- Email: [email protected]
Lesion growth and virus localization in leaves of Nicotiana tabacum cv. Xanthi nc. after inoculation with tobacco mosaic virus and incubation alternately at 22°C and 32°C
Physiological Plant Pathology (1981) 18,357-368
Lesion growth and virus localization in leaves of Nicotiana tabacum cv. Xanthi nc. after inoculation with tobacco mosaic virus and incubation alternately at 22°C and 32 °C EMMA
A.
WESTSTEIJN
Phytopathological Laboratory "lVillie Commelin Scholten", Javalaan 20, Baarn, The Netherlands
(Accepted/oT publication January 1981)
Factors which determine lesion size were studied with Nicotiana tabacum L. cv, Xanthi ne, and tobacco mosaic virus (TJ\IV). Inoculated plants were incubated alternately at 22 and 32 ·C. Incubation at 32 ·C was used as a condition under which multiplication and spread of the virus are uninhibited by the plant; incubation at 22 ·C for I day thereafter was necessary to visualize the spread of the virus at 32 ·C. Effect of different treatments on lesion size was compared in fully grown leaves of increasing age. Size of lesions formed at 22 ·C was leaf age dependent, as was size oflesions formed after incubation at 32 ·C followed by 22 ·C. Infectivity of the virus from the latter lesions was proportional to lesion area. Increase in size of these lesions at 22 ·C was also leaf age dependent. The width of the zone of damaged cells around lesions formed at 22 ·C depended on the age of the leaves and on the age of the lesions. Lesions formed at 22 ·C increased in size after incubation at 32 ·C followed by 22 ·C, and this increase depended on the age of the lesions. Furthermore, with older lesions, sectorial outgrowth occurred or lesions did not grow at all. For complete localization of the virus at least 12 days at 22 ·C were necessary. All effects mentioned here that were leaf or lesion age dependent were smaller with increasing leaf or lesion age. After interruption of the incubation at 32 ·C by short periods at 22 ·C necroses only appeared if the interval at 22 ·C was longer than 5 to 6 h; the size of these necroses was larger with increasing time interval at 22 ·C.
INTRODUCTION
The hypersensitive reaction (HR) of plants after infection with a virus is characterized by the appearance of necrotic local lesions. Usually this HR is accompanied by the localization of the virus. In Nicotiana tabacum L. cv. Xanthi ne. inoculated with tobacco mosaic virus (TMV), the lesions appear about 2 days after inoculation and increase in size until 8 to 10 days after inoculation. In this combination, lesion formation is temperature dependent. With increasing temperatures after inoculation up to about 27°C, lesion size increases, but at higher temperatures necrosis becomes inhibited. At 28 ·C only few necroses appear, which grow quickly, and the virus spreads through the whole plant. At 32°C no lesions develop at all. Instead, a systemic reaction occurs, comparable with the reaction at 22 °C of Nicotiana tabacum cv. Samsun infected with TMV [13]. However, extensive necrosis of the tissues invaded with virus occurs if the infected plants arc transferred from 32 to 22°C [6, 13]. The first signs of this necrosis become visible about 8 h after transfer [15]. After 3 to 6 h at 22°C, the induction of this necrosis is irreversible [4, 6]. 0048--4Q59/81/030357+12502.0010 © 1981 Academic Press Inc. (London) Limited
358
E. A. Weststeijn
Lesion size is also dependent on leaf age, i.e. with increasing age of fully grown leaves, lesion size decreases [1, 10, 12, 14], and also in leaves exhibiting systemic acquired resistance smaller lesions occur than in normal leaves [9]. The aim of the experiments described in this report, was to obtain more information about which factors determine lesion size. Lesion growth was analysed in inoculated plants after incubation alternately at 22 and 32°C. The effect of these treatments on lesions was studied in leaves of different ages. The temperature of 32 °C was used as a condition under which multiplication and spread of the virus are not inhibited by the plant, and 22°C for I day thereafter as a means to visualize the spread at 32 °C. MATERIALS AND METHODS
Tobacco plants (Nicoliana tabacum cv. Xanthi ne.) were grown in a glasshouse at about 22°C, with additional light during the winter for 16 h a day. TMV was purified from frozen TMV-infected Nicotiana tabacum cv. Samsun leaves with polyethylene glycol [3]. An appropriate dilution, inducing 50 to 150 lesions per leaf, served as inoculum. Inoculations were carried out by rubbing a drop of inoculum over the leaves with a finger, with carborundum as an abrasive. After inoculation the leaves were rinsed with tap water. Incubation at alternating temperatures included a first period, at 22°C, varying from 0 to 12 days after inoculation, a second period, at 32 °C,varying from 1 to 3 days, and a third period, again at 22°C, usually of 1 day, to evoke lesion formation. Incubations at 22°C were in the glasshouse with additional light during the winter for 16 h a day; incubations at 32°C were in growth chambers at 80% r.h. and a 16 h photoperiod. Size of lesions, if visible, was measured each time plants were transferred from one temperature to the other and in some experiments on some additional days after the transfer from 32 to 22°C. Lesion size was determined by measuring 2 perpendicular diameters with the aid of a magnifier with a micrometer. In case of sectorial outgrowth, the increase from the edge of the lesions formed at 22°C was measured. The infectivity of virus in and around lesions formed after incubation at 32°C followed by 22 °C for 1 day was used as a measure of virus multiplication at 32°C. Leaf disks, with a diameter of 9 mm with the lesion in the centre, were punched out with a cork borer. Each disk was homogenized separately with a mortar and pestle in I mI tap water and the homogenate inoculated on one-half leaf of Xanthi ne. tobacco, the other half receiving a standard TMV inoculum. Lesions were counted 3 days later and numbers were calibrated against the mean number on the control halves. Systemic acquired resistance was induced by inoculation of 3 fully grown leaves of Xanthi ne. tobacco plants with TMV and incubation at 22°C. Ten days later, 5 subsequent upper leaves were used as being resistant to TMV. Immediately after challenge inoculation, plants were incubated at 32°C for 2 days, and lesion size was measured I day after transfer of the plants to 22°C. Control plants were incubated at 22 °C for 7 days after challenge inoculation.
Lesion growth and virus localization
359
Damage of cells around lesions was investigated by staining sections through lesions and adjacent tissue with fluorescein diacetate (FDA), a vital stain, which is able to pass cell membranes. Inside the cell it is degraded to the green fluorescent compound, fluorescein, which cannot pass intact membranes. Intact cells retain the compound and damaged cells loose it [16]. Along a line perpendicular to the lesion edge, the number of palisade parenchyma cells, which had lost the stain were counted 15, 30, 45 and 60 min after administration of FDA. The number of destained cells did hardly increase, if at all, thereafter, Control tissue retained the stain for that period. RESULTS
Lesion growth at 22°C The general characteristics of lesion growth at 22 °C are shown in Fig. 1. Lesions grew steadily for approximately 7 days and reached their maximum size about 1 day later. Lesions in older leaves were smaller than those in younger leaves. (a)
(b)
3
E oS
~
° 2
•
°
E
o
'0
2
4
6
8
m u
Days after inoculaticn
2
3
4
5
leaf posilicn
FIG. I. Lesion size in leaves of different ages of Nicatiana tabacum cv. Xanthi ne, after inoculation with TMV and incubation at 22°C. (a) Development of lesion size in leaf I (.) and leaf 5 (0). (b) Lesion size 8 days after inoculation. Leaf I is the youngest fully grown leaf, the other numbers refer to successively older leaves. Each point is the mean from 15 leaves, 5 lesions per leaf, Lesion sizes for the different leaf positions are significantly different (trend test; day 3, a=O·OI; day 8, a=O·OOOI).
