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Experimental factors favouring infection of attached cucumber leaves by Didymella bryoniae
[ 89 ] Trans. Br. mycol. Soc. 71 (1) 89""97 (1978)
Printedin Great Britain
EXPERIMENTAL FACTORS FAVOURING INFECTION OF ATTACHED CUCUMBER LEAVES BY DIDYMELLA BRYONIAE By G. SVEDELIUS
AND
T. UNESTAM
University of Uppsala, Institute of Physiological Botany, Box 540, S-751 21 Uppsala, Sweden Didymella bryoniae (Auersw.) Rehm causes black rot infection in attached cucumber leaves. Environmental factors influencing infection were examined in the laboratory. Hydathode regions close to main veins were the most susceptible areas of the intact leaf to fungal infection. Mechanical injury increased susceptibility to conidial infection. Nutrients added to intact leaf surfaces increased susceptibility to the same extent as did injury. Germ-tube development from a conidial suspension on the leaf surface and subsequent infection of cucumber leaf tissue occurred only if the surface was kept wetted after inoculation. High light intensities during incubation increased the resistance of young cucumber plants to conidial infection; such intensities induced chlorosis and prevented the development of wet rot. Inoculation with a conidial suspension and incubation for 3-5 days caused more infection at 25°C than at 18° and 35° in mechanically injured leaves.
Cucumber growers in Sweden have tried to overcome cucumber black rot infections for the last 15 years. Infection is caused by Didymella bryoniae (Auersw.) Rehm, imperfect form Ascochyta cucumis Fautr. & Roum., and the disease was identified and surveyed for the first time in Sweden by Nyberg in 1962 (Nyberg, 1962, 1963). The fungus is also known as Mycosphaerella melonis (Pass.) Chiu & Walker and the nomenclature has been summarized by Chiu & Walker (1949a) and Muller & Arx (1962). The disease has increased in importance in recent years, as new cucumber cultivars and culture methods have been introduced. D. bryoniae infections on young cucumber seedlings were studied by Chiu & Walker (1949b), who found them to be resistant in the early seedling stage but the degree of infection varied between different cucumber cultivars. Hordijk & Goosen (1962) using spore inoculum, could infect old leaves but not younger ones, and showed that mechanical injury increased the susceptibility to infection. An attempt to relate conditions of cucumber culture and the degree of infection caused by D. bryoniae was made by Fletcher & Preece (1966). They studied the effects on infection of bedtype, training system, temperature regime, ventilation practice, date of planting and fungicide treatment in greenhouses. In order to analyse factors that may be important in the infectivity of D. bryoniae on cucumber plants, we have developed methods to infect attached leaves with conidial suspensions and
other types of inocula under controlled environmental conditions. MATERIAL
The fungus D. bryoniae, identified by the Commonwealth Mycological Institute, England, was isolated from infected cucumber leaves obtained from the Swedish Seed Association, Hammenhog. A single conidial strain was kept in stock culture under mineral oil at 5 °C on vegetable-extract agar (Biotta, Tagerwilen, Switzerland) at pH 6'0 during the research period. Conidia were obtained from 6- to ,-day-old cultures grown in daylight on autoclaved potato cubes in 150 ml Erlenmeyer flasks. The cultures were immersed for 30 min in sterile distilled water to release the conidia from pycnidia. There was usually a small percentage of ascospores (less than 5 %) in the suspension. However, conidia and ascospores of D. bryoniae were shown to be equally infective on watermelon (Chiu & Walker, 1949b) and our tests indicate that the same is true for the infection of cucumber leaves. The conidial suspension was first filtered through filterpaper (Munktell, NO.3) to remove mycelial and potato fragments and then washed twice in sterile distilled water using repeated centrifugation at 2000 g. The conidial concentration was measured with a haemacytorneter and adjusted to the desired level with distilled water. Cuttings of the cucumber cv. Bestseller OE were used as host plants. They were rooted and grown on fertilized peat moss in 9 ern plastic pots,
Fig. 1. (A) Mechanically injured cucumber leaf inoculated with droplets of conidial suspension of D. bryoniae. A gradient of inoculum concentration was tested on the same leaf. (B) The underside of the same leaf after incubation for 2 days in darkness. Conidia per site: zone a = 100; zone b = 30; zone c = 10. An area of wet rot has developed and is increasing with time. (C) Areas of wet rot surrounded by chlorotic tissue, developed at infected sites under intensive illumination (10 klx),
G. Svedelius and T. Unestam placed in a growth chamber with an 18 h light period at 5 klx (Osram 77/20 and Osram L20/20) at 28° and a 6 h dark period at 20° . The cuttings were kept free from attack by pests and other fungi. Attacks by fungal gnat maggots (Mycetophilidae) were prevented by adding Volaton (Bayer) to the soil. The rooted plants were allowed to develop three to four leaves before the infection experiments. METHODS
In most experiments the leaves were about 3 weeks old. In some experiments these were mechanically injured by pressing the surface with a 3 mm cork borer. In other experiments, with intact leaves, nutrient solution was sprayed on to the leaves and allowed to dry before inoculation. These nutrient solutions consisted of 0'05 % casein hydrolysate (Oxoid, London, England) with 0'1 % sucrose, or cucumber leaf exudate. Cucumber leaf exudate was produced by immersing leaves (mean fresh weight 3 g) in distilled water (50 ml per leaf) for 20 min. The exudate was then concentrated 40 times with low-pressure boiling at 45° and filtered aseptically (membrane filter, 0'2 pm) before use. The leaf was usually inoculated with 0'02 ml droplets containing suspended conidia. Unless otherwise indicated, 20 sites per leaf were inoculated by placing droplets on to the surface of intact or mechanically injured leaves. Microscopically, nearly all of the added conidia, applied as droplets of conidial suspension, had sedimented and stuck to the leaf surface (checked with running tap water) after less than 10 min . However, on leaves younger than 2 weeks old these droplets spread out on the surface, because of the undeveloped wax layer, which prevented the sedimentation of conidia on the sites of application. When a gradient of conidial concentrations was to be tested on the leaf surface, 5-10 sites per concentration were inoculated with conidia of each leaf. Infection of intact leaves was also tested by the application of 3 mm disks of vegetable extract agar with mycelium and 3 mm disks of infected cucumber leaves on to the surface. Five to 25 disks of each kind were applied to each leaf. About 15 min after application of droplets or disks the plants were sprayed carefully with an aerosol of distilled water until the leaf surface was completely wetted. The infected cucumber plants were incubated in clear plastic boxes (kept at 100 % humidity at 25°) which were placed in a moist chamber with a light period of 18 h (Osram 77R/80 and Osram
91
L20/8o) unless otherwise indicated. Leaf wetness was maintained by repeated spraying. L ight intensity was regulated by shading with black plastic sheets to obtain the desired illumination. Light intensity was measured with a lux meter (Lunasix 3, Gossen) and radiant energy with a quantum sensor (Li-185 Quantum-sensor, Lambda). Inoculated sites were inspected macroscopically for fungal infection and the percentage of infected sites calculated. Infected tissues were normally water-soaked and soft (wet rot) (Fig. 1). Illuminated plants developed chlorosis or wet rot surrounded by chloro sis (Fig. 1) at infected sites . Increased softness of the leaf surface at the wet rotted areas was tested by pressing with a blunted needle. RESULTS
It was not possible to achieve visible infection by D. bryoniae of cucumber leaves, intact or injured, unless the leaf surface was continuously wetted by repeated spraying until infection had developed. When the atmosphere was saturated, a water film on the leaf surface developed spontaneously along leaf margins, often close to hydathodes, and also on the parts of blades located close to other leaves. These same areas were sites for infection when inoculated cucumber plants were kept close to each other in moist chambers. The degree of infection in different regions of cucumber leaves after inoculation with conidial droplets on the surface was measured. Hydathode regions close to main veins were more susceptible to infection than other areas of the leaf blade when the percentage sites infected in each area were compared (Table 1). There was no significant difference of infection between leaf-blade areas and leaf margins between main veins, where hydathodes are also found.
