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Effect of sugar content of Poa pratensis on Helminthosporium leaf spot
Physiological
Plant
Patholopv
(1972)
2,279-287
Effect of sugar content of Pea pratensis on Helminthosporium leaf spot f A. F.
GIBBS
and ROY D.
Department of Plant Pathology, Uniuersity of Minnesota, St. Paul, (Accepted for publication
March
WILCOXSON Minnesota
55101,
U.S.A.
1972)
The sugar content of Merion Kentucky bluegrass leaves (Pea jmtemis) and the severity of symptoms caused by Helminthosprium dictyoides was varied by light, chemical treatments and grass lines. There was no consistent relationship between sugar in leaves and disease severity or incidence. The sugar content of leaves was lowest, and the disease the most severe under continuous low light intensity. The sugar content of leaves was the highest after spraying the plants with maleic hydrazide 14 days before inoculation, but the amount of disease was not significantly different from that in 2,4-D treated plants or untreated plants. The sugar content of leaves and the amount of disease in eight lines of bluegrass were different, but the correlation between the two was non-significant. Under the various light and chemical treatments, although symptom expression differed, the amount of infection was the same at 11 days after inoculation.
INTRODUCTION
Diseases of cereals and grasses caused by Helminthosporium species have been considered to be “low sugar” diseases, although there has been little supporting data, ever since Horsfall and Dimond [9] observed that sugar content of plant tissue may influence disease susceptibility. Lukens [13] suggested that melting-out of Kentucky bluegrass (Poa pratensis L.) was a low sugar disease because it was more severe when plants were managed with cultural practices that reduced the sugar content of leaves. On the other hand, Couch and Moore [4j found that the amount of leaf spot in Kentucky bluegrass caused by H. sativum was independent of the total non-structural carbohydrate content of leaf tissue. In the experiments reported here, attempts have been made to relate severity of leaf spot of Kentucky bluegrass caused by H. dictyoides Drechs, to the change in sugar content of leaves brought about by light treatments, chemicals and varieties. MATERIALS
AND
METHODS
Seed of varieties and lines of Kentucky bluegrass were obtained from the Department of Agronomy and Plant Genetics, University of Minnesota. Unless otherwise stated, plants were grown in No. 4 Wausau quartz sand (purchased from the Minnesota Mining and Manufacturing Co., St. Paul) in plastic pots. Plants were maintained in a greenhouse at about 20 “C. Supplementary lighting was provided by fluorescent lights between 5 a.m. and 11 p.m. daily. The light intensity at plant height varied from 1l,OOO& 1000 to 21,000 + 2000 lx depending on the time of day and cloud cover. 7 Paper No. 7872, Scientific Journal Series, of Minnesota, St. Paul, Minnesota 55 10 1.
Minnesota
Agricultural
Experiment
Station,
University
280
A. F. Gibbs and Roy D. Wilcoxson
Seedlings emerged 7 to 10 days after planting. They were thinned to approximately 20 plants/pot and watered by saturating the sand on alternate days with either tap water or standard Hoagland’s mineral salt, solution [SJ. Unless otherwise stated, we used the variety Merion because we thought it might best indicate the effects of sugar on disease. In the greenhouse typical leaf spots develop on Merion after inoculation with H. dictyoides and H. sativum but in the field the variety has field resistance, i.e. the plants become infected but they develop relatively less chlorosis around and necrosis in the lesions. One isolate of H. dictyoides was utilized throughout this investigation. It was maintained in infected Kentucky bluegrass plants in the greenhouse and in culture. Inoculum was obtained by growing the isolate on sucrose-asparagine-yeast agar containing six to eight surface sterilized pieces of bluegrass leaf Procedures in preparation of inoculum and cultures were essentially those described by Vargas and Wilcoxson [18]. A preliminary experiment showed that 3000 to 100,000 conidialml of distilled water caused about equal severities of disease. For this reason about 3000 conidia or more/ml of inoculum were used to inoculate plants. Two drops of Tween 20 were added to each 100 ml of inoculum. Plants were inoculated 6 to 10 weeks after emergence. Disease was evaluated 11 or 14 days after inoculation on a 1 to 5 scale, 1 representing the least and 5 the most disease. Three pots ofdiseased plants/treatment were used to judge disease severity. The methods used for the preparation of leaf samples and for extractions and determination of sugars were those described by Nalewaja and Smith [IS]. All experiments were repeated twice and for each treatment, three pots of inoculated and non-inoculated plants were harvested each time samples were taken. From each pot, three samples were harvested, two for carbohydrate determination and one for obtaining the dry weight. High light treatments in the greenhouse were provided by natural light supplemented by fluorescent lights hung 91.5 cm above the bench. The high light intensity varied from 11,000 & 1000 to 21,000 _+2000 lx. Low light intensities were provided by covering the plants with shade cloth which transmitted approximately 10% of the light of the high light treatment. The low light intensities varied from 1100 + 300 to 2000 + 500 lx. Maleic hydrazide (MH) (1,2-dihydropyridazine-3, b-dione) (1000 parts/million) and 2,4-dichlorophenoxyacetic acid (2,4-D) (1000 or 2000 parts/million) were sprayed onto the leaves of bluegrass 14 days before inoculation. The chemicals were applied until the liquid started to run from the leaves. Two drops of Tween 20 were added to the chemical to ensure that the leaves became completely wet. Dow-Formula 40 2,4-D and maleic acid hydrazide (practical), supplied by Eastern Organic Chemicals, were used. RESULTS Efect
of light on sugar content of Kentucky bluegrass leaves and the incidence of disease
The influence of high and low light intensity on the sugar content of Merion Kentucky bluegrass leaves and the incidence of disease were studied. One set of plants was exposed to high light for 4 days before inoculation and for 11 days afterwards. The
Helminthosporium
leaf
spot
281
in Poa pratensis
other set of plants was exposed to Iow light intensity for the same period. While the plants were being infected (during the 2 days after inoculation) they were kept in plastic bags on a greenhouse bench in the two light treatments. The experiment was repeated with similar results each time. On the day of inoculation the sugar content of leaves from plants grown under high light intensities was significantly higher than that of leaves from plants kept under low light intensity (Table 1). During the 2 days plants were in plastic bags the TABLE The sugar content
Trial 1
2
Time after inoculation (days)
(%)
of healthy Met-ion Kentucky dictyoides under di$erent
High Healthy
lighta Infected
1 bluegrass leaves and leaves infected light conditionst Low Healthy
lightb Infected
0 1 2 3 5 7 9 11
11.72 5.0" 4.72 7.82 9-32 ll*OE 8-7" 9-72
5.12 5.11 7.92 9.72 10.5’ 743" 10.6’
7.7" 5-2" 4.Z2 4*5y 4.9y 5.4y 5.0' 7.2y
4.5" 4.92 4.4' 4-5y 4.6' 4.2' 6.Ow
0 1 2 3 9 11
13.72 11.22 10.42 104" 9.7" 12-42
9.32 9.72 10.2" 10.32 12.0"
4*6y 5.8 5.3" 6-1y 6.8Y 6.8Y
5.2' 6.0' 5.5" 7.3' 6-3'
with
H.
