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The relationship of gibberellin content to cucumber mosaic virus infection of cucumber
Physiological
Plant
Pathology
(1974)
4, 73-79
The relationship of gibberellin content to cucumber mosaic virus infection of cucumber K. W.
BAILISS
Department of Biological Wye College (University (Accepted
for publication
Sciences,
of London), July
Wye, Ashford,
Kent
TN25
5AH,
U.K.
1973)
Early infection of cucumber by cucumber mosaic virus caused reduction in the net assimilation rate, pronounced stunting of the roots and reduced stem and leaf growth. Such stunting was associated with a reduction in the concentration of endogenous gibberellins. Evidence from paper and thin-layer chromatography suggested that the gibberellins involved were gibberellin A, and/or gibberellm As. Gibberellins could not be detected in root systems. No qualitative differences were found in the gibberellms of healthy and infected shoots. The reduction in the gibberellin content and the associated symptoms are discussed.
INTRODUCTION
Reduction in growth and abnormal growth forms are probably the commonest disease symptoms induced by viruses in plants. Many workers have considered the possibility that virus-induced alteration in hormone action may be involved in disease development [9, 131 and numerous investigations have been made on the influence of infection on hormone concentration [7, 181. So far, however, little attention has been paid to the role of gibberellins in virus-induced plant symptoms. Virus infection often causes plant stunting which may reflect an overall hormone imbalance involving gibberellins. Budagyan et al. [a found no difference in the amounts of gibberellins of healthy and tobacco mosaic virus-infected tobacco where stunting is not a main feature of the disease. Similar results were obtained with tomato infected with aspermy virus where stunting was a pronounced feature of the infected plant [2]. Russell & Kimmins [15], however, reported a reduction in endogenous gibberellins (possibly GA,) in stunted barley plants infected by barley yellow dwarf virus. It has also been concluded that a major factor in stunting was a reduction in mitotic activity [2, 151 which can be affected by gibberellins [16, 171. The present study was made to determine the gibberellin content of cucumber plants at various early times after infection with cucumber mosaic virus (CMV).
MATERIALS Virus
and
AND
METHODS
host
Cucumber leaves infected with CMV were ground (1 g leaf per 10 ml buffer) with a pestle and mortar in 0.07 M-potassium phosphate buffer (pH 7.7) and the filtered homogenate used to inoculate the “Celite” dusted cotyledons of experimental cucumber plants 7 to 10 days after sowing. Control plants were inoculated with sap
K. W. Bailiss
74
from healthy leaves. The cultivar used was Improved Telegraph (Dobie & Son Ltd, Chester). Plants were grown in a growth room illuminated by 80-W warm white fluorescent tubes providing a light intensity of 700 to 900 Im/ft2 and a 16-h photoperiod. Day and night temperatures were 24 and 22 “C respectively. - For the growth experiment plants were arranged in seven blocks each containing six healthy and six infected plants distributed at random. Each sample consisted of one healthy and one infected plant from each block. E&-action
of cucumber gibberellins
(i) Shoots and roots. Many methods have been described for the extraction of gibberellins from cucumber [I, 10, II]. It was felt essential to develop a reproducible technique for the plant material used in this study. Preliminary experiments using healthy shoots led to the adoption of the following scheme for healthy and infected material. Shoots cut at soil level were frozen at -20 “C for 6 h and extracted as described earlier [4]. The acidic ethyl acetate fraction was strip loaded onto thin-layer (t.1.c.) plates coated with ethanol washed silica gel G (without binder) 250 or 500 pm thick. Reference spots of authentic gibberellins were chromatographed at the same time as extracts. Chromatograms were developed with butan-1-ol-NH,OH (3 : 1 v/v) and the region between R, 0.0 and 0.4 eluted with 5% sodium bicarbonate. This active region corresponded with the GA, and GA, reference spots. Inhibitory materials, which caused root browning and hypocotyl twisting during assay, migrated ahead of the active zone. The bicarbonate eluent was acidified to pH 2.5, partitioned against ethyl acetate and after the removal of water, the ethyl acetate fraction was reduced in volume and chromatographed on solvent washed Whatman’s 3MM paper in a descending manner using