Table 1. Local susceptibility to D. bryoniae as percentage infection in different areas of the cucumber leaf surface (Inoculation with 105 conidia/site. Incubation for 6 days at 10 klx, 18 replicates.) Sites infected/sites inoculated x 100 in each area .-----------"'------------, Leaf blade 4'9±2'1
Leaf margin (between main veins) 9'2±3'S
Near hydathode (main vein ending) 2S'3±4'1***
*** Value significantly different from that of the blade surface, (P = 0'1 %).
Didymella and cucumber leaves
92 100
2e
.S
50
u
~
c:
"
"
10
103
10'
10·
10'
Inoculum(conidia/site)
_.-._
100
B
... _._. __. .... _._ .... .-._
,,I 6
~
50
,
I
I
"
~
J'
I
,I
r. ... .i->:
,j,
10
·f"
10'
103 104 Inoculum (conidia/site)
10"
Fig. 2. Effect of inoculum concentration and light intensity on infectivity of D. bryoniae on cucumber leaves. Mechanically injured (_._) and intact (--) leaves were incubated for (A) 2 and (B) 4 days. Il1umination for an 18 h light period with 10 klx (0), 5 klx (.), 1 klx (.) or in continuous darkness (D). Mean values of seven replicates and standard errors are given.
The ability of conidial suspensions of the fungus to infect intact blades of cucumber leaves was very low (Table 1), but the degree of infection increased with the concentration of conidia added (Figs . 2, 3). Infection levels were high when intact cucumber leaves were inoculated with infected leaf tissue or with mycelial disks from vegetable extract agar cultures. The disks of infected host tissue were more infective than the mycelial agar disks of about the same fungal age (Table 2). When different temperatures for incubation
were tested, infection of mechanically injured leaves was greatest at 25° after 3-5 days (Fig. 4). However, at 18° infection levels increased greatly between three and five days and the increase was much more pronounced than that at higher temperatures. The infection level at 18° had reached that at 25° on the fifth day. On intact leaves, the infection level was highest at 35°, but was low in comparison with that of injured leaves. The application of conidial suspensions of different concentration on to the leaf surface made it possible to calculate the effective dose causing
G. Suedelius and T. Unestam
93
100 , . . - - - - - - - - - - - - - - - - - - - - - - - ,
2 .e ti
50
~
t:
10' Inoculum (conidia/site)
10'
Fig. 3. Effect of nutrients and inoculum concentration gradient on infectivity of D. bryoniae on intact cucumber leaves. Plants were incubated for 4 days at 1 klx. •- . , Control; A-A, cucumber exudate; . - . , casein hydrolysate/sucrose. Means of seven replicates and standard errors are given.
Table 2. Infectivity ofD. bryoniae mycelium in (A) vegetable extract agar and (B) infected leaf tissue disks applied to cucumber leaves (Incubation for 5 days at 10 klx, 30 replicates.) Inoculum Infection ( %) (A) Infected cucumber leaf tissue 89'0 ± 2'5 (B) Vegetable extract agar 34'0 ± 6'1
50 % infection (ED so) of leaves in different treatments. Mechanically injured leaves were always significantly (P < 0'1) more susceptible to infection than intact leaves when inoculated with conidial suspensions (T able 3; Fig. 2). We found that slight injuries, such as broken trichomes, substantially increased infection . Also, necrotic tissues, originating from treatments other than mechanical injury, provided excellent sites for infection. There was a significant increase (P < 0'1) in susceptibility to infection when leaves were sprayed with the nutrient solution containing casein hydrolysate and sucrose (Fig . 3). Thus, the ED so values were lowered by addition of this solution to the leaf sur face (Table 4). When inoculated intact leaves were sprayed daily with the nutrient solution, the ED . o was almost as low as for those mechanically injured (Table 3). Also, leaf exudate sprayed on to the leaf surface before inoculation gave a slight increase (P < 5) in infectivity (Fig. 3). Conidial germination and hyphal growth were
examined in vitro in the presence of different nutrients (Table 5). Germination was almost 100 % in all solutions . In distilled water a short germ-tube was formed . Germ-tube growth was stimulated in cucumber leaf exudate but no hyphae were formed. In cucumber leaf exudate or distilled water, only germ-tubes were formed: these were thin and partly autolysed. Casein hydrolysate-sucrose solution and vegetable extract medium apparently allowed direct hyphal formation and growth without initial germ-tube formation. The hyphae were thick and filled with vacuoles and fat droplets, indicating good nutrient uptake from the nutrient solutions. However, the same average length was attained in all nutrient solutions except that containing casein hydrolysate without sucrose. Plants incubated in continuous darkness with mechanically injured leaves were infected by very low concentrations of conidia (Fig. 2). ED;o was in this case as low as 5-10 conidia per site after 2 days of incubation, and wet rot developed without any chlorosis (Fig. 1; Table 3). The use of different light intensities during incubation influenced the number of conidia needed for visible infection. Mechanically injured plants illuminated with 1 klx fluorescent light (12 ,uEinstein . rn" . S-I) had an ED so of 15 conidia per site after 2 days' incubation. The infected sites developed only a weak, peripheral chlorotic zone around the wet rot. Mechanically injured leaves illuminated with 5 klx (90/1E.m-2.s-l) and 10 klx (215 flE.m - 2 .s - l) gave ED so of 100 and
Didymella and cucumber leaves
94 100
c
o
'B
50
~
c
5
4
3
6
Incubation (days)
Fig. 4. Effect of temperature on infectivity of D. bryoniae on intact and injured cucumber leaves. Intact leaf surface (--) inoculated with 2'4 x lOS conidia/site and mechanically injured leaf surface (_._) with 8 x 10 2 conidia per site. Plants incubated for 3-6 days at 5 klx, Means of seven replicates and standard errors are given. Table 3. ED SO of D . bryoniae conidia infecting cucumber leaves: effects of light intensity (18 h light periotf), injury of the leaf surface, and time of incubation: ED so values from Fig. 2 ED so, conidia/site Light intensity (klx) 10 10