Low and highC Healthy Infected
9.52 10.521 11.5"
10.4" 9.7" 11.7"
t Each observation is the average from harvesting three pots, two samples being analysed from each pot. Like letters behind data indicate no differences, unlike letters indicate significant differences at 5% level. The comparisons are valid for readings on the same day only, not between readings on different days. a 11,000* 1000 to 21,000~2000 lx. b 1100 + 300 to 2000 + 500 lx. c Low ii’ght conditions for 4 days before and 2 days after inoculation, then 8 days under high light.
differences in sugar content of leaves from inoculated and non-inoculated plants disappeared. On the day the plants were removed from the plastic bags (day 3 after inoculation) and for the remainder of the experiment, significant differences again occurred in the sugar content of healthy leaves and of diseased leaves under high and low light intensities. For the period of the experiment no differences occurred in the sugar content of healthy and diseased leaves under each of the light treatments. Because the experiments were done in a greenhouse without precise control of the environment, no attempt was made to compare the sugar content of leaves on different days. Despite the failure to maintain plants with continuous high and low sugar content, the symptoms that developed varied with the light conditions. Under the conditions of high light intensity the lesions were either ovular or elliptical, mostly less than 1 mm in diameter, discrete with sharply defined, reddish brown borders
282
A. F. Gibbs
and
Roy D. Wilcoxson
surrounding whitish centers [Plate 1 (a)]. No chlorotic halos developed around the lesions. The symptoms were typical of the leaf spot associated with Merion in the field and the severity was rated 3 on the disease scale. The infected leaves kept under continuous low light intensity developed larger lesions and considerable chlorosis [Plate 1 (c)]. The lesions tended to merge to give a blotch effect which is atypical of field symptoms on Merion. Many leaves were chlorotic after 8 days and necrotic after 14 days. The severity was scored as the maximum 5 on the disease scale. Although the symptoms under the two light conditions were very different, the number of infections was approximately the same. Thus the sugar content and the severity of disease were both influenced by the light treatments. Disease was more severe, and the sugar content of leaves was lowest under continuous low light intensities. In the second experiment, high and low light intensities were provided as described in the first experiment, but from the time of inoculation and for 48 h afterwards, plants were placed in an air-conditioned humidity chamber illuminated with 11,000 + 1000 Ix of fluorescent light. This amount of light was considered the high light intensity for plants in the humidity chamber. Low light intensity of 1500 + 300 lx was provided by covering the plants with a layer of shade cloth. On the day of inoculation the sugar content of leaves was higher under high light treatments than under low light treatments (Table 1). This difference prevailed throughout the experiment for both healthy and diseased leaves. At no time did the sugar content of diseased leaves significantly vary from that of healthy leaves in any single light treatment. The sugar content of leaves under the low light + high light treatment changed from low to high as light intensity was changed from low to high. The experiment was repeated with similar results. Disease symptoms were different according to the light treatments. Typical leafspots developed under high light [Plate 1 (a)] and atypical leaf blotches developed under low light intensity [Plate 1 (c)l. Under the low light + high light treatment the symptoms were similar to those that developed under low light conditions [Plate 1 (b)]. The number of infections was approximately the same in all light treatments. Further experiments with light were not attempted because it seemed apparent that while both sugar content of tissue and the type of symptoms caused by H. dictyoides could be changed by the light intensity, the individual effects of sugar and of light on symptoms could not be clearly separated from each other. The effect of chemical treatments on the Sugar content of Kentucky of disease
bluegrass and the incidence
Merion Kentucky bluegrass was sprayed with maleic hydrazide (MH) (1000 parts/ million) or 2,4-dichlorophenoxyacetic acid (2,4-D) ( 1000 or 2000 parts/million), 14 days before inoculation with H. dictyoides. One day before inoculation all plants were placed in the air-conditioned humidity chamber. After inoculation plants remained in the chamber for 48 h and were then removed to the greenhouse bench where the high light intensities were maintained. The sugar content of healthy and diseased bluegrass leaves treated with MH was significantly greater than that of leaves treated with 2,4-D or that of untreated leaves. There was no difference in the sugar content of leaves treated with 2,4-D
PLATE 1. Symptoms produced in Kentucky bluegrass by H. d&aides when plants 11 days under (a) high light intensity, (b) low light intensity and (c) low light intensity foIlox by high light intensity for 8 days.
were kept for 3 days
PLATE 2. are exprrimrntal
Lesions incited by H. a’ictyoides lines, (h) is Me&n.
on eight
lines
of Kentucky
bluegrass.
