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Respiratory changes of barley leaves infected with Pyrenophora teres or affected by isolated toxins of this fungus
Physiological
Plant Pathology
(1977)
10, 213-220
Respiratory changes of barley leaves infected with Pyrenophora teres or affected by isolated toxins of this fungus V.
SMEDEG~RD-PETERSEN
Department of Plant Pathologv, llz Royal Thorvaldsensvej 40, DK-1871 Copenhagen (Accepted for publication
January
Veterinary and Agricultural V, Denmark
University,
1977)
Susceptible barley leaves infected with Pyrenofihora teres responded with a rapid and pronounced increase in the rate of respiration. The increase was significant within 16 h after inoculation and occurred far in advance of the appearance of visible symptoms. The respiration continued to increase until the fourth day after inoculation when it reached a maximum which was 322% higher than in healthy control leaves. It then sharply decreased and 7 days after inoculation the rate was lower in infected than in healthy leaves. Maximum respiration coincided with the appearance of visible necrosis. All evidence indicated that the major part of the increased oxygen uptake in infected leaves was contributed by the host tissues whereas the pathogen contributed only a little. Susceptible barley leaves which were allowed to take up solutions of purified P. teres toxins reacted with an increased rate of respiration similar to that of infected leaves. The increase was significant within 24 h after the start of toxin uptake. Leaves of non-host plants and resistant barley leaves reacted to toxin treatment with a toxin considerably lower increase in oxygen consumption than susceptible leaves. In addition, treatment failed to stimulate oxygen consumption further in infected leaves. Respiratory changes in plants are often considered to be a general response to disturbance Even so, the results and results on that subject should therefore be interpreted with caution. support previous results indicating that the toxins of P. teres may be a factor in the pathogenesis of net-spot blotch of barley.
INTRODUCTION
Visible symptoms of net-spot blotch of barley can be caused not only by the pathogen Pyrenophora teres Drechs. but also to a large extent by two toxins produced by this fungus. The detection and isolation of these toxins and evidence for their significance in the disease syndrome of net-spot blotch have been previously described [S]. The purpose of the present work was to study the effect of infection by P. tereson the respiratory rate of the host and to compare this effect with that incited by isolated toxins of the fungus. Such investigations might contribute to a better understanding of the significance of toxins in the physiological processes involved in pathogenesis of net-spot blotch. MATERIALS
AND
METHODS
The two barley cultivars “Wing” (highly susceptible) and “CI 9647” (resistant), the oat cultivar “Astor” and the wheat cultivar ‘Kranich” were used throughout the experiments.
214
V. Smedegbd-Petersen
In infection experiments 1Z-day-old barley seedlings grown under ordinary greenhouse conditions were inoculated with a mycelium suspension obtained from &dayold cultures of Pyrenophora teres f. teres, the ordinary form of the fungus which produces net-like necrotic lesions [5], grown on liquid medium. Occasionally spore suspensions obtained from sporulating leaves were used as inoculum. The inoculation procedure has been previously described [5]. Respiration measurements were performed with whole, detached leaves, about 6 cm in length. Only the first leaf of each plant was used in the tests. Before being placed in the Warburg reaction flasks, leaves were rinsed with water and gently rubbed with wet cotton in order to remove mycelial residue from the surface. Control leaves were treated in the same way. In experiments with toxin-treated leaves toxin solutions containing equal amounts of toxins A and B in pure form were used. The two toxins were isolated from culture filtrate by extraction, gel filtration and ion exchange chromatography as previously described [S]. Evidence for purity of the toxin preparations used in the tests was obtained by paper chromatography and thin-layer chromatography on cellulose and silica gel plates using four different solvent systems [6]. For toxin treatment the first leaves of IZday-old plants were excised, placed in small test tubes with the cut ends in the toxin solution and allowed to take up toxin for a specified time. For controls, comparable leaves were placed in test tubes with water. During the time of toxin uptake the test tubes with the leaves were placed in the laboratory and illuminated with a 100 W Osram bulb situated 60 cm above the leaves. In the following solutions of toxins A and B will be designated as P. teres toxins only. Oxygen uptake was determined with a Warburg apparatus using standard manometric techniques [7]. The Warburg reaction flasks (type A 05-72, 36 ml manufactured by Braun Melsungen, Germany) were designed for botanical work with whole leaves. Detached leaves, 6 cm in length, were placed in the reaction flasks on 6 cm2 pieces of filter paper moistened with O-5 ml of water. Carbon dioxide was absorbed by 0.2 ml 10% KOH applied to the centre well. The reaction flasks and manometers were allowed to equilibrate for 30 min and oxygen consumption measured at 25 “C in complete darkness to prevent photosynthesis. Readings were taken at intervals of 30 min during a period of 1 or 2 h. After completion of readings the leaves were dried at 110 “C for 24 h for dry matter determination. From three to six replications were carried out in all experiments. Respiration was calculated on a dry weight basis as 4 oxygen uptake per 10 mg dry weight of tissue per h, or as oxygen uptake per reaction flask. In the latter case special care was taken to ensure equal quantities of leaf tissue in each reaction flask. Microscopic examination of fungal penetration and development of hyphae in the tissue was performed at intervals of 24 h, starting 12 h after inoculation. Leaf sections of 1 cm length were cleared in a mixture of boiling glacial acetic acid and glycerol (1 : 1). After evaporation of the acetic acid, the sections were transferred to small vials with lactophenol-cotton blue (100 ml lactophenol + O-5 ml aniline blue) and gently heated for 1 min, then rinsed and mounted in lactophenol for microscopic examination.