Effect of 32°C treatment immediately after inoculation To investigate the uninhibited spread of the virus in relation to leaf age, plants were kept after inoculation at 32°C for 24, 48 or 72 h, followed by 22°C for 1 day. These treatments resulted in local lesions in the inoculated leaves. The size of these lesions increased with increasing time at 32°C and were about 2, 4·2 and 7·5 mm, respectively. Lesion size (in leaves at 32°C for 24 or 48 h) depended also on leaf age (Fig. 2), in the same manner as the size of lesions formed at 22°C (Fig. 1), i.e. lesion size decreased with increasing leaf age. Experiments with plants kept at 32°C for 72 h gave similar results, but because many lesions coalesced, statistical analysis was impossible. The diameter of these lesions was about 7 mm for leaf 5 and about 8 mm for leaf 1.
360
E. A. Weststeijn
5
2 3 4 leaf position
5
FIG. 2. Lesion size in leaves of different ages of Nicotiana tabaeum cv, Xanthi ne. after inoculation with Tl\1V and incubation at 32 QCfor 24 h (e) or 48 h (0) followed by 22 QC for I day. Leaf numbers as in Fig. 1. Each point is the mean from 5 leaves, 10 lesions per leaf. Lesion sizes for the different leafpositions are significantly different (trend test; a=O·OOI).
'0 140 .!! '0 s:
..... c
•
120
0
0;
-. .!!
100
0
L
.0
E
••
80
"c
e 0
0;
60
.!!
.. L
Q,
• •
40
~
~u
.!!
20
.s 1·5
20
2·5
30
Lesion area (mm Z )
FIG. 3. Infectivity of virus in and around lesions differing in size. Lesions were formed in leaves of different ages of Nicotiana tabacum cv. Xanthi ne. after inoculation with Tl\1V and incubation at 32 QC for 24 h followed by 22 QCfor I day. Each point is the mean from about 10 lesions in a particular leaf.
Lesion growth and virus localization
361
In addition, a linear relationship was found between the infectivity of the virus in and around these lesions in leaves of different ages and the area of the lesions (Fig. 3). A challenge inoculation of systemic resistant leaves followed by incubation at 22°C for 7 days yielded, as expected, smaller lesions as the same treatment of normal leaves (Table 1). A similar result was obtained after incubation at 32°C for 2 days followed by 22 °C for 1 day, except that the reduction in lesion size in the latter treatment was smaller than in the former one. TABLE I Lesion size in systemicresistant (SAR) and normal leaces ofNicotiana tab acum eo, Xanthi ne, after challenge inoculation with TMV and incubation at 22°Cfor 7 dtl)'s (22°C treatment) or at srcfor 2 daysfollowed by 2rCfor 1 day (32 °C treatment)
22°C Treatment
32 °C Treatment
Lesion diameter (mm)
Lesion diameter (mm)
Expt no.
Normal
SAR
Percentage reduction
Normal
SAR
I
2·40 2·09
0·90 1-16
62 45
3·53 3·15
2-54
2
2'81
Percentage reduction 20 19
4
E
3
E
.
0;
E 0 '5
2 0
c 0
&x; ..J
-4
-2
3
5
7
9
11
Leaf position
FIG. 4. Lesion size in all leaves of Nicotiana tabacum cv, Xanthi ne. after inoculation with TMV and incubation at 32 °C for 24 h and at 22 °C for I day (e) a nd 7 days (0). Leaf numbers as in Fig. 1; negative numbers refer to not yet fully grown leaves. Example of I plant, 6 others showing similar results. Each point is the mean from 15 lesions, except for the youngest leaves.
Relation beuoeen uninhibited spread of the virus at 32 QC and growlh of lesions at 22 QC oftertoards Lesion size in all leaves ofaXanthi ne. tobacco plant after inoculation with TMV and incubation at 32 QC for 24 h followed by 22 QC for I day, is shown in Fig. 4.
362
E. A. Weststeijn
This figure shows how the uninhibited spread of the virus depended on leaf age. Incubation of the same plant at 22 °0 for another 6 days resulted in an increase in lesion size (Fig. 4). Lesions in younger leaves grew faster than lesions of equal initial size in older leaves.
50
.!!! 0;
40
u
."
'" C
'0
on
30
'"
."
'0 ~
20
~
E ::l
Z
10
2 Days after inoculation
FIG. 5. Number of cells which lost the vital stain FDA, around lesions formed in leaves of Nicatlana tabacum cv. Xanthi ne. after inoculation with TMV and incubation at 22 cC for different periods. Investigated cells were palis ade parenchyma cells along a radial line extending from the lesion edge. Measurements were taken at 15 min (e), 30 min (0), 45 min (T) and 60 min ('1) after administration ofFDA. Each point is the mean of35 to 45 measurements.
Effect of 32 °G treatment after lesions haoe been formed at 22°G Ifplants kept at 22 °0 after inoculation were transferred to 32 °0, the lesions already present increased a little in size. In some experiments this increase was smaller with older lesions and also with lesions in older leaves compared with those in younger ones. In general, however, the increases were too small to measure accurately, the maximum radial increase being about 0'2 mm. As such an increase in lesion size can be considered to be due to the collapse of cells already irreversibly damaged at 22 °0, cell damage at 22 °0 was also investigated with the vital stain FDA. Results of one experiment are given in Fig. 5; 2 other experiments gave similar results. This figure shows that the nearer the cells were to the lesion edge, the sooner they lost their stain, i.e. the more damaged they were. Moreover, the zone of damaged cells was smaller when lesions were older. Statistical analysis of all observations of the three experiments with a trend test showed, that the zone of damaged cells was smaller around lesions in older leaves than around lesions in younger ones (a=O·OOl). The maximum number of destained cells, counted along a radial line, was about 60. As the width of one cell is approximately 20 urn, these 60 cells corresponded with 1'2 mm. The zone of irreversibly damaged cells, measured as increase
Lesion growth and virus localization
363
in lesion size at 32°C, was about 0·2 mm wide, corresponding with 10 cells. Apparently, in the more distant cells changes in the permeability of cell membranes had started at 22°C, but did not proceed at 32 °C. Lesions formed at 22°C increased in size after incubation at 32 °C for 24, 48 or 72 h followed by 22°C for I day, and this increase depended on the age of the lesio ns and the period at 32°C (Fig. 6). The older the lesions the smaller was the
10 9
e
'P
'P
° ~
3
_~
2
'<,
~
2
4
6
~
8
~
Days after lnocuIation (22'C1
FIG. 6. Increase in size oflesions formed at 22 ·C in leaves of Nieotiana tabacunt cv, Xanthi ne. infected with TMV, after a 32 ·C treatment. Inoculated plants were incubated at 22 ·C for different periods and subsequently at 32 ·C for 24 h (.),48 h (0) or 72 h (T) followed by incubation at 22 ·C for I day. Increase in lesion diameter is the difference between lesion diameters measured at the beginning and at the end of the second incubation period at 22 ·C. Each point is the mean from 5 plants, 5 leaves per plant and at least 5 lesions per leaf.
