- Email: [email protected]
Changes in cytokinin activity associated with the development of Verticillium wilt and water stress in cotton plants
Physialogieal
Plant Pathology
(1972)
2, 187-196
Changes in cytokinin activity associated with the development of Verficillium wilt and water stress in cotton plants I. MISAGHI, J. E. DEVAY Department University (Acceptedfor
of Plant Pathology, of California, Davis, publication
December
and T. KOSUGE Cal&
95616,
U.S.A.
1971)
Tracheal fluid and extract from leaves and stems of both healthy and Verticilliwn-infected cotton plants (Gossypium hirsutum L. var. Acala SJ-1) contain three cytokinin-active substances which can be separated from each other by thin-layer chromatography. There are significant reductions in the levels of cytokinins in tracheal fluid and extract from leaves and stems of Verticillium-infected cotton plants compared to that of healthy plants. A time course study showed that the changes in cytokinin activity in Verticillium-infected cotton occurred after development of leaf symptoms. The leaves and stems of cotton plants exposed to water stress contained less cytokinins than those from normal plants. However, there was a greater loss of cytokinin activity in extract from leaves and stems in Verticillium-infected plants than in plants subjected to water stress. The TLC Rf values of two of the factors from cotton were similar to those of zeatin mononucleotide and zeatin; however, their structural relatedness to zeatin or zeatin mononucleotide is unknown. The chromatographic behavior of the third factor from cotton was different from the known cytokinins.
INTRODUCTION Cotton plants infected with Verticillium albo-atrum often develop wilt symptoms which resemble an acceleration of leaf senescence. Since cytokinins appear to retard senescence in excised and intact leaves from certain plants [20, 51, it was hypothesized that the symptoms of Verticillium wilt in leaves of cotton might be due, at least in part, to changes in the activity of cytokinins. Changes in cytokinin activity in plants affected by several diseases have been studied. Although “green islands” surrounding fungal infection sites on senescing maple leaves had cytokinin activity, no cytokinin activity was detected in comparable amounts of green healthy leaf tissues [S]. Kiraly et al. [13] also reported that an increase in cytokinin activity in leaves of Phaseolus vulgaris and Vicia faba occurs as a consequence of infection by Uromycesphaseoli (rust) and that the formation of green islands around the infection centers might be due to the action of cytokinins. Tissues from club root galls on turnips also have higher cytokinin activity than tissues from healthy plants [I]. Samuels [21] found that loss of apical dominance in fasciation of peas could be induced by either Corynebacterium fascians or kinetin. That naturally occurring cytokinins are involved in fasciation of plants infected with C. fmciam was later confirmed by Klambt et al. [la] and Thimann & Sachs [26]. They isolated cytokinin-active substances from the bacterium; Helgeson & Leonard [9] subsequently determined the structure of these active compounds.
188
I. Misaghi
ef al.
In the foregoing examples the diseased plants contained higher concentrations of cytokinins than did healthy plants. However, in the case of Verticillium wilt of cotton a decrease in cytokinin activity is associated with disease development [17, 181. The object of the present study was to characterize more fully the changes in cytokininactive substances which occur in cotton following infection by V. albo-atrum. MATERIALS AND METHODS Biological materials and induction
of Verticillium
wilt symptoms in cotton plants
Go.s.#ium hirsutum L. var. Acala SJ-1 and a pathogenic, non-defoliating strain (SS4) of Verticillium albo-atrum Reinke & Berth [27] were used throughout this study. To prepare inoculum for plants, Petri dishes containing potato-dextrose agar (PDA) were inoculated with 1 ml of a conidial suspension of V. albo-atrum. After 4 days the agar and fungal colony of a single plate were minced and added to the soil of a 12.5 cm pot containing two 65-day-old plants. The inoculum was mixed with the top 1.5 cm of the soil using a spatula. Wilt symptoms, including chlorotic and necrotic lesions on the leaves of inoculated plants, developed within 12 to 18 days after inoculation in a greenhouse maintained at 23 to 28 “C. Isolation
and partial
purzjication
of cytokinins from tracheal jluids of cotton
When chlorotic areas in leaves of diseased plants began to necrose, healthy and diseased plants were excised below the cotyledonary node about 4 cm from the soil level. Tracheal fluid was collected from stumps according to the method of Schnathorst [24]. The pots were watered daily and the tracheal fluid that moved into the glass tube due to root pressure was collected with a sterile syringe at 8 a.m. and 5 p.m. daily for three consecutive days. The tracheal fluid was freeze-dried and the residue stored over calcium chloride under partial vacuum. For partial purification of cytokinins by thin-layer chromatography (TLC) an amount of residue corresponding to 100 ml of tracheal fluid was added to 100 ml of 950/, ethanol and the suspension was warmed to 60 “C. The mixture then was shaken vigorously and centrifuged at 10,000 g for 15 min at room temperature. The pellet was washed with warm (60 “C) 80% ethanol and then with warm 95% ethanol. The alcohol extracts were combined and the pellet was discarded. The volume of the alcohol extract was reduced to 2 to 3 ml under partial vacuum at 40 “C. This solution was used for TLC. Isolation
and partial
purification
of cytokinins from the leaves and stems of cotton
A method was developed by which a solution, low in pigments, with cytokinin activity could be extracted from the leaves of cotton. About 500 g of fresh leaves from 80-day-old cotton plants were washed and then immersed in distilled water at 22 “C under reduced pressure (40 to 60 mm) for 10 min. The water-soaked leaves were pressed gently between layers of paper towels, cut into l-in (25.4 mm) strips and placed into centrifuge tubes (6 cm x 10 cm, Sorval Polyethylene). A perforated rubber disk with a thickness of 1 cm placed at the bottom of a centrifuge tube allowed separation of sap from the leaves after centrifugation. The tubes were centrifuged for 20 min at 16,000 g at 4 “C and the collected sap was mixed immediately with three volumes of cold 95% ethanol.
