supply to meet the increased water consumption. On the other hand, the water potential values of the soil pots which were regulated at higher values than -10.0 kPa became unstable due to the large and frequent fluctuations of the water level in the reservoir. In the latter half of the growth period, it was necessary to fill the reservoirs with water to the top 2 to 4 times a day due to the high water consumption rate and small capacity of the reservoirs. The water consumption rate increased with the growth of the turnips. Namely in the first 10 d after seeding the values were less than 2 mm/d, from the 15th to 20th day they increased to 5 to 7 mm/d, and after 25 d they exceeded more than 10 mm/d with a maximum of 17 mm/d.
The purpose of this experiment was to examine the relationships between the soil water potential and the occurrence of clubroot disease on tap roots of turnips. The important processes of tap root infection with the pathogen were completed during the first 10 dafter seeding according to Katsura (1970) and Dobson et al. (1982). The latter half of growth was considered to correspond to the period during which the symptoms of the disease, clubroot development appeared distinctly. It was also considered that the decrease in the soil water potential of the pots in the latter half of the growth period prevented further infection and did not affect the relations between the soil water potential and the infection during the first half of the growth period.
Critical soil water matric potential for clubroot development Turnip seeds fully germinated within 2 d after seeding and grew well during 1 month
of cultivation except for pots L-l and L-4. The former pot experienced wet injury and the
Table l. Club disease occurrence and soil water matric potential.
NEWCIRC· Soil water matric potentialb Severity of clubroot Disease Wt. of root systems Pot No. (kPa) (pF) 0 1 2 3 indexC (g/4 plants)
(No. of plants) Ld -3.2 1.49 0 0 0 4 3.0 2.7e
2 -4.8 1.68 0 0 0 4 3.0 7.9 3 -6.3 1.80 0 0 0 4 3.0 13.6 4 -7.9 1.90 0 0 0 3 3.0 I. 7e
5 -9.2 1.96 0 2 1.8 13.8 Hd -9.2 1.96 I 1 1 1 1.5 8.8
2 -9.9 2.00 0 2 0 2 2.0 9.1 3 -11.2 2.05 2 0 0.7 17.8 4 -12.2 2.09 4 0 0 0 0.0 11.2 5 -13.8 2.14 4 0 0 0 0.0 17.8 6 -15.6 2.19 4 0 0 0 0.0 11.3 7 -16.5 2.22 4 0 0 0 0.0 13.4
• NEWCIRC = negative pressure water circulation. b Averaged during 10 d after seeding, I hPa= 1 mbar. C Disease index=}; (disease severity score X No. of plants)/total plants. d L: - 3.0 kPa at reservoir, H: -13.0 kPa at reservoir. e Experienced wet injury.
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Soil Water Potential and Infection 297
Fig. 3. Clubroot disease on turnip roots and soil water matric potential. Turnips were cultured for 1 month in soil pots (400 mL) infested with the resting spores of the pathogen at a concentration of 10' cm-3 . The clubroot symptoms did not develope on turnips grown at a soil water matric potential below - 12.2 kPa.
latter pot was affected by other soil-borne diseases. The relationships between the water matric potential and severity of clubroot disease
are ~hown in Table 1 and Fig. 3. The soil water matric potential values listed in Table 1 were averaged values during 10 d after seeding. Based on these results a soil water potential value between -11.2 kPa and -12.2 kPa was considered to correspond to the critical value for the initiation of clubroot disease.
DISCUSSION
The critical soil water matric potential for clubroot disease infection on humic volcanic ash soil was estimated at about -12 kPa in this experiment, a value slightly higher than the critical value, -15.0 kPa, reported by Dobson et al. (1982) on silty loam soil and sandy loam soil. The equivalent pore diameter of the capillary suction pressure value (-12.0 kPa) is 25)1m which is considered to be critical for the movement of the secondary zoospores. However, on muck soil with high moisture content characteristics, a lower critical soil water matric potential less than -20.0 kPa was reported by Dobson et al. (1982). Therefore, the critical soil water potential is supposed to be related to the pore diameter and also to the continuity and tortuosity of the pores, and to be changed to some extent with the soil moisture characteristics of each soil type.
The higher soil water potential (i.e. higher moisture content) required for the infection with secondary zoospores than that for the infection with primary zoospores, was adscribed by Dobson et al. (1982) to the fact that the size of the secondary zoospore is larger than that of the primary zoospore due to the plasma fusion of two zoospores. Tommerup and Ingram (1971) observed that the size of the secondary zoospore with a single nucleus was about 3.0 )1m in diameter and that of binucleate zoospore was about 6.0 )1m in diameter. In relation to the critical pore size, the authors examined the movement of the resting spores caused by capillary water flow in pot culture experiments. Layers of sand 2.5 em thick were used as filters. Diameter of sand particles ranged from 20 to 540 )1m. The resting spore suspension (spore concentration: lOB cm- 3 ) containing some of the germinated primary zoospores was absorbed by capillarity through the sand filters to soil pots in which turnip seedlings were planted. Clubroot symptoms were not observed in the soil pots on the sand filters with particle diameter of 50-100)1m (Table 2). Average equivalent pore diameter of the sand filter
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Table 2. Occurrence of clubroot disease on turnip roots by capillary upward movement of resting spores through sand filters.
