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Translocated wound stimuli affecting plant virus infections
VIROLOGY
20, 477-483
(1963)
Translocated
Wound
Stimuli
F. NIEXHAUS’ Department
oj Plant
Affecting
Accepted
Virus
Infections
C. E. YARWOOD”
AND
Pathology,
Plant
University
Apd
of
California,
Berkeley
1, 1963
Heating one primary Pinto bean leaf for 20 seconds at 70°C increased the number of lesions induced by the sweetpotato strain of tobacco mosaic virus in the opposite inoculated leaf to as many as 100 times the number in control plants. This translocated heat stimulus (THS) was greatest when the heat was applied within 1 hour after inoculation, but was clearly apparent when applied as much as 10 hours before or after inoculation. Manifestation of the THS was inhibited by high humidity, and this inhibition was complete when the heated and inoculated plants were held at. high humidity for 10 hours after inoculation. The THS was greatest in plants held at about 26”, was similar for plants held for 48 hours in darkness after inoculation and heating and for plants in natural light but was less for plants held in darkness for 48 hours before inoculation and heating. When the heated leaf was removed within 2 minutes of heating, no translocation of the stimulus was noted, but when the heated leaf was removed at 5 to 1440 minutes after heating a progressive and possibly pulsating increase in lesion numbers resulted. The stimulus moved through a l-mm column of water between the heated leaf and the stem. The rate of movement of the stimulus was estimated to be 4 mm per minute. When lesions manifested by starch staining, rather tha.n necrotic lesions visible in unstained leaves, were the criterion of infection, the THS was much less, and it is believed that the stimulus is primarily on the necrotic reaction of the host, rather than on the establishment of infection. A similar increase in infection resulted from freezing the noninoculated leaf or treating the noninoculated leaf with certain chemicals which caused severe injury.
response to the translocated heat, stimulus, the sweetpotato strain of TMV (STMV) (Elmer, 1960) produced in greenhousegrown tobacco (Nicotiana tabacum L. var. Turkish) was the virus principally used. Ground infected tobacco leaves diluted to 0.1% to 1% tissue in aqueous 1% K2HP04 usually served as inoculum. This was applied to the upper surface of Carborundum-dusted primary bean (Phase&s vulgar& L. var. Pinto) leaves with a poster brush at lo-14 days from planting. One leaf of each plant was inoculated. On treated plants the opposite leaf was immersed for 20 seconds in water at 70°C. This standard treatment killed the heated leaves but caused only slight translocated heat injury to these young plant’s, Control plant’s were treated similarly except that the opposite leaf was not heated. Lesions of STMV on bean were smaller than those of T&IV and
INTRODUCTION
Heat t,reatments which produced translocated injury to the host may cause marked increases in virus lesion formation (Yarwood, 1961; Yarwood et al., 1962)) and it has been suggested that a translocated wound hormone may be involved. The conditions for expression of this THS, or to manifestation of virus lesions, is further explored here. MATERIALS
AND
METHODS
Since the ordinary strain of tobacco mosaic virus (TMV) has not shown a clear ‘Aided by grants of the Deutsche Forschungsgemeinschaft and the Fulbright Commission. Permanent address : Institut fiir Pflanzenkrankheiten, Rhein. Fr. W. UniversitLt, Bonn, Germany. Director: Prof. Dr. H. Braun. *Aided by a grant (G9820) from the National Science Foundation. 477
NIESHAUS
478
AND
were usually counted at a magnification of 2.5 by means of transmitted light. The greenhouse at about 20”) with natural light and with a relative humidity ranging from 30 to 60% is considered the standard environment. To produce high humidity, estimated at close to loo%, the entire plants or individual leaves were enclosed in small met clear plastic bags. To produce low humidit,y, estimated at 5 to lo%, the entire plants or individual leaves were placed in plastic bags with dry powdered silica gel. To test movement of the stimulus through a water column, the petiole of the heated leaf was cut about midway along its length (15-20 mm) and the cut ends were inserted in the same small glass tube with about 1 mm of water between them. To test, movement across the stem, the stem was slit down the middle between the two primary leaves, and the cut parts were wrapped in aIuminum foil in some cases. The criterion of the magnitude of the translocated heat stimulus was the ratio of the number of lesions in heated plants to the number in control unheated plants. The higher this lesion ratio, the higher the apparent translocated stimulus. In caseswhere no infection occurred in the controls, but infection occurred in the heated plants, t’he number of lesions in the controls is assumed to be 1 for arithmetic purposes. Five to ten plants were treated in each trial unless otherwise noted. RESULTS
Heat
Treatment oculation
before and after
In-
Plants heated after inoculation produced more lesions than plants heat.ed before inoculation, but timing was not critical (Table 1). No clear effect on infection was detected if the heat was applied as much as 24 hours before or after inoculation. The time for the heating effect to be reduced to half its value at the time of inoculation was about 2 hours before inoculation and 15 hours after inoculation, Lhough neither value was adequately determined. The greatest effect on infection, 41- to 50-fold in this case, resulted from heating within 1 hour after inoculation.