propan-2-ol-NH,OH-H,O (10 : 1 : 1 v/v). The active zone again corresponded with the GA, and GA, reference spots. It is noteworthy that when the initial acidic ethyl acetate fraction was chromatographed on paper with propan-2-ol-NH,OH-H,O (10 : 1 : 1 v/v), by t.1.c. using butan-I-ol-propan-2-ol-NHdOH-H20 (2 : 6 : 1 : 2 v/v) or H,O, a single active region was obtained coinciding with the GA, and GA, reference spots but contaminated with inhibitory substances. In addition, examination of the methyl esters and silyl ethers of the methyl esters [S] of extracts by gas-liquid chromatography failed to reveal recognizable gibberellin peaks. Roots were sampled 7 and 14 days after inoculation from plants grown in compost or half-strength Hoagland’s nutrient solution. Samples of 60 to 140 root systems were used on each occasion and extracted as described for shoots. (ii) Dafusates. Two batches of40 healthy and 40 infected shoots 7 days after inoculation were cut 8 mm below the cotyledons. One group was immediately solvent extracted as described for shoots. The projecting hypocotyls of the second group were inserted into cylinders of 1 oh Ionagar No. 2 and diffusates collected for 24 h under continuous light. Agar cylinders were extracted by the method of Jones & Phillips [12] and chromatographed as for shoots. Assay
Extracts were examined for gibberellin-like activity by the lettuce hypocotyl assay [S]. Log transformation of assay responses to serially diluted extracts and GA, resulted in
Gibberellin
content
in cucumber
mosaic
virus
infection
75
satisfactory linearity of response and homogeneity of variance. Transformed assay data from each chromatogram were subjected to analysis of variance and chromatogram strips which gave rise to significant activity (P = O-05) located. Ross & Bradbeer [14] indicated that, as assayed by the pea epicotyl assay after t.l.c., only about 10% of GA, was re-extracted with water. Because of this the recovery of gibberellins known to occur in cucumber [IO, II] was estimated by assay after t.1.c. with butan-1-ol-NH,OH (3 : 1 v/v). About 80% of GA, and GA,, and 100% of GA, and GA, was re-extracted during the course of the lettuce hypocotyl assay. Total recovery of all four gibberellins was obtained after paper chromatography with propan-P-ol-NH,OH-H,O (10 : 1 : 1 v/v). RESULTS Effect of CMV
infection on the growth of cucumber
Following inoculation with CMV chlorotic lesions appeared on the cotyledons after 3 to 4 days followed by systemic chlorotic flecking of the first leaf after 5 to 6 days and a mosaic after 11 to 12 days. The effect on growth was measured every 5 days using several growth parameters for 25 days after inoculation. Some of these results are given in Table 1. Significant 1
TABLE
Effect of CMV-infection
of cucumber Time
Growth
parametersb
Stem length Stem fresh weight Stem dry weight Leaf area Leaf fresh weight Leaf dry weight Hypocotyl length Hypocotyl fresh weight Hypocotyl dry weight Root fresh weight Root dry weight
on selected growth in days
after
0 H
5 Ha
Da
H
-e -
-
3.60 0.11 4.00 0.07 10.00
27.20 0.10 13.00 3.78 0.15 4.00 0.43 36.00
22.80* 0.08* 11.00 3.81 0.15 4.00 0.21* 14.00*
1.04 0.19 11.00 172.90 2.47 270.00 3.93 0.45 20.00 1.67 140.00
parameters
inoculation 15 D
25 H
0+2* o-11* 4*00* 93.31* 1.26* 130*00* 3.87 O-32* 1 o.oo* 0*72* 58.00*
Q H = healthy control, D = CMV-infected. b All areas in ems; lengths in cm; dry weights in mg and fresh weights c Not measurable. * Mean significantly different from healthy control (P = 0.0 1).
2.99 0.79 53.00 382.12 6.91 840.00 4.34 0.94 56.00 5.21 460.00
D 1,91* 0.55* 30.00* 253.32* 4*19* 410.00* 4.01 0.56* 30.00* 2*11* 170*00*
in g.
effects 5 days after inoculation were a pronounced root stunting as shown by decreases in fresh and dry weight. Leaf area and fresh weight were also reduced but leaf dry weight was not reduced. Extreme stunting of the root system persisted, decreases in leaf dry matter accumulation and expansion became more pronounced and eventually infected stems became stunted. Although stem elongation was reduced after infection, hypocotyl elongation was not. The hypocotyls of healthy plants became progressively thicker than those of infected plants as shown by the reduced fresh and dry weight of
76
K. W. Bailiss
the latter (Table 1). those reported earlier A further effect of in the net assimilation
The effects of infection on cotyledon growth were similar to [3]. infection on host growth was demonstrated by the differences rates (NAR) of healthy and infected plants (Fig. 1). Both
‘i 0
5
IO Days after
1. Changes in net assimilation indicate the 95% confidence limits
FIG.