(a) to (g)
Helminthosporium
leaf spot
in
283
Poa pratensis
and untreated leaves (Table 2). These differences were maintained through the experiment. At no time did the sugar content of diseased leaves significantly vary from that of healthy leaves in any treatment. The sugar content of leaves treated with TABLE
2
7% sugar content (Oh) of healthy Merion blwgrass leavesand leaves infected with H. dictyoides aftir spraying them with maleic hydrazide (MH) and 2,4-O 14 days befme inoculation Time after inoculation (day4 0 : 3 9 11
Control Healthy 5.9” 6+ 11.52 13.5” 9.6z 13.8”
Leaf sugara treated” (%) Infected
Infected
2,4-D Healthy
7.12 9.9” 12.0” 9.52 13.2z
6.7” 6-6” 10.9” 12.3” 10.6” 13.92
6.2” 9.2” 11.0” 10.7” 13.92
MH Healthy
treatedb Infected
13*3y 13.8’ 15.6?J 17.3” 22.2” 23.3g
15.5” 13.7v 16.9” 21.0” 22.ay
a Each observation is the average of harvesting three pots, two samples being analysed from each pot. Like letters behind data indicate no difference, unlike letters indicate significant differences at 5% level. The comparisons are valid for readings on the same day only, not between readings on different days. b 1000 parts/million.
MH appeared to be less influenced by external variables as it continued to increase throughout the experiment. This was true for both healthy and diseased leaves. On the other hand, the sugar content of untreated leaves, and 2,4-D treated leaves, fluctuated from day to day. The experiment was repeated with similar results. The typical leaf spot [Plate 1 (a)] developed on plants treated with MH and 2,4-D as well as on untreated plants, though there was some slight chlorosis associated with lesions on the 2,4-D and untreated plants. In all three treatments the average disease rating was 3.5. The MH-treated plants appeared to be somewhat healthier than the 2,4-D-treated and untreated plants when viewed at a distance because they were a much darker green color, more upright and turgid, and faster growing. The sugar content of Kentucky bluegrass lines and the incidewe of disease
Seven lines of Kentucky bluegrass, resistant to powdery mildew and with some resistance to H. dictyoides and H. sorokinianum, were selected in the greenhouse from among 4000 lines during fall and winter of 1968169. The sugar content and disease reaction of these seven lines was compared with those of Merion. The plants were grown in soil in clay pots with the normal practices of fertilizing, cutting and watering. In all lines except line G (Table 3) the sugar content of leaves was greater in trial 2 than trial 1. The sugar content of leaves and the amount of disease varied among the lines but there was no correlation between the two. The correlation coefficients were 0.06 and O-0002 in trials 1 and 2, respectively. Line D had a low sugar content and low disease severity but line A had low sugar content and high disease severity. Lines 33 and F both had high leaf sugar content but line B had low disease severity and line F had high disease severity. These data suggest that when a number of
284
A. F. Gibbs
and
Roy
D. Wilcoxson
lines are considered there is no general relationship between sugar content and severity of disease. The symptoms developed on the eight lines are shown in Plate 2. Lines B and D show the most resistant reactions. All the other lines were susceptible. TABLE The
sugar
content
(%)
and incidence
Kentuchp Rank Line of grass
Leaf sugar(%)” Trial 1 Trial
A B C D E
6.3 15-3 7.9 6.9 12.0
10.0 16.5 10.2 II-7 13.4
fi Merion
15.0 13.6 8.5
17.1 12.2 13.4
2
Trial
for leaf sugar” 1 Trial
a 1 6 7 4 3 2 5
3
of leaf spot incited by H. dictyoides
2 6 3’ 1 5 3
on eight
lines of
bluegrass
2
Trial 3-o 1.0 3.5 2.0 3.0 4.5 4.5 4.0
Disease0 severity 1 Trial
2
Rank for disease Trial 1 Trial
2
5.0 1.0 2.0 1.0 4.0 4.0 4.5 4.0
@ Each observation is the average of harvesting two pots, two samples being analysed each pot on the day of inoculation. b l-highest, and a-lowest sugar percentage. G Mean of three readings, 14 days after inoculation; l-least, and 5-most disease.
from
DISCUSSION The hypothesis that sugar content of tissue influences disease reaction has received considerable attention since it was proposed in 1957 [9], and some controversy has followed. Some [7, 13, 141 have concluded that the quantity of soluble carbohydrate in tissue is strongly associated with disease but others [Z, 6, II, 121 have Inman [IO] was not able to arrive at a clear reached the opposite conclusion. conclusion about the importance of sugar to rust development in beans because the sugar concentration in tissue varied as the rust developed. With the exception of Luken’s report there are no data to support the idea that diseases caused by Helminthosporiurn sp. are low sugar diseases. The two examples cited by Horsfall and Dimond [9] were given as suggestions and they clearly called for data to support the hypothesis. Lukens interpreted his data to support the hypothesis but the data of Couch and Moore [4] and of this report do not support it. In the work presented here, the sugar content of Kentucky bluegrass was experimentally changed by the use of light, chemicals and different lines of grass, but the occurrence of leaf spot caused by H. dictyoides was not related to the sugar content of the leaves. This was true whether the number or type of lesion was used to evaluate the disease. It was not possible to clearly separate the effects of light itself from the possible effects of sugar on symptom development. Sugar content of leaves fluctuated with light intensity. When plants were high or low in sugar because of continuous exposure to high or low light intensities the type of symptom produced was distinct, but when sugar concentrations were caused to change from high to low during the 2 days after inoculation, the symptoms tended to be like those in plants containing
Helminthosporium
leaf
spot
in Poa pratensis
285