Cytokinin
activity
in Verticillium-infected
cotton
plants
189
The sap from the stems of plants was also collected by centrifugation. Fresh stems cut below the cotyledonary nodes were cut into 8-cm sections and placed into centrifuge tubes. The base of each section was rested on a perforated plastic cap placed near the bottom of each centrifuge tube to provide a small chamber for collection of the sap. These tubes were centrifuged for 20 min at 12,000 g at 4 “C. Sap collected from both stems and leaves was combined and centrifuged at 9000 g for 10 min. The pellet was discarded and the supernatant liquid was dried under reduced pressure at 40 “C. The residue was dissolved in 100 ml of warm water (50 “C). The solution was adjusted to pH 3.5 with 1-O N-HCl and shaken four times with one volume amounts of ethyl acetate to remove substances which interfered with the bioassay for cytokinins [15]. The ethyl acetate phases were discarded. The aqueous phase was concentrated by evaporation under reduced pressure to remove residual ethyl acetate, and the remaining solution was readjusted to pH 3.5. This preparation then was added to a cation exchange column (1.5 cm x 14 cm, Bio-Rad AG50W-X8 H+ form, 200-400 mesh) and the column was washed with 100 ml of distilled water [14, IS, 16-J. The effluent was adjusted to pH 9.0 with l-0 N-NaOH and passed through an anion exchange column (l-5 cm x 14 cm, BioRad AGl-X2, Cl- form, 200-400 mesh) [15, 161. The latter column was washed with 100 ml of distilled water and the effluent discarded. The cation and anion exchange columns were then eluted with 200 ml of 2-O N-ammonium hydroxide and 100 ml of 4-O N-foIXk acid, respectively. The ammonium hydroxide and formic acid eluates were evaporated individually to dryness under reduced pressure at 45 “C, the residues dissolved in 3 ml of 80% ethanol and then combined. This preparation was further purified by the use of TLC. The efficiency of the water infiltration-centrifugation method was compared with the ethanol-homogenization method for extraction of cytokinins. Frozen leaves and stems of cotton (500 g) were homogenized in 3.5 1 of 95% ethanol for 15 min at 24 “C. The homogenate was centrifuged at 16,000 g for 20 min. The volume of the supernatant liquid was reduced to about 400 ml under reduced pressure at 40 “C. The solution was freeze-dried, the residue was washed three times with 150-ml amounts of 80% ethanol (60 “C) and the combined washings centrifuged at 16,000 g for 15 min. After the ethanol was removed under reduced pressure at 40 “C, the remaining aqueous solution was diluted to 100 ml with water and adjusted to pH 3.5. The solution was then shaken four times with one volume amounts of ethyl acetate. Traces of ethyl acetate were removed from the aqueous phase by holding the solution under reduced pressure for 10 min at 40 “C; the pH of the solution was adjusted to 4-5. To precipitate the cytokinins from the solution 10 ml of 1-OM-silver nitrate solution were added and the mixture was left at 4 “C for 24 h [I4, 15, 221. The precipitate was centrifuged at 9000 g for 15 min at 4 “C. The pellet was resuspended in and washed with 150 ml of cold 0.05 M-silver nitrate three times. The precipitate was suspended in 100 ml of O-2 N-HCI and warmed to 50 “C. After 30 min, the suspension was centrifuged at 4500 g for 5 min at 25 “C. The supernatant liquid was saved. The suspension of precipitate and centrifugation were repeated using 60- and 30-ml portions of O-2 N-HCl, respectively. The supernatant fractions were combined and evaporated to dryness under reduced pressure at 45 “C. The residue was dissolved in 2 to 3 ml of 80% ethanol.
190
Separation
I. Misaghi
et
a/.
of cytokinins by TLC
The preparations of cytokinins from stems and leaves partially purified by ion exchange chromatography and from tracheal fluid were applied to an Eastman 6060 silica gel plate with fluorescent indicator. The plates were then developed with one of the following solvent systems: I-butanol-acetic acid-water, 12 : 3 : 5, v/v; 1-butanol-cont. ammonium hydroxide, 4 : 1, v/v; or water-saturated 2-butanol. After development, the chromatograms were dried and bands of silica gel, approximately 1.5 cm wide from the origin to the solvent front, were scraped off the plates and eluted twice with 15 ml of 80% ethanol. The ethanol was removed by evaporation under reduced pressure at 40 “C. The residue was taken up in 25 ml of warm water (50 “C) and bioassayed for cytokinin activity. Bioassay of cytokinins The soybean callus tissue test as reported by Miller [16] was used for the measurement of cytokinin activity. In some preliminary tests, activity of cytokinins was also measured by the chlorophyll retention bioassay [4] and by bud formation on Funaria hygrometrica [ 81. Effect of water stress on the cytokinin content of healthy cotton plants Since decreases in cytokinin activity may occur in water-stressed plant tissues [12], it was necessary to determine if the changes in cytokinin activity in cotton plants affected by Verticillium wilt may have been caused by water stress. This possibility was examined by comparing the cytokinin contents of healthy plants which were wilted or turgid. Cotton plants were grown in the greenhouse for 78 days and then were divided into two equal groups. One group was subsequently watered daily, whereas water was withheld from the other group. Approximately 24 h after the first visible wilting occurred, the degree of water stress in several individual leaves from both groups was estimated with a pressure chamber [2]. Immediately after the measurement of water stress, plants were excised just above the cotyledonary nodes and placed in plastic bags under dry ice. Cytokinins were isolated from the leaves and stems by the ethanol-homogenization method as described earlier and separated by TLC using water-saturated 2-butanol as the solvent and bioassayed. RESULTS
Evaluation
of cytokinin bioassay metho&
The chlorophyll retention bioassay was used for measuring the cytokinin activity in However, because of its low specificity [S] it was not used for preliminary studies. A bioassay based on the production of buds by Funaria quantitative analyses. hygrometrica (L.) Sibth. [8] proved useful for the detection of cytokinin activity in tracheal fluids but not for cytokinins in the stem and leaf extracts. The morphology of protonemata was changed after their exposure to cytokinin-active fractions from the stem and leaf extracts of cotton, i.e. cells which were normally long and narrow became short and barrel-shaped and no buds were formed from these protonemata. Consequently, Funaria was not used for the assay of cytokinins. The abnormal response of F. hygrometrica to the cytokinin factors in cotton extracts may have been caused by impurities in the final preparations of the leaves and stems. However,
Cytokinin
activity
in Verticilliom-infected
cotton
plants
191
the response ofF. hygrometrica was not improved by treating the cytokinin preparations from leaves and stems with cation and anion exchange resins or activated charcoal, extractions with chloroform, 1-butanol or ethyl acetate from basic solutions, barium acetate precipitation, treatment with PVP (Polyclar) or gel filtration (Bio-Gel P-2, 50-100 mesh). Preparations from leaves and stems were not inhibitory to the soybean callus tissue since the addition of different amounts of these extracts to soybean bioassay medium containing different concentrations of kinetin did not reduce the activity of this cytokinin. Cytokinins
in extracts of cotton
Three general areas with cytokinin activity were found on the thin-layer chromatograms of tracheal fluids and extracts from leaves and stems of both healthy and diseased plants. These areas are designated as I, II and III on Figs 1 and 2. For convenience the substances with cytokinin activity in these areas will be referred to as factors I, II and III, respectively. Based on their similar behavior in three different solvent systems used in TLC, the three factors isolated from tracheal fluid are probably identical to the three factors isolated from the leaves and stems,
Rf FIG. 1. Soybean callus bioassay of cytokinins in tracheal fluid from healthy and from Verticillium-infected Acala SJ-1 cotton plants. Cytokinins were separated by TLC using three solvents: (a) I-butanol-acetic acid-water; (b) I-butanol-ammonium hydroxide; (c) watersaturated P-butanol. O-O, Healthy; l - - - l , diseased; the horizontal broken lines at the lower part of the figures represent the average weight of soybean callus tissue in control flasks. The capital letters indicate the location on the chromatograms of adenosine-5’monophosphate (A), benzyladenine (B), dihydrozeatin (D), kinetin (K), 6-(y,y-dimethylallylamino)purine (P), zeatin (Z), zeatin riboside (R) and 6-methylaminopurine (S). Cytokinin active areas are marked by Roman numerals.
In another series of experiments the total cytokinin activity associated with each of the three factors was determined. There was a significant reduction in the activity of factor I in the tracheal fluid and in the leaf and stem extracts of cotton plants with Verticillium wilt when compared with healthy plants (Table 1). However, the differences in the total activity of either factor II or III in tracheal fluids and in extracts of leaves and stems of healthy and diseased plants were not significant.
192
I. Misaghi
et al.