.... "...: .
.(/:. "\~? ~ ."
.
.
e ..
f): /'::~::. . ...
d "
.'.\'".'" t!!!f4J .~)'~\::" ' . f!1J . ... .... .. -,
....
-,
'::
A
~
.·W . ··
.."
.:'.: :: .....
:/.'
.'.::'
FIG. 7. Schematical representation of Icsions in leaves of Nicotlana iabacum C\". Xanthi nc. inoculated with T11V and incubated at 22 cc for different periods , showing various types of sectorial outgrowth after incubation at 32 cC for 1,2 or:3 days followed by 1 day at 22 eu . ~,Lesion formed after first incubation at 22 cC ; t;i, Sectorial outgrowth after 32 ·C treatment.
364
E. A. Weststeijn
increase, and the longer the period at 32 °C the larger was the increase in lesion size. It was also found that the increase in size oflesions in older leaves was somewhat smaller than that in younger leaves. The increase in size of very young lesions after these treatments was uniform in all directions. Older lesions, however, often showed a sectorial outgrowth or no outgrowth at all (Fig. 7). These phenomena started to occur already with lesions present after 4 to 6 days of incubation at 22 °C, and occurred more frequently with older lesions (Table 2). The length of the sectorial outgrowth from old lesions varied largely, e.g, from about 0·1 to 5 mm after 3 days of incubation at 32 °C. Table 2 also shows that the relative frequency of the different types of outgrowth depended on the length of the incubation period at 32 °C. Irregular outgrowth was also observed to occur earlier in older leaves than in younger ones. TABLE 2 Percentages of lesions, formed at 22 ec in leases of Nicotiana tabacum cv. Xanthi ne. infected uitl: TMV, showing dijferent tJpes of outgrowth after 32°C treatments and percentage of the circumference ofsuchlesionsthat shotced outgrowth
Lesions (%)a Subsequent incubation periods (days) at 22°C, 32°C and 22 -o, respectively 3, 1, 1 3,2, 1 3,3, 1 4, 1, 1 4,2, 1 4,3, 1 5, 1, 1 5,2, 1 5,3, 1 6, 1, 1 6,2, 1 6,3, 1 7, 1, 1 7,2, 1 7,3, 1 8, 1, 1 8,2, 1 8,3,1 9, 1, 1 9,2,1 9,3, 1 10, 1, I 10,2, 1 10,3, I 12, I, 1 12,2, 1 12,3, 1 a b
With uniform outgrowth
With sectorial outgrowth
Without outgrowth
0 0 0 12 1·8 0 33 1 0 58 46 28 52 59 46 56
0 0 0 2 0·2 0 9 1 0 19 8 0 45 17 1 42
25 0·5 6
55 56 65
20 43·5 29
0·2 4 12
36 52 51
63 44 37
3 21
44 48
53 31
100 100 100 86 98 100 58 98 100 23 46 72 3 24 53 2
Circumference (%)b oflesions with outgrowth 100
100
93
78
15
9
1·5
0·1
0
Data are from the same experiment as in Fig. 6. Each figure is the mean from 5 plants, 5 leaves per plant, 50 to 150 lesions per leaf. Data are from a separate experiment. Each figure is the mean from 10 lesions in one leaf.
365
Lesion growth and virus localization
Treatments at 32°C of inoculated plants showed, by the absence of formation of top necrosis, that spread of the virus from inoculated leaves to top leaves was also inhibited with longer periods of preceding incubation at 22 °C (Table 3). TABLE 3 Number of Nicotiana tabacum cv. Xanthi ne, plants inoculated with TAfV and incubated at 22 QC fOT different periods, showing top necrosis after incubation at 32 QC for 24, 48 or 72 h followed by incubation at 22 QC
Incubation period at 22 QC (days) Hours at 32 QC
24 48 72
0
O·
5
5 5
5 5
2
3
4
5
5 5 5
5 5 5
5 5 5
I
5 5
7
8
I
o
o
5 5
4
3 5
6
5
9
o
o 4
10
o
o o
12
o
o o
• Five plants per treatment, 5 successive leaves per plant were inoculated.
(a)
(b)
(c)
FIG. 8. Schematical representation of necroses formed in leaves of Nlcotiana tabacum cv, Xanthi ne, after inoculation with T.MV and incubation at 22 QC for 3 days, followed by 32 QC for 3 days, 22 QC for I to 8·5 h and again 32 QC for 1 day. ~, Lesion formed after 3 days at 22 QC; ~,tissue collapsed after interrupted incubation at 32 QC; period of interruption: (a) less than 5 to 6 hat 22°C, (b) 6·5 h at 22°C, (c) 8·5 h at 22°C.
EjJect on lesion formation of interruption of the incubation at 32°C by short periods at 22 °C Plants were kept at 22°C for 3 days after inoculation, allowing small lesions to form. Subsequently, the plants were incubated at 32 °C for 3 days, returned to 22°C for a short period ranging from 1 to 8·5 h, and again incubated at 32°C for 1 day, and shape and size of the necroses were studied (Figs 8 and 9). Interrupting the 32°C incubation for periods at 22 °C shorter than 5 to 6 h did not induce the formation of necrosis around the lesions formed after the first incubation at 22°C. An interval of 6·5 h induced necrotic spots at a little distance (approx. 1·5 mm) from the lesions. Longer periods at 22°C led to the formation of a uniform necrotic zone around the lesions. The zones were wider with longer intervals at 22 °C.
366
E. A. Weststeijn
9
•
•
7
7.5
• 6
6.5
8
8.5
Interval at 22°C (h)
FIG. 9. Lesion size in leaves of Nicotiana iabacum cv, Xanthi ne. after inoculation with T1-1V and incubation at 22 cC for 3 days, followed by 32 cC for 3 days, 22 cC for 6·5 to 8·5 h and again 32°C for I day. Each point is the mean from 5 plants, 3 leaves per plant and 5 lesions per leaf. Lesion sizes are significantly different (trend test, a=O·OI).