YBRWOOD
The Influence of Humidity Low atmospheric humidity increases infection independently of THS (Yarwood, 1955). When heated plants were placed at continuous high humidity, little or no infection resulted. When heated plants were placed at high humidity for variable periods aft’er inoculation and heating, the amount of infection decreased as the period at high humidity increased (Table 2). The time necessary at high humidity for infection t,o be reduced to half that in heated plants not exposed to high humidity was about 1 hour. When plants were placed at high humidity immediately after heating, but inoculation was at the termination of the high humidity treatment, the time at high humidity necessary t’o reduce infection to half that in plants not exposed to high humidity was between 10 and 24 hours. When plants were inoculated, heated, and esposed to the standard environment before being placed at high humidity, a period of 10-24 hours in the standard environment was necessary for infection Lo reach half that which resulted from the heat keatment followed by the continuous st,andard environment. When the humidities surrounding the heated and inoculated leaves were separately manipulated, large differences in infection and in heat activation of infection resulted (Table 3). When the inoculated leaf was placed at high humidity, both infection and THS were reduced. When inoculated leaves of the control and heated plants were placed at low humidity, infection of both was increased, but THS was reduced. Placing the noninoculated leaf in water or paraffin oil had no apparent effect on infection of control plants or heated plants. EfJect of Temperature The optimum temperature for infection with TMV is about 31” (Harrison, 1956) (Table 4). The optimum temperature for manifestation of lesions induced by STMV in bean is apparently much lower (Table 4). With TMV, no clear evidence of THS was detected at, any temperature, and in most trials the heat treatment seemed to decrease infection, whether the inoculum was suspended in water or 1% KaHP04 .
TRMXSLOCATED
1Vit.h ST&IV, THS was greatest at. 26”, but this was clearly associated with low infection of the controls at this t,emperature. Light Most trials were in natural light. Placing plants in darkness aft,er inoculation reduced infection in control and heated plants but had no apparent effect on THS (Table 5). Placing plan& in darkness before inoculation so increased the infection in the cont’rols (Sa,muel a,nd Bald, 1933) that the THS was greatly reduced, even though placing heated plants in the dark also increased infection on these plants. Rate of Translocation
of
Heat
Stimulus
When the heated leaf was detached within 1 minute of heating, no THS was detected. As the time from heating to detachment of the heated leaf was increased, THS of infection was first det,ected when the detachment was at 2 minutes after heating, reached about half maximum value at 1.5 hours, and reached a maximum at between 5 and 24 hours (Table 6). When the heated leaf was detached at the base of the lamina THS was detected sooner than when it was detached at the base of the pet,iole, at least for detachments during the first 15 minutes. Since the time for equivalent’ response was about 5 minutes lat,er for detachment at the base of petiole than for detachment at base of lamina, and since the petiole was about 20 mm long, it is estimated that the heat stimulus moved through the petiole at a rate of about 4 mm per minute. The stepwise increase in THS associated with time from heating to detachment (5 minutes, 10 to 20 minutes, 25 to 50 minutes, and 1.5 to 5 hours) suggests an intermittent pulsation in the translocation of the stimulus. Translocation
across
the Stem
The THS was still manifest if the growing point of the bean plant was removed, if the plant was split bet’ween the primary leaves to the base of the hypocotyl, if the plant was cut off at the base of the hypocotyl, or if the stems were split longitudinally except for about 2 mm at the primary leaf node. In such trials the cut surfaces were covered
WOUKD
479
STIMULI TABLE
1
TREATMENT BEFORE vs. AFTER INOCULATIOX OS TRANSLOCATED HEAT STIXICLATION OF STMV INFECTION IN BEAK
EFFECT
Hours
OF
HEAT
from heating inoculation
to
Number of lesions in heated plants” Number of lesions in control plants 0.40 7.8 6.3 6.1 14 20r
24 10 5 3 2 1
19
0.17 Hours
from inoculation to heating 0.17 1 2 3
41 50 19 23 22 34 1.2
lo” 24 a The average trol plants was inoculation and inoculation.
number of lesions 34 for the plant’s 17 for the plants
in the 10 conheated before heated after
with plastic sheeting or aluminum foil to prevent translocation across or water loss from the cut surfaces, and cut stems were placed in water. If plants were detached and placed in water before heating and inoculation, infection was low but THS was clear. In another type of trial one half of the plant was detached by a longitudinal cut from the apex to the base and across the base, the leaf of the detached half was heated, the detached half was reatt,ached in its original position with aluminum foil, and the leaf on the opposite still-attached part of the plant was inoculated. In 10 such plants, 1120 lesions formed; in 10 plants similarly treat’ed, but without heating, 132 lesionswere formed; and in 10 nomnutilated plants heated in the st,andard way, 2154 lesions were formed. The stimulus from one leaf to another can apparently move across the stem at, the
480
NIENHAUS
AND
YARWOOD
TABLE EFFECT
OF HIGH
HUMIDITY
Time
Test humidity
High
(&
100%)
Standard
of exposure humidity
to test
STIMULATION
exposure exposure
Average lesions per leaf in control plants
Lesions in heated
and
0 2 5 10 24 72
30 29 31 9 12 23
35 8.8 5.0 0.9 0.17 1.3
Immediately after before inoculationa
heating,
but
0 1 2 5 10 24
46 50 32 52 50 9
14 5.7 6.6 12 11 1.1
Immediately
heating
and
1
0
2 5 10 24 72
0 21 25 60 35
after
the plants environment,
Test humidity substrate
High High Low Low Standard Hz0 Paraffin
oil
or
Inoculated leaf only Heated leaf only Inoculated leaf only Heated leaf only Both Heated leaf only Heated leaf only
3
Average Lesions on lesions per heated leaf on control Lesions plants on control 0.2
5
11
51
106
9
70
11
5 5
67 40
5
41
32 6.8 5.5 24 37
were returned to the standard environment. the plants were placed at high humidity.