Bars
15
20
25
inoculation
rate of healthy of the means.
( l ) and
CMV-infected
(0)
cucumber.
rates showed an upward trend during the first 10 days after inoculation followed by a steady decline. The NAR was always less in infected plants and the reduction greatest during the early stages of infection. Studies were made on endogenous gibberellins during this period. G’ibberellin
content of healthy and infected plants
(i) Shoots. Healthy and infected shoots were sampled at 0 (healthy only), 7, 14 and An equal number of healthy and infected shoots were 21 days after inoculation. sampled on each occasion. This varied from 87 to 120 shoots/sample. Graphs of assay data (Fig. 2) showed that extracts of healthy and infected shoots gave rise to a single region of activity corresponding to the GA, and GA, reference spots. The magnitude of the active zone declined as the plants aged indicating a progressive reduction in endogenous gibberellin content. Lower amounts of gibberellins were consistently recovered from infected compared with healthy shoots. No substances inhibiting the growth of the assay plant were found. A second, similar, experiment showed similar trends. As a difference in gibberellin content was apparent 7 days after inoculation further experiments examined gibberellin levels during the first week after infection. A typical result (Table 2) showed that the gibberellin content of infected shoots was less than healthy 4 and 7 days after inoculation.
Gibberellin
content
in cucumber
mosaic
virus
infection
77
21
r
0
o-2
I
I
I
I
o-4
0.6
0.8
I.0
GA,
I
0.2
I
I
0.4
0.6
RF
I
I
0.8
I.0
RF
FIG. 2. Response of lettuce hypocotyl assays to cucumber shoot extracts after final paper chromatography with propan-2-ol-NH40H-H,O (10 : 1 : 1 v/v). Healthy ( l ) and CMVinfected (0) extracts 0,7, 14 and 21 days after inoculation. Fiducial limits (5%) indicated either side of the control line (C). Gibberellin reference spots shown by horizontal bars.
TABLE
Gibberellin
acti&
of extracts Time 4
0.3-0.4 04-0.5 O-5-0.6
ofyoung
in days
Healthy
Infected
120” 207* 253*
118 145* 198*
after
cucumber
shoots
inoculation 7 Healthy Infected 134* 197” 220’
a Chromatography system; paper developed with propan-2-o1-NH,OH-HsO b Assay data; hypocotyl length expressed as 0h control. * Values significantly different from assay control (P = 0.05).
The content control) Solvent responses
112 138* 178* (10 : 1 : 1
V/V).
collection and extraction of diffusates confirmed the reduced gibberellin of infected compared with healthy shoots. Assay responses (as per cent to healthy and infected diffusate extracts were respectively 240 and 173%. extracted shoots of healthy and infected plants induced respectively assay of 201 and 135%. Gibberellin-like activity of the four extracts was confined
78
K.W. Bailiss
to R, 0.5 to O-6 after paper chromatography (propan-2-ol-NH,OH-HsO, 10 : 1 : 1 v/v) and corresponded to the GA, and GA, marker spots. (ii) Roots. As infection caused pronounced stunting of the roots attempts were made to quantify the endogenous gibberellin content of healthy and infected root systems. Assays of root extracts consistently failed to detect significant gibberellin-like activity. DISCUSSION
Gibberellin content was consistently lower in infected compared with healthy shoots and associated with CMV-induced stunting. A consistent reduction was apparent 4 and 7 days after inoculation which corresponded with the early manifestation of stunting and the greatest reduction in NAR. This work extends the association of virus-induced stunting and gibberellin depletion reported for barley infected with barley yellow dwarf virus [15]. In this study and that of Russell ‘& Kimmins [1.5] it was not possible to compare the gibberellin content of plants before the onset of stunting and so no causal relationship can be deduced. Nevertheless the decreases in absolute gibberellin levels in infected plants may be expected to have an adverse effect on growth and a considerable bearing on the stunting syndrome. Furthermore, the activity of gibberellins present in infected plants may be impaired by factors such as inefficient conversion(s) to an active gibberellin type(s) and increases in substances antagonistic to gibberellin synthesis and/or gibberellin regulated growth. In ‘contrast, stunting of tomatoes infected by aspermy virus was not associated with reduced ‘gibberellin content .