5 5 1 1
Darkness Darkness
Incubation Meehan. injured leaf (days) 2
4 2
4
Intact leaf
1 x lOS 1 X 10' 1 X 10 2
2
2 X 10 1
10~
4
10 1
10'
2
10 1 10 1
4
100000 conidia respectively after 2 days' incubation (Table 3; Fig. 2). In these leaves the infected sites were intensively chlorotic, with (Fig. 1) or without wet rot. There was an approximately logarithmic relationship between light intensity and ED so (Fig. 5) and a small increase in light intensity raised the ED so greatly . It appeared that the plants became more or less saturated with light, giving rise to resistance. After 4 days of incubation the levels of infection at all light intensities had increased in both intact and injured leaves (Fig. 2; Table 3). In order to investigate the actual light intensities which occur in commercial production of
Table 4. ED so of D. bryoniae conidia infecting cucumber leaves: effects of adding nutrients to the leaf surface (Incubation for 4 days at 1 klx,ED so valuesfrom Fig. 3 and additional data.) ED so Treatment (conidia/site) 10' Untreated leaf 10· Cucumber leaf exudate 10 3 Casein hydrolysate/sucrose 10 1 Caseinhydrolysate/sucrose twicea day
cucumbers, light intensity was measured during cloudy days in April in greenhouses in southern Sweden. At noon the top leaves received about 10 klx (daylight, estimated at 125 Elt .m- 2 • S-I) and the lower leaves of plants 2 klx (daylight, estimated at 25,uE. m- 2 •S-I). Such light conditions may persist for several weeks during spring. DISCUSSION
A film of free water seemed to be necessary during the initial phase of infection of leaves by conidia of D. bryoniae. After spot development 100 % humidity was required for further expansion of the tissue rot. Otherwise, infection ceased and a dry necrotic lesion was formed, Ch iu & Walker (1949b) obtained small chlorotic lesions when they incubated cucumber seedlings for 3 days after inoculation with conidial suspensions
G. Svedelius and T. Unestam
95
Table 5. Effect of nutrients on conidial germination and germ-tube or hyphal growth in vitro (Incubation for 12 h at
Mean values and standard errors were calculated from 50 sarnples .) Germination Length of germ tube Nutrients pH (%) or hypha (pm) 1 % vegetable extract 100 94'8 ± 8'3 (hypha) 5'0 0'05 % caseinhydrolysate 100 95'8 ± 10'3 (hypha) 5'5 1 % sucrose 0'05 % casein hydrolysate 39'S ± 5'2 (hypha) 5'5 98 100 Cucumberleaf exudate 6'0 93'8 ± 9'0 (germtube) Distilled water 9'4< 1'0 (germtube) 5'0 93 22 °.
fungus from which conidia had been removed by aseptic filtration. Condensation in greenhouses during the night occurs frequently and probably favours infection. ) We have observed that the spontaneous infection C} 1 5 level may be very low in greenhouses where old / dead leaves are left on the plants. Such leaves / • I probably absorb moisture during the night and / J4 thereby prevent water condensation and subseS / I;;; quent infection. Fewer wounds for fungal pene/ "" tration will also result when old leaves are not / I ~ . / 1 3 ~ removed The available nutrient supply on intact leaf / I e5 surfaces is obviously another restricting factor for / ~ leaf infect ion with conidia of D. bryoniae. Thus, / 0 J 2 .3 spraying the leaf with concentrated cucumber / I leaf exudate or casein hydrolysate supported / infection (Table 4). The raised infectivity is 0/ I probably the result of increased germ-tube or / 11 hyphal growth on the leaf surface, since this was the effect of such extracts in vitro (Table 5). I In the presence of cucumber leaf exudate, J0 10' germ-tube growth was increased but hyphae were not formed . The effect of leaf exudate is therefore 06--_~-_'---==:""-~--~ somewhat specific in relation to the other nutrient 10 5 solutions in which thick initial hyphae rather than Light intensity (k/xi germ-tubes seemed to develop directly from the Fig. 5. Relation between light intensity and ED so of conidia. D. bryoniae on mechanically injured cucumber leaves In our work, the hydathode regions close to the after 2 days incubation. Calculated from data in Fig. 2. main veins were the most readily infected parts of e, ED so; 0 , log sn.; the leaf (Table 1). It is probable that naturally occurring nutrients and water, originating from of D , bryoniae. Their results can be interpreted guttation droplets, stimulated germ-tube growth in different ways: a water film may, in fact, have on leaf surface and fungal infection. A relationship been present during incubation; enzymes and between the presence of guttation droplets pronutrients originating from the culture substrate viding water and nutrients and fungus infection may have been present in the inoculum and might was reported by Yarwood (1952). Nutrient have increased the ability of the fungus to infect leakage, especially from the main veins, may leavesj and it is also possible that a high concen- support fungal growth on specific areas of the leaf tration of such enzymes alone could cause the surface (Collins, 1976). Hydathode tissue is found observed lesions without fungal infection . We have to be a site for infection by the bacterium Xanthefound such enzyme effects on cucumber leaves monas campestris causing black rot of cabbage after only a few hours' application of droplets of (Stakman & Harrar, 1957). However, with parasolution taken from imm ersed cultures of the sitic fungi penetration and infection through J6 I