DISCUSSION
Size of lesions formed after incubation at 32°C followed by 22 °C was leaf age dependent (Fig. 2). The size differences may reflect leaf age dependent differences in multiplication and spread of the virus or in susceptibility to the necrotic reaction. Since the difference between the size of lesions formed after 24 and 48 h at 32°C was also leaf age dependent, and infectivity of the virus in and around these lesions was linearly related to lesion area (Fig. 3), it is most likely that the 32°C lesion size reflects multiplication and spread of the virus. However, the possibility that in younger leaves the virus had spread farther beyond the lesion edge than in older leaves cannot be excluded. Size of lesions formed at 22 °C was also leaf age dependent (Fig. 1). This size is determined by the rate ofmultiplication and spread of the virus and the rate of inhibition of these by the plant. Since uninhibited multiplication and spread of the virus was leaf age dependent, as was shown by 32°C treatments, at least part of the leaf age dependent differences in lesion size, as occurred at 22 °C, can be attribu- ted to differences in the multiplication and spread of the virus. The same may be concluded for the difference in lesion size between normal and systemic resistant leaves (Table I). Probably, there are also .leaf age dependent differences in the ability of the leaves to inhibit the virus, since increase in size of lesions formed after 32°C treatment, at 22 °C, was leaf age dependent, even when lesions were equal in size at the start of the incubation at 22°C (Fig. 4). Lesions formed at 22°C increased linearly for 6 to 7 days after inoculation (Fig. I) [12, 14], afterwards their growth gradually ceased and the virus became completely localized. Changes in the permeability of cell membranes were observed in a zone of cells around these lesions, extending gradually from much damage
Lesion growth and virus localization
367
in the cells just around the lesions to no damage at all at a distance. The width of this zone decreased with increasing lesion age, and was smaller around lesions in older leaves than around lesions in younger leaves (Fig. 5). Since permeability changes are one of the first detectable manifestations of the HR [15] this zone of damaged cells may give an indication of the spread of the virus. It seems less likely that these changes resulted from factors originating in the necrotic area, since the zone of damaged cells was widest around the youngest, smallest lesions. If this is true, it implies that the virus is present farther away from the edge of lesions in younger leaves than of lesions in older leaves and also farther from the edge of younger lesions than of older ones. Treatment at 32°C of lesions formed at 22 °C resulted in multiplication and spread of the virus. The amount of this decreased with increasing lesion age (Fig. 6) and with older lesions sectorial outgrowth was observed (Fig. 7 and Table 2). Therefore, the resistance against the virus, developed at 22°C in the cells around the lesions, was not nullified by the higher temperature. This was also shown for Samsun NN tobacco until 6 days after inoculation with TMV (13]. For complete localization of the virus, i.e. no multiplication and spread of the virus at 32°C, at least 12 days at 22 °C were necessary. The results mentioned here agree with the observation, made after 32°C treatment, that in Samsun NN tobacco with TMV, 5 days at 22°C after inoculation, localization in most leaves was not complete, but 15 days after inoculation more than 99% of the lesions showed complete localization [8]. However, sectorial outgrowth was not mentioned. Martin [6], however, using Xanthi ne, tobacco with TMV, stated that 30°C treatment after 15 days at 20 °C still resulted in necrotic rings around the lesions. Whether the resistance, visible as limited spread of the virus, is primarily due to inhibition of virus multiplication or to inhibition of the spread of the virus, is difficult to discern, as spread is a certain function of multiplication [7, 11]. The observation that with increasing lesion age increasing parts of the lesion edge did not show spread of the virus at 32°C, but that, if spread occurred, this was similar for the lesions of different ages (Fig. 6), may indicate that the final stage in localization is an inhibition of the spread of the virus. How virus multiplication leads to changes in the permeability of cell membranes is as yet a matter of speculation. A necrosis-inducing factor may be postulated, which is synthesized during virus multiplication, is functional at 22°C but not at 32 °C, and when non-functional is gradually broken down. Evidence for the existence of such a factor comes from the fact that a factor detrimental to protoplasts was shown to be present in infected, hypersensitively reacting tobacco, some 20h before lesions appeared, but not in systemically reacting tobacco [5], and may be inferred from the finding that the time at 22°C needed for irreversible induction of necrosis of 32 °C infected cells depended on the distance of the cells from the lesion edge (Figs 8 and 9). The infected cells closest to the lesion edge and those farthest away, which both needed 8·5 h at 22°C for collapse at 32 °C, would have less of this factor than the tissue in between, which needed 6·5 h at 22°C, the first zone, since some of this factor had already been broken down, the second zone, since not enough of this factor had been synthesized. This idea of synthesis and breakdown is supported by the observation that in Samsun NN tobacco with TMV, after incubation at
368
E. A. Weststeijn
32°C for 7 days followed by 22 °C for 24 h, necrotic rings appear around green islands [2], and that 30°C treatments for periods up to 7 days result in increasing amounts of necrosis in the whole plant, but longer periods result in decreasing amounts of necrosis, although much virus is present in the non-necrotic tissue [6]. I thank Dr ]. van den HeuveI for his valuable assistance in preparing the text. REFERENCES 1. BOZARTH, R. F. & Ross, A. F. (1964). Systemic resistance induced by localized virus infections. Extent of changes in virus infected plants. Virology 24, 446-455. 2. DA GRACA,J.V. & MARTIN, 11. M. (1976). An electron microscope study of hypersensitive tobacco infected with tobacco mosaic virus at 32 cc. Physiological Plant Pathology 8,215-219. 3. GOODL"iO, G. V. & HEDERT, T. T. (1967). A simple technique for purification of tobacco mosaic virus in large quantities. Phytopathology 57, 1285. 4. GUd'AS, A. & FARKAS, G. L. (1978). Is cell to cell contact necessary for the expression of the N-gene in Nicotiana tabacum cv. Xanthi ne, plants infected by TMV? Phytopathologische Zeitschrift 91, 182-187. 5. HOOLEY, R. & McCARTHY, D. (1980). Extracts from virus infected hypersensitive tobacco leaves are detrimental to protoplast survival. Physiological Plant Pathology 16, 25-38. 6. MARTIN, C. (1966). Contribution a I'etude du phenomene d'hypersensibilite du virus de la mosaique du tabac. Bulletin de la Sociltlfrancaise de Physiologie Vegetale 12, 345-354. 7. MA'ITlIEws, R. E. F. (1970). Plant Virology. Academic Press Inc. New York and London. 8. PRITClIARD, D. W. & Ross, A. F. (1973). Effect on virus localization of heat-treatments following inoculation of hypersensitive tobacco with tobacco mosaic virus. Abstracts of Papers 2nd International Congress of Plant Pathology, Unitersity rif Minnesota, Minneapolis, Minnesota, U.S.A., 5-12 September 1973. 9. Ross, A. F. (1961). Systemic acquired resistance induced by localized virus infections. Virology 14, 340-358. 10. TAKAlIASHl, T. (1972). Studies on viral pathogenesis in plant hosts Ill. Leaf age dependent susceptibility to tobacco mosaic virus infection in "Samsun NN" and "Samsun" tobacco plants. Phytopathologische Zeitschrift 75, 140-155. 11. TAKAlIASHI, T. (1973). Studies on viral pathogenesis in plant hosts IV. Comparison of early process of tobacco mosaic virus infection in the leaves of "Samsun NN" and "Samsun" tobacco plants. Phytopathologische Zeitschrift 77, 157-168. 12. TAKAHASllI, T. (1974). Studies on viral pathogenesis in plant hosts VI. The rate of primary lesion . growth in the leaves of "Samsun NN" tobacco to tobacco mosaic virus. Phytopathologische Zeitschrift 79, 53-66. 13. TAKAlIASlII, T. (1975). Studies on viral pathogenesis in plant hosts VIII. Systemic virus invasion and localization of infection in "Samsun 1\'N" tobacco plants resulting from tobacco mosaic virus infection. Phytopathologische Zeitschrift 84, 75-87. 14. WESTSTEIJN, E. A. (1976). Peroxidase activity in leaves of Nicotiana tabacum cv. Xanthi ne. before and after infection with tobacco mosaic virus. Physiological Plant Pathology 8, 63-71. 15. \VESTSTEIJN, E. A. (1978). Permeability changes in the hypersensitive reaction of Nicotiana tabacum C\'. Xanthi ne, after infection with tobacco mosaic virus, Physiological Plant Pathology 13,253-258. 16. \VIDUOLM, J. 11. (1972). The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technology 47, 189-194.
