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Growth and germination of fungal spores in guttation fluids of barley grown with different nitrogen sources
[ 239 ] Trans. Br. mycol. Soc. 78 (2), 239-245 (1982).
Printed in Great Britain
GROWTH AND GERMINATION OF FUNGAL SPORES IN GUTTATION FLUIDS OF BARLEY GROWN WITH DIFFERENT NITROGEN SOURCES By ROGER FROSSARD AND J. J. OERTLI* Botanisches Institut, S chimbeinstrasse 6, CH-4056 Basel, Switzerland Nitrogen nutrition of barle y had no uniform effect on conidial germination of Alternaria alternata , Botrytis cinerea and Cladosporium herbarum in guttation fluids in vitro or on leaves. Sterilized guttation fluids stimulated germination of C. herbarum and hyphal growth of all fungi in vitro, whereas non-sterile fluids lowered percentage germination of C. herbarum and sometimes inhibited hyphal growth of A. alternata and C. herbarum. In another experiment, non-sterile guttation fluids stimulated germination of all fungi. Non-sterile guttation fluid which promoted hyphal growth in vitro had less or no effect on germination on detached leaves. This suggests that factors other than plant nutrition are more important in determining fungal germination in guttation fluids. Guttation fluids have been shown to stimulate conidial germination in fungal pathogens and to increase the rate of infection of host plants (Weintraub, Miller & Schantz, 1958 ; Lewis, 1962; Endo & Amacher, 1964 ; Endo & Oertli, 1964). These effects are probably due to the presence in the fluids of a great variety of potential nutrients and growth factors (Goatley & Lewis , 1966). Nutrients are especially important in the phyllosphere, where micro-organisms compete for low amounts of carbohydrates and amino acids (Blakeman, 1978) and where certain epiphytic bacteria and yeasts are capable of inhibiting the germination of fungal conidia by rapid depletion of the available amino acids (Brodie & Blakeman, 1976; Blakeman & Brodie, 1977). In this connexion, new significance is given to a report by Curtis (1944), who described abundant exudation of glutamine in guttation fluids from plants previously treated with ammonium sulphate. If the amino acid content of guttation fluids is dependent on the nitrogen nutrition of the plant, one would also expect this to have an effect on fungal spores in guttation fluids. The aim of our study was, therefore, to investigate the influence of nitrogen nutrition of the plant on the germination and growth of some filamentous fungi in guttation fluids. MATERIALS AND METHODS
Plants Barley seeds (H ordeum v ulgare L. variety 'Uniongerste ') were surface sterilized for 2 min in o·2 %
* Present address: Institut fur Pflanzenbau, Versuch sstation Eschikon,
Switzerland.
CH-8307
Lindau-Eschikon,
formaldehyde solution, rinsed thoroughly with demineralized water, and soaked for 16 h in double-glass-distilled water under continuous aeration at 21·5 °C . The seeds were then spread on cheesecloth suspended over plastic racks standing in flat PVC trays containing nutrient solution. The level of the nutrient solution was periodically adjusted with demineralized water. The trays were placed in a special climate chamber at 230 and a relative humidity of 98 % (F rossard, 1979). Four Philips TLF 40 W / 34 fluorescent tubes provided continuous light with an intensity of 5200 Ix at seedling level. Nutrient solutions Nutrient solutions were based on Hoagland's solution no . 1 (Hewitt, 1966) and contained 1·0 mM I-I MgS0 4 and 0·5 mM I-I KH zP04. Other components were (mM 1-1) : Ca(NOa}z, 2'5 ; KNO a, 2·5 (N Oa-N solution); (NH 4)zS04' 3·75; CaCl z' 2'5; K ZS0 4, 1·25 (NH4-N solution); Ca(NO a)2' 0·25; CaCI 2 , 2·25; KzSO p 1·25 (low N solution). Total nitrogen concentrations were 7·5 mM 1-1 (N Oa-N and NHcN solution) and 0 ·5 mM 1-1 (low N solution). The pH of the NHcN solution was adjusted to 6·0 with 1 M-KOH twice a day, starting on the third day after the commencement of soaking of the seeds . Guttation fluids Guttation fluids were collected on day 4, 5 and 6 respectively, by means of a suction flask with exchangeable receptacle. Guttation droplets present on primary leaves in the morning were removed and discarded. Droplets appearing thereafter were collected separately every 2·5 h, four times each
240
Fungal growth in guttation fluids
day. Between collections receptacles were kept at 0-2°. After the last collection of the day, the guttation fluids were frozen and stored at - 20°. For spore germination tests, the fluids were thawed and the samples of the 3 days combined before measuring with a pH meter. One half of each sample was then sterilized by passage through a triacetate membrane filter with 0'2 pm pore diam (Skan, Basel) and subsamples distributed into small sterile test tubes. The other half was subdivided without sterilization. All samples were frozen again and kept at - 20° until germination tests were performed. Concentration of reducing sugars in the guttation fluids was determined by the method of Somogyi (1952) using the arsenomolybdate reagent of Nelson (1944) .
