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Infection of wheat seedlings by Septoria nodorum in relation to environmental factors
[ 493 ] TrtJflS. Br. mycol. Soc. 57 (3), 493-500 (1971) Printed in Grea: Britain
INFECTION OF WHEAT SEEDLINGS BY SEPTORIA NODORUM IN RELATION TO ENVIRONMENTAL FACTORS By S. J. 1. HOLMES
AND
Department of Cryptogamic Botany,
J. COLHOUN Universi!J of Manchester
(With Plate 41 and 3 Text-figures) A technique for the production of large quantities of spores of Septoria nodorum is described, this technique being also suitable for inducing copious sporulation of S. tritici, S. auenae and S. secalis. In glasshouse experiments very favourable conditions for infection of wheat coleoptiles by S. nodorum are provided in dry soil at a temperature of 12°C. Wetter soils and temperatures of 8° or 17° permit of the occurrence of less infection. Pycnidia were frequently produced on diseased coleoptiles in the glasshouse when high air humidities were provided after infection had occurred. It is suggested that cultivars differ in their reaction to S. nodorum in the seedling stage. Cappelle-Desprez has been shown to be very susceptible.
Recently Septaria diseases of cereals have assumed increasing importance in Britain. The significance of seed-borne inoculum and of other methods of survival of the fungus has received attention (Scharen, 1963; Weber, 1922). When infected seeds are sown, symptoms appear on the coleoptiles of the seedlings (Hopp, 1957; Kietreiber, 1961), but little is known about the factors which influence this stage of the development of the disease. An attempt has now been made to study the environmental factors affecting coleoptile infection which results when seeds naturally infected or inoculated with Septaria nodotum are sown in soil. MATERIALS AND METHODS
A single spore culture of S. nodorum isolated from wheat leaves and maintained on V8-Czapek-Dox medium was employed. Sporulation was induced by maintaining cultures at 15-17 °0 with constant illumination from blacklight lamps. Plates were inoculated by pouring over the surface of the medium I ml of spore suspension containing 2'5 x 106 spores per ml. Preliminary experiments showed the necessity of maintaining a high air humidity within plates. If this was not done sporulation greatly declined with repeated subculturing. An inexpensive incubator was constructed (Fig. I) which successfully maintained the required air temperature and humidity while supplying adequate light of the appropriate wavelength. This incubator is a large-scale modification of that used by Sanderson (1970) in work with Fusarium nivale and accommodates 90 plates in a single layer when the external dimensions are 2 13 cm long, 6 I em wide 31-2
494
Transactions British Mycological Society
and 76 ern high. This incubator was kept in a laboratory with normal working temperatures. From a culture in a plastic Petri dish (9 em diameter) a suspension containing 6 x 108 spores could be prepared 6 days after inoculation. The conditions provided have also induced sporulation of S. tritici, S. aoenae and S. secalis. Seeds were usually inoculated by immersion for about 16 h in an aqueous spore suspension containing 106 spores per ml. Immediately after the seeds were placed in the spore suspension the Universal bottles were thoroughly shaken.
rr===;r====r======r===;=====;r-- Wooden cabinet
Text-fig.
I.
Incubator used to induce sporulation of Septaria spp.
