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Estimation of resistance of three wheat cultivars to Septoria tritici using a chemical method for determination of fungal mycelium
[ 15 ] Trans. Br. mycol, Soc. 69 (1) 15-19 (1977)
Printedin Great Britain
ESTIMATION OF RESISTANCE OF THREE WHEAT CUL TIVARS TO SEPTORIA TRITICI USING A CHEMICAL METHOD FOR DETERMINATION OF FUNGAL MYCELIUM By K. M. HARROWER Botany Department, School of General Studies, Australian National University, P.O. Box 4, Canberra, 2600, Australia
A chemical method was employed to determine the mycelial content of wheat leaves infected by Septaria tritici Desm. A positive correlation was found between the latent period, incubation period, percentage of leaf area affected and the mycelial content of infected leaves. At certain times after inoculation the relative resistance of cultivars to S. tritici appeared to alter. The technique may be used to assess the reaction of wheat cultivars to this pathogen. Various methods have been employed to estimate the degree of resistance of wheat cultivars to foliar pathogens, including Septaria tritici Desm., the causal agent of speckled leaf blotch of wheat. Assessment keys for the proportion of foliar tissue bearing lesions have long been used but the problem of objectivity still exists. Assessments may also be made of the loss in grain yield due to foliar infection or of the loss in dry foliar tissue. Recently, a novel technique was reported for assessing the numbers of pycnidia of S. tritici in wheat leaves by means of electronic and photo-scanning equipment (Eyal & Brown, 1976). Many of the currently used methods of assessment express the resistance of a cultivar to a particular pathogen in terms of the quantity of visible symptoms which arise as the result of the host: pathogen interaction. The method described here is based on the premise that a strain of the pathogen will grow more slowly in a more resistant cultivar than in a less resistant cultivar. Many examples are cited in the literature in which the amount of hyphal material in stained, cleared leaves of resistant cultivars is less than that in those of lesser resistance. However, there may be times in the growth of a pathogen in host tissues when little or no increase in the degree of observable symptoms occurs and yet there is a significantly large increase in the quantity of pathogen mycelium. In the wheat: S. tritici system this occurs towards the end of the latent period when hyphae accumulate in the sub-stomatal cavities prior to pycnidium formation (Martin & Harrower, unpublished data). The formation of this 'reproductive' mycelium is, in fact, important in that there are several reports of a positive correlation being found between the level
of resistance of a cultivar and the quantity of pycnidia produced (Brokenshire, 1973). The method used here relies on a chemical estimation of fungal mycelium in the leaf tissues of wheat cultivars (Ride & Drysdale, 1972). MATERIALS AND METHODS
The isolate of S. tritici was from naturally infected wheat (cv. Robin) from Wagga Wagga, New South Wales. It was grown in a liquid medium of the following composition: NaN0 3 , 2'0 g; KH zP0 4 , 0'5 g; MgS0 4.7H zO, 0'5 g; xci, 0'5 g; FeS0 4 • 7HzO, 0'01 g; glucose, 20'0 g; trace element solution, 1'0 ml; distilled water, 1 1. This medium, gelled by the addition of 15'0 g of Oxoid No. 3 agar, was also used for producing inoculum for plant inoculations. Agar plates were inoculated with a pycnidiospore suspension of the pathogen obtained from glasshouse inoculated plants. Secondary conidia were produced in these cultures as described by Lee & Jones (1974) and were harvested after 8 days. A spore suspension of 105 spores per ml was prepared and two drops of Tween 20 per 100 ml of inoculum were added to ensure uniform wetting of the leaves. Leaves were inoculated by spraying till run-off and were then covered by clear polythene bags for 2 days to assist spore germination and penetration of the leaves. Seedlings of three wheat cultivars, Summit, Pinnacle and Teal, were grown until the second leaf stage when they were inoculated as described above. All plants were grown in a controlled environment maintained at 22° during a 12 h light period and at 19° during a 12 h dark period. The
16
Wheat resistance to Septoria tritici