Lesion growth and virus localization in leaves of Nicotiana tabacum cv. Xanthi nc. after inoculation with tobacco mosaic virus and incubation alternately at 22°C and 32 °C EMMA
A.
WESTSTEIJN
Phytopathological Laboratory "lVillie Commelin Scholten", Javalaan 20, Baarn, The Netherlands
(Accepted/oT publication January 1981)
Factors which determine lesion size were studied with Nicotiana tabacum L. cv, Xanthi ne, and tobacco mosaic virus (TJ\IV). Inoculated plants were incubated alternately at 22 and 32 ·C. Incubation at 32 ·C was used as a condition under which multiplication and spread of the virus are uninhibited by the plant; incubation at 22 ·C for I day thereafter was necessary to visualize the spread of the virus at 32 ·C. Effect of different treatments on lesion size was compared in fully grown leaves of increasing age. Size of lesions formed at 22 ·C was leaf age dependent, as was size oflesions formed after incubation at 32 ·C followed by 22 ·C. Infectivity of the virus from the latter lesions was proportional to lesion area. Increase in size of these lesions at 22 ·C was also leaf age dependent. The width of the zone of damaged cells around lesions formed at 22 ·C depended on the age of the leaves and on the age of the lesions. Lesions formed at 22 ·C increased in size after incubation at 32 ·C followed by 22 ·C, and this increase depended on the age of the lesions. Furthermore, with older lesions, sectorial outgrowth occurred or lesions did not grow at all. For complete localization of the virus at least 12 days at 22 ·C were necessary. All effects mentioned here that were leaf or lesion age dependent were smaller with increasing leaf or lesion age. After interruption of the incubation at 32 ·C by short periods at 22 ·C necroses only appeared if the interval at 22 ·C was longer than 5 to 6 h; the size of these necroses was larger with increasing time interval at 22 ·C.
INTRODUCTION
The hypersensitive reaction (HR) of plants after infection with a virus is characterized by the appearance of necrotic local lesions. Usually this HR is accompanied by the localization of the virus. In Nicotiana tabacum L. cv. Xanthi ne. inoculated with tobacco mosaic virus (TMV), the lesions appear about 2 days after inoculation and increase in size until 8 to 10 days after inoculation. In this combination, lesion formation is temperature dependent. With increasing temperatures after inoculation up to about 27°C, lesion size increases, but at higher temperatures necrosis becomes inhibited. At 28 ·C only few necroses appear, which grow quickly, and the virus spreads through the whole plant. At 32°C no lesions develop at all. Instead, a systemic reaction occurs, comparable with the reaction at 22 °C of Nicotiana tabacum cv. Samsun infected with TMV [13]. However, extensive necrosis of the tissues invaded with virus occurs if the infected plants arc transferred from 32 to 22°C [6, 13]. The first signs of this necrosis become visible about 8 h after transfer [15]. After 3 to 6 h at 22°C, the induction of this necrosis is irreversible [4, 6]. 0048--4Q59/81/030357+12502.0010 © 1981 Academic Press Inc. (London) Limited
358
E. A. Weststeijn
Lesion size is also dependent on leaf age, i.e. with increasing age of fully grown leaves, lesion size decreases [1, 10, 12, 14], and also in leaves exhibiting systemic acquired resistance smaller lesions occur than in normal leaves [9]. The aim of the experiments described in this report, was to obtain more information about which factors determine lesion size. Lesion growth was analysed in inoculated plants after incubation alternately at 22 and 32°C. The effect of these treatments on lesions was studied in leaves of different ages. The temperature of 32 °C was used as a condition under which multiplication and spread of the virus are not inhibited by the plant, and 22°C for I day thereafter as a means to visualize the spread at 32 °C. MATERIALS AND METHODS
Tobacco plants (Nicoliana tabacum cv. Xanthi ne.) were grown in a glasshouse at about 22°C, with additional light during the winter for 16 h a day. TMV was purified from frozen TMV-infected Nicotiana tabacum cv. Samsun leaves with polyethylene glycol [3]. An appropriate dilution, inducing 50 to 150 lesions per leaf, served as inoculum. Inoculations were carried out by rubbing a drop of inoculum over the leaves with a finger, with carborundum as an abrasive. After inoculation the leaves were rinsed with tap water. Incubation at alternating temperatures included a first period, at 22°C, varying from 0 to 12 days after inoculation, a second period, at 32 °C,varying from 1 to 3 days, and a third period, again at 22°C, usually of 1 day, to evoke lesion formation. Incubations at 22°C were in the glasshouse with additional light during the winter for 16 h a day; incubations at 32°C were in growth chambers at 80% r.h. and a 16 h photoperiod. Size of lesions, if visible, was measured each time plants were transferred from one temperature to the other and in some experiments on some additional days after the transfer from 32 to 22°C. Lesion size was determined by measuring 2 perpendicular diameters with the aid of a magnifier with a micrometer. In case of sectorial outgrowth, the increase from the edge of the lesions formed at 22°C was measured. The infectivity of virus in and around lesions formed after incubation at 32°C followed by 22 °C for 1 day was used as a measure of virus multiplication at 32°C. Leaf disks, with a diameter of 9 mm with the lesion in the centre, were punched out with a cork borer. Each disk was homogenized separately with a mortar and pestle in I mI tap water and the homogenate inoculated on one-half leaf of Xanthi ne. tobacco, the other half receiving a standard TMV inoculum. Lesions were counted 3 days later and numbers were calibrated against the mean number on the control halves. Systemic acquired resistance was induced by inoculation of 3 fully grown leaves of Xanthi ne. tobacco plants with TMV and incubation at 22°C. Ten days later, 5 subsequent upper leaves were used as being resistant to TMV. Immediately after challenge inoculation, plants were incubated at 32°C for 2 days, and lesion size was measured I day after transfer of the plants to 22°C. Control plants were incubated at 22 °C for 7 days after challenge inoculation.
Lesion growth and virus localization
359
Damage of cells around lesions was investigated by staining sections through lesions and adjacent tissue with fluorescein diacetate (FDA), a vital stain, which is able to pass cell membranes. Inside the cell it is degraded to the green fluorescent compound, fluorescein, which cannot pass intact membranes. Intact cells retain the compound and damaged cells loose it [16]. Along a line perpendicular to the lesion edge, the number of palisade parenchyma cells, which had lost the stain were counted 15, 30, 45 and 60 min after administration of FDA. The number of destained cells did hardly increase, if at all, thereafter, Control tissue retained the stain for that period. RESULTS
Lesion growth at 22°C The general characteristics of lesion growth at 22 °C are shown in Fig. 1. Lesions grew steadily for approximately 7 days and reached their maximum size about 1 day later. Lesions in older leaves were smaller than those in younger leaves. (a)
(b)
3
E oS
~
° 2
•
°
E
o
'0
2
4
6
8
m u
Days after inoculaticn
2
3
4
5
leaf posilicn
FIG. I. Lesion size in leaves of different ages of Nicatiana tabacum cv. Xanthi ne, after inoculation with TMV and incubation at 22°C. (a) Development of lesion size in leaf I (.) and leaf 5 (0). (b) Lesion size 8 days after inoculation. Leaf I is the youngest fully grown leaf, the other numbers refer to successively older leaves. Each point is the mean from 15 leaves, 5 lesions per leaf, Lesion sizes for the different leaf positions are significantly different (trend test; day 3, a=O·OI; day 8, a=O·OOOI).