EFFECT ON INFECTION AND ON TRANSLOCATED HEAT STIMULATION OF INFECTION BY ENVIRONMENTS SEPARATELY APPLIED TO INOCULATED AND HEATED LEAVES Leaf exposed to test humidity or substrate
Lesions in control
heating
to high humidity, to the standard TABLE
INFECTION
Hours at test humidity
inoculation*
a After b After
OF STMV
after
Immediately inoculationa
(30-60s)
2
ON TRANSLOCATED HEAT OF BEAN
primary leaf node or at the base of the hypocotyl. Tramlocation
through
Water
When a leaf was cut off midway along the petiole before it was heated, and this heated leaf was reconnected with the petiole by means of a glass tube with 1 mm of water between the two cut ends of the petiole, the THS moved through the water. In these trials heated inoculated plants with separated petioles were compared with heated inoculated plants with normal petioles (standard treatment) and with unheated inoculated plants (absolute controls). The water column of the plants with separated petioles was absorbed by the plants within about 2 hours, so the heated leaf of the plants given the standard treatment and uninoculated and unheated leaves of the absolute controls were cut off at 2 hours from the start of the test. In one trial with
TRANSLOCATED
WOUND
STIMULI
20 plants in each treatment, the average number of lesions per leaf was 25 in the unheated controls, 373 in plants given the standard heat treatment, and 119 in the plants with heated leaves and separated petioles. Of the 20 plants with heated leaves and separated petioles, 7 showed the stimulation of infection; in t,heset,he average number of lesions was 275, which was not significantly different’ from the number in standard treatment.
Natural
light
Virus Activity
Natural
light
in Inoculated Leaves
When tissues were assayed for STMV on i\‘icotiana glutinosa L. and on N. tabacum L. var xanthi, there was no clear difference in the number of lesions produced by inoculum from heated and control donor plants. In 3 trials with an average of 7 and 253 lesions per leaf count,ed in the control and heated donor plants, respectively, the numbers of lesions from these inocula were 203 and 172, respectively. The above results indicated either that each infection (lesion) in the control bean plants contained much more virus than each infection in the heated plants or that there were many more infections than lesions in the control plants. We believe the latter interpretation is correct and have been able TABLE
4
EFFECT OF TEMPERATURE AND VIRUS ON TRANSLOCATED HEAT STIMULATION OF INFECTION Inoculum
/ Temperature after inoculation (“C)
STMV in 1% KzHPOI
16 21 26 31
52 23 1 4
and result
T~Vi+# z
sions Heat per leaf on effecta control 7.7 17 110 3.8
STRAIX
TMV in Hz0
4
I Lesions
I
le%Zn control)
eff ecta lei/in
Heat
I Lesions
I Heat eff ecta
control
____ 254 391 563 591
0.15 0.28 0.49 0.45
47 895 3202 3618
a Average
lesions
per
leaf
on heated
plants
Average
lesions
per
leaf
on control
plants
2.1 0.82 0.64 0.47
481 TABLE
5
INFLUENCE OF LIGHT AND DARK ox TRM~SLOCATED HEAT STIMULATION OF INFECTION OF STMV IN BEAN
Exposure of plants after heat and inoculation
“xposure of ;u. n-+-r 5efore L ir--.-‘luculittion
48 hr.
dark
Lesions Average on lesions per ’neated ’ leaf on control Lesions r\,nntc yly*“LY on control
72 hr. natural light 48 hr. dark, 24 hr. light 72 hr. natural light
TABLE
4
52
2
69
73
6
EFFECT OF TIME REMOVAL OF HEATED TRANSLOCATED HEAT STIMULATION STMV INFECTION IN BEAN Time from heating and inoculation to removal of heated leaf Controla 5 10 15 20 25 30 35 40 45 50 1 1.5 2 3 4 5 12
min min min min min min min min min min hr hr hr hr hr hr hr
3.5
LEAF OF
ON
Position of detachment of heated leaf and number of lesions on inoculated leaf Base of lamina 6” 16b 45 63 37 162 127 157 121 105 136 227 337 551 589 545 471 780
Base of petiole
8 10 56 45 65 113 71 119 166 151 316 -
-
a Inoculated but not heated. * In another trial the numbers of lesions for control, 2 minutes, 3 minutes, and 5 minutes were 7, 6, 59, and 36, respectively.