exposed. The slides were immediately placed on glass rods in Petri dishes containing water, and each leaf was supplied with a single drop of test fluid and inoculum as described above. In order to prevent droplets from rolling off, sterile Tween 20 (0'04 %) was added to the test solution. Guttation fluids obtained from seedlings grown on a particular nutrient solution were applied to leaves from plants grown on the same type of solution only. After 20 h of incubation, the leaf pieces were heated at 45° for 1 h, cleared on filter papers saturated with lactophenol for 2 h, and mounted on glass slides. Samples were stained with cotton blue in lactophenol and gently heated in order to obtain clearly visible fungal structures for microscopic examination. For comparison, subsamples ofthe same guttation fluids with Tween 20 used on detached leaves were also tested in vitro at the same concentration. Spore germination Assessment of germination. Within each sample of Fungi and preparation of spore suspensions. guttation fluid or control, five replicates were Single conidial strains of Cladosporium herbarum examined. In each replicate, 100 conidia were Link ex Fr. and Alternaria alternata (Fr.) Keissler assessed for germination percentage. A conidium were isolated from beech leaves (Fagus sylvatica) was counted as germinated, if germ-tube length was and grown on 0'75 % malt extract agar. Botrytis at least as big as the largest diameter of the cinerea Pers. ex Pers. had originally been isolated conidium. Hyphal growth was also measured. For from Cassia angustifolia in India and was grown on C. herbarum the total hyphal length per conidium r- 5 % soybean flour (commercial grade) plus 1 % was measured for 40 conidia in each replicate. For malt extract agar. A. alternata and B. cinerea, a modified version of Conidia from 9- to 15-day-old cultures incubated the growth index method of Lewis (1962) was at 22'5° were collected with a loop and suspended applied. Instead of a square, we used a rectangular in 5 em" of sterile 0'1 % Tween 20. They were ocular insert, projecting an area of 330 by 220 pm washed four times by centrifuging at 1360 g for on the slides. Growth indices were then compared 5 min. The final suspension was adjusted to a with rectilinear standard curves, in which were concentration of 8 x 104 conidia cm> using a plotted the average growth indices for twelve haemocytometer. randomly chosen samples (five replicates each) Germination in vitro. Sterile and non-sterile against the mean actual hyphal length per field as guttation fluids were used for spore germination measured with the help of a projection microscope tests on the upper and lower halves of sterile 5 em and a map measurer. Hyphal growth of A. alternata Petri dishes (referred to as plastic slides). Drops and B. cinerea is therefore the total hyphal length (0'025 ern") of guttation fluids, sterile demineralized per unit area rather than per conidium. water, or sterile glucose solution (10 p.p.m.), were For statistical analysis, Duncan's multiple range 'inoculated' by adding 5 pI of spore suspension. test was applied on hyphal growth measurements The plastic slides with the drops were then placed and on arcsine values of germination percentages. in 9 em Petri dishes containing a little distilled water and enclosed in a transparent plastic bag to RESULTS maintain high humidity. After incubation for 20 h at room temperature, the drops were stained with Conidia of A. alternata and B. cinrea germinated cotton blue in lactophenol for microscopic equally well in water, glucose solution or guttation fluid in vitro, regardless of the nitrogen nutrition of examination. Germination on detached leaves. Non-sterile the plants from which the guttation fluid originated. guttation fluids were used for germination tests on Sterility of the fluids did not affect germination. By detached leaves. Barley seedlings were grown as contrast, percentage germination of C. herbarum described but at a relative humidity of 77 % to was considerably higher in sterilized guttation fluid reduce guttation, The apical 5 em of freshly cut than in water or glucose solution, whereas primary leaves from 6-day-old seedlings were non-sterile guttation fluid inhibited germination mounted on glass slides with their adaxial surfaces substantially (Table 1).
Table
1.