The unsterilized soil sample employed in glasshouse experiments was a sandy loam with a maximum water holding capacity (M.W.H.C.) of 34 % (Colhoun, 1953). The soil was dried to just below 30 % M.W.H.C., brought to the desired moisture content and standard weights were then placed in plastic boxes measuring 20 x 10 ern. Fifty equally spaced seeds were sown in each box at a depth of about 1·5 em. Two boxes constituted a unit for each treatment. The boxes were then placed in glasshouse cubicles or growth cabinets maintained at the appropriate temperatures. In the growth cabinets temperature did not fluctuate more than ± I ° at a setting of 8°. Even in summer the low-temperature glasshouse cubicle provided a temperature of 12° ± 1·0°. In the higher temperature glasshouse cubicle the temperature never fell below 17° but sometimes rose above this level due to solar radiation. During an experiment soil moisture was maintained by weighing each box on alternate days and bringing up to standard weight by addition of water. At the end of an experiment the percentage number of seedlings attacked by S. nodorum in each box was determined by visual observations. The diagnosis of the disease was assisted by the occurrence of protuberances on coleoptiles of infected plants as described by Kietreiber (1961). Such protuberances developed on infected plants of all varieties but were never present on more than 45 % of the diseased plants of any variety. S. nodorum was readily isolated from diseased plants bearing protuberances. Where only lesions were present it was necessary to plate out large numbers of seedlings on V8-Czapek-Dox or potato-dextrose media to ensure that only seedlings attacked by S. nodorum were included in the assessments. Seedlings were divided into four subgroups: (0) healthy, (I) slight infection with up to 25 % of the coleoptile bearing lesions, (2) moderate infection with 25-50 % of the coleoptile bearing lesions, and (3) severe infection with more than 50 % of the coleoptile bearing lesions (PI. 41,
Septoria nodorwn. S.]. 1. Holmes and]. Colhoun
495
fig. I). A disease index per box was obtained by multiplying the percentage of plants in each subgroup by the subgroup number and adding the products. Lesions sometimes developed on leaf sheaths and the first foliage leaf (PI. 41, fig. 2) and these assisted in disease diagnosis. RESULTS
Experiment I. Inoculated and uninoculated seeds of five wheat cultivars were sown in soil with three moisture levels at 12°. Disease indices for inoculated and control boxes were determined (Table I) when the seedlings reached the second leaf stage. A few diseased seedlings developing from uninoculated seed may be attributed to slight natural infection in the seed samples used. For each cultivar the most severe attack occurred in the driest soil and the least severe attack in the wet soil. The results suggest that the different cultivars vary in susceptibility with cv. Cappelle-Desprez being the most susceptible. Table
I.
Effect ofsoilmoisture on infection offive wheat cultivars Disease index
M.W.H.C.
of soil (%)
3 3° 5° 5° 7° 7° 0
Inoculum spores perml
106 Nil
10
6
Nil
106 Nil
I
CappelleDesprez
Maris Ensign
Maris Ranger
264 14 197
129 19 80 14 52 5
157 12 94 5 54 4
8
161 °
Kolibri
Troll
121 16 63 6 38
29 2 18 ° 3 °
I
Experiment 2. Two seed samples, cv. Cappelle-Desprez, were employed. Examination by the paper blotter test (Neergaard, 1970) showed that in samples 1 and 2 the percentages of seeds infected by S. nodorum were 12 and 38 respectively. After being inoculated by the standard technique seeds of sample 1 were sown in unsterilized soil at three moisture levels. Seeds of sample 2, without addition offurther inoculum, were also sown at the same levels of soil moisture. Equal numbers of boxes were maintained at three air temperatures. Seedlings were examined at the two-leaf stage and so those grown at different temperatures were of different ages at sampling time. With naturally infected seed temperature exercised little effect on the disease index, but most disease occurred in the driest soil (Fig. 2). As might be expected, there was more disease at any soil moisture when the seed was inoculated and the disease index declined sharply as the moisture content of the soil increased. With inoculated seed 17° was the least favourable for disease incidence. Experiment 3. Naturally infected seed of sample 2 used in Expt. 2 was sown in soil at the same temperature and moisture levels as employed in Expt. 2. When the second leaf of seedlings had just emerged the number of seedlings infected with S. nodorum in two boxes per treatment was determined (Table 2). At this time two additional boxes of soil with seedlings per treatment were enclosed in plastic bags and kept on the glasshouse
Transactions British Mycological Society
496
bench. An equal number of boxes per treatment was not enclosed in plastic bags and in these the seedlings continued to grow at lower air humidities than those within plastic bags. When the seedlings reached the three-leaf stage of growth, they were examined for the presence of pycnidia 280
240
.., .... :~"'"
220
..., ......
260
'e
'
200
.. .. .. '
"
'
OJ
"0
-',,,,
,,
\
'.
160
t:
..:l::l:
,,
,
....'lI.
180 x
e---e 7°C
.-_12°
,, ,,, , ,, \
\
140
\
,
'. 'e]
0 120
'\, ",.
,
100
Inoculated seed
~
80 60
_
40
• } Naturally infected ...",,:"~ seed
20 0
30 Soil moisture level
50
70
(%M.W.H.C.)