Effect of 32°C treatment immediately after inoculation To investigate the uninhibited spread of the virus in relation to leaf age, plants were kept after inoculation at 32°C for 24, 48 or 72 h, followed by 22°C for 1 day. These treatments resulted in local lesions in the inoculated leaves. The size of these lesions increased with increasing time at 32°C and were about 2, 4·2 and 7·5 mm, respectively. Lesion size (in leaves at 32°C for 24 or 48 h) depended also on leaf age (Fig. 2), in the same manner as the size of lesions formed at 22°C (Fig. 1), i.e. lesion size decreased with increasing leaf age. Experiments with plants kept at 32°C for 72 h gave similar results, but because many lesions coalesced, statistical analysis was impossible. The diameter of these lesions was about 7 mm for leaf 5 and about 8 mm for leaf 1.
360
E. A. Weststeijn
5
2 3 4 leaf position
5
FIG. 2. Lesion size in leaves of different ages of Nicotiana tabaeum cv, Xanthi ne. after inoculation with Tl\1V and incubation at 32 QCfor 24 h (e) or 48 h (0) followed by 22 QC for I day. Leaf numbers as in Fig. 1. Each point is the mean from 5 leaves, 10 lesions per leaf. Lesion sizes for the different leafpositions are significantly different (trend test; a=O·OOI).
'0 140 .!! '0 s:
..... c
•
120
0
0;
-. .!!
100
0
L
.0
E
••
80
"c
e 0
0;
60
.!!
.. L
Q,
• •
40
~
~u
.!!
20
.s 1·5
20
2·5
30
Lesion area (mm Z )
FIG. 3. Infectivity of virus in and around lesions differing in size. Lesions were formed in leaves of different ages of Nicotiana tabacum cv. Xanthi ne. after inoculation with Tl\1V and incubation at 32 QC for 24 h followed by 22 QCfor I day. Each point is the mean from about 10 lesions in a particular leaf.
Lesion growth and virus localization
361
In addition, a linear relationship was found between the infectivity of the virus in and around these lesions in leaves of different ages and the area of the lesions (Fig. 3). A challenge inoculation of systemic resistant leaves followed by incubation at 22°C for 7 days yielded, as expected, smaller lesions as the same treatment of normal leaves (Table 1). A similar result was obtained after incubation at 32°C for 2 days followed by 22 °C for 1 day, except that the reduction in lesion size in the latter treatment was smaller than in the former one. TABLE I Lesion size in systemicresistant (SAR) and normal leaces ofNicotiana tab acum eo, Xanthi ne, after challenge inoculation with TMV and incubation at 22°Cfor 7 dtl)'s (22°C treatment) or at srcfor 2 daysfollowed by 2rCfor 1 day (32 °C treatment)
22°C Treatment
32 °C Treatment
Lesion diameter (mm)
Lesion diameter (mm)
Expt no.
Normal
SAR
Percentage reduction
Normal
SAR
I
2·40 2·09
0·90 1-16
62 45
3·53 3·15
2-54
2
2'81
Percentage reduction 20 19
4
E
3
E
.
0;
E 0 '5
2 0
c 0
&x; ..J
-4
-2
3
5
7
9
11
Leaf position
FIG. 4. Lesion size in all leaves of Nicotiana tabacum cv, Xanthi ne. after inoculation with TMV and incubation at 32 °C for 24 h and at 22 °C for I day (e) a nd 7 days (0). Leaf numbers as in Fig. 1; negative numbers refer to not yet fully grown leaves. Example of I plant, 6 others showing similar results. Each point is the mean from 15 lesions, except for the youngest leaves.
Relation beuoeen uninhibited spread of the virus at 32 QC and growlh of lesions at 22 QC oftertoards Lesion size in all leaves ofaXanthi ne. tobacco plant after inoculation with TMV and incubation at 32 QC for 24 h followed by 22 QC for I day, is shown in Fig. 4.
362
E. A. Weststeijn
This figure shows how the uninhibited spread of the virus depended on leaf age. Incubation of the same plant at 22 °0 for another 6 days resulted in an increase in lesion size (Fig. 4). Lesions in younger leaves grew faster than lesions of equal initial size in older leaves.
50
.!!! 0;
40
u
."
'" C
'0
on
30
'"
."
'0 ~
20
~
E ::l
Z
10
2 Days after inoculation
FIG. 5. Number of cells which lost the vital stain FDA, around lesions formed in leaves of Nicatlana tabacum cv. Xanthi ne. after inoculation with TMV and incubation at 22 cC for different periods. Investigated cells were palis ade parenchyma cells along a radial line extending from the lesion edge. Measurements were taken at 15 min (e), 30 min (0), 45 min (T) and 60 min ('1) after administration ofFDA. Each point is the mean of35 to 45 measurements.
Effect of 32 °G treatment after lesions haoe been formed at 22°G Ifplants kept at 22 °0 after inoculation were transferred to 32 °0, the lesions already present increased a little in size. In some experiments this increase was smaller with older lesions and also with lesions in older leaves compared with those in younger ones. In general, however, the increases were too small to measure accurately, the maximum radial increase being about 0'2 mm. As such an increase in lesion size can be considered to be due to the collapse of cells already irreversibly damaged at 22 °0, cell damage at 22 °0 was also investigated with the vital stain FDA. Results of one experiment are given in Fig. 5; 2 other experiments gave similar results. This figure shows that the nearer the cells were to the lesion edge, the sooner they lost their stain, i.e. the more damaged they were. Moreover, the zone of damaged cells was smaller when lesions were older. Statistical analysis of all observations of the three experiments with a trend test showed, that the zone of damaged cells was smaller around lesions in older leaves than around lesions in younger ones (a=O·OOl). The maximum number of destained cells, counted along a radial line, was about 60. As the width of one cell is approximately 20 urn, these 60 cells corresponded with 1'2 mm. The zone of irreversibly damaged cells, measured as increase
Lesion growth and virus localization
363
in lesion size at 32°C, was about 0·2 mm wide, corresponding with 10 cells. Apparently, in the more distant cells changes in the permeability of cell membranes had started at 22°C, but did not proceed at 32 °C. Lesions formed at 22°C increased in size after incubation at 32 °C for 24, 48 or 72 h followed by 22°C for I day, and this increase depended on the age of the lesio ns and the period at 32°C (Fig. 6). The older the lesions the smaller was the
10 9
e
'P
'P
° ~
3
_~
2
'<,
~
2
4
6
~
8
~
Days after lnocuIation (22'C1
FIG. 6. Increase in size oflesions formed at 22 ·C in leaves of Nieotiana tabacunt cv, Xanthi ne. infected with TMV, after a 32 ·C treatment. Inoculated plants were incubated at 22 ·C for different periods and subsequently at 32 ·C for 24 h (.),48 h (0) or 72 h (T) followed by incubation at 22 ·C for I day. Increase in lesion diameter is the difference between lesion diameters measured at the beginning and at the end of the second incubation period at 22 ·C. Each point is the mean from 5 plants, 5 leaves per plant and at least 5 lesions per leaf.