Germination and hyphal growth of Alternaria alternata, Botrytis cinerea and Cladosporium herbarum in vitro in guttation fluids from barley seedlings grown in different nitrogen sources Sterilized
Alternaria Botrytis Cladosporium
Water
Glucose solution (8 p.p.m.)
N03
90 • 6a 93' 6a 54'4a
90' 0a 93'3 3 67' 6a
94'0a 95'2 a 96·8b
Guttation fluid NH. 10wN Germination (%) 91-8a 90-2a 96'8a 93'2a 97'8 b 88'ob
Hyphal growth (* ftm mm:";
Alternaria* Botrytis* Cladosporiumt
5487a 3333 a 9a
73143b 5472b 12a
9889bc 8234c 55 bc
::tl
Non-sterile
14190d 12565 d 61 c
t
8605 bC 9244c 47 b
Water
Glucose solution (8 p.p.m.)
90' 6a 89'0a 45-8 a
89'3 a 91'6a 64- 2b 7037b 7408b 14 b
~
Guttation fluid N03
NH.
10wN
9 1'6a
84'3 a 22·6 c
86'3 a 86'2 a 19' Oc
85' 8a 90' 0a 4' 2d
3332c 5606bc 73
52893 5108ac 7a
48353 5747 bc 10 3
ftm conidium:")
5268a 3536a lOa
Data are means of five replicates. Values in the same row within each treatment not followed by the same letter are significantly different at the 95 % level or better.
'" '"
~ ~
!:l.. ~
;:$
!:l..
~ ~
o ~ ~
-.... ....
N
...
.j::..
Fungal growth in guttation fluids
242
Table 2. Germination and hyphal growth of A. alternata, B. cinerea and C. herbarum in vitro in non-sterile guttation fluids from barley seedlings
Water
Glucose solution (8 p.p.m.)
Alternaria Botrytis Cladosporium
88"4a 97'0ab 44' 8a
87' 0a 95"4b 63-6 b
Alternaria* Botrytis* Cladosporiumt
3391a 2472a 12a
Guttation fluid N0 3
Germination (%) a 9 1"4 9 8-8" 94'4"
Hyphal growth (* {tm mm <; 4332 ab 6057" 3042a 7603 b 23 b 55"
t
{tm
NH 4
lowN
90' 8a 97'8 ab" 93'0"
90 -ga 9 8'2 ac 95-2"
conidium")
8158 d 12275" 90 d
5529bc 8234 b 82d
Data are means of five replicates. Values in the same row not followed by the same letter are significantly different at the 95 % level or better.
Sterilized guttation fluid stimulated hyphal growth of all three fungi. The response of C. herbarum was most pronounced, hyphal growth per conidium being on the average six times higher than in water. In non-sterile guttation fluid, a slight stimulation of hyphal growth occurred with B. cinerea while A. alternata and C. herbarum were not stimulated at all (Table 1). However, non-sterile guttation fluids collected from different N0 3 and NH 4 plants tested simultaneously with the above significantly inhibited the growth of A. alternata and C. herbarum. Otherwise, results were similar to those shown in Table 1, except that hyphal growth did not follow the same pattern of response towards the individual 'types' of guttation fluid. Growth of B. cinerea in sterilized fluid from NH 4 plants, for instance, was significantly lower than in fluid from N0 3 and low N plants. The pH values of the guttation fluids used varied between 6'1 and 6,6, so it seems unlikely that variation in pH could be responsible for these differences. On the whole, sterility of guttation fluids was obviously more important for conidial germination than any possible effects of plant nutrition. All non-sterile guttation fluid samples contained numerous bacteria and occasional yeasts growing around and/or between hyphae and conidia. We therefore concluded that the presence of other micro-organisms caused the general lack of stimulation of hyphal growth in non-sterile guttation fluids. However, when we tested a second collection of non-sterile guttation fluid in vitro we found that all fungi reacted in much the same way as they had done in sterilized guttation fluid in the earlier experiment. Percentage germination of C. herbarum was now stimulated and hyphal growth of all the
fungi was enhanced (Table 2) although numerous bacteria were seen again. These results suggest that not only the presence but also the level of antagonistic activity of other micro-organisms influence fungal development in guttation fluids. In order to find out whether the stimulating effect of non-sterile guttation fluids in the second experiment was perhaps due to a higher sugar content, we estimated the concentration of reducing sugars in all guttation fluids used. For the experiments in Table 1 these were equivalent to 28 flg glucose cm:" in fluid from N0 3 plants, 26'2 flg cm " in fluid from NH4 plants and 3 1'2 flg cm ? in fluid from low N plants. In the guttation fluids used in the second experiment (Table 2), we found 22'2, 29 and 44'2 flg glucose crn" respectively. The enhancement of germination in the second experiment could therefore not be merely attributed to a higher sugar content (preliminary chromatographic experiments had shown sucrose to be absent). The pH values of the guttation fluids were slightly higher in the second experiment, ranging from 6'75 to 7'45. It seems improbable, however, that this could have changed fungal behaviour to such an extent. Since leaf tissue has repeatedly been reported to contain inhibitors that affect fungal growth (Godfrey, 1976), we investigated the effects of guttation fluid on conidial germination on leaves. Again we used drops of sterile water or glucose solution as controls. The guttation fluids used were subsamples of the second collection of non-sterile fluids. Percentage germination of A. alternata and B. cinerea was high in all experiments and the few differences observed were of minor importance (Table 3). On leaves from NH 4 plants, C. herbarum germinated better in guttation fluid or glucose solution than in water. Similar results were
R. Frossard and J.J. Oertli
243
Table 3. Germination and hyphal growth of A. alternata, B. cinerea and C. herbarum in non-sterile guttation fluids on detach ed barley leaves NO. plants
NH. plants
Glucose Guttation solution fluid (8 p.p.m.) NO.