Text-fig. 2. Effects of soil moisture and temperature on the disease levels of wheat coleoptiles infected with S. nodorum.
Table
M.W.H.C.
of soil (%) 30 50
7°
2.
Efficts ofairhumidiry on development ofpycnidia of S. nodorum on infected coleoptiles
Mean air temperature (ec)
Mean %of infected coleoptiles at z-leaf stage
8 12 17 8 12 17 8 12 17
34 3° 42 25 32 29 30 27 25
Mean % of coleoptiles bearing pycnidia at 3-leaf stage , , High air Low air humidity humidity 0 I I
0 15 5 0
9 7
0 0 4 0 0 4 ° 0 0
Septoria nodorum. S.J. 1. Holmes andJ. Colhoun
497
of S. nodorum on the coleoptiles (Table 2). No pycnidia were produced at 8°, although considerable numbers of seedlings were infected at this temperature when examined at the two-leaf stage. Considerably more seedlings at 12° and 17° produced pycnidia when the air humidity after infection was higher (PI. 41, fig. 3). All infected coleoptiles enclosed in plastic bags did not produce pycnidia, and there appeared to be no close relationship between production of pycnidia and the extent of coleoptile infection. It was established that the snores nroduced in the pycnidia were viable. 300 280 260
- - Sterilized soil ...- --... Non-sterilized soil
240
,
220
....
200 180
. 160 .... 140 '" .. is 120 x
."
c:
....
, '.a. ........
.......
,,
,,
.... ....
........ .......... 10' sporesjml
,,
,,
.... ....
....
100
10'
80 60
'" .... .... ....
40
.... ....
'A
- - -...104 104 __ __ %Control
20
0
_
30 Soil moisture level
50
= 70Control
(% M.W.H.C.)
Text-fig. 3. Effects of spore concentration and soil moisture on disease levels on wheat coleoptiles inoculated with S. nodorum at 12°C.
Experiment 4. The development of disease at 12° in sterilized and unsterilized soil was studied using seed inoculated with three spore concentrations and uninoculated seed. Three levels of soil moisture were used. It was noted, 10 days after sowing, that as the inoculum level on the seed decreased, the number of seedlings which had emerged above soil level increased, in both sterilized and unsterilized soils. Twenty-three days after sowing, similar numbers of seedlings had emerged in all treatments, indicating that S. nodorum affects the speed of germination and seedling
498
Transactions British Mycological Society
growth rather than causes death of seedlings. Fig. 3 shows that the disease index is greatly influenced by the inoculum level on the seed, and that less disease occurs as the soil moisture content increases, The heights of all seedlings were measured 23 days after sowing and the mean values per treatment are presented in Table 3. The greatest effect of inoculum level on height of seedlings was shown in the driest soil where the two higher levels of inoculum very significantly reduced height in both sterilized and unsterilized soil. The effects of inoculum level in reducing plant height were less obvious in the wettest sterilized soil, although they were still marked in unsterilized soil of similar moisture content. Table 3. Effect ifSeptoria nodorum on seedling height (Expt. 3) Mean height of seedlings (mm)
M.W,H,C.
of soil (%)
Inoculum
3°
10"
50
1O· Nil 10"
\
Sterilized soil 106'1*** 140'9*
lOS
lSI'S
152'2 173"6 r7o,6 176'8 177'9 17°'3* 174'1 r8r'2 r80'7
IDS
7°
10· Nil 10· ro s ro· Nil
Unsterilized soil 125'6*** 137'0*** 145'1* 153'6 143'5*** 156.8 160·8 160'4 151'8*** r62·8· 168'0 177'r
Significance of difference between mean values for seedlings grown from inoculated and uninoculated seed: *** P = 0'001; ** P = o-or ; * P = 0'05.