Particle diameter (mm)
0.54 -0.38 0.38 -0.25 0.25 -0.15 0.175-0.10 0.10 -0.05 0.05 -0.02
Sand filters'
Pore diameter (mm)
0.18 0.13 0.08 0.055 0.030 0.014
Clubroot disease diseased plants/total
(plants/plants)
12/12 6/12
10/12 3/12 0/12 0/12
Occurrence percentb
(%)
100.0 50.0 83.3 25.0 0.0 0.0
• Soil pot consists of three layers as follows: a soil layer of 2 cm on the top, a coarse sand layer of 5 cm in the middle, and a filter sand layer 2.5 cm in thickness. The bottom of the pots was immersed in suspension of resting spores of the pathogen at a concentration of 108 cm-'. b Turnips were cultured for I month in a green house. Water was supplied by capillarity only from the bottom. Plant density: 4 plants per pot.
(particle diameter: 50-100 J.lm) corresponded to around 30 J.lm. This value was almost equal to the critical value of pore diameter, 25 J.lm, for the secondary zoospores estimated from the value of critical soil water potential. The size of the resting spore and that of the primary zoospore were 2.4-3.9 J.lm (av. 3.2 J.lm) and 2.4-6.0 J.lm respectively (Naiki 1987). For the swimming ability of zoospores of Phytophytora cryptogae, a soil water matric potential above - 5.0 kPa (equivalent diameter: 60 J.lm) was found to be critical by Duniway (1976). However, in his experiments, the zoospores had to swim over a considerable distance of more than 2-3 mm. These conditions which determine the critical pore diameter were different from the passive movement by water flow and the infection process from root hair to cortex of root.
Therefore, it is assumed that the large difference in the critical soil water matric potential between the primary and the secondary zoospore infection may be ascribed to the large size of secondary zoospore, and to the difference in the minimum distance necessary to complete the infection process. Namely, in the case of root-hair infection, since the root-hairs grow into bulk soil and come into contact with the resting spores, it may be possible for the primary zoospores to complete the infection process by covering a limited distance. On the other hand, in the case of the cortical infections, secondary zoospores have to cover larger distances, i.e., from the zoosporangium formed in root-hair to the cortex of roots. These considerations should be further examined.
Osozawa et al. (in press) studied further on the promotive effect of the CO2 content in soil air on the incidence of clubroot disease and also on the suppressive effect of a Low humic Andosol on the disease, using the negative pressure water circulation technique. In their experiments, the effects of drainage and moisture retention of the soils could be differentiated from other soil properties by precise soil moisture control. However, several improvements of the negative pressure water circulation system will be needed for more detailed analysis of the infection in the soil-root system. Instability of soil water potentials observed in our studies could be alleviated by the use of a more precise air pressure regulator, larger soil pots and also by the development of a water level regulator under suction pressure.
Acknowledgments. The authors are greatly indebted to Dr. N. Kobayashi, Nat!. Res. lnst. Vegetable, Ornamental Crops and Tea, Kurume Branch, and Dr. K. Sugawara, Tropical Agric. Res. Cent., Okinawa
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Branch, for their information on c1ubroot disease and microbiological experiments.
REFERENCES
Dobson, R.L. and Gabrielson, R.L. 1983: Role of primary and secondary zoospores of Plasmodiophora brassicae in the development of c1ubroot in Chinese cabbage. Phytopathology, 73, 559-561
Dobson, R.L., Gabrielson, R.L., and Baker, A.S. 1982: Soil water matric potential requirements for root-hair and cortical infection of Chinese cabbage by Plasmodiophora brassicae. Phytopathology, 72, 1598- 1 600
Duniway, J.M. 1976: Movement of zoospores of Phytophlhora cryptogea in soils of various textures and matric potentials. Phytopathology, 66, 877-882
Iwama, H., Kubota, T., Ushiroda, T., Osozawa, S., and Katou, H. 1991: Control of soil water potential using negative pressure water circulation technique. Soil Sci Plant Nutr., 37, 7-14
Katsura, K., Tamura, M., and Yamaguchi, N. 1970: Some observations on the infection of Plasmodiophora brassicae Woronin in root hair of Cruciferae plants. Proc. Kansai Plant Prot. Soc., No. 12, p. 23-29 (in Japanese with English summary)
Lipiec, J., Kubota, T., Iwama, H., and Hirose, 1. 1989: Measurement of plant water use under controlled soil moisture conditions by the negative pressure water circulation technique. Soil Sci. Plant Nutr., 34, 417-428
Monteith, J., Jr. 1924: Relation of soil temperature and soil moisture to infection by Plasmodiophora brassicae. J. Agric. Res., 28, 549-561
Naiki, T. 1987: Life cycle and control of Plasmodiophora brassicae, causing c1ubroot disease of cruciferous plants. Soil Microorg., No. 29, 25-39 (in Japanese with English summary)
Os ozawa, S., Iwama, H., Kanai, Y., and Kubota, T. 1994: The effect of soil aeration on incidence of club root disease of brassicae. Soil Sci. Plant Nutr., in press
Tommerup, I.e. and Ingram, D.S. 1971: The life-cycle of Plasmodiophora brassicae Woron. in brassica tissue culture and in intact roots. New Phytol., 70, 327-332
Yoshikawa, H., Ashizawa, M., and Hida, K. 1981: Studies on the breeding of c1ubroot-resistance in Cole crops. III The 'insertion' screening method for c1ubroot-resistance. Bull. Veg. Ornamental Crops Res. Stn., A18, 1-21 (in Japanese with English summary)
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