.... "...: .
.(/:. "\~? ~ ."
.
.
e ..
f): /'::~::. . ...
d "
.'.\'".'" t!!!f4J .~)'~\::" ' . f!1J . ... .... .. -,
....
-,
'::
A
~
.·W . ··
.."
.:'.: :: .....
:/.'
.'.::'
FIG. 7. Schematical representation of Icsions in leaves of Nicotlana iabacum C\". Xanthi nc. inoculated with T11V and incubated at 22 cc for different periods , showing various types of sectorial outgrowth after incubation at 32 cC for 1,2 or:3 days followed by 1 day at 22 eu . ~,Lesion formed after first incubation at 22 cC ; t;i, Sectorial outgrowth after 32 ·C treatment.
364
E. A. Weststeijn
increase, and the longer the period at 32 °C the larger was the increase in lesion size. It was also found that the increase in size oflesions in older leaves was somewhat smaller than that in younger leaves. The increase in size of very young lesions after these treatments was uniform in all directions. Older lesions, however, often showed a sectorial outgrowth or no outgrowth at all (Fig. 7). These phenomena started to occur already with lesions present after 4 to 6 days of incubation at 22 °C, and occurred more frequently with older lesions (Table 2). The length of the sectorial outgrowth from old lesions varied largely, e.g, from about 0·1 to 5 mm after 3 days of incubation at 32 °C. Table 2 also shows that the relative frequency of the different types of outgrowth depended on the length of the incubation period at 32 °C. Irregular outgrowth was also observed to occur earlier in older leaves than in younger ones. TABLE 2 Percentages of lesions, formed at 22 ec in leases of Nicotiana tabacum cv. Xanthi ne. infected uitl: TMV, showing dijferent tJpes of outgrowth after 32°C treatments and percentage of the circumference ofsuchlesionsthat shotced outgrowth
Lesions (%)a Subsequent incubation periods (days) at 22°C, 32°C and 22 -o, respectively 3, 1, 1 3,2, 1 3,3, 1 4, 1, 1 4,2, 1 4,3, 1 5, 1, 1 5,2, 1 5,3, 1 6, 1, 1 6,2, 1 6,3, 1 7, 1, 1 7,2, 1 7,3, 1 8, 1, 1 8,2, 1 8,3,1 9, 1, 1 9,2,1 9,3, 1 10, 1, I 10,2, 1 10,3, I 12, I, 1 12,2, 1 12,3, 1 a b
With uniform outgrowth
With sectorial outgrowth
Without outgrowth
0 0 0 12 1·8 0 33 1 0 58 46 28 52 59 46 56
0 0 0 2 0·2 0 9 1 0 19 8 0 45 17 1 42
25 0·5 6
55 56 65
20 43·5 29
0·2 4 12
36 52 51
63 44 37
3 21
44 48
53 31
100 100 100 86 98 100 58 98 100 23 46 72 3 24 53 2
Circumference (%)b oflesions with outgrowth 100
100
93
78
15
9
1·5
0·1
0
Data are from the same experiment as in Fig. 6. Each figure is the mean from 5 plants, 5 leaves per plant, 50 to 150 lesions per leaf. Data are from a separate experiment. Each figure is the mean from 10 lesions in one leaf.
365
Lesion growth and virus localization
Treatments at 32°C of inoculated plants showed, by the absence of formation of top necrosis, that spread of the virus from inoculated leaves to top leaves was also inhibited with longer periods of preceding incubation at 22 °C (Table 3). TABLE 3 Number of Nicotiana tabacum cv. Xanthi ne, plants inoculated with TAfV and incubated at 22 QC fOT different periods, showing top necrosis after incubation at 32 QC for 24, 48 or 72 h followed by incubation at 22 QC
Incubation period at 22 QC (days) Hours at 32 QC
24 48 72
0
O·
5
5 5
5 5
2
3
4
5
5 5 5
5 5 5
5 5 5
I
5 5
7
8
I
o
o
5 5
4
3 5
6
5
9
o
o 4
10
o
o o
12
o
o o
• Five plants per treatment, 5 successive leaves per plant were inoculated.
(a)
(b)
(c)
FIG. 8. Schematical representation of necroses formed in leaves of Nlcotiana tabacum cv, Xanthi ne, after inoculation with T.MV and incubation at 22 QC for 3 days, followed by 32 QC for 3 days, 22 QC for I to 8·5 h and again 32 QC for 1 day. ~, Lesion formed after 3 days at 22 QC; ~,tissue collapsed after interrupted incubation at 32 QC; period of interruption: (a) less than 5 to 6 hat 22°C, (b) 6·5 h at 22°C, (c) 8·5 h at 22°C.
EjJect on lesion formation of interruption of the incubation at 32°C by short periods at 22 °C Plants were kept at 22°C for 3 days after inoculation, allowing small lesions to form. Subsequently, the plants were incubated at 32 °C for 3 days, returned to 22°C for a short period ranging from 1 to 8·5 h, and again incubated at 32°C for 1 day, and shape and size of the necroses were studied (Figs 8 and 9). Interrupting the 32°C incubation for periods at 22 °C shorter than 5 to 6 h did not induce the formation of necrosis around the lesions formed after the first incubation at 22°C. An interval of 6·5 h induced necrotic spots at a little distance (approx. 1·5 mm) from the lesions. Longer periods at 22°C led to the formation of a uniform necrotic zone around the lesions. The zones were wider with longer intervals at 22 °C.
366
E. A. Weststeijn
9
•
•
7
7.5
• 6
6.5
8
8.5
Interval at 22°C (h)
FIG. 9. Lesion size in leaves of Nicotiana iabacum cv, Xanthi ne. after inoculation with T1-1V and incubation at 22 cC for 3 days, followed by 32 cC for 3 days, 22 cC for 6·5 to 8·5 h and again 32°C for I day. Each point is the mean from 5 plants, 3 leaves per plant and 5 lesions per leaf. Lesion sizes are significantly different (trend test, a=O·OI).