Didymella and cucumber leaves hydathodes is seldom observed and is considered comparatively unimportant (Wheeler, 1968). It is evident that the infectivity of D. bryoniae on cucumber is increased by the presence of injured or necrotic leaf tissue (T able 3). Mechanical injury facilitates fungal invasion because of release of nutrients from damaged cells rather than rupture of the protective cutin layer. Almost the same infectivity was obtained with intact cucumber leaves sprayed repeatedly with nutrients after inoculation (Table 4). Mycelium from infected host tissue or from vegetable extract agar gave high infection after inoculation on to intact cucumber leaves (Tables 1, 2) probably because of the production of extracellular enzymes as well as of the nutrient supply available for hyphal development. D . bryoniae was found to produce pectinase and cellulase in infected squash fruits (Current, 1969). Intense light apparently increased resistance in the cucumber leaves (Table 3). Under these conditions lesions were limited with or without wet rot and the infected sites were surrounded by chlorosis (Fig. 1). Calpouzos S: Stallknecht (1967) reported that when sugar beet was infected with Cercospora beticola, high light intensity (15-30 klx) resulted in lesions of limited size which did not increase with time; the spots had a sharp purple halo. On the other hand, under low intensity (1'5-3 klx) the lesions were usually larger, increasing in size with time and without purple discoloration. Similarly, in our tests lesions which developed in low light intensity and darkness were larger and without discoloration (F ig. 1). Preliminary tests showed that when one of three leaves of a cucumber plant was kept in darkness, all the leaves were equally resistant to infection and had the same tendency to form chlorotic tissue after inoculation with conidia. It therefore seems probable that the susceptibility of individual leaves depends upon the condition of the entire plant. Translocation between the leaves of assimilates or other substances may be involved in light-induced plant resistance. Low light intensity was recorded in greenhouses during cloudy weather in southern Sweden. This might encourage first infections of D. bryoniae on cucumber since in the laboratory there was a good correlation between low light intensity and infection (Table 3). In intact leaves the degree of infection obtained at 35° was found to be greater than at lower temperatures (Fig. 4). At th is temperature the plants also became gradually pale and chlorotic, in contrast with plants at 25° and 18°. The symptoms indicate that a light intensity of 5 klx was too low at 35° to maintain a positive energy
balance, result ing in starvation and an increased susceptibility to disease. However, preliminary tests in vivo have shown that 35° is above the optimum for germ-tube formation by D. bryoniae on the intact cucumber leaf surface: both germtube length and vitality seem to be affected. The infection level at 35° was therefore limited to 20-30 % ; probably by reduced germ-tube vitality (Fig. 4). This temperature effect also explains the consistently low infection levels on mechanically injured leaves at 35° compared with those at other temperatures (Fig. 4). The optimal temperatures for infection of watermelon and musk-melon plants by D. bryoniae were found to be 24° and 18° respectively after 2 days of incubation in the greenhouse (Chiu & Walker, 1949b ). A temperature of 24° was also reported as optimal for watermelon fruit rot development after one and 2 weeks' incubation (Luepschen, 1961). The present investigation indicates, however, that it is meaningless to propose an optimal temperature for plant infection after a certain period of incubation if critical environmental conditions, such as water and nutrient supply, light before and during the incubation, type of inoculum and host tissue, are not all described. This investigation was supported by grants from The Royal Academy of Agriculture and Forestry. We would like to thank Dr Gosta Karlsson and Dr Me Wellving, Swedish Seed Association, for their generous help with fungal material and advice on conidial production. Thanks are also due to Miss Birgitta Ericsson for measuring light intensity in greenhouses at Alnarp. The authors are grateful to Dr jannet Rowe for critically reviewing the manuscript. REFERENCES
CALPOUZOS, L. & STALLKNECHT, G. F. (1967). Symptoms of Cercospora leafspot of sugar beets influenced by light intensity. Phytopathology 57, 799-800. CHIU, W. F. & WALKER, J. C. (1949a). Morphology and variability of the cucurbit black rot fungus. Journal of Agricultural Research 78, 81-102.
CHIU, W. F. & WALKER, J. C. (1949b). Physiology and pathogenicity of the cucurbit black rot fungus. Journal of Agr icultural Research 78, 589-615.
COLLINS, M. A. (1976). Colonisation of leaves by phylloplane saprophytes and their interactions in this environment. In M icrobiology of Aerial Plant Surfa ces (ed. C. H. Dickinson and T. F. Preece), pp. 401-418 . London: Academic Press. CURRENT, T. (1969). Pectic and cellulolytic enzymes produced by Mycosphaerella citrullina and their relation to black rot of squash. Canadian Journal of Botany 47, 79 1-794.
G. Suedelius and T. Unestam FLETCHER, ] . T . & PREECE, T . F. (1966). My cosphaerella stem rot of cucumber in the Lea Valley. Annals of Applied Biology 58, 423-430. HORDIJK, C. V. & GOOSEN, P. G . (1962). Aanta sting van komkornmer door M ycosphaerella melonis. Tijd schrift cn'er Plantenzickten 68, 149. LUEPSCHEN, N. S. (1961). The de velopment of M y cosphaerella black rot and P ellicularia rolfsii rot of watermelon at various temperatures. Pl ant Disease Reporter 45, 557-559· M ULLER, E. & ARX, ] . A. (1962) . D ie Gattungen der didymosporen Pyrenomyceten, Beitrdge zur Kryptogamenflora der S chuieiz 11, 351-364.
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NYBERG, A. (1962 ). Svartprickrota - en for vllrt land ny sjukdorn p;\ gurka. V ax tsky ddsnotiser 7.7, 58-63. NYBERG, A. (1963). Svartprickrota pll gurka - iakttagelser och forsok. Vdxtskyddsnotiser 7.7, 76-82. STAKMAN, L. ]. & HARRAR, G. S. (1957). Prin ciples of Plant Pathology . New York: Ronald Press. WHEELER, B. E. ] . (1968 ). Fungal parasites of plants. In The Fungi - an advanced treatise, vol. III (ed. G. C. Ainsworth and A. S. Sussman), pp. 179-210. New York, London: Academic Press. YARWOOD, C. E. (1952). Guttation due to leaf presSure favours fungus infections. Phytopathology 42, 52 0 .