relative humidity within the cabinet varied between 72 and 91 % over the duration of the experiment. At various times after inoculation ten second leaves, each of approximately the same length, from replicate plants of each cultivar were removed and weighed prior to the assay procedure. The test leaves were cut into 1 ern lengths and homogenised in 5 ml acetone for 3 min in a Sunbeam blender at the fastest speed. The homogenate was transferred to a centrifuge tube and centrifuged at 1500 g for 10 min at 4°. The supernatant was removed and discarded and the remaining solids washed with 10 ml sterile distilled water before recentrifuging at 1500 g for 10 min. The solid residue was then mixed with 3 ml of KOH solution (120 g in 100 ml distilled water) and autoclaved at 130° for 1 h. After cooling the solution was mixed with 8 ml of 75 % ethanol and stored at 4° for 15 min. A celite suspension (0'9 ml), as prepared by Ride & Drysdale (1972), was layered on top and the tubes recentrifuged at 1500 g for 10 min at 4°. Further washings of the residue both with 40 % ethanol and with chilled distilled water followed. The final residue was diluted to i : 5 ml and assayed for chitosan at the r-5 ml level as described by Ride & Drysdale (1972). Control, uninoculated plants were also assessed for their chitin and chitosan content. Surface examination of these plants, which were grown adjacent to the treated plants, revealed the occa-
and inaccurate. Leaves bearing lesions, while still attached to the plant, were laid on an illuminated transparency viewer and then kept flat with a thin glass sheet. A sheet of clear plastic was laid over the glass and both the whole leaf outlines and the lesion outlines marked. The lesion outlines were then blacked out and the lesion area per leaf calculated using an automatic area meter (Hayashi Denko Co. Ltd, Tokyo). The entire leaf outline was then blacked out and the leaf area measured.
present in the infected leaves of Summit increased (Fig. 1). At about 22 days after inoculation an increased rate of mycelial proliferation became apparent, probably in the sub-stomatal cavities of leaflesions (Martin &Harrower, unpublished data). Microscopic examination of the leaves at that stage also showed the presence of many intercellular hyphae in the host tissues together with hyphal masses within the sub-stomatal cavities and in adjacent tissues. After the end of the latent period when mature pycnidia bearing pycnidiospores are present, the assay would also be partly assessing the multitude of hyphal and of spore cell walls present in the mature lesion. At this stage it would be unwise to assume that the level of pycnidial or of pycnidiospore cell wall material produced would be correlated with the extent of mycelial growth in
sional presence of saprophytic fungal mycelium
the leaves.
and spores (Martin &Harrower, unpublished data). In the calculations ofthe fungal content of inoculated leaves the probable level of chitosan due to these saprophytic fungi and to the basal glucosamine content of the leaves was taken into account by using values obtained from control uninoculated plants. The colorimetric assay employed was as used by Ride & Drysdale (1972). The level of sensitivity used was l' 5 ml and the extinction of the final reaction product was read at 650 nm. Cultures of the pathogen grown in the liquid medium were used to find the correlation between mycelial fresh weight and mycelial dry weight and then to correlate mycelial dry weight with chitin content. Solutions containing known quantities of glucosamine were assayed and a standard curve was produced to which all other readings could be related. The isolate of the pathogen used gave a conversion factor of 34'3 ± 2'7 flg glucosaminejmg dry weight fungus. A further batch of seedlings of each cultivar was assessed at various times after inoculation for the proportion of leaf area visibly affected, i.e. the percentage lesion area (% LAA). The use of leaf symptom assessment keys was found to be difficult
Fig. 1 also shows a gradual increase in lesion area. Towards the end of the sampling period there was no change in the rate of increase of lesion area. Observations made of infected leaves of Summit showed not only fresh lesions during the period of leaf colonization but also the coalescence of older lesions, notably those nearer the leaf tip. Those would then not make such a great contribution towards the net increase in lesion area. Such coalesced compound lesions gradually enlarged to eventually kill tissues further from the leaf tip. Newer formed lesions enlarged and may partly have compensated for the reduced rate of increase in lesion area in affected tissues caused by older lesions. At all stages of leaf colonization the level of assessed fungal dry weight takes into account not only that mycelium within the visible lesions but also any mycelium outside them. At the end of the sampling period there is a divergence between the assessed rate of increase of fungal dry weight in infected tissues and in the mean lesion area per leaf. This may be partly due to the formation of pycnidial primordia and the relatively more stable rate of appearance of infected tissues. Pycnidia
RESUL TS AND DISCUSSION
After inoculation, the quantity of fungal mycelium
K. M. Harrower
17
1500
300
'On 1000
200
,~
I
~
'-'
3> S ;:l
1 E
g
~ u
S '-
0:1
...'"0:1
0
l: 0
1: OIl
100
500
"03 ~
";;;
'"
-l
...