[2]. It is unlikely that methodology contributed significantly to this contrasting result as the techniques used to extract and assay gibberellins were identical to those employed in the present work and that of Russell & Kimmins [15]. It is probable that gibberellin imbalance is primarily determined by the interaction between the particular host and virus under investigation. Gibberellin imbalance in virus-infected plants is not well documented. More information is required on the relationship between the anatomical manifestation of stunting and gibberellm content and the efficiency of gibberellin activity and interaction with other plant growth regulators before the full significance of gibberellin imbalance and symptomatology can be ascertained. Work on these aspects is in progress. The author wishes to thank Dr T. A. Hill and Dr W. E. Peat for their advice and Miss M. Manwaring for her assistance. Gifts of gibberellins from Drs D. Broadbent and J. MacMillan are gratefully acknowledged. REFERENCES 1. ATSOM, D., LANG, .A. & LIGHT, E. N. (1968). Contents and recovery of gibberellins in monoecious and gynoecious cucumber plants. Plant Physiolog, Lancaster 43,806--810. 2. BAILISS, K. W. (1968). Gibberellins and the early disease syndrome of aspermy virus in tomato (Lycopersiconescukntum Mill.). Annals of Botany 32, 543-55 1, 3. BAILISS, K. W. (1970). Infection of cucumber cotyledons by cucumber mosaic virus and the participation of chlorophyllase in th.e development of chlorotic lesions. Annals of Botany 34, 647-655. 4. BAILISS, K. W. & WILSON, IRENE M. (1967). Growth hormones and the creeping thistle rust. Annuls of Botany 31, 195-211.
Gibberellin
content
in cucumber
mosaic
virus
infection
79
5.. BUDAGYAN, E. G., LOZHNIKOVA, V. N., GOL’DIN, M. I. & CHAILAKHYAN, M. K. (1964). On the effect of gibberellm-like substances on tobacco mosaic virus. Review of Applied Mycology 43, 333. 6. CAVELL, B. D., MACMILLAN, J., PRYCE, R. J. & SHEPPARD, A. C. (1967). Plant hormones-V. Thin layer and gas-liquid chromatography of the gibberellins; direct identification of the gibberellins in a crude plant extract by gas-liquid chromatography. Phytochemistry 6, 867-874. 7. DIENER, T. 0. ( 1963). Physiology of virus-infected plants. Annual Review of PhytopathoZology 1,197-Z 18. 8. FRANKLAND, B. & WAREING, P. F. (1960). Effect of gibberellic acid on hypocotyl growth of lettuce seedlings. Nature, London 185, 255-256. 9. GOODMAN, R. N., KIRALY, Z. & ZAITLIN, M. (1967). The Biochemistry and Physiology of Infectious Plant Disease. Van Nostrand, Princeton, New Jerse?. 10. HAYASHI, F., BOERNER, D., PETERSON, C. E. & SELL, H. M. (1971). The relative content of gibberellin in seedlings of gynoecious and monoecious cucumber (Cucumis sativus). Phytochemistry 10, 57-62. 11. HENIPHILL, D. D., BAKER, L. R. & SELL, H. M. (1972). Isolation and identification of the gibberellins of Cucz&s s&us &nd Cucumti melo. Planta (Berlin) 103, 241-248. s 12. JONES, R. L. & PHILLIPS, I. D. J. (1964). Agar-diffusion technique for estimating gibberellin production by plant organs. Nature, London 204, 497-499. 1% MATTHEWS, R. E. F. (1970). Plant Virology. Academic Press, New York. 14. Ross, J. D. & B~DBEER, J. W. (1971). Studies in seed dormancy. V. The content of endogenous gibberellins in seeds of Corylus’avellana L. Planta (Be&n) 100, 288-302. 15. RUSSELL, S. L. & KIMMINS, W. C. (1971). Growth regulators and the effect of barley yellow dwarf virus on barley {Hordeum uulgare L.). Annals of Bota?y 35, 1037-1043. 1.6. SACHS, R. M. & LANG, A. (1961). Shoot histogenesis and the subapical meristem: the action of gibberellic acid, AMO-1618 and maleic hydrazidk. In Plant Growth Regulation. Iowa State ss University Press, Ames; Iowa. 17. SACHS, R. M., LANG, A., BRETZ, C. F. & ROACH, J. (1960). Shoot histogenesis: subapical meristematic activity in a caulescent plant and the action of gibberellic acid and AMP-1618. American’ . Journal of Botany 47, 260-266. 18. SEQUEIRA, L. (1963). ‘Growth regulators in plant disease. Annual Review of’Phytobatholopy 1, 5-30.