Respiration
of
Pyrenophera feres infected
RESULTS The effect of infection leaves
barley
with Pyrenophora
215
leaves
teres on the respiratory
rate in susceptible barley
Oxygen consumption by infected and non-infected barley leaves was measured daily for 7 days after inoculation. The results are shown in Table 1. Already 16 h after inoculation oxygen uptake was 23.4% higher in the infected than in the non-infected leaves. The rate continued to increase until the fourth day after inoculation when it was 322.8% higher in the infected than in the non-infected control leaves. From the fourth day the rate sharply decreased and on the seventh day oxygen uptake in infected leaves was 26% lower than in the control leaves. TABLE
1
The effectof I?. teres infection on respiration of susceptiblebarlg leaves 0, Days after inoculation
uptake
Non-infected control
0.7 (16 h) :
14.1 16.8 14-9
3 4 5 6 7
13.3 12.7 13.9 16.4 16.9
(l~l/lO wt/h)
mg
dry
Infected 17.4 29.8 36.4 49.3 53.7 40.7 37.6 12.5
Increase ( y0 of non-infected)
Symptomsd
23.4b 77.4c 144*3c 270.7c 322.80 192.8c 129.3c - 26*OG
as 67 o Significant at 5, 1 and 0.1 o/0 probability level, respectively. d 0, no visible symptoms. 1, slightly developed dark brown lesions, slight chlorosis. 2, restricted dark brown spots or streaks, slight chlorosis. 3, dark brown spots or streaks, marked chlorosis and necrosis. 4, dark brown spots or streaks, extensive chlorosis and necrosis.
Time relationship between progress of infection, respiratory rate of susceptible barley leaves
symptom
development
and increase in the
In order to relate the increase in the respiratory rate of inoculated leaves with the progress of the invading pathogen, microscopic examination of cleared and stained leaf sections was carried out each day concurrently with respiratory measurements. In addition, daily notes were taken on symptoms using a rating scale from 0 to 4 (see footnote to Table 1). Sixteen h after inoculation the germinating spores or hyphal fragments used for inoculation had formed appressoria appearing as swellings on the germ tubes. Infection pegs were penetrating the cell wall either directly or through the stomata, but in no cases could infection hyphae be detected beneath the epidermal cells. The penetration sites were easiIy recognizable since they were always surrounded by a ring of densely stained tissue. One day (24 h) after inoculation many infection hyphae had completed penetration through the outer epidermal wall and formed large swellings (vesicles) in the
216
V. Smedeghd-Petersen
epidermal cells. In a few cases the hyphae had penetrated further into the mesophyll but the distance never exceeded two to three cells. Two days after inoculation the infection hyphae had penetrated into a depth of six to eight mesophyll cells. Hyphae had started to branch, but the single cells were short and swollen and always occurred intercellularly. The longest distance in the tissue any hyphae had advanced from the penetration site was 900 F. Three days after inoculation the hyphae had penetrated further into the mesophyll and had often grown to a distance beyond the stained areas surrounding the penetration sites. Seven days after inoculation mycelium was extensively present in large areas of the infected leaves. The first visible symptoms could be observed 2 to 3 days after inoculation as slight chlorosis and faint brown spots. After 5 days dark net lesions had appeared and 7 days after inoculation extensive chlorosis and necrosis occurred in large areas of the leaves. Comparing the respiratory rates with the progress of invading hyphae in the tissue, it is apparent that the host responded with an increase in respiration at a very early stage in the infection process, apparently already during the penetration of the epidermal cell and far before any visible symptoms could be detected. The maximum respiratory rate, occurring on the third and fourth day after inoculation, coincided with the appearance of the first visible symptoms. At this time the mycelium had only spread to a limited degree in the tissue. The rapid decline in respiration on the fifth and sixth day occurred at the same time as the beginning of necrosis. At this time mycelium was extensively present in large parts of the leaves. i3.e e$ectof th P. teres toxins on the resj&-atoryrate in susceptiblebarley leaves. The host response to the toxins of P. tereswas measured on excised leaves of the barley cultivar “Wing”. Leaves were placed with the cut end in a solution containing 300 pg pure toxin/ml and allowed to take up approximately 0.50 ml of solution/leaf. After uptake of toxin, leaves were transferred to water. The reason for using a toxin concentration of 300 &g/ml, which may seem exceedingly high considering that the threshold activity of the toxin is about 25 pg/ml, is that each leaf after uptake of 0.50 ml of toxin solution of this concentration will contain a toxin content which corresponds to the toxin content found in leaves heavily infected with the pathogen, namely about 400 pgglg fresh weight [S]. Measurements of oxygen uptake were made daily for 7 days after treatment of the leaves with the toxin solution. The results are shown in Table 2. One day after the start of treatment the oxygen uptake was 40.5% higher in toxin-treated leaves than in the water-treated control leaves. The maximum respiratory rate occurred 2 days after treatment with 73.6% higher oxygen uptake in toxin-treated leaves than in control leaves. From this date the respiratory rate gradually decreased in the toxin-treated leaves whereas it remained almost constant in the water-treated control leaves. Seven days after treatment the respiration in toxin-treated leaves was 43.5% lower than in the control leaves. As was the case in infected leaves, the increase in respiration appeared much in advance of the visible symptoms. The first visible symptoms were noted 3 days after
Respiration
of fyrenophera
feres
infected
barley
TABLE The effect of P. teres Days after start of toxin treatment
0, uptake Water-treated control
: 3 4 5 7
(pl/lO
19.5 17.8 19.8 18.6 17.5 16.8
217
leaves 2
toxin on respiration
of suxepible
ban$
leaves Symptoms toxin-treated leavesd
mg dry wt/h) Increase (% of control)
Toxin-treated
30.9 27.4 33.3 26.5 27.8 9.5
40.5b 73.60 68.2O 425” 58.9” - 43.50
ov b* 0 Significant at 5, 1 and 0.1 o/0 probability level, respectively. d 0, no visible symptoms. 1, slight chlorosis. 2, marked chlorosis, slight necrosis. 3, marked chlorosis and necrosis. 4, extensive chlorosis and necrosis. Toxin-treated leaves were allowed to take up 0.50 ml of a toxin pure toxin/ml and were then transferred to water.