continuous high sugar concentrations. Likewise when sugar content was changed from low to high the symptoms resembled those in plants containing continuous low sugar concentrations. While it is tempting to suggest that it was the sugar in the leaves at the time of infection that influenced symptom development, one cannot be certain that the light conditions alone did not produce some other effect that influenced symptom development. This possibility has been suggested for wheat stem rust [5], stripe rust of wheat and barley [17J and Cercosporaleaf spot of sugar beet [I], when symptoms were different under different light conditions. The experiments in which sugar content of the leaves was altered by maleic hydrazide indicated that the sugar content of leaves did not influence lesion number or type. The maleic hydrazide-treated plants had at least twice the sugar content of untreated plants or plants treated with 2,4-D but there was no difference in disease. When interpreting the maleic hydrazide experiments one should keep in mind that it is possible that maleic hydrazide had effects on the plants other than on sugar content which might have influenced disease development. Thus any subtle effects of sugar on disease might have been obscured in our experiments. The sugar content of leaves also varied in different lines of bluegrass but there was no correlation with development of leaf spot. It was clear that one could obtain any desired correlation by selecting particular lines. It is not likely that the lack of a correlation between sugar content of tissue and disease development was due to a change of the sugars into undetectable sugars as suggested by Inman [10] and Daly [S]. The sugars present in both healthy and diseased bluegrass leaves were sucrose, glucose and fructose, plus four unidentified sugars. The unidentified sugars had R, values lower than that of sucrose. While the precise amounts of the individual sugars were not determined, from qualitative observations it was obvious that the three sugars named were the major ones present. The overall conclusion that Welminthospori~m leaf spot of bluegrass is not a low sugar disease is also suggested by Couch and Moore [4J who concluded that the susceptibility of several cultivars of Kentucky bluegrass to H. sativum was independent of the carbohydrate content of tissue. We cannot definitely say why our results differ from those of Lukens [13]. It is possible, however, that the difference may have been due to the methods that we used: (a) our investigation was in the greenhouse and his was in the field; (b) our investigation was with the leaf spot and his with the melting out phase of the disease; (c) our investigation was with Merion and seven experimental lines selected because they had some resistance to leaf spot; Lukens studied susceptible and resistant varieties; (d) in our investigation doses of inoculum known to cause disease were used, Lukens used natural epidemics; and (e) it is possible that Lukens did not differentiate the effects of the environment from the effects of sugar on disease development. The terms high and low are too general to be used for designating the sugar status of plants or plant tissue unless the amounts are defined. The sugar content of grasses varies within wide limits [16], and even greater variation is found within the plant kingdom. Even in one clone, line or variety of a species the sugar content may vary considerably depending on light and temperature, season of the year, time of the day, age of the plant, chemical treatments and even with the conditions under which the sugar is extracted. For these reasons it is difficult to state clearly
286 what
A. F. Gibbs
are high
and low levels
of sugar,
particularly
when
and
considered
Roy
D. Wilcoxson
in relation
to
disease. It is also difficult to compare the results of different workers and the results obtained by the same individual at different times. Where perfect control does not exist over the many variables which cause fluctuations in the carbohydrate content of plants, the high sugar level in one experiment may correspond to a low level of another experiment. This was noted during our investigation when the sugar levels under high and low light intensities in one experiment were 9.3 to 13.7% and 4.6 to 7.3% respectively, and in another experiment 6.3 to 8.4% and 2.8 to 4.5%, respectively. The environmental conditions in the greenhouse during the experiments could not be controlled closely. Another objection to the hypothesis relating sugar to disease resistance and susceptibility occurs when a number of hosts are attacked by the same organism. According to Couch [3] some 100 species in the Gramineae are susceptible to H. sorokinianum. Considering just a few of the hosts, one could divide them into high, intermediate and low sugar species. A high sugar species would be
REFERENCES 1. CALPOUZOS, L. & STALLKNECHT, G. F. (1967). Symptoms of Cercospora leaf spot of sugar beets influenced by light intensity. PhytqbatholoQ 57, 799-800. 2. CHIBA, 0. (1964). Studies on the variation in susceptibility and the nature of resistance of poplars to the leaf rust caused by Melampsora Carici-populina Klebahn. Rings Shikenjo Ken&u Hokoku 166, 85-157. 3. COUCH, H. B. (1962). Diseases of ‘Turf Gasses. Reinhold Publishing Co., New York. 4. COUCH, H. B. & MOORE, L. D. (1971). Influence of nutrition and total nonstructural carbohydrate content on Helminthosporium satimm incited leaf spot of Kentucky bluegrasses. Phytopathology 61, 888 (Abstr.). 5. DALY, J. M. (1964). Pre- and postinoculation effects of light quality on infection intensity of stem rust of wheat. Phytopathalosy 54, 1342-1345. 6. DALY, J, M. (1967). Some metabolic consequences of infection by obligate parasites. In The Dynamic Role of Molecular Constituents in Plant-Parasite Interactions (C. J. Mirocha and I. Uritani, eds), pp. 144-161. American Phytopathology Society, St. Paul. 7. GRAINGER for fungal and bacterial parasites. Phyto-.I. (1962). r The host plant as a habitat pathology 52, 140-150. 8. HOAGLAND, D. R. & ARNON, D. I. (1950). The water-culture method for growing plants without soil. California Agricultural Experiment Station Circular 347. 9. HORSFALL, J. G. & DIMOND, A. E. (1957). Interactions of tissue sugar, growth substances and disease susceptibility.
Helminfhosporlom
leaf
spot
in Poa prafensis
14. LYLES, W. E., FUTRELL, M. C. & ATKINS, I. M. (1959).
Relation between reaction to race 15B rust and reducing sugars and sucrose in wheat. Phytopathology 49, 254-256. NALEWAJA, J. D. & SMITH, L. H. (1963). Standard procedure for the quantitative determination of individual su.gars and total soluble carbohydrate materials in plant extracts. Aeronomv I ” Journal 55, 523-525. OJIMA, K. & ISAWA, T. (1968). The variation in carbohydrates in various species of grasses and legumes. Canadian .Tournal of Botany 46, 1507-1511. STUB&, R. W. (1967).- Influence of lightintensity on the reactions of wheat and barley seedlings to Puccinia striijkmis. Phytopathology 57, 615-619. VARGAS,J.M. & WILCOXSON,R.D.(1969). S ome effects of temperature and radiation on sporulation by Helminthosporium dictyoides on agar media. PhytopatholoD 59, 1706-l 7 12. of stem
15.