;>,
Q
o
8
16
o
24
Time after inoculation (days)
Fig.
1.
The mycelial content and lesion area of wheat leaves (cv. Summit) at various times after inoculation with Septoria tritici.
2000 Summit
bii ,3 S ;:l
1500
~ o
;>,
S
1000
'0
1: OIl "03
~
...;>,
CI
500 Teal
o
10
20
30
40
Time after inoculation (days)
Fig.
2.
The mycelial content of three wheat cultivars at various times after inoculation with Septoria
tritici.
Wheat resistance to Septoria tritici
18
Table 1. Percentage of leaf area visibly affected at various times after inoculation of three wheat cultivars with S. tritici Days after inoculation 7 1S! 21 26
% Leaf area affected Sununit 1 22 S4 83
"
Pinnacle 0 11 38 67
Teal 0 4 20 36
Table 2. The incubation and latent periods of S. tritici on three wheat cultivars Incubation period (days) Latent period (days)
Sununit Pinnacle 11 6-7 37 41
Teal 13 47
Table 3. Relationships between the mycelial content of infected leaves and the percentage of leaf area visibly affected Teal Summit Pinnacle Day lS! Mycelial dry weight 360± 14'8 247± 12'9 167± 14'6 (pg) Relativemycelial 2'16 1'00 1'48 dry weight % Leaf area affected 22±2'7 11± 1'9 4±o'8 Relative % LAA 1'00 2'75 5'50 Ratio of mycelial dry 16'4 41"8 22'5 weight: % LAA Day 21 Mycelialdry weight 660±21'6 447± 16'9 273± 12'3 (pg) Relativemycelial 1'00 1'64 2'42 dry weight % Leaf area affected 54±3'4 38±2 '1 20± 1'7 Relative % LAA 1'00 2'70 1'90 11,8 Ratio of mycelial dry 12'2 13'7 weight: % LAA Day 26 Mycelialdry weight1480± 36'3 627±31'2 340± 19'3 (pg) Relativemycelial 1'00 1'84 4'35 dry weight % Leaf area affected 36±2'8 67±6'2 1,86 Relative % LAA 1'00 Ratio of mycelial dry 9'4 9'4 weight: % LAA were only observed to form in older lesions (Martin & Harrower, unpublished data).
Fig. 2 illustrates the different rate in mycelial growth of the pathogen in three wheat cultivars. J. Kuiper (pers. comm.) states that field trials indicate that, of the three wheat cultivars used in this study, Summit is the most susceptible to S. tritici and Teal is the least susceptible, and this is
borne out by the assessment of mycelial content of infected leaves of these three cultivars. Clearly, the rate of increase of the pathogen is greatest in Summit and least in Teal. The same ranking of susceptibilities could also be obtained in terms of the mean percentages of leaf area bearing lesions (Table 1) and in terms of the incubation and latent periods (Table 2). At each of the three sampling dates shown the order of susceptibilities is Summit > Pinnacle > Teal as judged by the estimated quantity of fungal mycelium found in infected tissues and by the mean leaf area bearing lesions for each cultivar (Table 3). The relative susceptibilities of the three cultivars, as judged by mycelial content, are approximately the same after both 1S! and 21 days after inoculation. However, after 26 days, there is an apparent increase in mycelial content in leaves of Summit which may be attributed to the large increase in mycelial content as dense regions of mycelium occur at various sites in the lesions due to the formation of pycnidial initials. In contrast, the ratios representing the % LAA at days 21 and 26 are very similar, In the most resistant cultivar, i.e, Teal, colonizing hyphae will move out from the lesion into healthy tissues. There is a lag period between this colonization and the appearance of chlorosis. In the least resistant cultivar this lag period will be short when compared to that for Teal. This view is further supported by the ratios of mycelial dry weight /% LAA. After 1St days from inoculation the largest ratio is observed in Teal and the smallest in Summit, This may suggest that much of the assessed mycelium in Teal is not within the assessed lesion area whereas in Summit there is relatively little mycelium in the tissues outside the visible periphery ofthe lesion, Thus, in Summit, a relatively shorter time exists between colonization of the healthy tissues and their metabolic disruption as assessed by the appearance of chlorosis. After 21 days from inoculation the ratio of mycelial dry weight /% LAA is very similar for each of the three cultivars. This may be due to the following: in Teal the observed lesion area has much external mycelium, whereas in Summit there is a larger lesion area with proliferation of hyphae within these lesions and little external hyphal material. After 26 days many of the older lesions in Summit have coalesced and can only enlarge towards the leaf base, In Teal, only 36 % of the total leaf area bears symptoms and there is still an adequate quantity of healthy tissue to colonize. The increase in mycelial dry weight /% LAA in Summit may be due to the proliferation of hyphae in the lesions and the formation of pycnidial initials .