Table 4. Infection ofseedlings by Septoria nodorum in the field andeffects onplant height No. infected plants Inoculum Nil Seed naturally infected Seed inoculated
(%)
Disease index
Mean height per plant (mm)
0 55'0 22'7
° 1I3 32
145'0 114'7 151'7
Mean fresh weight per seedling (mg) 39 26 34
Experiment 5. During 1970 a field experiment was made at the Botanical Experimental Grounds, Jodrell Bank, Cheshire. Two samples of seed of cv. Cappelle-Desprez were used. Examination of sample A by the paper blotter test showed that 62 % of the seeds were naturally infected by S. nodorum. Sample B was the same as sample 1 used in Expt. 2. Sowing was carried out on 28 September 1970 in drills 15 em apart and approximately 2'5 em deep in plots each 0'9 m square. Each plot was separated on all sides from other plots by a path 0'9 m in width. Duplicate plots were sown with inoculated and uninoculated seed of sample B and with the naturally infected seed of sample A. The standard method of seed inoculation was used. Three weeks after sowing time the seedlings in two drills (approximately 100 plants) were removed from one of each pair of duplicate plots.
Septoria nodorum. S.]. 1. Holmes and]. Colhoun
499
The heights of these plants were measured and a disease index calculated on the same basis as for glasshouse grown seedlings (Table 4). Naturally infected seeds produced the highest number of infected seedlings and the highest disease index. As compared with seedlings from uninoculated seed those produced by the naturally infected seed were very significantly reduced in height and fresh weight. DISCUSSION
When seeds contaminated with S. nodorum are sown in soil many seedlings show infection of the coleoptiles. The extent of such infection is determined by soil moisture and temperature. Fairly dry soils and an air temperature of about 12° are very favourable for infection. As soil moisture increases the number of infected seedlings decreases, although a considerable proportion may be diseased even in the wettest soils used. Temperatures which are not optimum still permit of a considerable amount of infection. When seeds are inoculated the inoculum level influences the amount of disease which results. When seedlings show infected coleoptiles lesions may also occur on the leaf sheaths which presumably have been directly infected from the coleoptile. In one glasshouse experiment it was shown that if high air humidities were provided around seedlings after infection had occurred more of them bore pycnidia of S. nodorum than did similar seedlings grown under drier air conditions after infection. It is likely that the air humidity near the bases of young seedlings in the field would be high enough to permit pycnidia to develop provided temperatures are high enough. The production of even relatively small numbers of pycnidia on coleoptiles would provide spores which could infect leaves. This suggestion is in agreement with observations made by Hopp (1957) regarding the occurrence of pycnidia on necrotic coleoptiles. Some evidence has been obtained to show that when coleoptile infection is considered wheat cultivars may differ in susceptibility to S. nodorum. Since, under favourable environmental conditions, high levels of seedling infection result when inoculated seed is sown in soil in both the glasshouse and the field there is now a basis for testing cultivars for resistance without having to employ naturally infected seed. Such observations on seedling infection would be a useful supplement to records of ear and leaf infection in field trials. Grateful acknowledgement is made of a grant in support of this work from the Agricultural Research Council. We thank Mr G. Grange for taking the photographs. REFERENCES
COLHOUN,J. (1953)' A study of the epidemiology of club root disease of Brassicae. Annals ofApplied Biology 40, 262-283. Hoss, H. (1957). Untersuchungen iiber die Braunfleckigkeit des Weizens und ihren Erreger Septoria nodorum Berk, (syn. Macrophoma hennebergii Kuhn). Phytopathologische :(,eitschrift 29,395-412.
500
Transactions British Mycological Society
KmTREmER, M. (1961). Die Erkennung des Septoria-Befalles von Weizenk6rnern bei der Saatgutpriifung. Pflanzenschutzberichte 26, 9-10, 129-157, NEERGAARD, P. (1970). Seed pathology, international co-operation and organization. Proceedings of the International Seed Testing Association 35, 19-42. SANDERSON, F. R. (1970). Fusarium diseases of cereals. VII. The effect oflight on sporulation ofF. nivale in culture. Transactions of the British Mycological Society 55, 131-135, SCHAREN, A. I. (1963). Epidemiology of glume blotch of wheat, Phytopathology 53, 747. WEBER, G. F, (1922), Septaria diseases of cereals. II. Septaria diseases of wheat. Phytopathology 12, 537-585. EXPLANATION OF PLATE
Fig.
I.
41
Wheat coleoptiles with (left to right) slight, moderate and severe infection by S. nodorum.
XI'S·
Fig. 2. Wheat seedling showing symptoms of infection by S. nodorum on sheath and first leaf. x 2'5. Fig. 3, Wheat coleoptile showing pycnidia of S. nodorum after growth in high air humidity. x 4.