16. 17. 18.
287
Plant
Patholopv
(1972)
2,279-287
Effect of sugar content of Pea pratensis on Helminthosporium leaf spot f A. F.
GIBBS
and ROY D.
Department of Plant Pathology, Uniuersity of Minnesota, St. Paul, (Accepted for publication
March
WILCOXSON Minnesota
55101,
U.S.A.
1972)
The sugar content of Merion Kentucky bluegrass leaves (Pea jmtemis) and the severity of symptoms caused by Helminthosprium dictyoides was varied by light, chemical treatments and grass lines. There was no consistent relationship between sugar in leaves and disease severity or incidence. The sugar content of leaves was lowest, and the disease the most severe under continuous low light intensity. The sugar content of leaves was the highest after spraying the plants with maleic hydrazide 14 days before inoculation, but the amount of disease was not significantly different from that in 2,4-D treated plants or untreated plants. The sugar content of leaves and the amount of disease in eight lines of bluegrass were different, but the correlation between the two was non-significant. Under the various light and chemical treatments, although symptom expression differed, the amount of infection was the same at 11 days after inoculation.
INTRODUCTION
Diseases of cereals and grasses caused by Helminthosporium species have been considered to be “low sugar” diseases, although there has been little supporting data, ever since Horsfall and Dimond [9] observed that sugar content of plant tissue may influence disease susceptibility. Lukens [13] suggested that melting-out of Kentucky bluegrass (Poa pratensis L.) was a low sugar disease because it was more severe when plants were managed with cultural practices that reduced the sugar content of leaves. On the other hand, Couch and Moore [4j found that the amount of leaf spot in Kentucky bluegrass caused by H. sativum was independent of the total non-structural carbohydrate content of leaf tissue. In the experiments reported here, attempts have been made to relate severity of leaf spot of Kentucky bluegrass caused by H. dictyoides Drechs, to the change in sugar content of leaves brought about by light treatments, chemicals and varieties. MATERIALS
AND
METHODS
Seed of varieties and lines of Kentucky bluegrass were obtained from the Department of Agronomy and Plant Genetics, University of Minnesota. Unless otherwise stated, plants were grown in No. 4 Wausau quartz sand (purchased from the Minnesota Mining and Manufacturing Co., St. Paul) in plastic pots. Plants were maintained in a greenhouse at about 20 “C. Supplementary lighting was provided by fluorescent lights between 5 a.m. and 11 p.m. daily. The light intensity at plant height varied from 1l,OOO& 1000 to 21,000 + 2000 lx depending on the time of day and cloud cover. 7 Paper No. 7872, Scientific Journal Series, of Minnesota, St. Paul, Minnesota 55 10 1.
Minnesota
Agricultural
Experiment
Station,
University
280
A. F. Gibbs and Roy D. Wilcoxson
Seedlings emerged 7 to 10 days after planting. They were thinned to approximately 20 plants/pot and watered by saturating the sand on alternate days with either tap water or standard Hoagland’s mineral salt, solution [SJ. Unless otherwise stated, we used the variety Merion because we thought it might best indicate the effects of sugar on disease. In the greenhouse typical leaf spots develop on Merion after inoculation with H. dictyoides and H. sativum but in the field the variety has field resistance, i.e. the plants become infected but they develop relatively less chlorosis around and necrosis in the lesions. One isolate of H. dictyoides was utilized throughout this investigation. It was maintained in infected Kentucky bluegrass plants in the greenhouse and in culture. Inoculum was obtained by growing the isolate on sucrose-asparagine-yeast agar containing six to eight surface sterilized pieces of bluegrass leaf Procedures in preparation of inoculum and cultures were essentially those described by Vargas and Wilcoxson [18]. A preliminary experiment showed that 3000 to 100,000 conidialml of distilled water caused about equal severities of disease. For this reason about 3000 conidia or more/ml of inoculum were used to inoculate plants. Two drops of Tween 20 were added to each 100 ml of inoculum. Plants were inoculated 6 to 10 weeks after emergence. Disease was evaluated 11 or 14 days after inoculation on a 1 to 5 scale, 1 representing the least and 5 the most disease. Three pots ofdiseased plants/treatment were used to judge disease severity. The methods used for the preparation of leaf samples and for extractions and determination of sugars were those described by Nalewaja and Smith [IS]. All experiments were repeated twice and for each treatment, three pots of inoculated and non-inoculated plants were harvested each time samples were taken. From each pot, three samples were harvested, two for carbohydrate determination and one for obtaining the dry weight. High light treatments in the greenhouse were provided by natural light supplemented by fluorescent lights hung 91.5 cm above the bench. The high light intensity varied from 11,000 & 1000 to 21,000 _+2000 lx. Low light intensities were provided by covering the plants with shade cloth which transmitted approximately 10% of the light of the high light treatment. The low light intensities varied from 1100 + 300 to 2000 + 500 lx. Maleic hydrazide (MH) (1,2-dihydropyridazine-3, b-dione) (1000 parts/million) and 2,4-dichlorophenoxyacetic acid (2,4-D) (1000 or 2000 parts/million) were sprayed onto the leaves of bluegrass 14 days before inoculation. The chemicals were applied until the liquid started to run from the leaves. Two drops of Tween 20 were added to the chemical to ensure that the leaves became completely wet. Dow-Formula 40 2,4-D and maleic acid hydrazide (practical), supplied by Eastern Organic Chemicals, were used. RESULTS Efect
of light on sugar content of Kentucky bluegrass leaves and the incidence of disease
The influence of high and low light intensity on the sugar content of Merion Kentucky bluegrass leaves and the incidence of disease were studied. One set of plants was exposed to high light for 4 days before inoculation and for 11 days afterwards. The
Helminthosporium
leaf
spot
281
in Poa pratensis
other set of plants was exposed to Iow light intensity for the same period. While the plants were being infected (during the 2 days after inoculation) they were kept in plastic bags on a greenhouse bench in the two light treatments. The experiment was repeated with similar results each time. On the day of inoculation the sugar content of leaves from plants grown under high light intensities was significantly higher than that of leaves from plants kept under low light intensity (Table 1). During the 2 days plants were in plastic bags the TABLE The sugar content
Trial 1
2
Time after inoculation (days)
(%)
of healthy Met-ion Kentucky dictyoides under di$erent
High Healthy
lighta Infected
1 bluegrass leaves and leaves infected light conditionst Low Healthy
lightb Infected
0 1 2 3 5 7 9 11
11.72 5.0" 4.72 7.82 9-32 ll*OE 8-7" 9-72
5.12 5.11 7.92 9.72 10.5’ 743" 10.6’
7.7" 5-2" 4.Z2 4*5y 4.9y 5.4y 5.0' 7.2y
4.5" 4.92 4.4' 4-5y 4.6' 4.2' 6.Ow
0 1 2 3 9 11
13.72 11.22 10.42 104" 9.7" 12-42
9.32 9.72 10.2" 10.32 12.0"
4*6y 5.8 5.3" 6-1y 6.8Y 6.8Y
5.2' 6.0' 5.5" 7.3' 6-3'
with
H.