Plant
Pathology
(1974)
4, 73-79
The relationship of gibberellin content to cucumber mosaic virus infection of cucumber K. W.
BAILISS
Department of Biological Wye College (University (Accepted
for publication
Sciences,
of London), July
Wye, Ashford,
Kent
TN25
5AH,
U.K.
1973)
Early infection of cucumber by cucumber mosaic virus caused reduction in the net assimilation rate, pronounced stunting of the roots and reduced stem and leaf growth. Such stunting was associated with a reduction in the concentration of endogenous gibberellins. Evidence from paper and thin-layer chromatography suggested that the gibberellins involved were gibberellin A, and/or gibberellm As. Gibberellins could not be detected in root systems. No qualitative differences were found in the gibberellms of healthy and infected shoots. The reduction in the gibberellin content and the associated symptoms are discussed.
INTRODUCTION
Reduction in growth and abnormal growth forms are probably the commonest disease symptoms induced by viruses in plants. Many workers have considered the possibility that virus-induced alteration in hormone action may be involved in disease development [9, 131 and numerous investigations have been made on the influence of infection on hormone concentration [7, 181. So far, however, little attention has been paid to the role of gibberellins in virus-induced plant symptoms. Virus infection often causes plant stunting which may reflect an overall hormone imbalance involving gibberellins. Budagyan et al. [a found no difference in the amounts of gibberellins of healthy and tobacco mosaic virus-infected tobacco where stunting is not a main feature of the disease. Similar results were obtained with tomato infected with aspermy virus where stunting was a pronounced feature of the infected plant [2]. Russell & Kimmins [15], however, reported a reduction in endogenous gibberellins (possibly GA,) in stunted barley plants infected by barley yellow dwarf virus. It has also been concluded that a major factor in stunting was a reduction in mitotic activity [2, 151 which can be affected by gibberellins [16, 171. The present study was made to determine the gibberellin content of cucumber plants at various early times after infection with cucumber mosaic virus (CMV).
MATERIALS Virus
and
AND
METHODS
host
Cucumber leaves infected with CMV were ground (1 g leaf per 10 ml buffer) with a pestle and mortar in 0.07 M-potassium phosphate buffer (pH 7.7) and the filtered homogenate used to inoculate the “Celite” dusted cotyledons of experimental cucumber plants 7 to 10 days after sowing. Control plants were inoculated with sap
K. W. Bailiss
74
from healthy leaves. The cultivar used was Improved Telegraph (Dobie & Son Ltd, Chester). Plants were grown in a growth room illuminated by 80-W warm white fluorescent tubes providing a light intensity of 700 to 900 Im/ft2 and a 16-h photoperiod. Day and night temperatures were 24 and 22 “C respectively. - For the growth experiment plants were arranged in seven blocks each containing six healthy and six infected plants distributed at random. Each sample consisted of one healthy and one infected plant from each block. E&-action
of cucumber gibberellins
(i) Shoots and roots. Many methods have been described for the extraction of gibberellins from cucumber [I, 10, II]. It was felt essential to develop a reproducible technique for the plant material used in this study. Preliminary experiments using healthy shoots led to the adoption of the following scheme for healthy and infected material. Shoots cut at soil level were frozen at -20 “C for 6 h and extracted as described earlier [4]. The acidic ethyl acetate fraction was strip loaded onto thin-layer (t.1.c.) plates coated with ethanol washed silica gel G (without binder) 250 or 500 pm thick. Reference spots of authentic gibberellins were chromatographed at the same time as extracts. Chromatograms were developed with butan-1-ol-NH,OH (3 : 1 v/v) and the region between R, 0.0 and 0.4 eluted with 5% sodium bicarbonate. This active region corresponded with the GA, and GA, reference spots. Inhibitory materials, which caused root browning and hypocotyl twisting during assay, migrated ahead of the active zone. The bicarbonate eluent was acidified to pH 2.5, partitioned against ethyl acetate and after the removal of water, the ethyl acetate fraction was reduced in volume and chromatographed on solvent washed Whatman’s 3MM paper in a descending manner using propan-2-ol-NH,OH-H,O (10 : 1 : 1 v/v). The active zone again corresponded with the