to activate these LLlatent” infections in the control plants by heat (Yarwood, 1958) and independently to demonstrate them by the starch iodine test (Holmes, 1931). In 4
482
NIEKHAUS~
AND YARWOOD
trials the average number of lesions per leaf in 11 leaves inoculated and not further treated was 9 whereas on 9 leaves which were inoculated and heated the corresponding number was 142. In two other trials the inoculated leaves were bleached with hot ethanol and stained with iodine. The average number of lesions per square centimeter counted in control and heated plants before staining was 5 and 77, respect.ively; after staining, the numbers were 105 and 157, respectively. The diameter of the starch lesions in the stained leaves ranged from 100 to 200 p for the control and 125 to 300 p for the heated plants. A similar demonstration of lesion formation by the U2 strain of TMV as manifested by iodine staining has been reported by Helms and McIntyre (1962). Translocated Stimulation of Infection by Chemical Injury and Cold Athough most studies have been with translocated stimulation by heat, similar effects on infection have been induced by treating the noninoculated bean leaves with glycerol, phenol, ethanol, toluol, pine oil, and carbon tetrachloride at room temperature; by cold liquid nitrogen; and by dry ice dissolved in methanol. Here, as with heat, increase in infection was correlated with injury to t,he treated and nontreated leaves. If complete immersion of the treated leaf cause severe injury to the nontreated leaf, this was reduced in some cases by reducing the concentration of the chemical, in some cases by treating only a portion of the leaf, and in some cases by removing the treated leaf at some time after treatment. In five trials with an average of 24 lesions in 7 control leaves, the average number of lesions was 81 for plants treated with carbon tetrachloride and 42 for plants treated with phenol. This study has not advanced to a stage suitable for a detailed report. DISCUSSION
In previous reports (Yarwood, 1961; Yarwood et al., 1962), the injury and increased members of local lesions of STMV
on one primary bean leaf as a result, of heating the opposite leaf was presumed to be an effect on the initiation of infection. In light of studies reported here, this presumption needs modification. Since the injury and the increased lesion formation in one leaf can be brought about by various chemicals and by freezing, as well as by heat applied to the opposite leaf, clearly the treatment causing the effect is not specific. Furthermore, the increased infection observed is primarily an activation of infection leading to necrosis, rather than an expression of the initiation of infection. Nevertheless, heating is the best method known to produce the stimulus, and manifestation of lesions is the method known for measuring response. Chemicals have the serious disadvantage that they leave residues which may themselves be translocated. Freezing with liquid nitrogen is a treatment which may not be generally available. Counting of lesions is more precise over a wide range of values than any other known method of recording data on the translocated stimulus, and effect,s on lesion numbers can be detected from treatment dosages which produce no other macroscopic evidence of a translocated stimulus. It is interesting that the same treatment which increases lesion formation by one strain of TMV may have little effect on lesions induced by another strain of TMV (Table 4) and may greatly decrease pustule formation by a nonrelated pathogen, the bean rust fungus (Yarwood, 1961). That the translocated stimulus is due to a chemical, not to some electrical phenomenon, is strongly indicated by its slow (4 mm/minute) rate of translocation and long-continued movement, by its movement through a water column, and by the detection of traumatin in extracts of treated plants (unpublished data). Differences in transpiration, osmot,ic pressure, sugars, amino acids, and phenols (unpublished data) induced in the inoculated leaf as a result of heating the opposite leaf are not known to have any causal relation to the translocated injury or to the manifestation of lesions, but they indicate that injury has
TRANSLOCATED
multiple effects in addition manifestation of lesions.
to injury
WOU’ND
and
REFERENCES ELMER, 0. H. (1960). Etiology and characteristics of sweetpotato mosaic. Phytopnthology 50, 744749. HARRISON, B. D. (1956). Studies on the effect of temperature on virus multiplication in inoculated leaves. Ann. Appl. Biol. 44, 215-226. HELENS, K., and MCINTYRE, G. A. (1962). Studies on size of lesions of tobacco mosaic virus on Pinto bean. ViTology 18, 535-545. HOLMES, F. 0. (1931). Local lesions of mosaic in
STIMULI
483
Nicotiuna tabacum L. Contrib. Boyce Thompson Inst. 3, 163-172. SAMUEL, G., and BALD, J. G. (1933). On the use of the primary lesions in quantitative work with two plant viruses. Ann. Appl. Biol. 20, 70-99. YARWOOD, C. E. (1955). Deleterious effects of water in plant virus inoculations. Virology 1, 268-285. YARWOOD, C. E. (1958). Heat activation of virus infections Phytopathology 48, 39-46. YARWOOD, C. E. (1961). Translocated heat injury. Plant Physiol. 36, 721-726. YARWOOD, C. E., RESCOSICH, E. C., and KADO, C. I. (1962). Translocated stimuli affecting plant virus infections. Virology 1’6, 414-418.