K. M. Harrower In using this technique consideration must be given to the pattern of development of the lesions, but it can provide another parameter by which the resistance of wheat cultivars to S. tritici can be assessed. However, a standard curve must be produced to relate the £650 values of glucosamine standards to mycelial dry weight, and this is impossible at present with rust, smut and mildew pathogens which cannot be grown adequately in culture. The author wishes to thank Mrs N. Plovanic and Mrs V. Keraitis for invaluable technical assistance and Mr J. Kuiper of the Agricultural Research Institute at Wagga Wagga for helpful discussion.
19 REFERENCES
BROKENSHIRE, T. (1973). Studies on Septaria tritici Desm. Ph.D. Thesis, University of Exeter. EYAL, Z. & BROWN, M. B. (1976). A quantitative method for estimating density of Septoria tritici pycnidia on wheat leaves. Phytopathology 66,11-14. LEE, N.-P. & JONES, D. G. (1974). Production of secondary conidia by Septaria tritici in culture. Transactions of the British Mycological Society 62, 212-213. RIDE, J. P. & DRYSDALE, R. B.. (1972). A rapid method for the chemical estimation of filamentous fungi in plant tissue. Physiological Plant Pathology 2, 7-15.
(Accepted for publication 13 December 1976)
Printedin Great Britain
ESTIMATION OF RESISTANCE OF THREE WHEAT CUL TIVARS TO SEPTORIA TRITICI USING A CHEMICAL METHOD FOR DETERMINATION OF FUNGAL MYCELIUM By K. M. HARROWER Botany Department, School of General Studies, Australian National University, P.O. Box 4, Canberra, 2600, Australia
A chemical method was employed to determine the mycelial content of wheat leaves infected by Septaria tritici Desm. A positive correlation was found between the latent period, incubation period, percentage of leaf area affected and the mycelial content of infected leaves. At certain times after inoculation the relative resistance of cultivars to S. tritici appeared to alter. The technique may be used to assess the reaction of wheat cultivars to this pathogen. Various methods have been employed to estimate the degree of resistance of wheat cultivars to foliar pathogens, including Septaria tritici Desm., the causal agent of speckled leaf blotch of wheat. Assessment keys for the proportion of foliar tissue bearing lesions have long been used but the problem of objectivity still exists. Assessments may also be made of the loss in grain yield due to foliar infection or of the loss in dry foliar tissue. Recently, a novel technique was reported for assessing the numbers of pycnidia of S. tritici in wheat leaves by means of electronic and photo-scanning equipment (Eyal & Brown, 1976). Many of the currently used methods of assessment express the resistance of a cultivar to a particular pathogen in terms of the quantity of visible symptoms which arise as the result of the host: pathogen interaction. The method described here is based on the premise that a strain of the pathogen will grow more slowly in a more resistant cultivar than in a less resistant cultivar. Many examples are cited in the literature in which the amount of hyphal material in stained, cleared leaves of resistant cultivars is less than that in those of lesser resistance. However, there may be times in the growth of a pathogen in host tissues when little or no increase in the degree of observable symptoms occurs and yet there is a significantly large increase in the quantity of pathogen mycelium. In the wheat: S. tritici system this occurs towards the end of the latent period when hyphae accumulate in the sub-stomatal cavities prior to pycnidium formation (Martin & Harrower, unpublished data). The formation of this 'reproductive' mycelium is, in fact, important in that there are several reports of a positive correlation being found between the level
of resistance of a cultivar and the quantity of pycnidia produced (Brokenshire, 1973). The method used here relies on a chemical estimation of fungal mycelium in the leaf tissues of wheat cultivars (Ride & Drysdale, 1972). MATERIALS AND METHODS