GA, and GA, reference spots. It is noteworthy that when the initial acidic ethyl acetate fraction was chromatographed on paper with propan-2-ol-NH,OH-H,O (10 : 1 : 1 v/v), by t.1.c. using butan-I-ol-propan-2-ol-NHdOH-H20 (2 : 6 : 1 : 2 v/v) or H,O, a single active region was obtained coinciding with the GA, and GA, reference spots but contaminated with inhibitory substances. In addition, examination of the methyl esters and silyl ethers of the methyl esters [S] of extracts by gas-liquid chromatography failed to reveal recognizable gibberellin peaks. Roots were sampled 7 and 14 days after inoculation from plants grown in compost or half-strength Hoagland’s nutrient solution. Samples of 60 to 140 root systems were used on each occasion and extracted as described for shoots. (ii) Dafusates. Two batches of40 healthy and 40 infected shoots 7 days after inoculation were cut 8 mm below the cotyledons. One group was immediately solvent extracted as described for shoots. The projecting hypocotyls of the second group were inserted into cylinders of 1 oh Ionagar No. 2 and diffusates collected for 24 h under continuous light. Agar cylinders were extracted by the method of Jones & Phillips [12] and chromatographed as for shoots. Assay
Extracts were examined for gibberellin-like activity by the lettuce hypocotyl assay [S]. Log transformation of assay responses to serially diluted extracts and GA, resulted in
Gibberellin
content
in cucumber
mosaic
virus
infection
75
satisfactory linearity of response and homogeneity of variance. Transformed assay data from each chromatogram were subjected to analysis of variance and chromatogram strips which gave rise to significant activity (P = O-05) located. Ross & Bradbeer [14] indicated that, as assayed by the pea epicotyl assay after t.l.c., only about 10% of GA, was re-extracted with water. Because of this the recovery of gibberellins known to occur in cucumber [IO, II] was estimated by assay after t.1.c. with butan-1-ol-NH,OH (3 : 1 v/v). About 80% of GA, and GA,, and 100% of GA, and GA, was re-extracted during the course of the lettuce hypocotyl assay. Total recovery of all four gibberellins was obtained after paper chromatography with propan-P-ol-NH,OH-H,O (10 : 1 : 1 v/v). RESULTS Effect of CMV
infection on the growth of cucumber
Following inoculation with CMV chlorotic lesions appeared on the cotyledons after 3 to 4 days followed by systemic chlorotic flecking of the first leaf after 5 to 6 days and a mosaic after 11 to 12 days. The effect on growth was measured every 5 days using several growth parameters for 25 days after inoculation. Some of these results are given in Table 1. Significant 1
TABLE
Effect of CMV-infection
of cucumber Time
Growth
parametersb
Stem length Stem fresh weight Stem dry weight Leaf area Leaf fresh weight Leaf dry weight Hypocotyl length Hypocotyl fresh weight Hypocotyl dry weight Root fresh weight Root dry weight
on selected growth in days
after
0 H
5 Ha
Da
H
-e -
-
3.60 0.11 4.00 0.07 10.00
27.20 0.10 13.00 3.78 0.15 4.00 0.43 36.00
22.80* 0.08* 11.00 3.81 0.15 4.00 0.21* 14.00*
1.04 0.19 11.00 172.90 2.47 270.00 3.93 0.45 20.00 1.67 140.00
parameters
inoculation 15 D
25 H
0+2* o-11* 4*00* 93.31* 1.26* 130*00* 3.87 O-32* 1 o.oo* 0*72* 58.00*
Q H = healthy control, D = CMV-infected. b All areas in ems; lengths in cm; dry weights in mg and fresh weights c Not measurable. * Mean significantly different from healthy control (P = 0.0 1).
2.99 0.79 53.00 382.12 6.91 840.00 4.34 0.94 56.00 5.21 460.00
D 1,91* 0.55* 30.00* 253.32* 4*19* 410.00* 4.01 0.56* 30.00* 2*11* 170*00*
in g.
effects 5 days after inoculation were a pronounced root stunting as shown by decreases in fresh and dry weight. Leaf area and fresh weight were also reduced but leaf dry weight was not reduced. Extreme stunting of the root system persisted, decreases in leaf dry matter accumulation and expansion became more pronounced and eventually infected stems became stunted. Although stem elongation was reduced after infection, hypocotyl elongation was not. The hypocotyls of healthy plants became progressively thicker than those of infected plants as shown by the reduced fresh and dry weight of
76
K. W. Bailiss
the latter (Table 1). those reported earlier A further effect of in the net assimilation
The effects of infection on cotyledon growth were similar to [3]. infection on host growth was demonstrated by the differences rates (NAR) of healthy and infected plants (Fig. 1). Both
‘i 0
5
IO Days after
1. Changes in net assimilation indicate the 95% confidence limits
FIG.