The total amount of cytokinin activity recovered from the leaves and stems of cotton, calculated from the data in Table 1, was 20.9 rig/g (dry weight) of healthy tissue and 15.2 rig/g (dry weight) of Verticillium-infected tissue. The corresponding figures for tracheal fluids of healthy and Verticillium-infected cotton plants, calculated from the data in Table 1, were 21.6 ng and 17.1 “g/ml of sap, respectively. In diseased plants there was greater reduction in cytokinin activity in the leaves and stems than in the tracheal fluids. The percentage of recovery of kinetin, benzyladenine and zeatin added to leaf extract, as measured by gas-liquid chromatography, was found to be from 83 to 90 [2.5].
FIG. 2. Soybean callus bioassay of cytokinins in leaves and stems from healthy and from Verticillium-infected Acala SJ-1 cotton plants. Cytokinins were isolated by the water infiltrationcentrifirgation method and separated by TLC using three solvents: (a) I-butanol-acetic acid-water; (b) 1-butanol-ammonium hydroxide; (c) water-saturated P-butanol. o-0, Healthy; l - - - 0, diseased; the horizontal broken lines at the lower part of the figures represent the average weights of soybean callus tissue in control flasks. Cytokinin-active areas are marked by Roman numerals. TABLE Amounts
1
of three factors with cytokinin activity in the tracheal&id stems of healthy and Verticillium-infected Acala Kinetin equivalent (rig/ml of tracheal fluid)a
Solvent systemsb
Cytokinin factors
A
5.9kO.2 3*5+0.2 5.8kO.l
3.1 1.7 0.3
I
9.550.2 5.9kO.l 7.3 + 0.3
6.9 + 0.3 38+0.2 6.9kO.3
2.6 2.1 0.4
9*4+ 0.4 2.9+0.1 6.2 & 0.3
2.6 0.6 0.0
I II III
Difference
9.02 0.3 5.2 + 0.3 6.1 +O.l
II III c
Verticilliuminfected plants
Kinetin weight)
I II III
B
Healthy plants
12.OkO.4 3.5kO.2 6.2iO.2
and in the extract ST-I cotton plants
of leaves and
equivalent of leaves
Healthy plants
Verticilliuminfected plants
10.3kO.4 3.7kO.2 68kO.3
6+0+0*3 2.8+0.1 6.920.3
9.750.4 3.3kO.l 6.2 + 0.2 11.2+0.4 3.9kO.l 7.7kO.l
a Average of three replications using the soybean callus tissue bioassay. b A: I-butanol-acetic acid-water, 12 : 3 : 5; B: I-butanol-ammonium 4 : 1; C: water-saturated P-butanol. c More activity in infected tissue.
(rig/g (dry and stems)a
Difference 4.3 0.9 -O.lC
6.3 + 0.3 Z-8+0*1 4.9kO.l
3.4 0.5 1.3
6.6 + 0.3 3.2kO.l 6.1 +O.l
4.6 0.7 1.6
hydroxide
(cont.),
Cytokinin
activity
in Verticilliom-infected
cotton
193
plants
The extracts from leaves and stems prepared by the water infiltrationcentrifugation method [Fig. 2c] yielded slightly more cytokinins and significantly less pigment than the extracts obtained by the ethanol-homogenization method (Fig. 3). However, the amount of cytokinins in healthy and diseased tissues were proportionally the same in both extraction procedures.
0
05
I-O
FIG. 3. Soybean callus bioassay of cytokinins in leaves and stems from healthy and from Cytokinins were isolated by the ethanolVe&cillium-infected Acala SJ-1 cotton plants. homogenization method and separated by TLC using water-saturated 2-butanol as solvent. O-O, Healthy; l - - - l , diseased; the horizontal broken line at the lower part of the figure represents the average weights of soybean callus tissue in control flasks. Cytokinin active areas are marked by Roman numerals.
Chromatography of naturally occurring cytokinins from cotton with several known cytokinins and adenosine-5’-monophosphate (AMP) is shown in Fig. 1. The R, value of factor I in water-saturated P-butanol using TLC is similar to a factor in corn kernel which was a mononucleotide of zeatin [ls]. The chromatographic behavior of this factor in water-saturated 2-butanol also is similar to a major cotton fruit cytokinin prepared by Sandstedt [ZZ, 231. Experiments so far with alkaline phosphatase to test the possible nucleotide nature of factor I have not been conclusive, mainly because of the limited amounts of factor I available. The R, values of factor II in the TLC solvent systems were different from those of other known cytokinins tested but were somewhat similar to those of factor II isolated from seeds of pumpkin [7]. The R, values of factor III in all TLC solvent systems tested were closer to dihydrozeatin and zeatin than to other naturally occurring cytokinins tested (Fig. 1). Relation of changes in cytokinin factors to time of symptom development in Verticillium cotton
wilt in
Experiments were made to find out how soon after infection the observed changes in activity of cytokinins were detectable. The cytokinin contents of leaves and stems of healthy cotton plants were compared with those of infected plants at 2, 6, 11 and 16 days after inoculation with V. albo-atrum. The activities of the three factors were measured after their preparation by the water infiltration-centrifugation method and their purification by TLC with 1-butanol-ammonium hydroxide. The results showed that a reduction in the activity of the cytokinin factors in infected plants was detectable only after advanced development of the symptoms (Table 2).
194
1. Misaghi TABLE Amounts of three factors cotton plants on dyerent
with cytokinin days following Kinetin
Days after inoculation 2 6 lib 16
activity isolated from the leaves and sterns inoculation with Verticillium albo-atrum
equivalent
Factor I Healthy Inoculated 8.6 9.3 9.8 9-9
(rig/g
a Average of three replications using the soybean b Leaf symptoms (chlorotic lesions) were evident.
lj”ect
(dry
weight)
Factor
II Inoculated
Healthy
8.8 9.5 6.1 6.9
et al.
2
3.1 3.3 2.7 3.2 callus
of leaves
2.9 3.5 2.1 2.6 tissue
of Acala S3-I (SS4)
and stemsja Factor Healthy 5.8 6.0 6.0 5.7
III Inoculated 5.8 5,8 5.7 4.4
bioassay.
of water stress on activity of cytokinins
Figure 4 shows that there was some decrease in the cytokinin activity of waterstressed healthy cotton. However, the pattern of change in the activity of the three factors in water-stressed cotton plants was different from that found in cotton plants with Verticillium wilt. Verticillium infection caused a reduction in the activity of factor I in contrast to water stress (Figs 2, 4). On the other hand, water stress, but not Verticillium infection, caused a reduction in the activity of factor III. Estimated water potentials were between - 11 and - 13 bars in the well-watered and turgid plants, and were between - 19 and - 22 bars in the plants from which water was withheld. Wilting of cotton leaves was consistently observed when the water potential was lower than - 15 bars.
FIO. 4. Soybean callus bioassay of cytokinins in leaves and stems from normal and from water-stressed Acala SJ-1 cotton plants. Cytokinins were isolated by the ethanol-homogenization method and separated by TLC, using water-saturated P-butanol as solvent. O-O, Normal; A - - - A, water stressed; the horizontal broken line at the lower part of the figure represents the average weights of soybean callus tissue in control flasks. Cytokinin-active areas are marked by Roman numerals. DISCUSSION
A comparison of methods for extraction of cytokinin from leaves and stems and their partial purification by TLC indicated that highest total yields were obtained with the water infiltration-centrifugation method and with water-saturated 2-butanol as the irrigating solvent in TLC. There is a reduction in the level of cytokinins as a result of Verticillium infection. The reason for the reduced cytokinin activity in infected plants is not clearly known.