The isolate of S. tritici was from naturally infected wheat (cv. Robin) from Wagga Wagga, New South Wales. It was grown in a liquid medium of the following composition: NaN0 3 , 2'0 g; KH zP0 4 , 0'5 g; MgS0 4.7H zO, 0'5 g; xci, 0'5 g; FeS0 4 • 7HzO, 0'01 g; glucose, 20'0 g; trace element solution, 1'0 ml; distilled water, 1 1. This medium, gelled by the addition of 15'0 g of Oxoid No. 3 agar, was also used for producing inoculum for plant inoculations. Agar plates were inoculated with a pycnidiospore suspension of the pathogen obtained from glasshouse inoculated plants. Secondary conidia were produced in these cultures as described by Lee & Jones (1974) and were harvested after 8 days. A spore suspension of 105 spores per ml was prepared and two drops of Tween 20 per 100 ml of inoculum were added to ensure uniform wetting of the leaves. Leaves were inoculated by spraying till run-off and were then covered by clear polythene bags for 2 days to assist spore germination and penetration of the leaves. Seedlings of three wheat cultivars, Summit, Pinnacle and Teal, were grown until the second leaf stage when they were inoculated as described above. All plants were grown in a controlled environment maintained at 22° during a 12 h light period and at 19° during a 12 h dark period. The
16
Wheat resistance to Septoria tritici
relative humidity within the cabinet varied between 72 and 91 % over the duration of the experiment. At various times after inoculation ten second leaves, each of approximately the same length, from replicate plants of each cultivar were removed and weighed prior to the assay procedure. The test leaves were cut into 1 ern lengths and homogenised in 5 ml acetone for 3 min in a Sunbeam blender at the fastest speed. The homogenate was transferred to a centrifuge tube and centrifuged at 1500 g for 10 min at 4°. The supernatant was removed and discarded and the remaining solids washed with 10 ml sterile distilled water before recentrifuging at 1500 g for 10 min. The solid residue was then mixed with 3 ml of KOH solution (120 g in 100 ml distilled water) and autoclaved at 130° for 1 h. After cooling the solution was mixed with 8 ml of 75 % ethanol and stored at 4° for 15 min. A celite suspension (0'9 ml), as prepared by Ride & Drysdale (1972), was layered on top and the tubes recentrifuged at 1500 g for 10 min at 4°. Further washings of the residue both with 40 % ethanol and with chilled distilled water followed. The final residue was diluted to i : 5 ml and assayed for chitosan at the r-5 ml level as described by Ride & Drysdale (1972). Control, uninoculated plants were also assessed for their chitin and chitosan content. Surface examination of these plants, which were grown adjacent to the treated plants, revealed the occa-
and inaccurate. Leaves bearing lesions, while still attached to the plant, were laid on an illuminated transparency viewer and then kept flat with a thin glass sheet. A sheet of clear plastic was laid over the glass and both the whole leaf outlines and the lesion outlines marked. The lesion outlines were then blacked out and the lesion area per leaf calculated using an automatic area meter (Hayashi Denko Co. Ltd, Tokyo). The entire leaf outline was then blacked out and the leaf area measured.
present in the infected leaves of Summit increased (Fig. 1). At about 22 days after inoculation an increased rate of mycelial proliferation became apparent, probably in the sub-stomatal cavities of leaflesions (Martin &Harrower, unpublished data). Microscopic examination of the leaves at that stage also showed the presence of many intercellular hyphae in the host tissues together with hyphal masses within the sub-stomatal cavities and in adjacent tissues. After the end of the latent period when mature pycnidia bearing pycnidiospores are present, the assay would also be partly assessing the multitude of hyphal and of spore cell walls present in the mature lesion. At this stage it would be unwise to assume that the level of pycnidial or of pycnidiospore cell wall material produced would be correlated with the extent of mycelial growth in
sional presence of saprophytic fungal mycelium
the leaves.