(Acceptedfor publication
20
May 1971)
Trans. Br, mycol. Soc.
Vol. 57.
Plate 4 I
J /
2
3 (Facing p. 500)
INFECTION OF WHEAT SEEDLINGS BY SEPTORIA NODORUM IN RELATION TO ENVIRONMENTAL FACTORS By S. J. 1. HOLMES
AND
Department of Cryptogamic Botany,
J. COLHOUN Universi!J of Manchester
(With Plate 41 and 3 Text-figures) A technique for the production of large quantities of spores of Septoria nodorum is described, this technique being also suitable for inducing copious sporulation of S. tritici, S. auenae and S. secalis. In glasshouse experiments very favourable conditions for infection of wheat coleoptiles by S. nodorum are provided in dry soil at a temperature of 12°C. Wetter soils and temperatures of 8° or 17° permit of the occurrence of less infection. Pycnidia were frequently produced on diseased coleoptiles in the glasshouse when high air humidities were provided after infection had occurred. It is suggested that cultivars differ in their reaction to S. nodorum in the seedling stage. Cappelle-Desprez has been shown to be very susceptible.
Recently Septaria diseases of cereals have assumed increasing importance in Britain. The significance of seed-borne inoculum and of other methods of survival of the fungus has received attention (Scharen, 1963; Weber, 1922). When infected seeds are sown, symptoms appear on the coleoptiles of the seedlings (Hopp, 1957; Kietreiber, 1961), but little is known about the factors which influence this stage of the development of the disease. An attempt has now been made to study the environmental factors affecting coleoptile infection which results when seeds naturally infected or inoculated with Septaria nodotum are sown in soil. MATERIALS AND METHODS
A single spore culture of S. nodorum isolated from wheat leaves and maintained on V8-Czapek-Dox medium was employed. Sporulation was induced by maintaining cultures at 15-17 °0 with constant illumination from blacklight lamps. Plates were inoculated by pouring over the surface of the medium I ml of spore suspension containing 2'5 x 106 spores per ml. Preliminary experiments showed the necessity of maintaining a high air humidity within plates. If this was not done sporulation greatly declined with repeated subculturing. An inexpensive incubator was constructed (Fig. I) which successfully maintained the required air temperature and humidity while supplying adequate light of the appropriate wavelength. This incubator is a large-scale modification of that used by Sanderson (1970) in work with Fusarium nivale and accommodates 90 plates in a single layer when the external dimensions are 2 13 cm long, 6 I em wide 31-2
494
Transactions British Mycological Society
and 76 ern high. This incubator was kept in a laboratory with normal working temperatures. From a culture in a plastic Petri dish (9 em diameter) a suspension containing 6 x 108 spores could be prepared 6 days after inoculation. The conditions provided have also induced sporulation of S. tritici, S. aoenae and S. secalis. Seeds were usually inoculated by immersion for about 16 h in an aqueous spore suspension containing 106 spores per ml. Immediately after the seeds were placed in the spore suspension the Universal bottles were thoroughly shaken.
rr===;r====r======r===;=====;r-- Wooden cabinet
Text-fig.
I.
Incubator used to induce sporulation of Septaria spp.