20, 477-483
(1963)
Translocated
Wound
Stimuli
F. NIEXHAUS’ Department
oj Plant
Affecting
Accepted
Virus
Infections
C. E. YARWOOD”
AND
Pathology,
Plant
University
Apd
of
California,
Berkeley
1, 1963
Heating one primary Pinto bean leaf for 20 seconds at 70°C increased the number of lesions induced by the sweetpotato strain of tobacco mosaic virus in the opposite inoculated leaf to as many as 100 times the number in control plants. This translocated heat stimulus (THS) was greatest when the heat was applied within 1 hour after inoculation, but was clearly apparent when applied as much as 10 hours before or after inoculation. Manifestation of the THS was inhibited by high humidity, and this inhibition was complete when the heated and inoculated plants were held at. high humidity for 10 hours after inoculation. The THS was greatest in plants held at about 26”, was similar for plants held for 48 hours in darkness after inoculation and heating and for plants in natural light but was less for plants held in darkness for 48 hours before inoculation and heating. When the heated leaf was removed within 2 minutes of heating, no translocation of the stimulus was noted, but when the heated leaf was removed at 5 to 1440 minutes after heating a progressive and possibly pulsating increase in lesion numbers resulted. The stimulus moved through a l-mm column of water between the heated leaf and the stem. The rate of movement of the stimulus was estimated to be 4 mm per minute. When lesions manifested by starch staining, rather tha.n necrotic lesions visible in unstained leaves, were the criterion of infection, the THS was much less, and it is believed that the stimulus is primarily on the necrotic reaction of the host, rather than on the establishment of infection. A similar increase in infection resulted from freezing the noninoculated leaf or treating the noninoculated leaf with certain chemicals which caused severe injury.
response to the translocated heat, stimulus, the sweetpotato strain of TMV (STMV) (Elmer, 1960) produced in greenhousegrown tobacco (Nicotiana tabacum L. var. Turkish) was the virus principally used. Ground infected tobacco leaves diluted to 0.1% to 1% tissue in aqueous 1% K2HP04 usually served as inoculum. This was applied to the upper surface of Carborundum-dusted primary bean (Phase&s vulgar& L. var. Pinto) leaves with a poster brush at lo-14 days from planting. One leaf of each plant was inoculated. On treated plants the opposite leaf was immersed for 20 seconds in water at 70°C. This standard treatment killed the heated leaves but caused only slight translocated heat injury to these young plant’s, Control plant’s were treated similarly except that the opposite leaf was not heated. Lesions of STMV on bean were smaller than those of T&IV and
INTRODUCTION
Heat t,reatments which produced translocated injury to the host may cause marked increases in virus lesion formation (Yarwood, 1961; Yarwood et al., 1962)) and it has been suggested that a translocated wound hormone may be involved. The conditions for expression of this THS, or to manifestation of virus lesions, is further explored here. MATERIALS
AND
METHODS
Since the ordinary strain of tobacco mosaic virus (TMV) has not shown a clear ‘Aided by grants of the Deutsche Forschungsgemeinschaft and the Fulbright Commission. Permanent address : Institut fiir Pflanzenkrankheiten, Rhein. Fr. W. UniversitLt, Bonn, Germany. Director: Prof. Dr. H. Braun. *Aided by a grant (G9820) from the National Science Foundation. 477
NIESHAUS
478
AND
were usually counted at a magnification of 2.5 by means of transmitted light. The greenhouse at about 20”) with natural light and with a relative humidity ranging from 30 to 60% is considered the standard environment. To produce high humidity, estimated at close to loo%, the entire plants or individual leaves were enclosed in small met clear plastic bags. To produce low humidit,y, estimated at 5 to lo%, the entire plants or individual leaves were placed in plastic bags with dry powdered silica gel. To test movement of the stimulus through a water column, the petiole of the heated leaf was cut about midway along its length (15-20 mm) and the cut ends were inserted in the same small glass tube with about 1 mm of water between them. To test, movement across the stem, the stem was slit down the middle between the two primary leaves, and the cut parts were wrapped in aIuminum foil in some cases. The criterion of the magnitude of the translocated heat stimulus was the ratio of the number of lesions in heated plants to the number in control unheated plants. The higher this lesion ratio, the higher the apparent translocated stimulus. In caseswhere no infection occurred in the controls, but infection occurred in the heated plants, t’he number of lesions in the controls is assumed to be 1 for arithmetic purposes. Five to ten plants were treated in each trial unless otherwise noted. RESULTS
Heat
Treatment oculation
before and after
In-
Plants heated after inoculation produced more lesions than plants heat.ed before inoculation, but timing was not critical (Table 1). No clear effect on infection was detected if the heat was applied as much as 24 hours before or after inoculation. The time for the heating effect to be reduced to half its value at the time of inoculation was about 2 hours before inoculation and 15 hours after inoculation, Lhough neither value was adequately determined. The greatest effect on infection, 41- to 50-fold in this case, resulted from heating within 1 hour after inoculation.