Water Alternaria Botrytis Cladosporium
93'8& 95'8& 66'2&
95'0& 96'0& 89.8&
96"4& 95'2& 9 1.8&
Alternaria" Botrytis" Cladosporiumt
6173& 4029& 16'
7167' 4860' 17'
8603 b 7044b 25'
Water
Glucose solution (8 p .p.rn.)
low N plants Guttation fluid NH.
Germination (%) 96'0&b 96'8& 94'0b 96"4& 95'2& 96'4& 77'0& 92·8b 97'0 b
Water 95'0& 95'4& 88'2&
Glucose Guttation solution fluid (8 p .p.m.) 10wN 96'2& 96'0& 88'4&
98'6 b 94'4& 90'2&
7933' 4144' 16b
6900' 6784b 19'
H yphal growth (* pm mm" ; t I'm conidium" ) 7702' 4297' 11 a
8485& 4588' 18b
11680& 8230b 47c
7900' 3895' 17· b
Data are means of five replicates. Values within each nitrogen treatment not followed by the same letter are significantly different at the 95 % level or better.
obtained on leaves from N0 3 plants; the differences, however, were not significant due to high variation between replicates in the water control. Similarly, variability of hyphaI growth of A . alternata and C. herbarum was high between replicates on leaves . A significant stimulation of A. alternata by guttation fluid was found, however, on leaves from NO. plants and of C. herbarum on leaves from NH. plants. B. cinerea, on the other hand, was stimulated by guttation fluid in all three treatments. No consistent correlation between nitrogen nutrition of the plants and conidial germination could be detected. Both water controls and glucose solutions supported some bacterial growth on leaf surfaces; when compared with guttation fluids, however, it was judged as low . DISCUSSION
Fertilization of grasses and other herbaceous plants with ammonium salts may enhance exudation of glutamine, and probably other amino acids, either through bleeding from cut surfaces or through guttation (Greenhill & Chibnall, 1934; Curtis, 1944; Raleigh, 1946). Starting from the hypothesis that amino acids may be limiting nutrients restricting conidial germination (cf. Blakeman, 1978), a marked increase of hyphal growth in guttation fluid from NH. plants was to be expected. Nevertheless, we found no unequivocal correlation between the type or amount of nitrogen supplied to barley seedlings and the stimulation of growth and germination of A. alt ernata, B. cinerea and C. herbarum conidia in guttation fluid. The stimulation of hyphaI growth of A. alternata and B . cinerea by sterilized guttation fluid from NH. plants could not be confirmed when the experiment
was repeated with another series of fluids (results not reported here). Yet in our second experiment, with non-sterile fluids, we observed this tendency again. Apparently the chemical composition of guttation fluids is subject to considerable variation, which in turn affects fungal growth. This observation is supported by results obtained with Helminthosporium sorokinianum (Endo & Amacher, 1964) and Saccharomyces cerevisiae (Perrin, 1972) in guttation fluids . Different fungi will not necessarily respond alike towards the same sample of guttation fluid as is seen when one compares hyphal growth of C. herbarum with that of the other fungi tested here. C. herbarum grew equally well in fluid from low N plants as in fluid from NH. plants (T able 2). This may have been due to the high sugar content of the guttation fluid of the low N plants. C. herbarum has been reported to show increasing germination with increasing sugar concentrations (M cBride & Hayes, 1979). The presence of a natural microflora clearly lowered percentage germination or hyphal growth (T able 1). The inhibitory effect of bacteria and yeasts on fungal germination in media with a low nutrient supply is well documented (Blakeman & Fraser, 1971; Brodie & Blakeman, 1976; Blakeman & Brodie, 1977; McBride & Hayes, 1979 ; Sharman & Heale, 1979). It is generally assumed that competition for nutrients is the most important cause of inhibition, although other mechanisms, for example the production of antibiot ic substances, cannot be ruled out (Blakeman & Brodie, 1976; Fokkema, 1976; Sharman & Heale, 1979). In our second experiment non-sterile guttation fluids failed to inhibit germination of the test fungi