(Accepted for publication 18 January 1978)
~IYC
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Printedin Great Britain
EXPERIMENTAL FACTORS FAVOURING INFECTION OF ATTACHED CUCUMBER LEAVES BY DIDYMELLA BRYONIAE By G. SVEDELIUS
AND
T. UNESTAM
University of Uppsala, Institute of Physiological Botany, Box 540, S-751 21 Uppsala, Sweden Didymella bryoniae (Auersw.) Rehm causes black rot infection in attached cucumber leaves. Environmental factors influencing infection were examined in the laboratory. Hydathode regions close to main veins were the most susceptible areas of the intact leaf to fungal infection. Mechanical injury increased susceptibility to conidial infection. Nutrients added to intact leaf surfaces increased susceptibility to the same extent as did injury. Germ-tube development from a conidial suspension on the leaf surface and subsequent infection of cucumber leaf tissue occurred only if the surface was kept wetted after inoculation. High light intensities during incubation increased the resistance of young cucumber plants to conidial infection; such intensities induced chlorosis and prevented the development of wet rot. Inoculation with a conidial suspension and incubation for 3-5 days caused more infection at 25°C than at 18° and 35° in mechanically injured leaves.
Cucumber growers in Sweden have tried to overcome cucumber black rot infections for the last 15 years. Infection is caused by Didymella bryoniae (Auersw.) Rehm, imperfect form Ascochyta cucumis Fautr. & Roum., and the disease was identified and surveyed for the first time in Sweden by Nyberg in 1962 (Nyberg, 1962, 1963). The fungus is also known as Mycosphaerella melonis (Pass.) Chiu & Walker and the nomenclature has been summarized by Chiu & Walker (1949a) and Muller & Arx (1962). The disease has increased in importance in recent years, as new cucumber cultivars and culture methods have been introduced. D. bryoniae infections on young cucumber seedlings were studied by Chiu & Walker (1949b), who found them to be resistant in the early seedling stage but the degree of infection varied between different cucumber cultivars. Hordijk & Goosen (1962) using spore inoculum, could infect old leaves but not younger ones, and showed that mechanical injury increased the susceptibility to infection. An attempt to relate conditions of cucumber culture and the degree of infection caused by D. bryoniae was made by Fletcher & Preece (1966). They studied the effects on infection of bedtype, training system, temperature regime, ventilation practice, date of planting and fungicide treatment in greenhouses. In order to analyse factors that may be important in the infectivity of D. bryoniae on cucumber plants, we have developed methods to infect attached leaves with conidial suspensions and
other types of inocula under controlled environmental conditions. MATERIAL
The fungus D. bryoniae, identified by the Commonwealth Mycological Institute, England, was isolated from infected cucumber leaves obtained from the Swedish Seed Association, Hammenhog. A single conidial strain was kept in stock culture under mineral oil at 5 °C on vegetable-extract agar (Biotta, Tagerwilen, Switzerland) at pH 6'0 during the research period. Conidia were obtained from 6- to ,-day-old cultures grown in daylight on autoclaved potato cubes in 150 ml Erlenmeyer flasks. The cultures were immersed for 30 min in sterile distilled water to release the conidia from pycnidia. There was usually a small percentage of ascospores (less than 5 %) in the suspension. However, conidia and ascospores of D. bryoniae were shown to be equally infective on watermelon (Chiu & Walker, 1949b) and our tests indicate that the same is true for the infection of cucumber leaves. The conidial suspension was first filtered through filterpaper (Munktell, NO.3) to remove mycelial and potato fragments and then washed twice in sterile distilled water using repeated centrifugation at 2000 g. The conidial concentration was measured with a haemacytorneter and adjusted to the desired level with distilled water. Cuttings of the cucumber cv. Bestseller OE were used as host plants. They were rooted and grown on fertilized peat moss in 9 ern plastic pots,
Fig. 1. (A) Mechanically injured cucumber leaf inoculated with droplets of conidial suspension of D. bryoniae. A gradient of inoculum concentration was tested on the same leaf. (B) The underside of the same leaf after incubation for 2 days in darkness. Conidia per site: zone a = 100; zone b = 30; zone c = 10. An area of wet rot has developed and is increasing with time. (C) Areas of wet rot surrounded by chlorotic tissue, developed at infected sites under intensive illumination (10 klx),
G. Svedelius and T. Unestam placed in a growth chamber with an 18 h light period at 5 klx (Osram 77/20 and Osram L20/20) at 28° and a 6 h dark period at 20° . The cuttings were kept free from attack by pests and other fungi. Attacks by fungal gnat maggots (Mycetophilidae) were prevented by adding Volaton (Bayer) to the soil. The rooted plants were allowed to develop three to four leaves before the infection experiments. METHODS
In most experiments the leaves were about 3 weeks old. In some experiments these were mechanically injured by pressing the surface with a 3 mm cork borer. In other experiments, with intact leaves, nutrient solution was sprayed on to the leaves and allowed to dry before inoculation. These nutrient solutions consisted of 0'05 % casein hydrolysate (Oxoid, London, England) with 0'1 % sucrose, or cucumber leaf exudate. Cucumber leaf exudate was produced by immersing leaves (mean fresh weight 3 g) in distilled water (50 ml per leaf) for 20 min. The exudate was then concentrated 40 times with low-pressure boiling at 45° and filtered aseptically (membrane filter, 0'2 pm) before use. The leaf was usually inoculated with 0'02 ml droplets containing suspended conidia. Unless otherwise indicated, 20 sites per leaf were inoculated by placing droplets on to the surface of intact or mechanically injured leaves. Microscopically, nearly all of the added conidia, applied as droplets of conidial suspension, had sedimented and stuck to the leaf surface (checked with running tap water) after less than 10 min . However, on leaves younger than 2 weeks old these droplets spread out on the surface, because of the undeveloped wax layer, which prevented the sedimentation of conidia on the sites of application. When a gradient of conidial concentrations was to be tested on the leaf surface, 5-10 sites per concentration were inoculated with conidia of each leaf. Infection of intact leaves was also tested by the application of 3 mm disks of vegetable extract agar with mycelium and 3 mm disks of infected cucumber leaves on to the surface. Five to 25 disks of each kind were applied to each leaf. About 15 min after application of droplets or disks the plants were sprayed carefully with an aerosol of distilled water until the leaf surface was completely wetted. The infected cucumber plants were incubated in clear plastic boxes (kept at 100 % humidity at 25°) which were placed in a moist chamber with a light period of 18 h (Osram 77R/80 and Osram
91
L20/8o) unless otherwise indicated. Leaf wetness was maintained by repeated spraying. L ight intensity was regulated by shading with black plastic sheets to obtain the desired illumination. Light intensity was measured with a lux meter (Lunasix 3, Gossen) and radiant energy with a quantum sensor (Li-185 Quantum-sensor, Lambda). Inoculated sites were inspected macroscopically for fungal infection and the percentage of infected sites calculated. Infected tissues were normally water-soaked and soft (wet rot) (Fig. 1). Illuminated plants developed chlorosis or wet rot surrounded by chloro sis (Fig. 1) at infected sites . Increased softness of the leaf surface at the wet rotted areas was tested by pressing with a blunted needle. RESULTS
It was not possible to achieve visible infection by D. bryoniae of cucumber leaves, intact or injured, unless the leaf surface was continuously wetted by repeated spraying until infection had developed. When the atmosphere was saturated, a water film on the leaf surface developed spontaneously along leaf margins, often close to hydathodes, and also on the parts of blades located close to other leaves. These same areas were sites for infection when inoculated cucumber plants were kept close to each other in moist chambers. The degree of infection in different regions of cucumber leaves after inoculation with conidial droplets on the surface was measured. Hydathode regions close to main veins were more susceptible to infection than other areas of the leaf blade when the percentage sites infected in each area were compared (Table 1). There was no significant difference of infection between leaf-blade areas and leaf margins between main veins, where hydathodes are also found.