Cytokinin
activity
in Verficillium-infected
cotton
plants
195
However, on the basis of this study the fungus could play a partial role. The reduction of factor I in diseased plants compared with healthy plants could be due to the activity of a fungal that could convert this material to factor II or factor III such as the one detected in the fungal culture (I. Misaghi et al., unpublished results). Water stress, which may play a major role in symptom development in Verticillium wilt of cotton, could also cause a reduction in cytokinin activity. A significant reduction in cytokinin activity in tracheal fluid has been reported when plant roots have been subjected to water stress [II] and to high salinity [IO]. Water stress in the plant shoot also reduced cytokinin activity in an extract from xylem and leaves of Nicotiana rustica [12]. In the present study, the leaves and stems of the cotton plants exposed to water stress yielded less cytokinins than those from normal plants. However, this reduction in cytokinin activity was not as severe as that caused by the fungal infection. Moreover, the relative changes in activity of the three factors in water-stressed and in fungus-infected plants were not the same. It should be noted that the type and degree of water stress caused by infection may not be comparable to that induced by withholding water, and the physiological changes caused by these two types of wilting may be different. Thus, the results presented here do not preclude entirely the possibility that water stress reduces the cytokinin activity of plants infected with Verticillium. Although the decrease in activities of cytokinins and the apparent early senescence of the leaves of Verticillium-infected cotton plants might suggest a cause-and-effect relationship, the existence of such a relationship is not supported by this study since the reduction in the activity of cytokinins in plants infected with Verticillium occurred after the onset of wilt symptoms. The defoliation of cotton plants infected by defoliating strains of V. albo-atrum, such as T-9, is apparently related to the differential alterations in abscisic acid and ethylene levels [271. The usual symptoms of wilt caused by non-defoliating strains of V. albo-atrum, such as SS4, are apparently not directly related to changes in cytokinin activity. However, the changes in cytokinin activity in Verticillium-infected cotton plants may indirectly affect the activity of the other growth regulators which could contribute to development of wilt symptoms. The authors gratefully acknowledge the support of this work by the Cooperative State Research Service, United States Department of Agriculture, Grant No. 016-15-06. We also thank Dr J. M. Duniway for assistance in determining moisture stress in leaf tissues, Dr C. Miller for cultures of soybean callus tissue and Mr Jeff Hall for help with photographs. REFERENCES 1. DEKHUIJZEN, H. M. & OVEREEM, J. Cl. (1971). The role of cytokinins in clubroot formation. Physiological Plant PatholoQ 1, 151-161. 2. DUNIWAY, J. M. (1971). Comparison of pressure chamber and thermocouple psychrometer determinations of leaf water status in tomato. Plant Physiology, Lancaster 48, 106-107. 3. ENGELBRECHT, L. (1968). Cytokinin in den “grtinen Inseln” des Herbstlaubes. Flora 159,208-2 14. 4. FAWCETT, C. H. & WRIGHT, S. T. C. (1968). Cytokinin activity in a homologous series of whydroxypolymethyleneamino purines. Phytachemistry 7, 1719-1725. 5. FLETCHER, R. A. (1969). Retardation of senescence by benzyladenine in intact bean plants. Planta, Berlin 89, l-8.
196
I. Misaghi
et al.
6. FLETCHER, R. A. & OSBORNE, D. J. (1966). Gibberellin, as a regulator of protein and ribonucleic acid synthesis during senescence in leaf cells of Taraxacum oj%inale. Canadian Journal of Botany 44, 739-745. 7. GUPTA, G. R. P. & MAHESHWARI, S. C. (1970). Cytokinins in seeds of pumpkin. Plant PhysioloQ, Lancaster 45, 14-18. 8. HEINZ, H. & BOPP, M. (1968). A cytokinin test with high specificity. Planta, Berlin 83, 115-I 18. 9. HELGESON, J. P. & LEONARD, N. J. (1966). Cytokinins: identification of compounds isolated from Co7ynebacterium fasciaas. Proceedings of the National Academy of Sciences of the United States of America 56,60-63. 10. ITAI, C., RICHMOND, A., & VAADIA, Y. (1968). The role of root cytokinins during water and salinity stress. Israel 3ournal of Botany 17, 187-193. 11, ITAI, Cl. & VAADIA, Y. (1965). Kinetin-like activity in root exudate of water-stressed sunflower plants. Physiologia plantarum 18, 941-944. 12. ITAI, C. & VAADIA, Y. (1971). Cytokinin activity in water-stressed shoots. Plant Physiology, Luncaster 47, 87-90. 13. KIRALY, Z., EL HAMMADY, M. & Poz&, B. I. (1967). Increased cytokinin activity of rustinfected bean and broad bean leaves. Phytopatholopy 57, 93-94. 14. KL.&BT, D., Tnras, G. & SKOOG, F. (1966). Isolation of cytokinins from Copebacterium fascians. Proceedings of &he JVational Academy of Sciences of the United States of America 56, 52-59. 15. LETHAM, D. S. (1966). Regulators of cell division in plant tissues. II. A cytokinin in plant extracts: isolation and interaction with other growth regulators. Phytochemistry 5, 269-286. 16. MILLER, C. 0. (1965). Evidence for the natural occurrence of zeatin and derivatives: compounds from maize which promote cell division. Proceedings of the flational Academy of Sciences of the United States of America 54, 1052-1058. 17. MISAGHI, I. & DEVAY, J. E. (1970). Isolation and assay of cytokinins in healthy and Vertic-illiumwilted cotton plants. In Proceedings of the 1970 Beltwide Cotton Production Research Conferences, Houston, Texas, p. 6. 30th Cotton Disease Council, Natn. Cotton Council, Memphis, Tenn. 18. MISAGHI, I., DEVAY, J. E. & RAVENSCROFT, A. (1969). Cytokinin activity in cotton plants infected by Verticillium albo-atrum. Phytopathology 59, 1041 (Abstr.). of cytokinins. Journal 19. MOST, B. H., WILLIAMS, J. C. & PARKER, K. J. (1968). G as chromatography of Chromatography 38, 136-l 38. 20. MOTHES, K. & ENGELBRECHT, L. (1961). Kinetin-induced directed transport of substances in excised leaves in the dark. Phytochemistry 1, 58-62. 21, SAMUELS, R. M. (1961). Bacterial-induced fasciation in Pisum satiuum var. Alaska. Ph.D. i%sis Indiana University, Bloomington, Indiana. 22. SANDSTEDT, R. (1970). Partial purification of a cytokinin from immature cotton fruit. In Proceedings of the I970 Beltwide Cotton Production Research Conferences, Houston, Texas, pp. 29-31. 30th Cotton Disease Council, Natn. Cotton Council, Memphis, Tenn. 23. SANDSTEDT, R, (1971). Cytokinin activity during development of cotton fruit. Physiologia Plantarum 24,408-410. 24. SCHNATHORST, W. C. (1970). Obtaining xylem fluid from Gossypium hirsutum and its uses in studies on vascular pathogens. Phytopatholopy 60, 175-l 76. 25. SHINDY, W. W., AECHER, T., MISAGHI, I. & DEVAY, J. E. (1970). Gas-liquid chromatography of cytokinins in plant extract. Plant Physiology, Lancaster 46 (suppl.), 8. 26. THIMANN, K. V. & SACHS, T. (1966). The role of cytokinins in the “fasciation” disease caused by Corynebacterium fascians. American Journal of Botany 53, 73 I-739. 27. WIESE, M. V. & DEVAY, J, E. (1970). Growth regulator changes in cotton associated with dePlant Physiology, Lancaster 45, 304-309. foliating caused by Verticillium albo-atrum.