solution
of
0 1 1-2 2 4
containing
300 pg
treatment, whereas a significant increase in oxygen uptake occurred within 24 h after start of toxin treatment (Table 2). Maximum oxygen uptake occurred just before the onset of visible necrosis. Comparing Tables 1 and 2 it appears that the host tissue responds similarly to infection and toxin treatment. The less pronounced rise in oxygen consumption after toxin treatment than after infection is presumably related to the toxin concentration employed. That a direct relationship exists between the rate of respiration and the toxin concentration can be seen in Table 3 which shows the influence of two toxin concentrations of 300 and 600 pg/ml on respiration of YVing” barley leaves. It is seen that the two toxin concentrations increased the respiratory rate by 35-9 and 60.5 %, respectively. These data clearly demonstrate that the respiratory rate increases with increasing toxin concentration. A similar relationship was also found to exist between toxin concentration and the severity of visible symptoms. TABLE
3
The effect of P. teres toxin on respiration Disease reaction by inoculation with P. ten+ Barley, “Wig” (susceptible) Barley ,“CI 9647” (resistant) Wheat Oats
0, Water-treated control
of bar&,
wheat and oats
uptake (ul/lO mg dry wt/h) 300 (rg Increase 600 pg toxin/ml (%) toxin/ml 22.7
4
16.7
l-2
18.0
21.5
19*5c
1 0
15.2 17.8
18.7 18.5
23.0” 3.9
a* ba c Significant at 5, 1 and 0.1 y0 probability level, respectively. 6 See footnote to Table 1. Toxin-treated leaves were allowed to take up toxin solutions in the Warburg reaction flasks.
35.9”
for 24 h before
26.8
Increase (%)
being
60.5c
placed
218
V. Smedeghd-Petersen
The similar respiratory response in barley leaves to infection and toxin treatment indicates that the toxins may be involved in the pathologically enhanced respiration in barley leaves infected with P. teres. The e$ect of the P. teres toxins on the respiratory rate of host plants resistance
with &yering
degrees of
Studies were performed to investigate whether the toxins exert a selective respiratory effect on hosts possessing different, degrees of resistance against P. teres. The barley cultivars “Wing” and “CI 9647” were chosen as highly susceptible and resistant test plants, respectively. Wheat and oats were chosen as highly resistant and non-host plants, respectively. Wheat and oats are usually both considered to be non-hosts for P. teres. However, many infection experiments by the present author revealed that wheat upon artificial inoculation is slightly susceptible reacting with slight chlorosis but not with the dark net lesions which are typical for barley. The fungus could be reisolated from infected wheat leaves and weak sporulation was occasionally observed on diseased leaves placed on agar. Oats occasionally showed a very slight chlorosis upon heavy inoculation but the fungus could not be isolated from inoculated leaves and this species was consequently considered as a non-host. Table 3 shows the effect of 24 h of toxin uptake (600 pg toxin/ml) on tissue with differing degree of resistance. The susceptible barley cultivar “Wing” reacted with an increase in oxygen uptake of 60.5%, whereas the resistant cultivar “CI 9647” showed only a 19.5 o/o increase. Wheat (highly resistant) reacted with an increase of 23.0% and oats (non-host) with an increase of 3.9%. These results suggest that highly susceptible tissue responds to the toxins with a more pronounced increase in respiration than resistant tissue, and that non-host tissue is almost unaffected by the toxin. More extensive studies comprising a wider range of host and non-host plants may be needed before conclusions can be drawn as to the host specificity of the P. teres toxins. However, the results are in agreement with previous results [6] demonstrating a good, although not complete, correlation between the host range of the pathogen and that of the toxins. The effect of P. teres toxins on respiration
of barley leaves previously infected with P. teres
The fact that P. teres pathogen and its isolated toxins cause similar increases in the rate of respiration of susceptible barley leaves suggests that the toxins constitute a causal factor in the pathologically enhanced respiration. If this assumption is correct, the addition of moderate concentrations of purified toxins would not be expected to cause further respiratory increases in barley leaves previously infected with the pathogen. Table 4 shows that infected leaves which were allowed to take up water for 24 h exhibited an increase in oxygen consumption amounting to 2OO*9o/oof healthy leaves treated in the same way. Infected leaves which were allowed to take up a toxin solution (600 pg purified toxins/ml) for 24 h exhibited an increase amounting to 205.6% of the healthy leaves. The difference of 4.7% is not significant. The fact that toxin treatment failed to stimulate oxygen uptake in leaves previously infected with P. teres supports the view that the toxins produced by this
Respiration
of Pyrenophera
feres infected
barley
TABLE 77te effeckctof the P. teres toxins
on respiration
Healthy Infected Infected
leaves + water leaves + water leaves + toxin
4
of barley leaves previousb pathogen 0,
Treatment
219
leaves
(~1/10
uptake mg dry
Wh)
10.7 32.2 32.7
infected
with the P. teres
Increase (% of healthy)
200.9 205.6”
Four days after inoculation infected leaves were excised and allowed to take up deionized water or toxin solution (600 pg purified toxin/ml) for 24 h before being placed in the Warburg reaction flasks. a The difference between infected leaves+water and infected leaves+ toxin was not significant.