YBRWOOD
The Influence of Humidity Low atmospheric humidity increases infection independently of THS (Yarwood, 1955). When heated plants were placed at continuous high humidity, little or no infection resulted. When heated plants were placed at high humidity for variable periods aft’er inoculation and heating, the amount of infection decreased as the period at high humidity increased (Table 2). The time necessary at high humidity for infection t,o be reduced to half that in heated plants not exposed to high humidity was about 1 hour. When plants were placed at high humidity immediately after heating, but inoculation was at the termination of the high humidity treatment, the time at high humidity necessary t’o reduce infection to half that in plants not exposed to high humidity was between 10 and 24 hours. When plants were inoculated, heated, and esposed to the standard environment before being placed at high humidity, a period of 10-24 hours in the standard environment was necessary for infection Lo reach half that which resulted from the heat keatment followed by the continuous st,andard environment. When the humidities surrounding the heated and inoculated leaves were separately manipulated, large differences in infection and in heat activation of infection resulted (Table 3). When the inoculated leaf was placed at high humidity, both infection and THS were reduced. When inoculated leaves of the control and heated plants were placed at low humidity, infection of both was increased, but THS was reduced. Placing the noninoculated leaf in water or paraffin oil had no apparent effect on infection of control plants or heated plants. EfJect of Temperature The optimum temperature for infection with TMV is about 31” (Harrison, 1956) (Table 4). The optimum temperature for manifestation of lesions induced by STMV in bean is apparently much lower (Table 4). With TMV, no clear evidence of THS was detected at, any temperature, and in most trials the heat treatment seemed to decrease infection, whether the inoculum was suspended in water or 1% KaHP04 .
TRMXSLOCATED
1Vit.h ST&IV, THS was greatest at. 26”, but this was clearly associated with low infection of the controls at this t,emperature. Light Most trials were in natural light. Placing plants in darkness aft,er inoculation reduced infection in control and heated plants but had no apparent effect on THS (Table 5). Placing plan& in darkness before inoculation so increased the infection in the cont’rols (Sa,muel a,nd Bald, 1933) that the THS was greatly reduced, even though placing heated plants in the dark also increased infection on these plants. Rate of Translocation
of
Heat
Stimulus
When the heated leaf was detached within 1 minute of heating, no THS was detected. As the time from heating to detachment of the heated leaf was increased, THS of infection was first det,ected when the detachment was at 2 minutes after heating, reached about half maximum value at 1.5 hours, and reached a maximum at between 5 and 24 hours (Table 6). When the heated leaf was detached at the base of the lamina THS was detected sooner than when it was detached at the base of the pet,iole, at least for detachments during the first 15 minutes. Since the time for equivalent’ response was about 5 minutes lat,er for detachment at the base of petiole than for detachment at base of lamina, and since the petiole was about 20 mm long, it is estimated that the heat stimulus moved through the petiole at a rate of about 4 mm per minute. The stepwise increase in THS associated with time from heating to detachment (5 minutes, 10 to 20 minutes, 25 to 50 minutes, and 1.5 to 5 hours) suggests an intermittent pulsation in the translocation of the stimulus. Translocation
across
the Stem
The THS was still manifest if the growing point of the bean plant was removed, if the plant was split bet’ween the primary leaves to the base of the hypocotyl, if the plant was cut off at the base of the hypocotyl, or if the stems were split longitudinally except for about 2 mm at the primary leaf node. In such trials the cut surfaces were covered
WOUKD
479
STIMULI TABLE
1
TREATMENT BEFORE vs. AFTER INOCULATIOX OS TRANSLOCATED HEAT STIXICLATION OF STMV INFECTION IN BEAK
EFFECT
Hours
OF
HEAT
from heating inoculation
to
Number of lesions in heated plants” Number of lesions in control plants 0.40 7.8 6.3 6.1 14 20r
24 10 5 3 2 1
19
0.17 Hours
from inoculation to heating 0.17 1 2 3
41 50 19 23 22 34 1.2
lo” 24 a The average trol plants was inoculation and inoculation.
number of lesions 34 for the plant’s 17 for the plants
in the 10 conheated before heated after
with plastic sheeting or aluminum foil to prevent translocation across or water loss from the cut surfaces, and cut stems were placed in water. If plants were detached and placed in water before heating and inoculation, infection was low but THS was clear. In another type of trial one half of the plant was detached by a longitudinal cut from the apex to the base and across the base, the leaf of the detached half was heated, the detached half was reatt,ached in its original position with aluminum foil, and the leaf on the opposite still-attached part of the plant was inoculated. In 10 such plants, 1120 lesions formed; in 10 plants similarly treat’ed, but without heating, 132 lesionswere formed; and in 10 nomnutilated plants heated in the st,andard way, 2154 lesions were formed. The stimulus from one leaf to another can apparently move across the stem at, the
480
NIENHAUS
AND
YARWOOD
TABLE EFFECT
OF HIGH
HUMIDITY
Time
Test humidity
High
(&
100%)
Standard
of exposure humidity
to test
STIMULATION
exposure exposure
Average lesions per leaf in control plants
Lesions in heated
and
0 2 5 10 24 72
30 29 31 9 12 23
35 8.8 5.0 0.9 0.17 1.3
Immediately after before inoculationa
heating,
but
0 1 2 5 10 24
46 50 32 52 50 9
14 5.7 6.6 12 11 1.1
Immediately
heating
and
1
0
2 5 10 24 72
0 21 25 60 35
after
the plants environment,
Test humidity substrate
High High Low Low Standard Hz0 Paraffin
oil
or
Inoculated leaf only Heated leaf only Inoculated leaf only Heated leaf only Both Heated leaf only Heated leaf only
3
Average Lesions on lesions per heated leaf on control Lesions plants on control 0.2
5
11
51
106
9
70
11
5 5
67 40
5
41
32 6.8 5.5 24 37
were returned to the standard environment. the plants were placed at high humidity.