Plant Pathology
(1972)
2, 187-196
Changes in cytokinin activity associated with the development of Verficillium wilt and water stress in cotton plants I. MISAGHI, J. E. DEVAY Department University (Acceptedfor
of Plant Pathology, of California, Davis, publication
December
and T. KOSUGE Cal&
95616,
U.S.A.
1971)
Tracheal fluid and extract from leaves and stems of both healthy and Verticilliwn-infected cotton plants (Gossypium hirsutum L. var. Acala SJ-1) contain three cytokinin-active substances which can be separated from each other by thin-layer chromatography. There are significant reductions in the levels of cytokinins in tracheal fluid and extract from leaves and stems of Verticillium-infected cotton plants compared to that of healthy plants. A time course study showed that the changes in cytokinin activity in Verticillium-infected cotton occurred after development of leaf symptoms. The leaves and stems of cotton plants exposed to water stress contained less cytokinins than those from normal plants. However, there was a greater loss of cytokinin activity in extract from leaves and stems in Verticillium-infected plants than in plants subjected to water stress. The TLC Rf values of two of the factors from cotton were similar to those of zeatin mononucleotide and zeatin; however, their structural relatedness to zeatin or zeatin mononucleotide is unknown. The chromatographic behavior of the third factor from cotton was different from the known cytokinins.
INTRODUCTION Cotton plants infected with Verticillium albo-atrum often develop wilt symptoms which resemble an acceleration of leaf senescence. Since cytokinins appear to retard senescence in excised and intact leaves from certain plants [20, 51, it was hypothesized that the symptoms of Verticillium wilt in leaves of cotton might be due, at least in part, to changes in the activity of cytokinins. Changes in cytokinin activity in plants affected by several diseases have been studied. Although “green islands” surrounding fungal infection sites on senescing maple leaves had cytokinin activity, no cytokinin activity was detected in comparable amounts of green healthy leaf tissues [S]. Kiraly et al. [13] also reported that an increase in cytokinin activity in leaves of Phaseolus vulgaris and Vicia faba occurs as a consequence of infection by Uromycesphaseoli (rust) and that the formation of green islands around the infection centers might be due to the action of cytokinins. Tissues from club root galls on turnips also have higher cytokinin activity than tissues from healthy plants [I]. Samuels [21] found that loss of apical dominance in fasciation of peas could be induced by either Corynebacterium fascians or kinetin. That naturally occurring cytokinins are involved in fasciation of plants infected with C. fmciam was later confirmed by Klambt et al. [la] and Thimann & Sachs [26]. They isolated cytokinin-active substances from the bacterium; Helgeson & Leonard [9] subsequently determined the structure of these active compounds.
188
I. Misaghi
ef al.
In the foregoing examples the diseased plants contained higher concentrations of cytokinins than did healthy plants. However, in the case of Verticillium wilt of cotton a decrease in cytokinin activity is associated with disease development [17, 181. The object of the present study was to characterize more fully the changes in cytokininactive substances which occur in cotton following infection by V. albo-atrum. MATERIALS AND METHODS Biological materials and induction
of Verticillium
wilt symptoms in cotton plants
Go.s.#ium hirsutum L. var. Acala SJ-1 and a pathogenic, non-defoliating strain (SS4) of Verticillium albo-atrum Reinke & Berth [27] were used throughout this study. To prepare inoculum for plants, Petri dishes containing potato-dextrose agar (PDA) were inoculated with 1 ml of a conidial suspension of V. albo-atrum. After 4 days the agar and fungal colony of a single plate were minced and added to the soil of a 12.5 cm pot containing two 65-day-old plants. The inoculum was mixed with the top 1.5 cm of the soil using a spatula. Wilt symptoms, including chlorotic and necrotic lesions on the leaves of inoculated plants, developed within 12 to 18 days after inoculation in a greenhouse maintained at 23 to 28 “C. Isolation
and partial
purzjication
of cytokinins from tracheal jluids of cotton
When chlorotic areas in leaves of diseased plants began to necrose, healthy and diseased plants were excised below the cotyledonary node about 4 cm from the soil level. Tracheal fluid was collected from stumps according to the method of Schnathorst [24]. The pots were watered daily and the tracheal fluid that moved into the glass tube due to root pressure was collected with a sterile syringe at 8 a.m. and 5 p.m. daily for three consecutive days. The tracheal fluid was freeze-dried and the residue stored over calcium chloride under partial vacuum. For partial purification of cytokinins by thin-layer chromatography (TLC) an amount of residue corresponding to 100 ml of tracheal fluid was added to 100 ml of 950/, ethanol and the suspension was warmed to 60 “C. The mixture then was shaken vigorously and centrifuged at 10,000 g for 15 min at room temperature. The pellet was washed with warm (60 “C) 80% ethanol and then with warm 95% ethanol. The alcohol extracts were combined and the pellet was discarded. The volume of the alcohol extract was reduced to 2 to 3 ml under partial vacuum at 40 “C. This solution was used for TLC. Isolation
and partial
purification
of cytokinins from the leaves and stems of cotton
A method was developed by which a solution, low in pigments, with cytokinin activity could be extracted from the leaves of cotton. About 500 g of fresh leaves from 80-day-old cotton plants were washed and then immersed in distilled water at 22 “C under reduced pressure (40 to 60 mm) for 10 min. The water-soaked leaves were pressed gently between layers of paper towels, cut into l-in (25.4 mm) strips and placed into centrifuge tubes (6 cm x 10 cm, Sorval Polyethylene). A perforated rubber disk with a thickness of 1 cm placed at the bottom of a centrifuge tube allowed separation of sap from the leaves after centrifugation. The tubes were centrifuged for 20 min at 16,000 g at 4 “C and the collected sap was mixed immediately with three volumes of cold 95% ethanol.