and spores (Martin &Harrower, unpublished data). In the calculations ofthe fungal content of inoculated leaves the probable level of chitosan due to these saprophytic fungi and to the basal glucosamine content of the leaves was taken into account by using values obtained from control uninoculated plants. The colorimetric assay employed was as used by Ride & Drysdale (1972). The level of sensitivity used was l' 5 ml and the extinction of the final reaction product was read at 650 nm. Cultures of the pathogen grown in the liquid medium were used to find the correlation between mycelial fresh weight and mycelial dry weight and then to correlate mycelial dry weight with chitin content. Solutions containing known quantities of glucosamine were assayed and a standard curve was produced to which all other readings could be related. The isolate of the pathogen used gave a conversion factor of 34'3 ± 2'7 flg glucosaminejmg dry weight fungus. A further batch of seedlings of each cultivar was assessed at various times after inoculation for the proportion of leaf area visibly affected, i.e. the percentage lesion area (% LAA). The use of leaf symptom assessment keys was found to be difficult
Fig. 1 also shows a gradual increase in lesion area. Towards the end of the sampling period there was no change in the rate of increase of lesion area. Observations made of infected leaves of Summit showed not only fresh lesions during the period of leaf colonization but also the coalescence of older lesions, notably those nearer the leaf tip. Those would then not make such a great contribution towards the net increase in lesion area. Such coalesced compound lesions gradually enlarged to eventually kill tissues further from the leaf tip. Newer formed lesions enlarged and may partly have compensated for the reduced rate of increase in lesion area in affected tissues caused by older lesions. At all stages of leaf colonization the level of assessed fungal dry weight takes into account not only that mycelium within the visible lesions but also any mycelium outside them. At the end of the sampling period there is a divergence between the assessed rate of increase of fungal dry weight in infected tissues and in the mean lesion area per leaf. This may be partly due to the formation of pycnidial primordia and the relatively more stable rate of appearance of infected tissues. Pycnidia
RESUL TS AND DISCUSSION
After inoculation, the quantity of fungal mycelium
K. M. Harrower
17
1500
300
'On 1000
200
,~
I
~
'-'
3> S ;:l
1 E
g
~ u
S '-
0:1
...'"0:1
0
l: 0
1: OIl
100
500
"03 ~
";;;
'"
-l
...
;>,
Q
o
8
16
o
24
Time after inoculation (days)
Fig.
1.
The mycelial content and lesion area of wheat leaves (cv. Summit) at various times after inoculation with Septoria tritici.
2000 Summit
bii ,3 S ;:l
1500
~ o
;>,
S
1000
'0
1: OIl "03
~
...;>,
CI
500 Teal
o
10
20
30
40
Time after inoculation (days)
Fig.
2.
The mycelial content of three wheat cultivars at various times after inoculation with Septoria
tritici.
Wheat resistance to Septoria tritici
18
Table 1. Percentage of leaf area visibly affected at various times after inoculation of three wheat cultivars with S. tritici Days after inoculation 7 1S! 21 26
% Leaf area affected Sununit 1 22 S4 83
"
Pinnacle 0 11 38 67
Teal 0 4 20 36
Table 2. The incubation and latent periods of S. tritici on three wheat cultivars Incubation period (days) Latent period (days)
Sununit Pinnacle 11 6-7 37 41
Teal 13 47
Table 3. Relationships between the mycelial content of infected leaves and the percentage of leaf area visibly affected Teal Summit Pinnacle Day lS! Mycelial dry weight 360± 14'8 247± 12'9 167± 14'6 (pg) Relativemycelial 2'16 1'00 1'48 dry weight % Leaf area affected 22±2'7 11± 1'9 4±o'8 Relative % LAA 1'00 2'75 5'50 Ratio of mycelial dry 16'4 41"8 22'5 weight: % LAA Day 21 Mycelialdry weight 660±21'6 447± 16'9 273± 12'3 (pg) Relativemycelial 1'00 1'64 2'42 dry weight % Leaf area affected 54±3'4 38±2 '1 20± 1'7 Relative % LAA 1'00 2'70 1'90 11,8 Ratio of mycelial dry 12'2 13'7 weight: % LAA Day 26 Mycelialdry weight1480± 36'3 627±31'2 340± 19'3 (pg) Relativemycelial 1'00 1'84 4'35 dry weight % Leaf area affected 36±2'8 67±6'2 1,86 Relative % LAA 1'00 Ratio of mycelial dry 9'4 9'4 weight: % LAA were only observed to form in older lesions (Martin & Harrower, unpublished data).