Bars
15
20
25
inoculation
rate of healthy of the means.
( l ) and
CMV-infected
(0)
cucumber.
rates showed an upward trend during the first 10 days after inoculation followed by a steady decline. The NAR was always less in infected plants and the reduction greatest during the early stages of infection. Studies were made on endogenous gibberellins during this period. G’ibberellin
content of healthy and infected plants
(i) Shoots. Healthy and infected shoots were sampled at 0 (healthy only), 7, 14 and An equal number of healthy and infected shoots were 21 days after inoculation. sampled on each occasion. This varied from 87 to 120 shoots/sample. Graphs of assay data (Fig. 2) showed that extracts of healthy and infected shoots gave rise to a single region of activity corresponding to the GA, and GA, reference spots. The magnitude of the active zone declined as the plants aged indicating a progressive reduction in endogenous gibberellin content. Lower amounts of gibberellins were consistently recovered from infected compared with healthy shoots. No substances inhibiting the growth of the assay plant were found. A second, similar, experiment showed similar trends. As a difference in gibberellin content was apparent 7 days after inoculation further experiments examined gibberellin levels during the first week after infection. A typical result (Table 2) showed that the gibberellin content of infected shoots was less than healthy 4 and 7 days after inoculation.
Gibberellin
content
in cucumber
mosaic
virus
infection
77
21
r
0
o-2
I
I
I
I
o-4
0.6
0.8
I.0
GA,
I
0.2
I
I
0.4
0.6
RF
I
I
0.8
I.0
RF
FIG. 2. Response of lettuce hypocotyl assays to cucumber shoot extracts after final paper chromatography with propan-2-ol-NH40H-H,O (10 : 1 : 1 v/v). Healthy ( l ) and CMVinfected (0) extracts 0,7, 14 and 21 days after inoculation. Fiducial limits (5%) indicated either side of the control line (C). Gibberellin reference spots shown by horizontal bars.
TABLE
Gibberellin
acti&
of extracts Time 4
0.3-0.4 04-0.5 O-5-0.6
ofyoung
in days
Healthy
Infected
120” 207* 253*
118 145* 198*
after
cucumber
shoots
inoculation 7 Healthy Infected 134* 197” 220’
a Chromatography system; paper developed with propan-2-o1-NH,OH-HsO b Assay data; hypocotyl length expressed as 0h control. * Values significantly different from assay control (P = 0.05).
The content control) Solvent responses
112 138* 178* (10 : 1 : 1
V/V).
collection and extraction of diffusates confirmed the reduced gibberellin of infected compared with healthy shoots. Assay responses (as per cent to healthy and infected diffusate extracts were respectively 240 and 173%. extracted shoots of healthy and infected plants induced respectively assay of 201 and 135%. Gibberellin-like activity of the four extracts was confined
78
K.W. Bailiss
to R, 0.5 to O-6 after paper chromatography (propan-2-ol-NH,OH-HsO, 10 : 1 : 1 v/v) and corresponded to the GA, and GA, marker spots. (ii) Roots. As infection caused pronounced stunting of the roots attempts were made to quantify the endogenous gibberellin content of healthy and infected root systems. Assays of root extracts consistently failed to detect significant gibberellin-like activity. DISCUSSION
Gibberellin content was consistently lower in infected compared with healthy shoots and associated with CMV-induced stunting. A consistent reduction was apparent 4 and 7 days after inoculation which corresponded with the early manifestation of stunting and the greatest reduction in NAR. This work extends the association of virus-induced stunting and gibberellin depletion reported for barley infected with barley yellow dwarf virus [15]. In this study and that of Russell ‘& Kimmins [1.5] it was not possible to compare the gibberellin content of plants before the onset of stunting and so no causal relationship can be deduced. Nevertheless the decreases in absolute gibberellin levels in infected plants may be expected to have an adverse effect on growth and a considerable bearing on the stunting syndrome. Furthermore, the activity of gibberellins present in infected plants may be impaired by factors such as inefficient conversion(s) to an active gibberellin type(s) and increases in substances antagonistic to gibberellin synthesis and/or gibberellin regulated growth. In ‘contrast, stunting of tomatoes infected by aspermy virus was not associated with reduced ‘gibberellin content .