Table 1. Local susceptibility to D. bryoniae as percentage infection in different areas of the cucumber leaf surface (Inoculation with 105 conidia/site. Incubation for 6 days at 10 klx, 18 replicates.) Sites infected/sites inoculated x 100 in each area .-----------"'------------, Leaf blade 4'9±2'1
Leaf margin (between main veins) 9'2±3'S
Near hydathode (main vein ending) 2S'3±4'1***
*** Value significantly different from that of the blade surface, (P = 0'1 %).
Didymella and cucumber leaves
92 100
2e
.S
50
u
~
c:
"
"
10
103
10'
10·
10'
Inoculum(conidia/site)
_.-._
100
B
... _._. __. .... _._ .... .-._
,,I 6
~
50
,
I
I
"
~
J'
I
,I
r. ... .i->:
,j,
10
·f"
10'
103 104 Inoculum (conidia/site)
10"
Fig. 2. Effect of inoculum concentration and light intensity on infectivity of D. bryoniae on cucumber leaves. Mechanically injured (_._) and intact (--) leaves were incubated for (A) 2 and (B) 4 days. Il1umination for an 18 h light period with 10 klx (0), 5 klx (.), 1 klx (.) or in continuous darkness (D). Mean values of seven replicates and standard errors are given.
The ability of conidial suspensions of the fungus to infect intact blades of cucumber leaves was very low (Table 1), but the degree of infection increased with the concentration of conidia added (Figs . 2, 3). Infection levels were high when intact cucumber leaves were inoculated with infected leaf tissue or with mycelial disks from vegetable extract agar cultures. The disks of infected host tissue were more infective than the mycelial agar disks of about the same fungal age (Table 2). When different temperatures for incubation
were tested, infection of mechanically injured leaves was greatest at 25° after 3-5 days (Fig. 4). However, at 18° infection levels increased greatly between three and five days and the increase was much more pronounced than that at higher temperatures. The infection level at 18° had reached that at 25° on the fifth day. On intact leaves, the infection level was highest at 35°, but was low in comparison with that of injured leaves. The application of conidial suspensions of different concentration on to the leaf surface made it possible to calculate the effective dose causing
G. Suedelius and T. Unestam
93
100 , . . - - - - - - - - - - - - - - - - - - - - - - - ,
2 .e ti
50
~
t:
10' Inoculum (conidia/site)
10'
Fig. 3. Effect of nutrients and inoculum concentration gradient on infectivity of D. bryoniae on intact cucumber leaves. Plants were incubated for 4 days at 1 klx. •- . , Control; A-A, cucumber exudate; . - . , casein hydrolysate/sucrose. Means of seven replicates and standard errors are given.
Table 2. Infectivity ofD. bryoniae mycelium in (A) vegetable extract agar and (B) infected leaf tissue disks applied to cucumber leaves (Incubation for 5 days at 10 klx, 30 replicates.) Inoculum Infection ( %) (A) Infected cucumber leaf tissue 89'0 ± 2'5 (B) Vegetable extract agar 34'0 ± 6'1
50 % infection (ED so) of leaves in different treatments. Mechanically injured leaves were always significantly (P < 0'1) more susceptible to infection than intact leaves when inoculated with conidial suspensions (T able 3; Fig. 2). We found that slight injuries, such as broken trichomes, substantially increased infection . Also, necrotic tissues, originating from treatments other than mechanical injury, provided excellent sites for infection. There was a significant increase (P < 0'1) in susceptibility to infection when leaves were sprayed with the nutrient solution containing casein hydrolysate and sucrose (Fig . 3). Thus, the ED so values were lowered by addition of this solution to the leaf sur face (Table 4). When inoculated intact leaves were sprayed daily with the nutrient solution, the ED . o was almost as low as for those mechanically injured (Table 3). Also, leaf exudate sprayed on to the leaf surface before inoculation gave a slight increase (P < 5) in infectivity (Fig. 3). Conidial germination and hyphal growth were
examined in vitro in the presence of different nutrients (Table 5). Germination was almost 100 % in all solutions . In distilled water a short germ-tube was formed . Germ-tube growth was stimulated in cucumber leaf exudate but no hyphae were formed. In cucumber leaf exudate or distilled water, only germ-tubes were formed: these were thin and partly autolysed. Casein hydrolysate-sucrose solution and vegetable extract medium apparently allowed direct hyphal formation and growth without initial germ-tube formation. The hyphae were thick and filled with vacuoles and fat droplets, indicating good nutrient uptake from the nutrient solutions. However, the same average length was attained in all nutrient solutions except that containing casein hydrolysate without sucrose. Plants incubated in continuous darkness with mechanically injured leaves were infected by very low concentrations of conidia (Fig. 2). ED;o was in this case as low as 5-10 conidia per site after 2 days of incubation, and wet rot developed without any chlorosis (Fig. 1; Table 3). The use of different light intensities during incubation influenced the number of conidia needed for visible infection. Mechanically injured plants illuminated with 1 klx fluorescent light (12 ,uEinstein . rn" . S-I) had an ED so of 15 conidia per site after 2 days' incubation. The infected sites developed only a weak, peripheral chlorotic zone around the wet rot. Mechanically injured leaves illuminated with 5 klx (90/1E.m-2.s-l) and 10 klx (215 flE.m - 2 .s - l) gave ED so of 100 and
Didymella and cucumber leaves
94 100
c
o
'B
50
~
c
5
4
3
6
Incubation (days)
Fig. 4. Effect of temperature on infectivity of D. bryoniae on intact and injured cucumber leaves. Intact leaf surface (--) inoculated with 2'4 x lOS conidia/site and mechanically injured leaf surface (_._) with 8 x 10 2 conidia per site. Plants incubated for 3-6 days at 5 klx, Means of seven replicates and standard errors are given. Table 3. ED SO of D . bryoniae conidia infecting cucumber leaves: effects of light intensity (18 h light periotf), injury of the leaf surface, and time of incubation: ED so values from Fig. 2 ED so, conidia/site Light intensity (klx) 10 10