The unsterilized soil sample employed in glasshouse experiments was a sandy loam with a maximum water holding capacity (M.W.H.C.) of 34 % (Colhoun, 1953). The soil was dried to just below 30 % M.W.H.C., brought to the desired moisture content and standard weights were then placed in plastic boxes measuring 20 x 10 ern. Fifty equally spaced seeds were sown in each box at a depth of about 1·5 em. Two boxes constituted a unit for each treatment. The boxes were then placed in glasshouse cubicles or growth cabinets maintained at the appropriate temperatures. In the growth cabinets temperature did not fluctuate more than ± I ° at a setting of 8°. Even in summer the low-temperature glasshouse cubicle provided a temperature of 12° ± 1·0°. In the higher temperature glasshouse cubicle the temperature never fell below 17° but sometimes rose above this level due to solar radiation. During an experiment soil moisture was maintained by weighing each box on alternate days and bringing up to standard weight by addition of water. At the end of an experiment the percentage number of seedlings attacked by S. nodorum in each box was determined by visual observations. The diagnosis of the disease was assisted by the occurrence of protuberances on coleoptiles of infected plants as described by Kietreiber (1961). Such protuberances developed on infected plants of all varieties but were never present on more than 45 % of the diseased plants of any variety. S. nodorum was readily isolated from diseased plants bearing protuberances. Where only lesions were present it was necessary to plate out large numbers of seedlings on V8-Czapek-Dox or potato-dextrose media to ensure that only seedlings attacked by S. nodorum were included in the assessments. Seedlings were divided into four subgroups: (0) healthy, (I) slight infection with up to 25 % of the coleoptile bearing lesions, (2) moderate infection with 25-50 % of the coleoptile bearing lesions, and (3) severe infection with more than 50 % of the coleoptile bearing lesions (PI. 41,
Septoria nodorwn. S.]. 1. Holmes and]. Colhoun
495
fig. I). A disease index per box was obtained by multiplying the percentage of plants in each subgroup by the subgroup number and adding the products. Lesions sometimes developed on leaf sheaths and the first foliage leaf (PI. 41, fig. 2) and these assisted in disease diagnosis. RESULTS
Experiment I. Inoculated and uninoculated seeds of five wheat cultivars were sown in soil with three moisture levels at 12°. Disease indices for inoculated and control boxes were determined (Table I) when the seedlings reached the second leaf stage. A few diseased seedlings developing from uninoculated seed may be attributed to slight natural infection in the seed samples used. For each cultivar the most severe attack occurred in the driest soil and the least severe attack in the wet soil. The results suggest that the different cultivars vary in susceptibility with cv. Cappelle-Desprez being the most susceptible. Table
I.
Effect ofsoilmoisture on infection offive wheat cultivars Disease index
M.W.H.C.
of soil (%)
3 3° 5° 5° 7° 7° 0
Inoculum spores perml
106 Nil
10
6
Nil
106 Nil
I
CappelleDesprez
Maris Ensign
Maris Ranger
264 14 197
129 19 80 14 52 5
157 12 94 5 54 4
8
161 °
Kolibri
Troll
121 16 63 6 38
29 2 18 ° 3 °
I
Experiment 2. Two seed samples, cv. Cappelle-Desprez, were employed. Examination by the paper blotter test (Neergaard, 1970) showed that in samples 1 and 2 the percentages of seeds infected by S. nodorum were 12 and 38 respectively. After being inoculated by the standard technique seeds of sample 1 were sown in unsterilized soil at three moisture levels. Seeds of sample 2, without addition offurther inoculum, were also sown at the same levels of soil moisture. Equal numbers of boxes were maintained at three air temperatures. Seedlings were examined at the two-leaf stage and so those grown at different temperatures were of different ages at sampling time. With naturally infected seed temperature exercised little effect on the disease index, but most disease occurred in the driest soil (Fig. 2). As might be expected, there was more disease at any soil moisture when the seed was inoculated and the disease index declined sharply as the moisture content of the soil increased. With inoculated seed 17° was the least favourable for disease incidence. Experiment 3. Naturally infected seed of sample 2 used in Expt. 2 was sown in soil at the same temperature and moisture levels as employed in Expt. 2. When the second leaf of seedlings had just emerged the number of seedlings infected with S. nodorum in two boxes per treatment was determined (Table 2). At this time two additional boxes of soil with seedlings per treatment were enclosed in plastic bags and kept on the glasshouse
Transactions British Mycological Society
496
bench. An equal number of boxes per treatment was not enclosed in plastic bags and in these the seedlings continued to grow at lower air humidities than those within plastic bags. When the seedlings reached the three-leaf stage of growth, they were examined for the presence of pycnidia 280
240
.., .... :~"'"
220
..., ......
260
'e
'
200
.. .. .. '
"
'
OJ
"0
-',,,,
,,
\
'.
160
t:
..:l::l:
,,
,
....'lI.
180 x
e---e 7°C
.-_12°
,, ,,, , ,, \
\
140
\
,
'. 'e]
0 120
'\, ",.
,
100
Inoculated seed
~
80 60
_
40
• } Naturally infected ...",,:"~ seed
20 0
30 Soil moisture level
50
70
(%M.W.H.C.)
Text-fig. 2. Effects of soil moisture and temperature on the disease levels of wheat coleoptiles infected with S. nodorum.