pathogen may constitute a cause of the pathologically leaves with net-spot blotch.
enhanced respiration of barley
DISCUSSION
Already 16 h after inoculation the respiratory rate of susceptible barley leaves significantly exceeded that of non-inoculated control leaves. At that time the infection hyphae had hardly more than penetrated the epidermal cells and had not moved into the mesophyll to any appreciable extent. This suggests that the Pyrenophora teres toxins move in advance of the hyphae and stimulate the metabolic apparatus of the tissue to an increased rate of respiration. Presumably the toxin is released during penetration of the epidermal wall. The assumption that the early metabolic response to infection is due to toxins released from the penetrating hyphae is supported by the fact that the purified P. teres toxins, when produced in vitro and introduced into healthy leaves in concentrations comparable to those found in leaves heavily infected with the pathogen, cause an increased rate of respiration. The early metabolic response to infection with P. teres is in contrast to the situation found in most other host-pathogen combinations involving non-obligate parasites. In oats infected with Helminthosporium victoriae a marked increase occurred on the third day after inoculation and coincided with the appearance of visible symptoms [3]. Similar conditions were found in maize leaves infected with Helminthosporium carbonum, where Kuo & Scheffer [4] noted a pathological rise in oxygen uptake starting 2 days after inoculation. The question naturally arises concerning how much the pathogen itself contributes to the increased respiratory rate. This aspect has been studied in other diseases, e.g. on cereals infected with powdery mildew and rust [I, 2, 81. Although the contribution by the pathogen is generally considered to be very limited, the results and conclusions from these investigations are contradictory. In the present host-pathogen combination there are several lines of evidence to suggest that the pathological increase in respiration is contributed almost exclusively by the host and that the pathogen only contributes a little. Thus the increase in respiration occurred during the first 4 days after inoculation during which period the 18
220
V. Smedeghd-Petersen
amount of mycelium in the host tissue was very limited. After the onset of necrosis the rate sharply decreased whereas the amount of mycelium in the tissue rapidly increased. Furthermore, the rise in the respiratory rate started within 16 h after inoculation and at this time the infection hyphae had hardly more than penetrated the epidermal cells. As the leaves were rinsed with water and gently rubbed with wet cotton before being placed in the Warburg reaction flasks in order to remove spores and surplus mycelial fragments applied by inoculation, the amount of mycelium in the leaves 16 and 24 h after inoculation may be considered negligible. Finally, a respiratory increase could be induced without the presence of the pathogen by applying biological concentrations of purified toxins to the leaves. Previous results [6] suggest that the toxins of P. teres induce the most important visible symptoms of the disease. The present results suggest that the toxins also induce respiratory changes in susceptible tissue which parallel those induced by the pathogen. The data further indicate that highly susceptible host tissue reacts with a greater increase in respiration than resistant tissue in response to P. teres toxins, and that non-host tissue is almost unaffected by both the pathogen and its toxins. It is well known that increased respiration is a common phenomenon in diseased plants and that many factors including chemicals and wounding may cause respiration to increase. Hence results on that subject should be interpreted with caution. Even so, the data presented in this paper seem to support previous results [6] indicating that the toxins of P. teres may be a factor in the pathogenesis of net-spot blotch of barley. The author is indebted to Dr Kurt Christiansen, Department of Plant Physiology, for advice and discussions. The investigations were supported by the Danish Agricultural and Veterinary Research Council. REFERENCES I. ALLEN, P. J. & GODDARD, D. R. ( 1938). A respiratory study of powdery mildew of wheat. American Journal of Botany 25, 6 13-62 1. 2. BUSHNELL, W. R. & ALLEN, P. J. (1962). Respiratory changes in barley leaves produced by single colonies of powdery mildew. Plant Physioloa 37, 751-758. 3. GRIMM, R. B. & WHEELER, H. (1963). Respiratory and enzymatic changes in Victoria blight of oats. Phytopathology 53, 436-440. 4. Kuo, M. & SGHEFFER, R. P. ( 1970). Comparative effects of host-specific toxins and Helminthosporium infections on respiration and carboxylation by host tissue. Phytopathology 60, 1391-1394. 5. SMEDEG~D-PETERSEN, V. (197 1). Pyrenophora teres f. maxulata f. nov. and Pyrenophora teres f. teres on barley in Denmark. Royal Veterinary and Agricultural Universi~ Yearbook 1971, 1241144. 6. SMEDEG~RD-PETERSEN, V. (1977). Isolation of two toxins produced by Pyrenophora teres and their significance in disease development of net-spot blotch of barley. Physiological Plant Pathology 10, 203-211. 7. UMBREIT, W. W., BURRIS, R. H. & STAUPFER, J. F. (1964). M mom&c Techniques, 305 pp. Burgess Publishing Co., Minneapolis, Minn. 8. YARWOOD, C. E. (1953). Heat of respiration of injured and diseased leaves. Phytopatholop 43, 675-681.