244
Fungal growth in guttation fluids
(Table 2) although microscopic observation clearly REFERENCES showed large numbers of bacteria and some yeasts. Unfortunately, sterile subsamples of the same BLAKEMAN, J. P. (1978). Microbial competition for nutrients and germination of fungal spores. Annals of guttation fluids could not be tested due to the Applied Biology 89, 151-155. amount available. It may therefore be possible that hypha I growth would have been stimulated even BLAKEMAN, J. P. & ATKINSON, P. (1976). Evidence for a spore germination inhibitor co-extracted with wax from more had the fluids been sterilized. Nevertheless, leaves. In Microbiology of Aerial Plant Surfaces (ed. the microflora present in these specific samples was e. H. Dickinson & T. F. Preece), pp. 441-449. less effective in inhibiting conidial germination. London: Academic Press. Previous investigations on the effects of guttation BLAKEMAN, J. P. & BRODIE, I. D. S. (1976). Inhibition of fluids on fungi have almost exclusively been made pathogens by epiphytic bacteria on aerial plant with sterilized guttation fluids (Lewis, 1962; Endo surfaces. In Microbiology of Aerial Plant Surfaces (ed. C. H. Dickinson & T. F. Preece), pp. 529-557. & Amacher, 1964; Endo & Oertli, 1964; Healy & London: Academic Press. Britton, 1968; Potbury & Drysdale, 1969; Perrin, 1972). Our results demonstrate that conclusions BLAKEMAN, J. P. & BRODIE, I. D. S. (1977). Competition for nutrients between epiphytic micro-organisms and obtained with sterilized samples will not necessarily germination of spores of plant pathogens on beetroot apply to the actual situation in the field. leaves. Physiological Plant Pathology 10, 29-42. On detached barley leaves, C. herbarum had BLAKEMAN, J. P. & FRASER, A. K. (1971). Inhibition of higher germination percentages in water or glucose Botrytis cinerea spores by bacteria on the surface of solution than in vitro, suggesting that nutrients, chrysanthemum leaves. PhysiologicalPlant Pathology 1, leached out from the leaf tissue stimulated 45-54· germination. Similarly, hyphal growth of A. BRODIE, I. D. S. & BLAKEMAN, J. P. (1976). Competition for exogenous substrates in vitro by leaf surface alternata in water droplets on leaves was of the same micro-organisms and germination of conidia of Botrytis order of magnitude as in the non-sterile guttation cinerea. Physiological Plant Pathology 9, 227-239. fluids in vitro. B. cinerea and C. herbarum also grew CURTIS, L. c. (1944). The exudation of glutamine from better in water droplets on leaves, but absolute lawn grass. Plant Physiology 19, 1-5. values of hypha I lengths in guttation fluids on leaves DIX, N. J, (1974). Identification ofa water-soluble fungal were less than in vitro. Though a direct comparison inhibitor in the leaves of Acer platanoides L. Annals of between results on leaves and in vitro is only of Botany 38, 505-514. limited value, because the experiments were carried ENDO, R. M. & AMACHER, R. H. (1964). Influence of guttation fluid on infection structures of Helminthoout with different spore suspensions and at a sporium sorokinianum. Phytopathology 54, 1327-1334. different time, we may assume that the in vitro stimulation of germination by guttation fluids was ENDO, R. M. & OERTLI,J. J. (1964). Stimulationoffungal infection of bentgrass. Nature, London 201, 313. reduced or even absent on detached leaves. FOKKEMA, N. J. (1976). Antagonism between fungal The negative influence of the leaf tissue could be saprophytes and pathogens on aerial plant surfaces. due to the selective stimulation of the natural In Microbiology of Aerial Plant Surfaces (ed. e. H. microflora in