EFFECT ON INFECTION AND ON TRANSLOCATED HEAT STIMULATION OF INFECTION BY ENVIRONMENTS SEPARATELY APPLIED TO INOCULATED AND HEATED LEAVES Leaf exposed to test humidity or substrate
Lesions in control
heating
to high humidity, to the standard TABLE
INFECTION
Hours at test humidity
inoculation*
a After b After
OF STMV
after
Immediately inoculationa
(30-60s)
2
ON TRANSLOCATED HEAT OF BEAN
primary leaf node or at the base of the hypocotyl. Tramlocation
through
Water
When a leaf was cut off midway along the petiole before it was heated, and this heated leaf was reconnected with the petiole by means of a glass tube with 1 mm of water between the two cut ends of the petiole, the THS moved through the water. In these trials heated inoculated plants with separated petioles were compared with heated inoculated plants with normal petioles (standard treatment) and with unheated inoculated plants (absolute controls). The water column of the plants with separated petioles was absorbed by the plants within about 2 hours, so the heated leaf of the plants given the standard treatment and uninoculated and unheated leaves of the absolute controls were cut off at 2 hours from the start of the test. In one trial with
TRANSLOCATED
WOUND
STIMULI
20 plants in each treatment, the average number of lesions per leaf was 25 in the unheated controls, 373 in plants given the standard heat treatment, and 119 in the plants with heated leaves and separated petioles. Of the 20 plants with heated leaves and separated petioles, 7 showed the stimulation of infection; in t,heset,he average number of lesions was 275, which was not significantly different’ from the number in standard treatment.
Natural
light
Virus Activity
Natural
light
in Inoculated Leaves
When tissues were assayed for STMV on i\‘icotiana glutinosa L. and on N. tabacum L. var xanthi, there was no clear difference in the number of lesions produced by inoculum from heated and control donor plants. In 3 trials with an average of 7 and 253 lesions per leaf count,ed in the control and heated donor plants, respectively, the numbers of lesions from these inocula were 203 and 172, respectively. The above results indicated either that each infection (lesion) in the control bean plants contained much more virus than each infection in the heated plants or that there were many more infections than lesions in the control plants. We believe the latter interpretation is correct and have been able TABLE
4
EFFECT OF TEMPERATURE AND VIRUS ON TRANSLOCATED HEAT STIMULATION OF INFECTION Inoculum
/ Temperature after inoculation (“C)
STMV in 1% KzHPOI
16 21 26 31
52 23 1 4
and result
T~Vi+# z
sions Heat per leaf on effecta control 7.7 17 110 3.8
STRAIX
TMV in Hz0
4
I Lesions
I
le%Zn control)
eff ecta lei/in
Heat
I Lesions
I Heat eff ecta
control
____ 254 391 563 591
0.15 0.28 0.49 0.45
47 895 3202 3618
a Average
lesions
per
leaf
on heated
plants
Average
lesions
per
leaf
on control
plants
2.1 0.82 0.64 0.47
481 TABLE
5
INFLUENCE OF LIGHT AND DARK ox TRM~SLOCATED HEAT STIMULATION OF INFECTION OF STMV IN BEAN
Exposure of plants after heat and inoculation
“xposure of ;u. n-+-r 5efore L ir--.-‘luculittion
48 hr.
dark
Lesions Average on lesions per ’neated ’ leaf on control Lesions r\,nntc yly*“LY on control
72 hr. natural light 48 hr. dark, 24 hr. light 72 hr. natural light
TABLE
4
52
2
69
73
6
EFFECT OF TIME REMOVAL OF HEATED TRANSLOCATED HEAT STIMULATION STMV INFECTION IN BEAN Time from heating and inoculation to removal of heated leaf Controla 5 10 15 20 25 30 35 40 45 50 1 1.5 2 3 4 5 12
min min min min min min min min min min hr hr hr hr hr hr hr
3.5
LEAF OF
ON
Position of detachment of heated leaf and number of lesions on inoculated leaf Base of lamina 6” 16b 45 63 37 162 127 157 121 105 136 227 337 551 589 545 471 780
Base of petiole
8 10 56 45 65 113 71 119 166 151 316 -
-
a Inoculated but not heated. * In another trial the numbers of lesions for control, 2 minutes, 3 minutes, and 5 minutes were 7, 6, 59, and 36, respectively.