Cytokinin
activity
in Verticillium-infected
cotton
plants
189
The sap from the stems of plants was also collected by centrifugation. Fresh stems cut below the cotyledonary nodes were cut into 8-cm sections and placed into centrifuge tubes. The base of each section was rested on a perforated plastic cap placed near the bottom of each centrifuge tube to provide a small chamber for collection of the sap. These tubes were centrifuged for 20 min at 12,000 g at 4 “C. Sap collected from both stems and leaves was combined and centrifuged at 9000 g for 10 min. The pellet was discarded and the supernatant liquid was dried under reduced pressure at 40 “C. The residue was dissolved in 100 ml of warm water (50 “C). The solution was adjusted to pH 3.5 with 1-O N-HCl and shaken four times with one volume amounts of ethyl acetate to remove substances which interfered with the bioassay for cytokinins [15]. The ethyl acetate phases were discarded. The aqueous phase was concentrated by evaporation under reduced pressure to remove residual ethyl acetate, and the remaining solution was readjusted to pH 3.5. This preparation then was added to a cation exchange column (1.5 cm x 14 cm, Bio-Rad AG50W-X8 H+ form, 200-400 mesh) and the column was washed with 100 ml of distilled water [14, IS, 16-J. The effluent was adjusted to pH 9.0 with l-0 N-NaOH and passed through an anion exchange column (l-5 cm x 14 cm, BioRad AGl-X2, Cl- form, 200-400 mesh) [15, 161. The latter column was washed with 100 ml of distilled water and the effluent discarded. The cation and anion exchange columns were then eluted with 200 ml of 2-O N-ammonium hydroxide and 100 ml of 4-O N-foIXk acid, respectively. The ammonium hydroxide and formic acid eluates were evaporated individually to dryness under reduced pressure at 45 “C, the residues dissolved in 3 ml of 80% ethanol and then combined. This preparation was further purified by the use of TLC. The efficiency of the water infiltration-centrifugation method was compared with the ethanol-homogenization method for extraction of cytokinins. Frozen leaves and stems of cotton (500 g) were homogenized in 3.5 1 of 95% ethanol for 15 min at 24 “C. The homogenate was centrifuged at 16,000 g for 20 min. The volume of the supernatant liquid was reduced to about 400 ml under reduced pressure at 40 “C. The solution was freeze-dried, the residue was washed three times with 150-ml amounts of 80% ethanol (60 “C) and the combined washings centrifuged at 16,000 g for 15 min. After the ethanol was removed under reduced pressure at 40 “C, the remaining aqueous solution was diluted to 100 ml with water and adjusted to pH 3.5. The solution was then shaken four times with one volume amounts of ethyl acetate. Traces of ethyl acetate were removed from the aqueous phase by holding the solution under reduced pressure for 10 min at 40 “C; the pH of the solution was adjusted to 4-5. To precipitate the cytokinins from the solution 10 ml of 1-OM-silver nitrate solution were added and the mixture was left at 4 “C for 24 h [I4, 15, 221. The precipitate was centrifuged at 9000 g for 15 min at 4 “C. The pellet was resuspended in and washed with 150 ml of cold 0.05 M-silver nitrate three times. The precipitate was suspended in 100 ml of O-2 N-HCI and warmed to 50 “C. After 30 min, the suspension was centrifuged at 4500 g for 5 min at 25 “C. The supernatant liquid was saved. The suspension of precipitate and centrifugation were repeated using 60- and 30-ml portions of O-2 N-HCl, respectively. The supernatant fractions were combined and evaporated to dryness under reduced pressure at 45 “C. The residue was dissolved in 2 to 3 ml of 80% ethanol.
190
Separation
I. Misaghi
et
a/.
of cytokinins by TLC
The preparations of cytokinins from stems and leaves partially purified by ion exchange chromatography and from tracheal fluid were applied to an Eastman 6060 silica gel plate with fluorescent indicator. The plates were then developed with one of the following solvent systems: I-butanol-acetic acid-water, 12 : 3 : 5, v/v; 1-butanol-cont. ammonium hydroxide, 4 : 1, v/v; or water-saturated 2-butanol. After development, the chromatograms were dried and bands of silica gel, approximately 1.5 cm wide from the origin to the solvent front, were scraped off the plates and eluted twice with 15 ml of 80% ethanol. The ethanol was removed by evaporation under reduced pressure at 40 “C. The residue was taken up in 25 ml of warm water (50 “C) and bioassayed for cytokinin activity. Bioassay of cytokinins The soybean callus tissue test as reported by Miller [16] was used for the measurement of cytokinin activity. In some preliminary tests, activity of cytokinins was also measured by the chlorophyll retention bioassay [4] and by bud formation on Funaria hygrometrica [ 81. Effect of water stress on the cytokinin content of healthy cotton plants Since decreases in cytokinin activity may occur in water-stressed plant tissues [12], it was necessary to determine if the changes in cytokinin activity in cotton plants affected by Verticillium wilt may have been caused by water stress. This possibility was examined by comparing the cytokinin contents of healthy plants which were wilted or turgid. Cotton plants were grown in the greenhouse for 78 days and then were divided into two equal groups. One group was subsequently watered daily, whereas water was withheld from the other group. Approximately 24 h after the first visible wilting occurred, the degree of water stress in several individual leaves from both groups was estimated with a pressure chamber [2]. Immediately after the measurement of water stress, plants were excised just above the cotyledonary nodes and placed in plastic bags under dry ice. Cytokinins were isolated from the leaves and stems by the ethanol-homogenization method as described earlier and separated by TLC using water-saturated 2-butanol as the solvent and bioassayed. RESULTS
Evaluation
of cytokinin bioassay metho&
The chlorophyll retention bioassay was used for measuring the cytokinin activity in However, because of its low specificity [S] it was not used for preliminary studies. A bioassay based on the production of buds by Funaria quantitative analyses. hygrometrica (L.) Sibth. [8] proved useful for the detection of cytokinin activity in tracheal fluids but not for cytokinins in the stem and leaf extracts. The morphology of protonemata was changed after their exposure to cytokinin-active fractions from the stem and leaf extracts of cotton, i.e. cells which were normally long and narrow became short and barrel-shaped and no buds were formed from these protonemata. Consequently, Funaria was not used for the assay of cytokinins. The abnormal response of F. hygrometrica to the cytokinin factors in cotton extracts may have been caused by impurities in the final preparations of the leaves and stems. However,
Cytokinin
activity
in Verticilliom-infected
cotton
plants
191
the response ofF. hygrometrica was not improved by treating the cytokinin preparations from leaves and stems with cation and anion exchange resins or activated charcoal, extractions with chloroform, 1-butanol or ethyl acetate from basic solutions, barium acetate precipitation, treatment with PVP (Polyclar) or gel filtration (Bio-Gel P-2, 50-100 mesh). Preparations from leaves and stems were not inhibitory to the soybean callus tissue since the addition of different amounts of these extracts to soybean bioassay medium containing different concentrations of kinetin did not reduce the activity of this cytokinin. Cytokinins
in extracts of cotton
Three general areas with cytokinin activity were found on the thin-layer chromatograms of tracheal fluids and extracts from leaves and stems of both healthy and diseased plants. These areas are designated as I, II and III on Figs 1 and 2. For convenience the substances with cytokinin activity in these areas will be referred to as factors I, II and III, respectively. Based on their similar behavior in three different solvent systems used in TLC, the three factors isolated from tracheal fluid are probably identical to the three factors isolated from the leaves and stems,
Rf FIG. 1. Soybean callus bioassay of cytokinins in tracheal fluid from healthy and from Verticillium-infected Acala SJ-1 cotton plants. Cytokinins were separated by TLC using three solvents: (a) I-butanol-acetic acid-water; (b) I-butanol-ammonium hydroxide; (c) watersaturated P-butanol. O-O, Healthy; l - - - l , diseased; the horizontal broken lines at the lower part of the figures represent the average weight of soybean callus tissue in control flasks. The capital letters indicate the location on the chromatograms of adenosine-5’monophosphate (A), benzyladenine (B), dihydrozeatin (D), kinetin (K), 6-(y,y-dimethylallylamino)purine (P), zeatin (Z), zeatin riboside (R) and 6-methylaminopurine (S). Cytokinin active areas are marked by Roman numerals.
In another series of experiments the total cytokinin activity associated with each of the three factors was determined. There was a significant reduction in the activity of factor I in the tracheal fluid and in the leaf and stem extracts of cotton plants with Verticillium wilt when compared with healthy plants (Table 1). However, the differences in the total activity of either factor II or III in tracheal fluids and in extracts of leaves and stems of healthy and diseased plants were not significant.
192
I. Misaghi
et al.
The total amount of cytokinin activity recovered from the leaves and stems of cotton, calculated from the data in Table 1, was 20.9 rig/g (dry weight) of healthy tissue and 15.2 rig/g (dry weight) of Verticillium-infected tissue. The corresponding figures for tracheal fluids of healthy and Verticillium-infected cotton plants, calculated from the data in Table 1, were 21.6 ng and 17.1 “g/ml of sap, respectively. In diseased plants there was greater reduction in cytokinin activity in the leaves and stems than in the tracheal fluids. The percentage of recovery of kinetin, benzyladenine and zeatin added to leaf extract, as measured by gas-liquid chromatography, was found to be from 83 to 90 [2.5].