5 5 1 1
Darkness Darkness
Incubation Meehan. injured leaf (days) 2
4 2
4
Intact leaf
1 x lOS 1 X 10' 1 X 10 2
2
2 X 10 1
10~
4
10 1
10'
2
10 1 10 1
4
100000 conidia respectively after 2 days' incubation (Table 3; Fig. 2). In these leaves the infected sites were intensively chlorotic, with (Fig. 1) or without wet rot. There was an approximately logarithmic relationship between light intensity and ED so (Fig. 5) and a small increase in light intensity raised the ED so greatly . It appeared that the plants became more or less saturated with light, giving rise to resistance. After 4 days of incubation the levels of infection at all light intensities had increased in both intact and injured leaves (Fig. 2; Table 3). In order to investigate the actual light intensities which occur in commercial production of
Table 4. ED so of D. bryoniae conidia infecting cucumber leaves: effects of adding nutrients to the leaf surface (Incubation for 4 days at 1 klx,ED so valuesfrom Fig. 3 and additional data.) ED so Treatment (conidia/site) 10' Untreated leaf 10· Cucumber leaf exudate 10 3 Casein hydrolysate/sucrose 10 1 Caseinhydrolysate/sucrose twicea day
cucumbers, light intensity was measured during cloudy days in April in greenhouses in southern Sweden. At noon the top leaves received about 10 klx (daylight, estimated at 125 Elt .m- 2 • S-I) and the lower leaves of plants 2 klx (daylight, estimated at 25,uE. m- 2 •S-I). Such light conditions may persist for several weeks during spring. DISCUSSION
A film of free water seemed to be necessary during the initial phase of infection of leaves by conidia of D. bryoniae. After spot development 100 % humidity was required for further expansion of the tissue rot. Otherwise, infection ceased and a dry necrotic lesion was formed, Ch iu & Walker (1949b) obtained small chlorotic lesions when they incubated cucumber seedlings for 3 days after inoculation with conidial suspensions
G. Svedelius and T. Unestam
95
Table 5. Effect of nutrients on conidial germination and germ-tube or hyphal growth in vitro (Incubation for 12 h at
Mean values and standard errors were calculated from 50 sarnples .) Germination Length of germ tube Nutrients pH (%) or hypha (pm) 1 % vegetable extract 100 94'8 ± 8'3 (hypha) 5'0 0'05 % caseinhydrolysate 100 95'8 ± 10'3 (hypha) 5'5 1 % sucrose 0'05 % casein hydrolysate 39'S ± 5'2 (hypha) 5'5 98 100 Cucumberleaf exudate 6'0 93'8 ± 9'0 (germtube) Distilled water 9'4< 1'0 (germtube) 5'0 93 22 °.
fungus from which conidia had been removed by aseptic filtration. Condensation in greenhouses during the night occurs frequently and probably favours infection. ) We have observed that the spontaneous infection C} 1 5 level may be very low in greenhouses where old / dead leaves are left on the plants. Such leaves / • I probably absorb moisture during the night and / J4 thereby prevent water condensation and subseS / I;;; quent infection. Fewer wounds for fungal pene/ "" tration will also result when old leaves are not / I ~ . / 1 3 ~ removed The available nutrient supply on intact leaf / I e5 surfaces is obviously another restricting factor for / ~ leaf infect ion with conidia of D. bryoniae. Thus, / 0 J 2 .3 spraying the leaf with concentrated cucumber / I leaf exudate or casein hydrolysate supported / infection (Table 4). The raised infectivity is 0/ I probably the result of increased germ-tube or / 11 hyphal growth on the leaf surface, since this was the effect of such extracts in vitro (Table 5). I In the presence of cucumber leaf exudate, J0 10' germ-tube growth was increased but hyphae were not formed . The effect of leaf exudate is therefore 06--_~-_'---==:""-~--~ somewhat specific in relation to the other nutrient 10 5 solutions in which thick initial hyphae rather than Light intensity (k/xi germ-tubes seemed to develop directly from the Fig. 5. Relation between light intensity and ED so of conidia. D. bryoniae on mechanically injured cucumber leaves In our work, the hydathode regions close to the after 2 days incubation. Calculated from data in Fig. 2. main veins were the most readily infected parts of e, ED so; 0 , log sn.; the leaf (Table 1). It is probable that naturally occurring nutrients and water, originating from of D , bryoniae. Their results can be interpreted guttation droplets, stimulated germ-tube growth in different ways: a water film may, in fact, have on leaf surface and fungal infection. A relationship been present during incubation; enzymes and between the presence of guttation droplets pronutrients originating from the culture substrate viding water and nutrients and fungus infection may have been present in the inoculum and might was reported by Yarwood (1952). Nutrient have increased the ability of the fungus to infect leakage, especially from the main veins, may leavesj and it is also possible that a high concen- support fungal growth on specific areas of the leaf tration of such enzymes alone could cause the surface (Collins, 1976). Hydathode tissue is found observed lesions without fungal infection . We have to be a site for infection by the bacterium Xanthefound such enzyme effects on cucumber leaves monas campestris causing black rot of cabbage after only a few hours' application of droplets of (Stakman & Harrar, 1957). However, with parasolution taken from imm ersed cultures of the sitic fungi penetration and infection through J6 I