Fig. 2 illustrates the different rate in mycelial growth of the pathogen in three wheat cultivars. J. Kuiper (pers. comm.) states that field trials indicate that, of the three wheat cultivars used in this study, Summit is the most susceptible to S. tritici and Teal is the least susceptible, and this is
borne out by the assessment of mycelial content of infected leaves of these three cultivars. Clearly, the rate of increase of the pathogen is greatest in Summit and least in Teal. The same ranking of susceptibilities could also be obtained in terms of the mean percentages of leaf area bearing lesions (Table 1) and in terms of the incubation and latent periods (Table 2). At each of the three sampling dates shown the order of susceptibilities is Summit > Pinnacle > Teal as judged by the estimated quantity of fungal mycelium found in infected tissues and by the mean leaf area bearing lesions for each cultivar (Table 3). The relative susceptibilities of the three cultivars, as judged by mycelial content, are approximately the same after both 1S! and 21 days after inoculation. However, after 26 days, there is an apparent increase in mycelial content in leaves of Summit which may be attributed to the large increase in mycelial content as dense regions of mycelium occur at various sites in the lesions due to the formation of pycnidial initials. In contrast, the ratios representing the % LAA at days 21 and 26 are very similar, In the most resistant cultivar, i.e, Teal, colonizing hyphae will move out from the lesion into healthy tissues. There is a lag period between this colonization and the appearance of chlorosis. In the least resistant cultivar this lag period will be short when compared to that for Teal. This view is further supported by the ratios of mycelial dry weight /% LAA. After 1St days from inoculation the largest ratio is observed in Teal and the smallest in Summit, This may suggest that much of the assessed mycelium in Teal is not within the assessed lesion area whereas in Summit there is relatively little mycelium in the tissues outside the visible periphery ofthe lesion, Thus, in Summit, a relatively shorter time exists between colonization of the healthy tissues and their metabolic disruption as assessed by the appearance of chlorosis. After 21 days from inoculation the ratio of mycelial dry weight /% LAA is very similar for each of the three cultivars. This may be due to the following: in Teal the observed lesion area has much external mycelium, whereas in Summit there is a larger lesion area with proliferation of hyphae within these lesions and little external hyphal material. After 26 days many of the older lesions in Summit have coalesced and can only enlarge towards the leaf base, In Teal, only 36 % of the total leaf area bears symptoms and there is still an adequate quantity of healthy tissue to colonize. The increase in mycelial dry weight /% LAA in Summit may be due to the proliferation of hyphae in the lesions and the formation of pycnidial initials .
K. M. Harrower In using this technique consideration must be given to the pattern of development of the lesions, but it can provide another parameter by which the resistance of wheat cultivars to S. tritici can be assessed. However, a standard curve must be produced to relate the £650 values of glucosamine standards to mycelial dry weight, and this is impossible at present with rust, smut and mildew pathogens which cannot be grown adequately in culture. The author wishes to thank Mrs N. Plovanic and Mrs V. Keraitis for invaluable technical assistance and Mr J. Kuiper of the Agricultural Research Institute at Wagga Wagga for helpful discussion.
19 REFERENCES
BROKENSHIRE, T. (1973). Studies on Septaria tritici Desm. Ph.D. Thesis, University of Exeter. EYAL, Z. & BROWN, M. B. (1976). A quantitative method for estimating density of Septoria tritici pycnidia on wheat leaves. Phytopathology 66,11-14. LEE, N.-P. & JONES, D. G. (1974). Production of secondary conidia by Septaria tritici in culture. Transactions of the British Mycological Society 62, 212-213. RIDE, J. P. & DRYSDALE, R. B.. (1972). A rapid method for the chemical estimation of filamentous fungi in plant tissue. Physiological Plant Pathology 2, 7-15.
(Accepted for publication 13 December 1976)