FIG. 2. Soybean callus bioassay of cytokinins in leaves and stems from healthy and from Verticillium-infected Acala SJ-1 cotton plants. Cytokinins were isolated by the water infiltrationcentrifirgation method and separated by TLC using three solvents: (a) I-butanol-acetic acid-water; (b) 1-butanol-ammonium hydroxide; (c) water-saturated P-butanol. o-0, Healthy; l - - - 0, diseased; the horizontal broken lines at the lower part of the figures represent the average weights of soybean callus tissue in control flasks. Cytokinin-active areas are marked by Roman numerals. TABLE Amounts
1
of three factors with cytokinin activity in the tracheal&id stems of healthy and Verticillium-infected Acala Kinetin equivalent (rig/ml of tracheal fluid)a
Solvent systemsb
Cytokinin factors
A
5.9kO.2 3*5+0.2 5.8kO.l
3.1 1.7 0.3
I
9.550.2 5.9kO.l 7.3 + 0.3
6.9 + 0.3 38+0.2 6.9kO.3
2.6 2.1 0.4
9*4+ 0.4 2.9+0.1 6.2 & 0.3
2.6 0.6 0.0
I II III
Difference
9.02 0.3 5.2 + 0.3 6.1 +O.l
II III c
Verticilliuminfected plants
Kinetin weight)
I II III
B
Healthy plants
12.OkO.4 3.5kO.2 6.2iO.2
and in the extract ST-I cotton plants
of leaves and
equivalent of leaves
Healthy plants
Verticilliuminfected plants
10.3kO.4 3.7kO.2 68kO.3
6+0+0*3 2.8+0.1 6.920.3
9.750.4 3.3kO.l 6.2 + 0.2 11.2+0.4 3.9kO.l 7.7kO.l
a Average of three replications using the soybean callus tissue bioassay. b A: I-butanol-acetic acid-water, 12 : 3 : 5; B: I-butanol-ammonium 4 : 1; C: water-saturated P-butanol. c More activity in infected tissue.
(rig/g (dry and stems)a
Difference 4.3 0.9 -O.lC
6.3 + 0.3 Z-8+0*1 4.9kO.l
3.4 0.5 1.3
6.6 + 0.3 3.2kO.l 6.1 +O.l
4.6 0.7 1.6
hydroxide
(cont.),
Cytokinin
activity
in Verticilliom-infected
cotton
193
plants
The extracts from leaves and stems prepared by the water infiltrationcentrifugation method [Fig. 2c] yielded slightly more cytokinins and significantly less pigment than the extracts obtained by the ethanol-homogenization method (Fig. 3). However, the amount of cytokinins in healthy and diseased tissues were proportionally the same in both extraction procedures.
0
05
I-O
FIG. 3. Soybean callus bioassay of cytokinins in leaves and stems from healthy and from Cytokinins were isolated by the ethanolVe&cillium-infected Acala SJ-1 cotton plants. homogenization method and separated by TLC using water-saturated 2-butanol as solvent. O-O, Healthy; l - - - l , diseased; the horizontal broken line at the lower part of the figure represents the average weights of soybean callus tissue in control flasks. Cytokinin active areas are marked by Roman numerals.
Chromatography of naturally occurring cytokinins from cotton with several known cytokinins and adenosine-5’-monophosphate (AMP) is shown in Fig. 1. The R, value of factor I in water-saturated P-butanol using TLC is similar to a factor in corn kernel which was a mononucleotide of zeatin [ls]. The chromatographic behavior of this factor in water-saturated 2-butanol also is similar to a major cotton fruit cytokinin prepared by Sandstedt [ZZ, 231. Experiments so far with alkaline phosphatase to test the possible nucleotide nature of factor I have not been conclusive, mainly because of the limited amounts of factor I available. The R, values of factor II in the TLC solvent systems were different from those of other known cytokinins tested but were somewhat similar to those of factor II isolated from seeds of pumpkin [7]. The R, values of factor III in all TLC solvent systems tested were closer to dihydrozeatin and zeatin than to other naturally occurring cytokinins tested (Fig. 1). Relation of changes in cytokinin factors to time of symptom development in Verticillium cotton
wilt in
Experiments were made to find out how soon after infection the observed changes in activity of cytokinins were detectable. The cytokinin contents of leaves and stems of healthy cotton plants were compared with those of infected plants at 2, 6, 11 and 16 days after inoculation with V. albo-atrum. The activities of the three factors were measured after their preparation by the water infiltration-centrifugation method and their purification by TLC with 1-butanol-ammonium hydroxide. The results showed that a reduction in the activity of the cytokinin factors in infected plants was detectable only after advanced development of the symptoms (Table 2).
194
1. Misaghi TABLE Amounts of three factors cotton plants on dyerent
with cytokinin days following Kinetin
Days after inoculation 2 6 lib 16
activity isolated from the leaves and sterns inoculation with Verticillium albo-atrum
equivalent
Factor I Healthy Inoculated 8.6 9.3 9.8 9-9
(rig/g
a Average of three replications using the soybean b Leaf symptoms (chlorotic lesions) were evident.
lj”ect
(dry
weight)
Factor
II Inoculated
Healthy
8.8 9.5 6.1 6.9
et al.
2
3.1 3.3 2.7 3.2 callus
of leaves
2.9 3.5 2.1 2.6 tissue
of Acala S3-I (SS4)
and stemsja Factor Healthy 5.8 6.0 6.0 5.7
III Inoculated 5.8 5,8 5.7 4.4
bioassay.
of water stress on activity of cytokinins
Figure 4 shows that there was some decrease in the cytokinin activity of waterstressed healthy cotton. However, the pattern of change in the activity of the three factors in water-stressed cotton plants was different from that found in cotton plants with Verticillium wilt. Verticillium infection caused a reduction in the activity of factor I in contrast to water stress (Figs 2, 4). On the other hand, water stress, but not Verticillium infection, caused a reduction in the activity of factor III. Estimated water potentials were between - 11 and - 13 bars in the well-watered and turgid plants, and were between - 19 and - 22 bars in the plants from which water was withheld. Wilting of cotton leaves was consistently observed when the water potential was lower than - 15 bars.
FIO. 4. Soybean callus bioassay of cytokinins in leaves and stems from normal and from water-stressed Acala SJ-1 cotton plants. Cytokinins were isolated by the ethanol-homogenization method and separated by TLC, using water-saturated P-butanol as solvent. O-O, Normal; A - - - A, water stressed; the horizontal broken line at the lower part of the figure represents the average weights of soybean callus tissue in control flasks. Cytokinin-active areas are marked by Roman numerals. DISCUSSION
A comparison of methods for extraction of cytokinin from leaves and stems and their partial purification by TLC indicated that highest total yields were obtained with the water infiltration-centrifugation method and with water-saturated 2-butanol as the irrigating solvent in TLC. There is a reduction in the level of cytokinins as a result of Verticillium infection. The reason for the reduced cytokinin activity in infected plants is not clearly known.