Table
M.W.H.C.
of soil (%) 30 50
7°
2.
Efficts ofairhumidiry on development ofpycnidia of S. nodorum on infected coleoptiles
Mean air temperature (ec)
Mean %of infected coleoptiles at z-leaf stage
8 12 17 8 12 17 8 12 17
34 3° 42 25 32 29 30 27 25
Mean % of coleoptiles bearing pycnidia at 3-leaf stage , , High air Low air humidity humidity 0 I I
0 15 5 0
9 7
0 0 4 0 0 4 ° 0 0
Septoria nodorum. S.J. 1. Holmes andJ. Colhoun
497
of S. nodorum on the coleoptiles (Table 2). No pycnidia were produced at 8°, although considerable numbers of seedlings were infected at this temperature when examined at the two-leaf stage. Considerably more seedlings at 12° and 17° produced pycnidia when the air humidity after infection was higher (PI. 41, fig. 3). All infected coleoptiles enclosed in plastic bags did not produce pycnidia, and there appeared to be no close relationship between production of pycnidia and the extent of coleoptile infection. It was established that the snores nroduced in the pycnidia were viable. 300 280 260
- - Sterilized soil ...- --... Non-sterilized soil
240
,
220
....
200 180
. 160 .... 140 '" .. is 120 x
."
c:
....
, '.a. ........
.......
,,
,,
.... ....
........ .......... 10' sporesjml
,,
,,
.... ....
....
100
10'
80 60
'" .... .... ....
40
.... ....
'A
- - -...104 104 __ __ %Control
20
0
_
30 Soil moisture level
50
= 70Control
(% M.W.H.C.)
Text-fig. 3. Effects of spore concentration and soil moisture on disease levels on wheat coleoptiles inoculated with S. nodorum at 12°C.
Experiment 4. The development of disease at 12° in sterilized and unsterilized soil was studied using seed inoculated with three spore concentrations and uninoculated seed. Three levels of soil moisture were used. It was noted, 10 days after sowing, that as the inoculum level on the seed decreased, the number of seedlings which had emerged above soil level increased, in both sterilized and unsterilized soils. Twenty-three days after sowing, similar numbers of seedlings had emerged in all treatments, indicating that S. nodorum affects the speed of germination and seedling
498
Transactions British Mycological Society
growth rather than causes death of seedlings. Fig. 3 shows that the disease index is greatly influenced by the inoculum level on the seed, and that less disease occurs as the soil moisture content increases, The heights of all seedlings were measured 23 days after sowing and the mean values per treatment are presented in Table 3. The greatest effect of inoculum level on height of seedlings was shown in the driest soil where the two higher levels of inoculum very significantly reduced height in both sterilized and unsterilized soil. The effects of inoculum level in reducing plant height were less obvious in the wettest sterilized soil, although they were still marked in unsterilized soil of similar moisture content. Table 3. Effect ifSeptoria nodorum on seedling height (Expt. 3) Mean height of seedlings (mm)
M.W,H,C.
of soil (%)
Inoculum
3°
10"
50
1O· Nil 10"
\
Sterilized soil 106'1*** 140'9*
lOS
lSI'S
152'2 173"6 r7o,6 176'8 177'9 17°'3* 174'1 r8r'2 r80'7
IDS
7°
10· Nil 10· ro s ro· Nil
Unsterilized soil 125'6*** 137'0*** 145'1* 153'6 143'5*** 156.8 160·8 160'4 151'8*** r62·8· 168'0 177'r
Significance of difference between mean values for seedlings grown from inoculated and uninoculated seed: *** P = 0'001; ** P = o-or ; * P = 0'05.