Low and highC Healthy Infected
9.52 10.521 11.5"
10.4" 9.7" 11.7"
t Each observation is the average from harvesting three pots, two samples being analysed from each pot. Like letters behind data indicate no differences, unlike letters indicate significant differences at 5% level. The comparisons are valid for readings on the same day only, not between readings on different days. a 11,000* 1000 to 21,000~2000 lx. b 1100 + 300 to 2000 + 500 lx. c Low ii’ght conditions for 4 days before and 2 days after inoculation, then 8 days under high light.
differences in sugar content of leaves from inoculated and non-inoculated plants disappeared. On the day the plants were removed from the plastic bags (day 3 after inoculation) and for the remainder of the experiment, significant differences again occurred in the sugar content of healthy leaves and of diseased leaves under high and low light intensities. For the period of the experiment no differences occurred in the sugar content of healthy and diseased leaves under each of the light treatments. Because the experiments were done in a greenhouse without precise control of the environment, no attempt was made to compare the sugar content of leaves on different days. Despite the failure to maintain plants with continuous high and low sugar content, the symptoms that developed varied with the light conditions. Under the conditions of high light intensity the lesions were either ovular or elliptical, mostly less than 1 mm in diameter, discrete with sharply defined, reddish brown borders
282
A. F. Gibbs
and
Roy D. Wilcoxson
surrounding whitish centers [Plate 1 (a)]. No chlorotic halos developed around the lesions. The symptoms were typical of the leaf spot associated with Merion in the field and the severity was rated 3 on the disease scale. The infected leaves kept under continuous low light intensity developed larger lesions and considerable chlorosis [Plate 1 (c)]. The lesions tended to merge to give a blotch effect which is atypical of field symptoms on Merion. Many leaves were chlorotic after 8 days and necrotic after 14 days. The severity was scored as the maximum 5 on the disease scale. Although the symptoms under the two light conditions were very different, the number of infections was approximately the same. Thus the sugar content and the severity of disease were both influenced by the light treatments. Disease was more severe, and the sugar content of leaves was lowest under continuous low light intensities. In the second experiment, high and low light intensities were provided as described in the first experiment, but from the time of inoculation and for 48 h afterwards, plants were placed in an air-conditioned humidity chamber illuminated with 11,000 + 1000 Ix of fluorescent light. This amount of light was considered the high light intensity for plants in the humidity chamber. Low light intensity of 1500 + 300 lx was provided by covering the plants with a layer of shade cloth. On the day of inoculation the sugar content of leaves was higher under high light treatments than under low light treatments (Table 1). This difference prevailed throughout the experiment for both healthy and diseased leaves. At no time did the sugar content of diseased leaves significantly vary from that of healthy leaves in any single light treatment. The sugar content of leaves under the low light + high light treatment changed from low to high as light intensity was changed from low to high. The experiment was repeated with similar results. Disease symptoms were different according to the light treatments. Typical leafspots developed under high light [Plate 1 (a)] and atypical leaf blotches developed under low light intensity [Plate 1 (c)l. Under the low light + high light treatment the symptoms were similar to those that developed under low light conditions [Plate 1 (b)]. The number of infections was approximately the same in all light treatments. Further experiments with light were not attempted because it seemed apparent that while both sugar content of tissue and the type of symptoms caused by H. dictyoides could be changed by the light intensity, the individual effects of sugar and of light on symptoms could not be clearly separated from each other. The effect of chemical treatments on the Sugar content of Kentucky of disease
bluegrass and the incidence
Merion Kentucky bluegrass was sprayed with maleic hydrazide (MH) (1000 parts/ million) or 2,4-dichlorophenoxyacetic acid (2,4-D) ( 1000 or 2000 parts/million), 14 days before inoculation with H. dictyoides. One day before inoculation all plants were placed in the air-conditioned humidity chamber. After inoculation plants remained in the chamber for 48 h and were then removed to the greenhouse bench where the high light intensities were maintained. The sugar content of healthy and diseased bluegrass leaves treated with MH was significantly greater than that of leaves treated with 2,4-D or that of untreated leaves. There was no difference in the sugar content of leaves treated with 2,4-D
PLATE 1. Symptoms produced in Kentucky bluegrass by H. d&aides when plants 11 days under (a) high light intensity, (b) low light intensity and (c) low light intensity foIlox by high light intensity for 8 days.
were kept for 3 days
PLATE 2. are exprrimrntal
Lesions incited by H. a’ictyoides lines, (h) is Me&n.
on eight
lines
of Kentucky
bluegrass.