guttation fluid through physical or Dickinson & T. F. Preece), pp. 487-506. London: chemical factors associated with the leaf surface, Academic Press. which would enhance the antagonistic capacity of FROSSARD, R. (1979). Einfluss der Pflanzenernahrung auf die Guttation und die Zusammensetzung der Guttathe microflora towards the fungi or to the presence tionsflussigkeit von Gerste und deren Bedeutung fur of inhibitory substances in the wax layer (Blakeman die Keimung von saprophytischen Pilzen in vitro und & Atkinson, 1976; Parbery & Blakeman, 1978) or auf bgeschnittenen Blattern. Ph.D. Thesis, University in the leaf tissue (Dix, 1974; Godfrey, 1976; of Basel, Switzerland. Rossall & Mansfield, 1980). GOATLEY, J. L. & LEWIS, R. W. (1966). Composition of Our findings show that the nitrogen nutrition of guttation fluid from rye, wheat, and barley seedlings. plants is not of great significance to fungal Plant Physiology 41, 373-375. development in guttation fluid. Other factors have GODFREY, B. E. S. (1976). Leachates from aerial parts of more influence and probably mask any direct effects plants and their relation to plant surface microbial populations. In Microbiology of Aerial Plant Surfaces of plant nutrition. Research on the effects of (ed. C. H. Dickinson & T. F. Preece), pp. 433-439. guttation fluids must therefore be seen in the London: Academic Press. context of other environmental conditions before any definite conclusions about ecological signifi- GREENHILL, A. W. & CHIBNALL, A. C. (1934). The exudation of glutamine from perennial rye-grass. cance are drawn. We wish to thank Dr F. Barlocher for supplying the fungi we used and for helpful discussions.
Biochemical Journal 28, 1422-1427. HEALY, M. J. & BRITTON, M. P. (1968). Infection and development of Helminthosporium sorokinianum in Agrostis palustris. Phytopathology 58, 273-276.
R. Frossard and HEWITT, E. J. ( 1966). Sand and Water Culture Methods used in the Study of Plant Nutrition , and ed . Farnham Royal (Bucks.), U .K . : Commonwealth Agricultural Bureaux. LEWIS, R. W. ( 1962). Guttation fluid : effects on growth of Claviceps purpur ea in vitro. Sci ence 138, 690-691. McBRIDE,R . P. & HAYES, A. J. (1979). The germination of Cladosporium herbarum spores in relation to environmental factors in the larch ph ylloplane. N ova Hed wigia 31, 497-506. NELSON, N . ( 1944) . A ph otometric adaptation of the Somogyi method for the determination of glucose . J ournal of Biological Chemistry 153, 375-380. PARBERY, D . G. & BLAKEMAN, J. P. ( 1978). Effect of substances associated with leaf surfaces on appressorium formation by Colletotrichum aCUlatum . Transa ctions of the Br it ish My cological So ciety 70, 7-19. PERRIN, A. (1972). Contribution ill'etu de de I 'organisation et du fonctionnement des hydathodes: recherches anatomiques, ultrastructurales et physiologiques. Ph.D. Thesis, University Claude Bernard, Lyon, France.
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POTBURY, M . & DRYSDALE, R. B. ( 1969). Effects of guttation fluid from rye and barley on growth of Clauiceps purpurea in vitro. Tran sactions of the British Mycological Society 53, 137-138. RALEIGH, G. J. ( 1946). Glutamine from rye gra ss. S cience 1°3, 206--207. ROSSALL, S. & MANSFIELD, J. W. (1980). Investigation of the cause s of poor germination of B otrytis spp . on broad bean leaves ( V icia faba L. ). Phy siological Plant Path ology 16, 369"-382. SHARMAN, S. & HEALE, J. B. (1979). Germination studies on B otry tis cinerea infecting intact carrot (Daucus carola ) roots . Transactions of the Br itish Mycological S ociety 73, 147-154. SOMOGYI, M. (1952). Notes on sugar determination. J ournal of Biological Chemistry 195, 19-23. WEINTRAUB, R. L., MILLER, W . E. & SCHANTZ, E . J. (1958). Chemical stimulation of germination of spores of Piricularia oryzae. Phytopathology 48, 7-10.