Plant Pathology
(1977)
10, 213-220
Respiratory changes of barley leaves infected with Pyrenophora teres or affected by isolated toxins of this fungus V.
SMEDEG~RD-PETERSEN
Department of Plant Pathologv, llz Royal Thorvaldsensvej 40, DK-1871 Copenhagen (Accepted for publication
January
Veterinary and Agricultural V, Denmark
University,
1977)
Susceptible barley leaves infected with Pyrenofihora teres responded with a rapid and pronounced increase in the rate of respiration. The increase was significant within 16 h after inoculation and occurred far in advance of the appearance of visible symptoms. The respiration continued to increase until the fourth day after inoculation when it reached a maximum which was 322% higher than in healthy control leaves. It then sharply decreased and 7 days after inoculation the rate was lower in infected than in healthy leaves. Maximum respiration coincided with the appearance of visible necrosis. All evidence indicated that the major part of the increased oxygen uptake in infected leaves was contributed by the host tissues whereas the pathogen contributed only a little. Susceptible barley leaves which were allowed to take up solutions of purified P. teres toxins reacted with an increased rate of respiration similar to that of infected leaves. The increase was significant within 24 h after the start of toxin uptake. Leaves of non-host plants and resistant barley leaves reacted to toxin treatment with a toxin considerably lower increase in oxygen consumption than susceptible leaves. In addition, treatment failed to stimulate oxygen consumption further in infected leaves. Respiratory changes in plants are often considered to be a general response to disturbance Even so, the results and results on that subject should therefore be interpreted with caution. support previous results indicating that the toxins of P. teres may be a factor in the pathogenesis of net-spot blotch of barley.
INTRODUCTION
Visible symptoms of net-spot blotch of barley can be caused not only by the pathogen Pyrenophora teres Drechs. but also to a large extent by two toxins produced by this fungus. The detection and isolation of these toxins and evidence for their significance in the disease syndrome of net-spot blotch have been previously described [S]. The purpose of the present work was to study the effect of infection by P. tereson the respiratory rate of the host and to compare this effect with that incited by isolated toxins of the fungus. Such investigations might contribute to a better understanding of the significance of toxins in the physiological processes involved in pathogenesis of net-spot blotch. MATERIALS
AND
METHODS
The two barley cultivars “Wing” (highly susceptible) and “CI 9647” (resistant), the oat cultivar “Astor” and the wheat cultivar ‘Kranich” were used throughout the experiments.
214
V. Smedegbd-Petersen
In infection experiments 1Z-day-old barley seedlings grown under ordinary greenhouse conditions were inoculated with a mycelium suspension obtained from &dayold cultures of Pyrenophora teres f. teres, the ordinary form of the fungus which produces net-like necrotic lesions [5], grown on liquid medium. Occasionally spore suspensions obtained from sporulating leaves were used as inoculum. The inoculation procedure has been previously described [5]. Respiration measurements were performed with whole, detached leaves, about 6 cm in length. Only the first leaf of each plant was used in the tests. Before being placed in the Warburg reaction flasks, leaves were rinsed with water and gently rubbed with wet cotton in order to remove mycelial residue from the surface. Control leaves were treated in the same way. In experiments with toxin-treated leaves toxin solutions containing equal amounts of toxins A and B in pure form were used. The two toxins were isolated from culture filtrate by extraction, gel filtration and ion exchange chromatography as previously described [S]. Evidence for purity of the toxin preparations used in the tests was obtained by paper chromatography and thin-layer chromatography on cellulose and silica gel plates using four different solvent systems [6]. For toxin treatment the first leaves of IZday-old plants were excised, placed in small test tubes with the cut ends in the toxin solution and allowed to take up toxin for a specified time. For controls, comparable leaves were placed in test tubes with water. During the time of toxin uptake the test tubes with the leaves were placed in the laboratory and illuminated with a 100 W Osram bulb situated 60 cm above the leaves. In the following solutions of toxins A and B will be designated as P. teres toxins only. Oxygen uptake was determined with a Warburg apparatus using standard manometric techniques [7]. The Warburg reaction flasks (type A 05-72, 36 ml manufactured by Braun Melsungen, Germany) were designed for botanical work with whole leaves. Detached leaves, 6 cm in length, were placed in the reaction flasks on 6 cm2 pieces of filter paper moistened with O-5 ml of water. Carbon dioxide was absorbed by 0.2 ml 10% KOH applied to the centre well. The reaction flasks and manometers were allowed to equilibrate for 30 min and oxygen consumption measured at 25 “C in complete darkness to prevent photosynthesis. Readings were taken at intervals of 30 min during a period of 1 or 2 h. After completion of readings the leaves were dried at 110 “C for 24 h for dry matter determination. From three to six replications were carried out in all experiments. Respiration was calculated on a dry weight basis as 4 oxygen uptake per 10 mg dry weight of tissue per h, or as oxygen uptake per reaction flask. In the latter case special care was taken to ensure equal quantities of leaf tissue in each reaction flask. Microscopic examination of fungal penetration and development of hyphae in the tissue was performed at intervals of 24 h, starting 12 h after inoculation. Leaf sections of 1 cm length were cleared in a mixture of boiling glacial acetic acid and glycerol (1 : 1). After evaporation of the acetic acid, the sections were transferred to small vials with lactophenol-cotton blue (100 ml lactophenol + O-5 ml aniline blue) and gently heated for 1 min, then rinsed and mounted in lactophenol for microscopic examination.