(a) to (g)
Helminthosporium
leaf spot
in
283
Poa pratensis
and untreated leaves (Table 2). These differences were maintained through the experiment. At no time did the sugar content of diseased leaves significantly vary from that of healthy leaves in any treatment. The sugar content of leaves treated with TABLE
2
7% sugar content (Oh) of healthy Merion blwgrass leavesand leaves infected with H. dictyoides aftir spraying them with maleic hydrazide (MH) and 2,4-O 14 days befme inoculation Time after inoculation (day4 0 : 3 9 11
Control Healthy 5.9” 6+ 11.52 13.5” 9.6z 13.8”
Leaf sugara treated” (%) Infected
Infected
2,4-D Healthy
7.12 9.9” 12.0” 9.52 13.2z
6.7” 6-6” 10.9” 12.3” 10.6” 13.92
6.2” 9.2” 11.0” 10.7” 13.92
MH Healthy
treatedb Infected
13*3y 13.8’ 15.6?J 17.3” 22.2” 23.3g
15.5” 13.7v 16.9” 21.0” 22.ay
a Each observation is the average of harvesting three pots, two samples being analysed from each pot. Like letters behind data indicate no difference, unlike letters indicate significant differences at 5% level. The comparisons are valid for readings on the same day only, not between readings on different days. b 1000 parts/million.
MH appeared to be less influenced by external variables as it continued to increase throughout the experiment. This was true for both healthy and diseased leaves. On the other hand, the sugar content of untreated leaves, and 2,4-D treated leaves, fluctuated from day to day. The experiment was repeated with similar results. The typical leaf spot [Plate 1 (a)] developed on plants treated with MH and 2,4-D as well as on untreated plants, though there was some slight chlorosis associated with lesions on the 2,4-D and untreated plants. In all three treatments the average disease rating was 3.5. The MH-treated plants appeared to be somewhat healthier than the 2,4-D-treated and untreated plants when viewed at a distance because they were a much darker green color, more upright and turgid, and faster growing. The sugar content of Kentucky bluegrass lines and the incidewe of disease
Seven lines of Kentucky bluegrass, resistant to powdery mildew and with some resistance to H. dictyoides and H. sorokinianum, were selected in the greenhouse from among 4000 lines during fall and winter of 1968169. The sugar content and disease reaction of these seven lines was compared with those of Merion. The plants were grown in soil in clay pots with the normal practices of fertilizing, cutting and watering. In all lines except line G (Table 3) the sugar content of leaves was greater in trial 2 than trial 1. The sugar content of leaves and the amount of disease varied among the lines but there was no correlation between the two. The correlation coefficients were 0.06 and O-0002 in trials 1 and 2, respectively. Line D had a low sugar content and low disease severity but line A had low sugar content and high disease severity. Lines 33 and F both had high leaf sugar content but line B had low disease severity and line F had high disease severity. These data suggest that when a number of
284
A. F. Gibbs
and
Roy
D. Wilcoxson
lines are considered there is no general relationship between sugar content and severity of disease. The symptoms developed on the eight lines are shown in Plate 2. Lines B and D show the most resistant reactions. All the other lines were susceptible. TABLE The
sugar
content
(%)
and incidence
Kentuchp Rank Line of grass
Leaf sugar(%)” Trial 1 Trial
A B C D E
6.3 15-3 7.9 6.9 12.0
10.0 16.5 10.2 II-7 13.4
fi Merion
15.0 13.6 8.5
17.1 12.2 13.4
2
Trial
for leaf sugar” 1 Trial
a 1 6 7 4 3 2 5
3
of leaf spot incited by H. dictyoides
2 6 3’ 1 5 3
on eight
lines of
bluegrass
2
Trial 3-o 1.0 3.5 2.0 3.0 4.5 4.5 4.0
Disease0 severity 1 Trial
2
Rank for disease Trial 1 Trial
2
5.0 1.0 2.0 1.0 4.0 4.0 4.5 4.0
@ Each observation is the average of harvesting two pots, two samples being analysed each pot on the day of inoculation. b l-highest, and a-lowest sugar percentage. G Mean of three readings, 14 days after inoculation; l-least, and 5-most disease.
from
DISCUSSION The hypothesis that sugar content of tissue influences disease reaction has received considerable attention since it was proposed in 1957 [9], and some controversy has followed. Some [7, 13, 141 have concluded that the quantity of soluble carbohydrate in tissue is strongly associated with disease but others [Z, 6, II, 121 have Inman [IO] was not able to arrive at a clear reached the opposite conclusion. conclusion about the importance of sugar to rust development in beans because the sugar concentration in tissue varied as the rust developed. With the exception of Luken’s report there are no data to support the idea that diseases caused by Helminthosporiurn sp. are low sugar diseases. The two examples cited by Horsfall and Dimond [9] were given as suggestions and they clearly called for data to support the hypothesis. Lukens interpreted his data to support the hypothesis but the data of Couch and Moore [4] and of this report do not support it. In the work presented here, the sugar content of Kentucky bluegrass was experimentally changed by the use of light, chemicals and different lines of grass, but the occurrence of leaf spot caused by H. dictyoides was not related to the sugar content of the leaves. This was true whether the number or type of lesion was used to evaluate the disease. It was not possible to clearly separate the effects of light itself from the possible effects of sugar on symptom development. Sugar content of leaves fluctuated with light intensity. When plants were high or low in sugar because of continuous exposure to high or low light intensities the type of symptom produced was distinct, but when sugar concentrations were caused to change from high to low during the 2 days after inoculation, the symptoms tended to be like those in plants containing
Helminthosporium
leaf
spot
in Poa pratensis
285