(Received for publication 19 February 1981)
Printed in Great Britain
GROWTH AND GERMINATION OF FUNGAL SPORES IN GUTTATION FLUIDS OF BARLEY GROWN WITH DIFFERENT NITROGEN SOURCES By ROGER FROSSARD AND J. J. OERTLI* Botanisches Institut, S chimbeinstrasse 6, CH-4056 Basel, Switzerland Nitrogen nutrition of barle y had no uniform effect on conidial germination of Alternaria alternata , Botrytis cinerea and Cladosporium herbarum in guttation fluids in vitro or on leaves. Sterilized guttation fluids stimulated germination of C. herbarum and hyphal growth of all fungi in vitro, whereas non-sterile fluids lowered percentage germination of C. herbarum and sometimes inhibited hyphal growth of A. alternata and C. herbarum. In another experiment, non-sterile guttation fluids stimulated germination of all fungi. Non-sterile guttation fluid which promoted hyphal growth in vitro had less or no effect on germination on detached leaves. This suggests that factors other than plant nutrition are more important in determining fungal germination in guttation fluids. Guttation fluids have been shown to stimulate conidial germination in fungal pathogens and to increase the rate of infection of host plants (Weintraub, Miller & Schantz, 1958 ; Lewis, 1962; Endo & Amacher, 1964 ; Endo & Oertli, 1964). These effects are probably due to the presence in the fluids of a great variety of potential nutrients and growth factors (Goatley & Lewis , 1966). Nutrients are especially important in the phyllosphere, where micro-organisms compete for low amounts of carbohydrates and amino acids (Blakeman, 1978) and where certain epiphytic bacteria and yeasts are capable of inhibiting the germination of fungal conidia by rapid depletion of the available amino acids (Brodie & Blakeman, 1976; Blakeman & Brodie, 1977). In this connexion, new significance is given to a report by Curtis (1944), who described abundant exudation of glutamine in guttation fluids from plants previously treated with ammonium sulphate. If the amino acid content of guttation fluids is dependent on the nitrogen nutrition of the plant, one would also expect this to have an effect on fungal spores in guttation fluids. The aim of our study was, therefore, to investigate the influence of nitrogen nutrition of the plant on the germination and growth of some filamentous fungi in guttation fluids. MATERIALS AND METHODS
Plants Barley seeds (H ordeum v ulgare L. variety 'Uniongerste ') were surface sterilized for 2 min in o·2 %
* Present address: Institut fur Pflanzenbau, Versuch sstation Eschikon,
Switzerland.
CH-8307
Lindau-Eschikon,
formaldehyde solution, rinsed thoroughly with demineralized water, and soaked for 16 h in double-glass-distilled water under continuous aeration at 21·5 °C . The seeds were then spread on cheesecloth suspended over plastic racks standing in flat PVC trays containing nutrient solution. The level of the nutrient solution was periodically adjusted with demineralized water. The trays were placed in a special climate chamber at 230 and a relative humidity of 98 % (F rossard, 1979). Four Philips TLF 40 W / 34 fluorescent tubes provided continuous light with an intensity of 5200 Ix at seedling level. Nutrient solutions Nutrient solutions were based on Hoagland's solution no . 1 (Hewitt, 1966) and contained 1·0 mM I-I MgS0 4 and 0·5 mM I-I KH zP04. Other components were (mM 1-1) : Ca(NOa}z, 2'5 ; KNO a, 2·5 (N Oa-N solution); (NH 4)zS04' 3·75; CaCl z' 2'5; K ZS0 4, 1·25 (NH4-N solution); Ca(NO a)2' 0·25; CaCI 2 , 2·25; KzSO p 1·25 (low N solution). Total nitrogen concentrations were 7·5 mM 1-1 (N Oa-N and NHcN solution) and 0 ·5 mM 1-1 (low N solution). The pH of the NHcN solution was adjusted to 6·0 with 1 M-KOH twice a day, starting on the third day after the commencement of soaking of the seeds . Guttation fluids Guttation fluids were collected on day 4, 5 and 6 respectively, by means of a suction flask with exchangeable receptacle. Guttation droplets present on primary leaves in the morning were removed and discarded. Droplets appearing thereafter were collected separately every 2·5 h, four times each
240
Fungal growth in guttation fluids
day. Between collections receptacles were kept at 0-2°. After the last collection of the day, the guttation fluids were frozen and stored at - 20°. For spore germination tests, the fluids were thawed and the samples of the 3 days combined before measuring with a pH meter. One half of each sample was then sterilized by passage through a triacetate membrane filter with 0'2 pm pore diam (Skan, Basel) and subsamples distributed into small sterile test tubes. The other half was subdivided without sterilization. All samples were frozen again and kept at - 20° until germination tests were performed. Concentration of reducing sugars in the guttation fluids was determined by the method of Somogyi (1952) using the arsenomolybdate reagent of Nelson (1944) .