[2]. It is unlikely that methodology contributed significantly to this contrasting result as the techniques used to extract and assay gibberellins were identical to those employed in the present work and that of Russell & Kimmins [15]. It is probable that gibberellin imbalance is primarily determined by the interaction between the particular host and virus under investigation. Gibberellin imbalance in virus-infected plants is not well documented. More information is required on the relationship between the anatomical manifestation of stunting and gibberellm content and the efficiency of gibberellin activity and interaction with other plant growth regulators before the full significance of gibberellin imbalance and symptomatology can be ascertained. Work on these aspects is in progress. The author wishes to thank Dr T. A. Hill and Dr W. E. Peat for their advice and Miss M. Manwaring for her assistance. Gifts of gibberellins from Drs D. Broadbent and J. MacMillan are gratefully acknowledged. REFERENCES 1. ATSOM, D., LANG, .A. & LIGHT, E. N. (1968). Contents and recovery of gibberellins in monoecious and gynoecious cucumber plants. Plant Physiolog, Lancaster 43,806--810. 2. BAILISS, K. W. (1968). Gibberellins and the early disease syndrome of aspermy virus in tomato (Lycopersiconescukntum Mill.). Annals of Botany 32, 543-55 1, 3. BAILISS, K. W. (1970). Infection of cucumber cotyledons by cucumber mosaic virus and the participation of chlorophyllase in th.e development of chlorotic lesions. Annals of Botany 34, 647-655. 4. BAILISS, K. W. & WILSON, IRENE M. (1967). Growth hormones and the creeping thistle rust. Annuls of Botany 31, 195-211.
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5.. BUDAGYAN, E. G., LOZHNIKOVA, V. N., GOL’DIN, M. I. & CHAILAKHYAN, M. K. (1964). On the effect of gibberellm-like substances on tobacco mosaic virus. Review of Applied Mycology 43, 333. 6. CAVELL, B. D., MACMILLAN, J., PRYCE, R. J. & SHEPPARD, A. C. (1967). Plant hormones-V. Thin layer and gas-liquid chromatography of the gibberellins; direct identification of the gibberellins in a crude plant extract by gas-liquid chromatography. Phytochemistry 6, 867-874. 7. DIENER, T. 0. ( 1963). Physiology of virus-infected plants. Annual Review of PhytopathoZology 1,197-Z 18. 8. FRANKLAND, B. & WAREING, P. F. (1960). Effect of gibberellic acid on hypocotyl growth of lettuce seedlings. Nature, London 185, 255-256. 9. GOODMAN, R. N., KIRALY, Z. & ZAITLIN, M. (1967). The Biochemistry and Physiology of Infectious Plant Disease. Van Nostrand, Princeton, New Jerse?. 10. HAYASHI, F., BOERNER, D., PETERSON, C. E. & SELL, H. M. (1971). The relative content of gibberellin in seedlings of gynoecious and monoecious cucumber (Cucumis sativus). Phytochemistry 10, 57-62. 11. HENIPHILL, D. D., BAKER, L. R. & SELL, H. M. (1972). Isolation and identification of the gibberellins of Cucz&s s&us &nd Cucumti melo. Planta (Berlin) 103, 241-248. s 12. JONES, R. L. & PHILLIPS, I. D. J. (1964). Agar-diffusion technique for estimating gibberellin production by plant organs. Nature, London 204, 497-499. 1% MATTHEWS, R. E. F. (1970). Plant Virology. Academic Press, New York. 14. Ross, J. D. & B~DBEER, J. W. (1971). Studies in seed dormancy. V. The content of endogenous gibberellins in seeds of Corylus’avellana L. Planta (Be&n) 100, 288-302. 15. RUSSELL, S. L. & KIMMINS, W. C. (1971). Growth regulators and the effect of barley yellow dwarf virus on barley {Hordeum uulgare L.). Annals of Bota?y 35, 1037-1043. 1.6. SACHS, R. M. & LANG, A. (1961). Shoot histogenesis and the subapical meristem: the action of gibberellic acid, AMO-1618 and maleic hydrazidk. In Plant Growth Regulation. Iowa State ss University Press, Ames; Iowa. 17. SACHS, R. M., LANG, A., BRETZ, C. F. & ROACH, J. (1960). Shoot histogenesis: subapical meristematic activity in a caulescent plant and the action of gibberellic acid and AMP-1618. American’ . Journal of Botany 47, 260-266. 18. SEQUEIRA, L. (1963). ‘Growth regulators in plant disease. Annual Review of’Phytobatholopy 1, 5-30.