continuous high sugar concentrations. Likewise when sugar content was changed from low to high the symptoms resembled those in plants containing continuous low sugar concentrations. While it is tempting to suggest that it was the sugar in the leaves at the time of infection that influenced symptom development, one cannot be certain that the light conditions alone did not produce some other effect that influenced symptom development. This possibility has been suggested for wheat stem rust [5], stripe rust of wheat and barley [17J and Cercosporaleaf spot of sugar beet [I], when symptoms were different under different light conditions. The experiments in which sugar content of the leaves was altered by maleic hydrazide indicated that the sugar content of leaves did not influence lesion number or type. The maleic hydrazide-treated plants had at least twice the sugar content of untreated plants or plants treated with 2,4-D but there was no difference in disease. When interpreting the maleic hydrazide experiments one should keep in mind that it is possible that maleic hydrazide had effects on the plants other than on sugar content which might have influenced disease development. Thus any subtle effects of sugar on disease might have been obscured in our experiments. The sugar content of leaves also varied in different lines of bluegrass but there was no correlation with development of leaf spot. It was clear that one could obtain any desired correlation by selecting particular lines. It is not likely that the lack of a correlation between sugar content of tissue and disease development was due to a change of the sugars into undetectable sugars as suggested by Inman [10] and Daly [S]. The sugars present in both healthy and diseased bluegrass leaves were sucrose, glucose and fructose, plus four unidentified sugars. The unidentified sugars had R, values lower than that of sucrose. While the precise amounts of the individual sugars were not determined, from qualitative observations it was obvious that the three sugars named were the major ones present. The overall conclusion that Welminthospori~m leaf spot of bluegrass is not a low sugar disease is also suggested by Couch and Moore [4J who concluded that the susceptibility of several cultivars of Kentucky bluegrass to H. sativum was independent of the carbohydrate content of tissue. We cannot definitely say why our results differ from those of Lukens [13]. It is possible, however, that the difference may have been due to the methods that we used: (a) our investigation was in the greenhouse and his was in the field; (b) our investigation was with the leaf spot and his with the melting out phase of the disease; (c) our investigation was with Merion and seven experimental lines selected because they had some resistance to leaf spot; Lukens studied susceptible and resistant varieties; (d) in our investigation doses of inoculum known to cause disease were used, Lukens used natural epidemics; and (e) it is possible that Lukens did not differentiate the effects of the environment from the effects of sugar on disease development. The terms high and low are too general to be used for designating the sugar status of plants or plant tissue unless the amounts are defined. The sugar content of grasses varies within wide limits [16], and even greater variation is found within the plant kingdom. Even in one clone, line or variety of a species the sugar content may vary considerably depending on light and temperature, season of the year, time of the day, age of the plant, chemical treatments and even with the conditions under which the sugar is extracted. For these reasons it is difficult to state clearly
286 what
A. F. Gibbs
are high
and low levels
of sugar,
particularly
when
and
considered
Roy
D. Wilcoxson
in relation
to
disease. It is also difficult to compare the results of different workers and the results obtained by the same individual at different times. Where perfect control does not exist over the many variables which cause fluctuations in the carbohydrate content of plants, the high sugar level in one experiment may correspond to a low level of another experiment. This was noted during our investigation when the sugar levels under high and low light intensities in one experiment were 9.3 to 13.7% and 4.6 to 7.3% respectively, and in another experiment 6.3 to 8.4% and 2.8 to 4.5%, respectively. The environmental conditions in the greenhouse during the experiments could not be controlled closely. Another objection to the hypothesis relating sugar to disease resistance and susceptibility occurs when a number of hosts are attacked by the same organism. According to Couch [3] some 100 species in the Gramineae are susceptible to H. sorokinianum. Considering just a few of the hosts, one could divide them into high, intermediate and low sugar species. A high sugar species would be
REFERENCES 1. CALPOUZOS, L. & STALLKNECHT, G. F. (1967). Symptoms of Cercospora leaf spot of sugar beets influenced by light intensity. PhytqbatholoQ 57, 799-800. 2. CHIBA, 0. (1964). Studies on the variation in susceptibility and the nature of resistance of poplars to the leaf rust caused by Melampsora Carici-populina Klebahn. Rings Shikenjo Ken&u Hokoku 166, 85-157. 3. COUCH, H. B. (1962). Diseases of ‘Turf Gasses. Reinhold Publishing Co., New York. 4. COUCH, H. B. & MOORE, L. D. (1971). Influence of nutrition and total nonstructural carbohydrate content on Helminthosporium satimm incited leaf spot of Kentucky bluegrasses. Phytopathology 61, 888 (Abstr.). 5. DALY, J. M. (1964). Pre- and postinoculation effects of light quality on infection intensity of stem rust of wheat. Phytopathalosy 54, 1342-1345. 6. DALY, J, M. (1967). Some metabolic consequences of infection by obligate parasites. In The Dynamic Role of Molecular Constituents in Plant-Parasite Interactions (C. J. Mirocha and I. Uritani, eds), pp. 144-161. American Phytopathology Society, St. Paul. 7. GRAINGER for fungal and bacterial parasites. Phyto-.I. (1962). r The host plant as a habitat pathology 52, 140-150. 8. HOAGLAND, D. R. & ARNON, D. I. (1950). The water-culture method for growing plants without soil. California Agricultural Experiment Station Circular 347. 9. HORSFALL, J. G. & DIMOND, A. E. (1957). Interactions of tissue sugar, growth substances and disease susceptibility.
Helminfhosporlom
leaf
spot
in Poa prafensis
14. LYLES, W. E., FUTRELL, M. C. & ATKINS, I. M. (1959).
Relation between reaction to race 15B rust and reducing sugars and sucrose in wheat. Phytopathology 49, 254-256. NALEWAJA, J. D. & SMITH, L. H. (1963). Standard procedure for the quantitative determination of individual su.gars and total soluble carbohydrate materials in plant extracts. Aeronomv I ” Journal 55, 523-525. OJIMA, K. & ISAWA, T. (1968). The variation in carbohydrates in various species of grasses and legumes. Canadian .Tournal of Botany 46, 1507-1511. STUB&, R. W. (1967).- Influence of lightintensity on the reactions of wheat and barley seedlings to Puccinia striijkmis. Phytopathology 57, 615-619. VARGAS,J.M. & WILCOXSON,R.D.(1969). S ome effects of temperature and radiation on sporulation by Helminthosporium dictyoides on agar media. PhytopatholoD 59, 1706-l 7 12. of stem
15.
16. 17. 18.
287