Didymella and cucumber leaves hydathodes is seldom observed and is considered comparatively unimportant (Wheeler, 1968). It is evident that the infectivity of D. bryoniae on cucumber is increased by the presence of injured or necrotic leaf tissue (T able 3). Mechanical injury facilitates fungal invasion because of release of nutrients from damaged cells rather than rupture of the protective cutin layer. Almost the same infectivity was obtained with intact cucumber leaves sprayed repeatedly with nutrients after inoculation (Table 4). Mycelium from infected host tissue or from vegetable extract agar gave high infection after inoculation on to intact cucumber leaves (Tables 1, 2) probably because of the production of extracellular enzymes as well as of the nutrient supply available for hyphal development. D . bryoniae was found to produce pectinase and cellulase in infected squash fruits (Current, 1969). Intense light apparently increased resistance in the cucumber leaves (Table 3). Under these conditions lesions were limited with or without wet rot and the infected sites were surrounded by chlorosis (Fig. 1). Calpouzos S: Stallknecht (1967) reported that when sugar beet was infected with Cercospora beticola, high light intensity (15-30 klx) resulted in lesions of limited size which did not increase with time; the spots had a sharp purple halo. On the other hand, under low intensity (1'5-3 klx) the lesions were usually larger, increasing in size with time and without purple discoloration. Similarly, in our tests lesions which developed in low light intensity and darkness were larger and without discoloration (F ig. 1). Preliminary tests showed that when one of three leaves of a cucumber plant was kept in darkness, all the leaves were equally resistant to infection and had the same tendency to form chlorotic tissue after inoculation with conidia. It therefore seems probable that the susceptibility of individual leaves depends upon the condition of the entire plant. Translocation between the leaves of assimilates or other substances may be involved in light-induced plant resistance. Low light intensity was recorded in greenhouses during cloudy weather in southern Sweden. This might encourage first infections of D. bryoniae on cucumber since in the laboratory there was a good correlation between low light intensity and infection (Table 3). In intact leaves the degree of infection obtained at 35° was found to be greater than at lower temperatures (Fig. 4). At th is temperature the plants also became gradually pale and chlorotic, in contrast with plants at 25° and 18°. The symptoms indicate that a light intensity of 5 klx was too low at 35° to maintain a positive energy
balance, result ing in starvation and an increased susceptibility to disease. However, preliminary tests in vivo have shown that 35° is above the optimum for germ-tube formation by D. bryoniae on the intact cucumber leaf surface: both germtube length and vitality seem to be affected. The infection level at 35° was therefore limited to 20-30 % ; probably by reduced germ-tube vitality (Fig. 4). This temperature effect also explains the consistently low infection levels on mechanically injured leaves at 35° compared with those at other temperatures (Fig. 4). The optimal temperatures for infection of watermelon and musk-melon plants by D. bryoniae were found to be 24° and 18° respectively after 2 days of incubation in the greenhouse (Chiu & Walker, 1949b ). A temperature of 24° was also reported as optimal for watermelon fruit rot development after one and 2 weeks' incubation (Luepschen, 1961). The present investigation indicates, however, that it is meaningless to propose an optimal temperature for plant infection after a certain period of incubation if critical environmental conditions, such as water and nutrient supply, light before and during the incubation, type of inoculum and host tissue, are not all described. This investigation was supported by grants from The Royal Academy of Agriculture and Forestry. We would like to thank Dr Gosta Karlsson and Dr Me Wellving, Swedish Seed Association, for their generous help with fungal material and advice on conidial production. Thanks are also due to Miss Birgitta Ericsson for measuring light intensity in greenhouses at Alnarp. The authors are grateful to Dr jannet Rowe for critically reviewing the manuscript. REFERENCES
CALPOUZOS, L. & STALLKNECHT, G. F. (1967). Symptoms of Cercospora leafspot of sugar beets influenced by light intensity. Phytopathology 57, 799-800. CHIU, W. F. & WALKER, J. C. (1949a). Morphology and variability of the cucurbit black rot fungus. Journal of Agricultural Research 78, 81-102.
CHIU, W. F. & WALKER, J. C. (1949b). Physiology and pathogenicity of the cucurbit black rot fungus. Journal of Agr icultural Research 78, 589-615.
COLLINS, M. A. (1976). Colonisation of leaves by phylloplane saprophytes and their interactions in this environment. In M icrobiology of Aerial Plant Surfa ces (ed. C. H. Dickinson and T. F. Preece), pp. 401-418 . London: Academic Press. CURRENT, T. (1969). Pectic and cellulolytic enzymes produced by Mycosphaerella citrullina and their relation to black rot of squash. Canadian Journal of Botany 47, 79 1-794.
G. Suedelius and T. Unestam FLETCHER, ] . T . & PREECE, T . F. (1966). My cosphaerella stem rot of cucumber in the Lea Valley. Annals of Applied Biology 58, 423-430. HORDIJK, C. V. & GOOSEN, P. G . (1962). Aanta sting van komkornmer door M ycosphaerella melonis. Tijd schrift cn'er Plantenzickten 68, 149. LUEPSCHEN, N. S. (1961). The de velopment of M y cosphaerella black rot and P ellicularia rolfsii rot of watermelon at various temperatures. Pl ant Disease Reporter 45, 557-559· M ULLER, E. & ARX, ] . A. (1962) . D ie Gattungen der didymosporen Pyrenomyceten, Beitrdge zur Kryptogamenflora der S chuieiz 11, 351-364.
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NYBERG, A. (1962 ). Svartprickrota - en for vllrt land ny sjukdorn p;\ gurka. V ax tsky ddsnotiser 7.7, 58-63. NYBERG, A. (1963). Svartprickrota pll gurka - iakttagelser och forsok. Vdxtskyddsnotiser 7.7, 76-82. STAKMAN, L. ]. & HARRAR, G. S. (1957). Prin ciples of Plant Pathology . New York: Ronald Press. WHEELER, B. E. ] . (1968 ). Fungal parasites of plants. In The Fungi - an advanced treatise, vol. III (ed. G. C. Ainsworth and A. S. Sussman), pp. 179-210. New York, London: Academic Press. YARWOOD, C. E. (1952). Guttation due to leaf presSure favours fungus infections. Phytopathology 42, 52 0 .
(Accepted for publication 18 January 1978)
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