Cytokinin
activity
in Verficillium-infected
cotton
plants
195
However, on the basis of this study the fungus could play a partial role. The reduction of factor I in diseased plants compared with healthy plants could be due to the activity of a fungal that could convert this material to factor II or factor III such as the one detected in the fungal culture (I. Misaghi et al., unpublished results). Water stress, which may play a major role in symptom development in Verticillium wilt of cotton, could also cause a reduction in cytokinin activity. A significant reduction in cytokinin activity in tracheal fluid has been reported when plant roots have been subjected to water stress [II] and to high salinity [IO]. Water stress in the plant shoot also reduced cytokinin activity in an extract from xylem and leaves of Nicotiana rustica [12]. In the present study, the leaves and stems of the cotton plants exposed to water stress yielded less cytokinins than those from normal plants. However, this reduction in cytokinin activity was not as severe as that caused by the fungal infection. Moreover, the relative changes in activity of the three factors in water-stressed and in fungus-infected plants were not the same. It should be noted that the type and degree of water stress caused by infection may not be comparable to that induced by withholding water, and the physiological changes caused by these two types of wilting may be different. Thus, the results presented here do not preclude entirely the possibility that water stress reduces the cytokinin activity of plants infected with Verticillium. Although the decrease in activities of cytokinins and the apparent early senescence of the leaves of Verticillium-infected cotton plants might suggest a cause-and-effect relationship, the existence of such a relationship is not supported by this study since the reduction in the activity of cytokinins in plants infected with Verticillium occurred after the onset of wilt symptoms. The defoliation of cotton plants infected by defoliating strains of V. albo-atrum, such as T-9, is apparently related to the differential alterations in abscisic acid and ethylene levels [271. The usual symptoms of wilt caused by non-defoliating strains of V. albo-atrum, such as SS4, are apparently not directly related to changes in cytokinin activity. However, the changes in cytokinin activity in Verticillium-infected cotton plants may indirectly affect the activity of the other growth regulators which could contribute to development of wilt symptoms. The authors gratefully acknowledge the support of this work by the Cooperative State Research Service, United States Department of Agriculture, Grant No. 016-15-06. We also thank Dr J. M. Duniway for assistance in determining moisture stress in leaf tissues, Dr C. Miller for cultures of soybean callus tissue and Mr Jeff Hall for help with photographs. REFERENCES 1. DEKHUIJZEN, H. M. & OVEREEM, J. Cl. (1971). The role of cytokinins in clubroot formation. Physiological Plant PatholoQ 1, 151-161. 2. DUNIWAY, J. M. (1971). Comparison of pressure chamber and thermocouple psychrometer determinations of leaf water status in tomato. Plant Physiology, Lancaster 48, 106-107. 3. ENGELBRECHT, L. (1968). Cytokinin in den “grtinen Inseln” des Herbstlaubes. Flora 159,208-2 14. 4. FAWCETT, C. H. & WRIGHT, S. T. C. (1968). Cytokinin activity in a homologous series of whydroxypolymethyleneamino purines. Phytachemistry 7, 1719-1725. 5. FLETCHER, R. A. (1969). Retardation of senescence by benzyladenine in intact bean plants. Planta, Berlin 89, l-8.
196
I. Misaghi
et al.
6. FLETCHER, R. A. & OSBORNE, D. J. (1966). Gibberellin, as a regulator of protein and ribonucleic acid synthesis during senescence in leaf cells of Taraxacum oj%inale. Canadian Journal of Botany 44, 739-745. 7. GUPTA, G. R. P. & MAHESHWARI, S. C. (1970). Cytokinins in seeds of pumpkin. Plant PhysioloQ, Lancaster 45, 14-18. 8. HEINZ, H. & BOPP, M. (1968). A cytokinin test with high specificity. Planta, Berlin 83, 115-I 18. 9. HELGESON, J. P. & LEONARD, N. J. (1966). Cytokinins: identification of compounds isolated from Co7ynebacterium fasciaas. Proceedings of the National Academy of Sciences of the United States of America 56,60-63. 10. ITAI, C., RICHMOND, A., & VAADIA, Y. (1968). The role of root cytokinins during water and salinity stress. Israel 3ournal of Botany 17, 187-193. 11, ITAI, Cl. & VAADIA, Y. (1965). Kinetin-like activity in root exudate of water-stressed sunflower plants. Physiologia plantarum 18, 941-944. 12. ITAI, C. & VAADIA, Y. (1971). Cytokinin activity in water-stressed shoots. Plant Physiology, Luncaster 47, 87-90. 13. KIRALY, Z., EL HAMMADY, M. & Poz&, B. I. (1967). Increased cytokinin activity of rustinfected bean and broad bean leaves. Phytopatholopy 57, 93-94. 14. KL.&BT, D., Tnras, G. & SKOOG, F. (1966). Isolation of cytokinins from Copebacterium fascians. Proceedings of &he JVational Academy of Sciences of the United States of America 56, 52-59. 15. LETHAM, D. S. (1966). Regulators of cell division in plant tissues. II. A cytokinin in plant extracts: isolation and interaction with other growth regulators. Phytochemistry 5, 269-286. 16. MILLER, C. 0. (1965). Evidence for the natural occurrence of zeatin and derivatives: compounds from maize which promote cell division. Proceedings of the flational Academy of Sciences of the United States of America 54, 1052-1058. 17. MISAGHI, I. & DEVAY, J. E. (1970). Isolation and assay of cytokinins in healthy and Vertic-illiumwilted cotton plants. In Proceedings of the 1970 Beltwide Cotton Production Research Conferences, Houston, Texas, p. 6. 30th Cotton Disease Council, Natn. Cotton Council, Memphis, Tenn. 18. MISAGHI, I., DEVAY, J. E. & RAVENSCROFT, A. (1969). Cytokinin activity in cotton plants infected by Verticillium albo-atrum. Phytopathology 59, 1041 (Abstr.). of cytokinins. Journal 19. MOST, B. H., WILLIAMS, J. C. & PARKER, K. J. (1968). G as chromatography of Chromatography 38, 136-l 38. 20. MOTHES, K. & ENGELBRECHT, L. (1961). Kinetin-induced directed transport of substances in excised leaves in the dark. Phytochemistry 1, 58-62. 21, SAMUELS, R. M. (1961). Bacterial-induced fasciation in Pisum satiuum var. Alaska. Ph.D. i%sis Indiana University, Bloomington, Indiana. 22. SANDSTEDT, R. (1970). Partial purification of a cytokinin from immature cotton fruit. In Proceedings of the I970 Beltwide Cotton Production Research Conferences, Houston, Texas, pp. 29-31. 30th Cotton Disease Council, Natn. Cotton Council, Memphis, Tenn. 23. SANDSTEDT, R, (1971). Cytokinin activity during development of cotton fruit. Physiologia Plantarum 24,408-410. 24. SCHNATHORST, W. C. (1970). Obtaining xylem fluid from Gossypium hirsutum and its uses in studies on vascular pathogens. Phytopatholopy 60, 175-l 76. 25. SHINDY, W. W., AECHER, T., MISAGHI, I. & DEVAY, J. E. (1970). Gas-liquid chromatography of cytokinins in plant extract. Plant Physiology, Lancaster 46 (suppl.), 8. 26. THIMANN, K. V. & SACHS, T. (1966). The role of cytokinins in the “fasciation” disease caused by Corynebacterium fascians. American Journal of Botany 53, 73 I-739. 27. WIESE, M. V. & DEVAY, J, E. (1970). Growth regulator changes in cotton associated with dePlant Physiology, Lancaster 45, 304-309. foliating caused by Verticillium albo-atrum.