Respiration
of
Pyrenophera feres infected
RESULTS The effect of infection leaves
barley
with Pyrenophora
215
leaves
teres on the respiratory
rate in susceptible barley
Oxygen consumption by infected and non-infected barley leaves was measured daily for 7 days after inoculation. The results are shown in Table 1. Already 16 h after inoculation oxygen uptake was 23.4% higher in the infected than in the non-infected leaves. The rate continued to increase until the fourth day after inoculation when it was 322.8% higher in the infected than in the non-infected control leaves. From the fourth day the rate sharply decreased and on the seventh day oxygen uptake in infected leaves was 26% lower than in the control leaves. TABLE
1
The effectof I?. teres infection on respiration of susceptiblebarlg leaves 0, Days after inoculation
uptake
Non-infected control
0.7 (16 h) :
14.1 16.8 14-9
3 4 5 6 7
13.3 12.7 13.9 16.4 16.9
(l~l/lO wt/h)
mg
dry
Infected 17.4 29.8 36.4 49.3 53.7 40.7 37.6 12.5
Increase ( y0 of non-infected)
Symptomsd
23.4b 77.4c 144*3c 270.7c 322.80 192.8c 129.3c - 26*OG
as 67 o Significant at 5, 1 and 0.1 o/0 probability level, respectively. d 0, no visible symptoms. 1, slightly developed dark brown lesions, slight chlorosis. 2, restricted dark brown spots or streaks, slight chlorosis. 3, dark brown spots or streaks, marked chlorosis and necrosis. 4, dark brown spots or streaks, extensive chlorosis and necrosis.
Time relationship between progress of infection, respiratory rate of susceptible barley leaves
symptom
development
and increase in the
In order to relate the increase in the respiratory rate of inoculated leaves with the progress of the invading pathogen, microscopic examination of cleared and stained leaf sections was carried out each day concurrently with respiratory measurements. In addition, daily notes were taken on symptoms using a rating scale from 0 to 4 (see footnote to Table 1). Sixteen h after inoculation the germinating spores or hyphal fragments used for inoculation had formed appressoria appearing as swellings on the germ tubes. Infection pegs were penetrating the cell wall either directly or through the stomata, but in no cases could infection hyphae be detected beneath the epidermal cells. The penetration sites were easiIy recognizable since they were always surrounded by a ring of densely stained tissue. One day (24 h) after inoculation many infection hyphae had completed penetration through the outer epidermal wall and formed large swellings (vesicles) in the
216
V. Smedeghd-Petersen
epidermal cells. In a few cases the hyphae had penetrated further into the mesophyll but the distance never exceeded two to three cells. Two days after inoculation the infection hyphae had penetrated into a depth of six to eight mesophyll cells. Hyphae had started to branch, but the single cells were short and swollen and always occurred intercellularly. The longest distance in the tissue any hyphae had advanced from the penetration site was 900 F. Three days after inoculation the hyphae had penetrated further into the mesophyll and had often grown to a distance beyond the stained areas surrounding the penetration sites. Seven days after inoculation mycelium was extensively present in large areas of the infected leaves. The first visible symptoms could be observed 2 to 3 days after inoculation as slight chlorosis and faint brown spots. After 5 days dark net lesions had appeared and 7 days after inoculation extensive chlorosis and necrosis occurred in large areas of the leaves. Comparing the respiratory rates with the progress of invading hyphae in the tissue, it is apparent that the host responded with an increase in respiration at a very early stage in the infection process, apparently already during the penetration of the epidermal cell and far before any visible symptoms could be detected. The maximum respiratory rate, occurring on the third and fourth day after inoculation, coincided with the appearance of the first visible symptoms. At this time the mycelium had only spread to a limited degree in the tissue. The rapid decline in respiration on the fifth and sixth day occurred at the same time as the beginning of necrosis. At this time mycelium was extensively present in large parts of the leaves. i3.e e$ectof th P. teres toxins on the resj&-atoryrate in susceptiblebarley leaves. The host response to the toxins of P. tereswas measured on excised leaves of the barley cultivar “Wing”. Leaves were placed with the cut end in a solution containing 300 pg pure toxin/ml and allowed to take up approximately 0.50 ml of solution/leaf. After uptake of toxin, leaves were transferred to water. The reason for using a toxin concentration of 300 &g/ml, which may seem exceedingly high considering that the threshold activity of the toxin is about 25 pg/ml, is that each leaf after uptake of 0.50 ml of toxin solution of this concentration will contain a toxin content which corresponds to the toxin content found in leaves heavily infected with the pathogen, namely about 400 pgglg fresh weight [S]. Measurements of oxygen uptake were made daily for 7 days after treatment of the leaves with the toxin solution. The results are shown in Table 2. One day after the start of treatment the oxygen uptake was 40.5% higher in toxin-treated leaves than in the water-treated control leaves. The maximum respiratory rate occurred 2 days after treatment with 73.6% higher oxygen uptake in toxin-treated leaves than in control leaves. From this date the respiratory rate gradually decreased in the toxin-treated leaves whereas it remained almost constant in the water-treated control leaves. Seven days after treatment the respiration in toxin-treated leaves was 43.5% lower than in the control leaves. As was the case in infected leaves, the increase in respiration appeared much in advance of the visible symptoms. The first visible symptoms were noted 3 days after
Respiration
of fyrenophera
feres
infected
barley
TABLE The effect of P. teres Days after start of toxin treatment
0, uptake Water-treated control
: 3 4 5 7
(pl/lO
19.5 17.8 19.8 18.6 17.5 16.8
217
leaves 2
toxin on respiration
of suxepible
ban$
leaves Symptoms toxin-treated leavesd
mg dry wt/h) Increase (% of control)
Toxin-treated
30.9 27.4 33.3 26.5 27.8 9.5
40.5b 73.60 68.2O 425” 58.9” - 43.50
ov b* 0 Significant at 5, 1 and 0.1 o/0 probability level, respectively. d 0, no visible symptoms. 1, slight chlorosis. 2, marked chlorosis, slight necrosis. 3, marked chlorosis and necrosis. 4, extensive chlorosis and necrosis. Toxin-treated leaves were allowed to take up 0.50 ml of a toxin pure toxin/ml and were then transferred to water.