DISCUSSION
Size of lesions formed after incubation at 32°C followed by 22 °C was leaf age dependent (Fig. 2). The size differences may reflect leaf age dependent differences in multiplication and spread of the virus or in susceptibility to the necrotic reaction. Since the difference between the size of lesions formed after 24 and 48 h at 32°C was also leaf age dependent, and infectivity of the virus in and around these lesions was linearly related to lesion area (Fig. 3), it is most likely that the 32°C lesion size reflects multiplication and spread of the virus. However, the possibility that in younger leaves the virus had spread farther beyond the lesion edge than in older leaves cannot be excluded. Size of lesions formed at 22 °C was also leaf age dependent (Fig. 1). This size is determined by the rate ofmultiplication and spread of the virus and the rate of inhibition of these by the plant. Since uninhibited multiplication and spread of the virus was leaf age dependent, as was shown by 32°C treatments, at least part of the leaf age dependent differences in lesion size, as occurred at 22 °C, can be attribu- ted to differences in the multiplication and spread of the virus. The same may be concluded for the difference in lesion size between normal and systemic resistant leaves (Table I). Probably, there are also .leaf age dependent differences in the ability of the leaves to inhibit the virus, since increase in size of lesions formed after 32°C treatment, at 22 °C, was leaf age dependent, even when lesions were equal in size at the start of the incubation at 22°C (Fig. 4). Lesions formed at 22°C increased linearly for 6 to 7 days after inoculation (Fig. I) [12, 14], afterwards their growth gradually ceased and the virus became completely localized. Changes in the permeability of cell membranes were observed in a zone of cells around these lesions, extending gradually from much damage
Lesion growth and virus localization
367
in the cells just around the lesions to no damage at all at a distance. The width of this zone decreased with increasing lesion age, and was smaller around lesions in older leaves than around lesions in younger leaves (Fig. 5). Since permeability changes are one of the first detectable manifestations of the HR [15] this zone of damaged cells may give an indication of the spread of the virus. It seems less likely that these changes resulted from factors originating in the necrotic area, since the zone of damaged cells was widest around the youngest, smallest lesions. If this is true, it implies that the virus is present farther away from the edge of lesions in younger leaves than of lesions in older leaves and also farther from the edge of younger lesions than of older ones. Treatment at 32°C of lesions formed at 22 °C resulted in multiplication and spread of the virus. The amount of this decreased with increasing lesion age (Fig. 6) and with older lesions sectorial outgrowth was observed (Fig. 7 and Table 2). Therefore, the resistance against the virus, developed at 22°C in the cells around the lesions, was not nullified by the higher temperature. This was also shown for Samsun NN tobacco until 6 days after inoculation with TMV (13]. For complete localization of the virus, i.e. no multiplication and spread of the virus at 32°C, at least 12 days at 22 °C were necessary. The results mentioned here agree with the observation, made after 32°C treatment, that in Samsun NN tobacco with TMV, 5 days at 22°C after inoculation, localization in most leaves was not complete, but 15 days after inoculation more than 99% of the lesions showed complete localization [8]. However, sectorial outgrowth was not mentioned. Martin [6], however, using Xanthi ne, tobacco with TMV, stated that 30°C treatment after 15 days at 20 °C still resulted in necrotic rings around the lesions. Whether the resistance, visible as limited spread of the virus, is primarily due to inhibition of virus multiplication or to inhibition of the spread of the virus, is difficult to discern, as spread is a certain function of multiplication [7, 11]. The observation that with increasing lesion age increasing parts of the lesion edge did not show spread of the virus at 32°C, but that, if spread occurred, this was similar for the lesions of different ages (Fig. 6), may indicate that the final stage in localization is an inhibition of the spread of the virus. How virus multiplication leads to changes in the permeability of cell membranes is as yet a matter of speculation. A necrosis-inducing factor may be postulated, which is synthesized during virus multiplication, is functional at 22°C but not at 32 °C, and when non-functional is gradually broken down. Evidence for the existence of such a factor comes from the fact that a factor detrimental to protoplasts was shown to be present in infected, hypersensitively reacting tobacco, some 20h before lesions appeared, but not in systemically reacting tobacco [5], and may be inferred from the finding that the time at 22°C needed for irreversible induction of necrosis of 32 °C infected cells depended on the distance of the cells from the lesion edge (Figs 8 and 9). The infected cells closest to the lesion edge and those farthest away, which both needed 8·5 h at 22°C for collapse at 32 °C, would have less of this factor than the tissue in between, which needed 6·5 h at 22°C, the first zone, since some of this factor had already been broken down, the second zone, since not enough of this factor had been synthesized. This idea of synthesis and breakdown is supported by the observation that in Samsun NN tobacco with TMV, after incubation at
368
E. A. Weststeijn
32°C for 7 days followed by 22 °C for 24 h, necrotic rings appear around green islands [2], and that 30°C treatments for periods up to 7 days result in increasing amounts of necrosis in the whole plant, but longer periods result in decreasing amounts of necrosis, although much virus is present in the non-necrotic tissue [6]. I thank Dr ]. van den HeuveI for his valuable assistance in preparing the text. REFERENCES 1. BOZARTH, R. F. & Ross, A. F. (1964). Systemic resistance induced by localized virus infections. Extent of changes in virus infected plants. Virology 24, 446-455. 2. DA GRACA,J.V. & MARTIN, 11. M. (1976). An electron microscope study of hypersensitive tobacco infected with tobacco mosaic virus at 32 cc. Physiological Plant Pathology 8,215-219. 3. GOODL"iO, G. V. & HEDERT, T. T. (1967). A simple technique for purification of tobacco mosaic virus in large quantities. Phytopathology 57, 1285. 4. GUd'AS, A. & FARKAS, G. L. (1978). Is cell to cell contact necessary for the expression of the N-gene in Nicotiana tabacum cv. Xanthi ne, plants infected by TMV? Phytopathologische Zeitschrift 91, 182-187. 5. HOOLEY, R. & McCARTHY, D. (1980). Extracts from virus infected hypersensitive tobacco leaves are detrimental to protoplast survival. Physiological Plant Pathology 16, 25-38. 6. MARTIN, C. (1966). Contribution a I'etude du phenomene d'hypersensibilite du virus de la mosaique du tabac. Bulletin de la Sociltlfrancaise de Physiologie Vegetale 12, 345-354. 7. MA'ITlIEws, R. E. F. (1970). Plant Virology. Academic Press Inc. New York and London. 8. PRITClIARD, D. W. & Ross, A. F. (1973). Effect on virus localization of heat-treatments following inoculation of hypersensitive tobacco with tobacco mosaic virus. Abstracts of Papers 2nd International Congress of Plant Pathology, Unitersity rif Minnesota, Minneapolis, Minnesota, U.S.A., 5-12 September 1973. 9. Ross, A. F. (1961). Systemic acquired resistance induced by localized virus infections. Virology 14, 340-358. 10. TAKAlIASHl, T. (1972). Studies on viral pathogenesis in plant hosts Ill. Leaf age dependent susceptibility to tobacco mosaic virus infection in "Samsun NN" and "Samsun" tobacco plants. Phytopathologische Zeitschrift 75, 140-155. 11. TAKAlIASHI, T. (1973). Studies on viral pathogenesis in plant hosts IV. Comparison of early process of tobacco mosaic virus infection in the leaves of "Samsun NN" and "Samsun" tobacco plants. Phytopathologische Zeitschrift 77, 157-168. 12. TAKAHASllI, T. (1974). Studies on viral pathogenesis in plant hosts VI. The rate of primary lesion . growth in the leaves of "Samsun NN" tobacco to tobacco mosaic virus. Phytopathologische Zeitschrift 79, 53-66. 13. TAKAlIASlII, T. (1975). Studies on viral pathogenesis in plant hosts VIII. Systemic virus invasion and localization of infection in "Samsun 1\'N" tobacco plants resulting from tobacco mosaic virus infection. Phytopathologische Zeitschrift 84, 75-87. 14. WESTSTEIJN, E. A. (1976). Peroxidase activity in leaves of Nicotiana tabacum cv. Xanthi ne. before and after infection with tobacco mosaic virus. Physiological Plant Pathology 8, 63-71. 15. \VESTSTEIJN, E. A. (1978). Permeability changes in the hypersensitive reaction of Nicotiana tabacum C\'. Xanthi ne, after infection with tobacco mosaic virus, Physiological Plant Pathology 13,253-258. 16. \VIDUOLM, J. 11. (1972). The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technology 47, 189-194.