to activate these LLlatent” infections in the control plants by heat (Yarwood, 1958) and independently to demonstrate them by the starch iodine test (Holmes, 1931). In 4
482
NIEKHAUS~
AND YARWOOD
trials the average number of lesions per leaf in 11 leaves inoculated and not further treated was 9 whereas on 9 leaves which were inoculated and heated the corresponding number was 142. In two other trials the inoculated leaves were bleached with hot ethanol and stained with iodine. The average number of lesions per square centimeter counted in control and heated plants before staining was 5 and 77, respect.ively; after staining, the numbers were 105 and 157, respectively. The diameter of the starch lesions in the stained leaves ranged from 100 to 200 p for the control and 125 to 300 p for the heated plants. A similar demonstration of lesion formation by the U2 strain of TMV as manifested by iodine staining has been reported by Helms and McIntyre (1962). Translocated Stimulation of Infection by Chemical Injury and Cold Athough most studies have been with translocated stimulation by heat, similar effects on infection have been induced by treating the noninoculated bean leaves with glycerol, phenol, ethanol, toluol, pine oil, and carbon tetrachloride at room temperature; by cold liquid nitrogen; and by dry ice dissolved in methanol. Here, as with heat, increase in infection was correlated with injury to t,he treated and nontreated leaves. If complete immersion of the treated leaf cause severe injury to the nontreated leaf, this was reduced in some cases by reducing the concentration of the chemical, in some cases by treating only a portion of the leaf, and in some cases by removing the treated leaf at some time after treatment. In five trials with an average of 24 lesions in 7 control leaves, the average number of lesions was 81 for plants treated with carbon tetrachloride and 42 for plants treated with phenol. This study has not advanced to a stage suitable for a detailed report. DISCUSSION
In previous reports (Yarwood, 1961; Yarwood et al., 1962), the injury and increased members of local lesions of STMV
on one primary bean leaf as a result, of heating the opposite leaf was presumed to be an effect on the initiation of infection. In light of studies reported here, this presumption needs modification. Since the injury and the increased lesion formation in one leaf can be brought about by various chemicals and by freezing, as well as by heat applied to the opposite leaf, clearly the treatment causing the effect is not specific. Furthermore, the increased infection observed is primarily an activation of infection leading to necrosis, rather than an expression of the initiation of infection. Nevertheless, heating is the best method known to produce the stimulus, and manifestation of lesions is the method known for measuring response. Chemicals have the serious disadvantage that they leave residues which may themselves be translocated. Freezing with liquid nitrogen is a treatment which may not be generally available. Counting of lesions is more precise over a wide range of values than any other known method of recording data on the translocated stimulus, and effect,s on lesion numbers can be detected from treatment dosages which produce no other macroscopic evidence of a translocated stimulus. It is interesting that the same treatment which increases lesion formation by one strain of TMV may have little effect on lesions induced by another strain of TMV (Table 4) and may greatly decrease pustule formation by a nonrelated pathogen, the bean rust fungus (Yarwood, 1961). That the translocated stimulus is due to a chemical, not to some electrical phenomenon, is strongly indicated by its slow (4 mm/minute) rate of translocation and long-continued movement, by its movement through a water column, and by the detection of traumatin in extracts of treated plants (unpublished data). Differences in transpiration, osmot,ic pressure, sugars, amino acids, and phenols (unpublished data) induced in the inoculated leaf as a result of heating the opposite leaf are not known to have any causal relation to the translocated injury or to the manifestation of lesions, but they indicate that injury has
TRANSLOCATED
multiple effects in addition manifestation of lesions.
to injury
WOU’ND
and
REFERENCES ELMER, 0. H. (1960). Etiology and characteristics of sweetpotato mosaic. Phytopnthology 50, 744749. HARRISON, B. D. (1956). Studies on the effect of temperature on virus multiplication in inoculated leaves. Ann. Appl. Biol. 44, 215-226. HELENS, K., and MCINTYRE, G. A. (1962). Studies on size of lesions of tobacco mosaic virus on Pinto bean. ViTology 18, 535-545. HOLMES, F. 0. (1931). Local lesions of mosaic in
STIMULI
483
Nicotiuna tabacum L. Contrib. Boyce Thompson Inst. 3, 163-172. SAMUEL, G., and BALD, J. G. (1933). On the use of the primary lesions in quantitative work with two plant viruses. Ann. Appl. Biol. 20, 70-99. YARWOOD, C. E. (1955). Deleterious effects of water in plant virus inoculations. Virology 1, 268-285. YARWOOD, C. E. (1958). Heat activation of virus infections Phytopathology 48, 39-46. YARWOOD, C. E. (1961). Translocated heat injury. Plant Physiol. 36, 721-726. YARWOOD, C. E., RESCOSICH, E. C., and KADO, C. I. (1962). Translocated stimuli affecting plant virus infections. Virology 1’6, 414-418.