solution
of
0 1 1-2 2 4
containing
300 pg
treatment, whereas a significant increase in oxygen uptake occurred within 24 h after start of toxin treatment (Table 2). Maximum oxygen uptake occurred just before the onset of visible necrosis. Comparing Tables 1 and 2 it appears that the host tissue responds similarly to infection and toxin treatment. The less pronounced rise in oxygen consumption after toxin treatment than after infection is presumably related to the toxin concentration employed. That a direct relationship exists between the rate of respiration and the toxin concentration can be seen in Table 3 which shows the influence of two toxin concentrations of 300 and 600 pg/ml on respiration of YVing” barley leaves. It is seen that the two toxin concentrations increased the respiratory rate by 35-9 and 60.5 %, respectively. These data clearly demonstrate that the respiratory rate increases with increasing toxin concentration. A similar relationship was also found to exist between toxin concentration and the severity of visible symptoms. TABLE
3
The effect of P. teres toxin on respiration Disease reaction by inoculation with P. ten+ Barley, “Wig” (susceptible) Barley ,“CI 9647” (resistant) Wheat Oats
0, Water-treated control
of bar&,
wheat and oats
uptake (ul/lO mg dry wt/h) 300 (rg Increase 600 pg toxin/ml (%) toxin/ml 22.7
4
16.7
l-2
18.0
21.5
19*5c
1 0
15.2 17.8
18.7 18.5
23.0” 3.9
a* ba c Significant at 5, 1 and 0.1 y0 probability level, respectively. 6 See footnote to Table 1. Toxin-treated leaves were allowed to take up toxin solutions in the Warburg reaction flasks.
35.9”
for 24 h before
26.8
Increase (%)
being
60.5c
placed
218
V. Smedeghd-Petersen
The similar respiratory response in barley leaves to infection and toxin treatment indicates that the toxins may be involved in the pathologically enhanced respiration in barley leaves infected with P. teres. The e$ect of the P. teres toxins on the respiratory rate of host plants resistance
with &yering
degrees of
Studies were performed to investigate whether the toxins exert a selective respiratory effect on hosts possessing different, degrees of resistance against P. teres. The barley cultivars “Wing” and “CI 9647” were chosen as highly susceptible and resistant test plants, respectively. Wheat and oats were chosen as highly resistant and non-host plants, respectively. Wheat and oats are usually both considered to be non-hosts for P. teres. However, many infection experiments by the present author revealed that wheat upon artificial inoculation is slightly susceptible reacting with slight chlorosis but not with the dark net lesions which are typical for barley. The fungus could be reisolated from infected wheat leaves and weak sporulation was occasionally observed on diseased leaves placed on agar. Oats occasionally showed a very slight chlorosis upon heavy inoculation but the fungus could not be isolated from inoculated leaves and this species was consequently considered as a non-host. Table 3 shows the effect of 24 h of toxin uptake (600 pg toxin/ml) on tissue with differing degree of resistance. The susceptible barley cultivar “Wing” reacted with an increase in oxygen uptake of 60.5%, whereas the resistant cultivar “CI 9647” showed only a 19.5 o/o increase. Wheat (highly resistant) reacted with an increase of 23.0% and oats (non-host) with an increase of 3.9%. These results suggest that highly susceptible tissue responds to the toxins with a more pronounced increase in respiration than resistant tissue, and that non-host tissue is almost unaffected by the toxin. More extensive studies comprising a wider range of host and non-host plants may be needed before conclusions can be drawn as to the host specificity of the P. teres toxins. However, the results are in agreement with previous results [6] demonstrating a good, although not complete, correlation between the host range of the pathogen and that of the toxins. The effect of P. teres toxins on respiration
of barley leaves previously infected with P. teres
The fact that P. teres pathogen and its isolated toxins cause similar increases in the rate of respiration of susceptible barley leaves suggests that the toxins constitute a causal factor in the pathologically enhanced respiration. If this assumption is correct, the addition of moderate concentrations of purified toxins would not be expected to cause further respiratory increases in barley leaves previously infected with the pathogen. Table 4 shows that infected leaves which were allowed to take up water for 24 h exhibited an increase in oxygen consumption amounting to 2OO*9o/oof healthy leaves treated in the same way. Infected leaves which were allowed to take up a toxin solution (600 pg purified toxins/ml) for 24 h exhibited an increase amounting to 205.6% of the healthy leaves. The difference of 4.7% is not significant. The fact that toxin treatment failed to stimulate oxygen uptake in leaves previously infected with P. teres supports the view that the toxins produced by this
Respiration
of Pyrenophera
feres infected
barley
TABLE 77te effeckctof the P. teres toxins
on respiration
Healthy Infected Infected
leaves + water leaves + water leaves + toxin
4
of barley leaves previousb pathogen 0,
Treatment
219
leaves
(~1/10
uptake mg dry
Wh)
10.7 32.2 32.7
infected
with the P. teres
Increase (% of healthy)
200.9 205.6”
Four days after inoculation infected leaves were excised and allowed to take up deionized water or toxin solution (600 pg purified toxin/ml) for 24 h before being placed in the Warburg reaction flasks. a The difference between infected leaves+water and infected leaves+ toxin was not significant.