Table 4. Infection ofseedlings by Septoria nodorum in the field andeffects onplant height No. infected plants Inoculum Nil Seed naturally infected Seed inoculated
(%)
Disease index
Mean height per plant (mm)
0 55'0 22'7
° 1I3 32
145'0 114'7 151'7
Mean fresh weight per seedling (mg) 39 26 34
Experiment 5. During 1970 a field experiment was made at the Botanical Experimental Grounds, Jodrell Bank, Cheshire. Two samples of seed of cv. Cappelle-Desprez were used. Examination of sample A by the paper blotter test showed that 62 % of the seeds were naturally infected by S. nodorum. Sample B was the same as sample 1 used in Expt. 2. Sowing was carried out on 28 September 1970 in drills 15 em apart and approximately 2'5 em deep in plots each 0'9 m square. Each plot was separated on all sides from other plots by a path 0'9 m in width. Duplicate plots were sown with inoculated and uninoculated seed of sample B and with the naturally infected seed of sample A. The standard method of seed inoculation was used. Three weeks after sowing time the seedlings in two drills (approximately 100 plants) were removed from one of each pair of duplicate plots.
Septoria nodorum. S.]. 1. Holmes and]. Colhoun
499
The heights of these plants were measured and a disease index calculated on the same basis as for glasshouse grown seedlings (Table 4). Naturally infected seeds produced the highest number of infected seedlings and the highest disease index. As compared with seedlings from uninoculated seed those produced by the naturally infected seed were very significantly reduced in height and fresh weight. DISCUSSION
When seeds contaminated with S. nodorum are sown in soil many seedlings show infection of the coleoptiles. The extent of such infection is determined by soil moisture and temperature. Fairly dry soils and an air temperature of about 12° are very favourable for infection. As soil moisture increases the number of infected seedlings decreases, although a considerable proportion may be diseased even in the wettest soils used. Temperatures which are not optimum still permit of a considerable amount of infection. When seeds are inoculated the inoculum level influences the amount of disease which results. When seedlings show infected coleoptiles lesions may also occur on the leaf sheaths which presumably have been directly infected from the coleoptile. In one glasshouse experiment it was shown that if high air humidities were provided around seedlings after infection had occurred more of them bore pycnidia of S. nodorum than did similar seedlings grown under drier air conditions after infection. It is likely that the air humidity near the bases of young seedlings in the field would be high enough to permit pycnidia to develop provided temperatures are high enough. The production of even relatively small numbers of pycnidia on coleoptiles would provide spores which could infect leaves. This suggestion is in agreement with observations made by Hopp (1957) regarding the occurrence of pycnidia on necrotic coleoptiles. Some evidence has been obtained to show that when coleoptile infection is considered wheat cultivars may differ in susceptibility to S. nodorum. Since, under favourable environmental conditions, high levels of seedling infection result when inoculated seed is sown in soil in both the glasshouse and the field there is now a basis for testing cultivars for resistance without having to employ naturally infected seed. Such observations on seedling infection would be a useful supplement to records of ear and leaf infection in field trials. Grateful acknowledgement is made of a grant in support of this work from the Agricultural Research Council. We thank Mr G. Grange for taking the photographs. REFERENCES
COLHOUN,J. (1953)' A study of the epidemiology of club root disease of Brassicae. Annals ofApplied Biology 40, 262-283. Hoss, H. (1957). Untersuchungen iiber die Braunfleckigkeit des Weizens und ihren Erreger Septoria nodorum Berk, (syn. Macrophoma hennebergii Kuhn). Phytopathologische :(,eitschrift 29,395-412.
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Transactions British Mycological Society
KmTREmER, M. (1961). Die Erkennung des Septoria-Befalles von Weizenk6rnern bei der Saatgutpriifung. Pflanzenschutzberichte 26, 9-10, 129-157, NEERGAARD, P. (1970). Seed pathology, international co-operation and organization. Proceedings of the International Seed Testing Association 35, 19-42. SANDERSON, F. R. (1970). Fusarium diseases of cereals. VII. The effect oflight on sporulation ofF. nivale in culture. Transactions of the British Mycological Society 55, 131-135, SCHAREN, A. I. (1963). Epidemiology of glume blotch of wheat, Phytopathology 53, 747. WEBER, G. F, (1922), Septaria diseases of cereals. II. Septaria diseases of wheat. Phytopathology 12, 537-585. EXPLANATION OF PLATE
Fig.
I.
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Wheat coleoptiles with (left to right) slight, moderate and severe infection by S. nodorum.
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Fig. 2. Wheat seedling showing symptoms of infection by S. nodorum on sheath and first leaf. x 2'5. Fig. 3, Wheat coleoptile showing pycnidia of S. nodorum after growth in high air humidity. x 4.
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Vol. 57.
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