pathogen may constitute a cause of the pathologically leaves with net-spot blotch.
enhanced respiration of barley
DISCUSSION
Already 16 h after inoculation the respiratory rate of susceptible barley leaves significantly exceeded that of non-inoculated control leaves. At that time the infection hyphae had hardly more than penetrated the epidermal cells and had not moved into the mesophyll to any appreciable extent. This suggests that the Pyrenophora teres toxins move in advance of the hyphae and stimulate the metabolic apparatus of the tissue to an increased rate of respiration. Presumably the toxin is released during penetration of the epidermal wall. The assumption that the early metabolic response to infection is due to toxins released from the penetrating hyphae is supported by the fact that the purified P. teres toxins, when produced in vitro and introduced into healthy leaves in concentrations comparable to those found in leaves heavily infected with the pathogen, cause an increased rate of respiration. The early metabolic response to infection with P. teres is in contrast to the situation found in most other host-pathogen combinations involving non-obligate parasites. In oats infected with Helminthosporium victoriae a marked increase occurred on the third day after inoculation and coincided with the appearance of visible symptoms [3]. Similar conditions were found in maize leaves infected with Helminthosporium carbonum, where Kuo & Scheffer [4] noted a pathological rise in oxygen uptake starting 2 days after inoculation. The question naturally arises concerning how much the pathogen itself contributes to the increased respiratory rate. This aspect has been studied in other diseases, e.g. on cereals infected with powdery mildew and rust [I, 2, 81. Although the contribution by the pathogen is generally considered to be very limited, the results and conclusions from these investigations are contradictory. In the present host-pathogen combination there are several lines of evidence to suggest that the pathological increase in respiration is contributed almost exclusively by the host and that the pathogen only contributes a little. Thus the increase in respiration occurred during the first 4 days after inoculation during which period the 18
220
V. Smedeghd-Petersen
amount of mycelium in the host tissue was very limited. After the onset of necrosis the rate sharply decreased whereas the amount of mycelium in the tissue rapidly increased. Furthermore, the rise in the respiratory rate started within 16 h after inoculation and at this time the infection hyphae had hardly more than penetrated the epidermal cells. As the leaves were rinsed with water and gently rubbed with wet cotton before being placed in the Warburg reaction flasks in order to remove spores and surplus mycelial fragments applied by inoculation, the amount of mycelium in the leaves 16 and 24 h after inoculation may be considered negligible. Finally, a respiratory increase could be induced without the presence of the pathogen by applying biological concentrations of purified toxins to the leaves. Previous results [6] suggest that the toxins of P. teres induce the most important visible symptoms of the disease. The present results suggest that the toxins also induce respiratory changes in susceptible tissue which parallel those induced by the pathogen. The data further indicate that highly susceptible host tissue reacts with a greater increase in respiration than resistant tissue in response to P. teres toxins, and that non-host tissue is almost unaffected by both the pathogen and its toxins. It is well known that increased respiration is a common phenomenon in diseased plants and that many factors including chemicals and wounding may cause respiration to increase. Hence results on that subject should be interpreted with caution. Even so, the data presented in this paper seem to support previous results [6] indicating that the toxins of P. teres may be a factor in the pathogenesis of net-spot blotch of barley. The author is indebted to Dr Kurt Christiansen, Department of Plant Physiology, for advice and discussions. The investigations were supported by the Danish Agricultural and Veterinary Research Council. REFERENCES I. ALLEN, P. J. & GODDARD, D. R. ( 1938). A respiratory study of powdery mildew of wheat. American Journal of Botany 25, 6 13-62 1. 2. BUSHNELL, W. R. & ALLEN, P. J. (1962). Respiratory changes in barley leaves produced by single colonies of powdery mildew. Plant Physioloa 37, 751-758. 3. GRIMM, R. B. & WHEELER, H. (1963). Respiratory and enzymatic changes in Victoria blight of oats. Phytopathology 53, 436-440. 4. Kuo, M. & SGHEFFER, R. P. ( 1970). Comparative effects of host-specific toxins and Helminthosporium infections on respiration and carboxylation by host tissue. Phytopathology 60, 1391-1394. 5. SMEDEG~D-PETERSEN, V. (197 1). Pyrenophora teres f. maxulata f. nov. and Pyrenophora teres f. teres on barley in Denmark. Royal Veterinary and Agricultural Universi~ Yearbook 1971, 1241144. 6. SMEDEG~RD-PETERSEN, V. (1977). Isolation of two toxins produced by Pyrenophora teres and their significance in disease development of net-spot blotch of barley. Physiological Plant Pathology 10, 203-211. 7. UMBREIT, W. W., BURRIS, R. H. & STAUPFER, J. F. (1964). M mom&c Techniques, 305 pp. Burgess Publishing Co., Minneapolis, Minn. 8. YARWOOD, C. E. (1953). Heat of respiration of injured and diseased leaves. Phytopatholop 43, 675-681.