exposed. The slides were immediately placed on glass rods in Petri dishes containing water, and each leaf was supplied with a single drop of test fluid and inoculum as described above. In order to prevent droplets from rolling off, sterile Tween 20 (0'04 %) was added to the test solution. Guttation fluids obtained from seedlings grown on a particular nutrient solution were applied to leaves from plants grown on the same type of solution only. After 20 h of incubation, the leaf pieces were heated at 45° for 1 h, cleared on filter papers saturated with lactophenol for 2 h, and mounted on glass slides. Samples were stained with cotton blue in lactophenol and gently heated in order to obtain clearly visible fungal structures for microscopic examination. For comparison, subsamples ofthe same guttation fluids with Tween 20 used on detached leaves were also tested in vitro at the same concentration. Spore germination Assessment of germination. Within each sample of Fungi and preparation of spore suspensions. guttation fluid or control, five replicates were Single conidial strains of Cladosporium herbarum examined. In each replicate, 100 conidia were Link ex Fr. and Alternaria alternata (Fr.) Keissler assessed for germination percentage. A conidium were isolated from beech leaves (Fagus sylvatica) was counted as germinated, if germ-tube length was and grown on 0'75 % malt extract agar. Botrytis at least as big as the largest diameter of the cinerea Pers. ex Pers. had originally been isolated conidium. Hyphal growth was also measured. For from Cassia angustifolia in India and was grown on C. herbarum the total hyphal length per conidium r- 5 % soybean flour (commercial grade) plus 1 % was measured for 40 conidia in each replicate. For malt extract agar. A. alternata and B. cinerea, a modified version of Conidia from 9- to 15-day-old cultures incubated the growth index method of Lewis (1962) was at 22'5° were collected with a loop and suspended applied. Instead of a square, we used a rectangular in 5 em" of sterile 0'1 % Tween 20. They were ocular insert, projecting an area of 330 by 220 pm washed four times by centrifuging at 1360 g for on the slides. Growth indices were then compared 5 min. The final suspension was adjusted to a with rectilinear standard curves, in which were concentration of 8 x 104 conidia cm> using a plotted the average growth indices for twelve haemocytometer. randomly chosen samples (five replicates each) Germination in vitro. Sterile and non-sterile against the mean actual hyphal length per field as guttation fluids were used for spore germination measured with the help of a projection microscope tests on the upper and lower halves of sterile 5 em and a map measurer. Hyphal growth of A. alternata Petri dishes (referred to as plastic slides). Drops and B. cinerea is therefore the total hyphal length (0'025 ern") of guttation fluids, sterile demineralized per unit area rather than per conidium. water, or sterile glucose solution (10 p.p.m.), were For statistical analysis, Duncan's multiple range 'inoculated' by adding 5 pI of spore suspension. test was applied on hyphal growth measurements The plastic slides with the drops were then placed and on arcsine values of germination percentages. in 9 em Petri dishes containing a little distilled water and enclosed in a transparent plastic bag to RESULTS maintain high humidity. After incubation for 20 h at room temperature, the drops were stained with Conidia of A. alternata and B. cinrea germinated cotton blue in lactophenol for microscopic equally well in water, glucose solution or guttation fluid in vitro, regardless of the nitrogen nutrition of examination. Germination on detached leaves. Non-sterile the plants from which the guttation fluid originated. guttation fluids were used for germination tests on Sterility of the fluids did not affect germination. By detached leaves. Barley seedlings were grown as contrast, percentage germination of C. herbarum described but at a relative humidity of 77 % to was considerably higher in sterilized guttation fluid reduce guttation, The apical 5 em of freshly cut than in water or glucose solution, whereas primary leaves from 6-day-old seedlings were non-sterile guttation fluid inhibited germination mounted on glass slides with their adaxial surfaces substantially (Table 1).
Table
1.
Germination and hyphal growth of Alternaria alternata, Botrytis cinerea and Cladosporium herbarum in vitro in guttation fluids from barley seedlings grown in different nitrogen sources Sterilized
Alternaria Botrytis Cladosporium
Water
Glucose solution (8 p.p.m.)
N03
90 • 6a 93' 6a 54'4a
90' 0a 93'3 3 67' 6a
94'0a 95'2 a 96·8b
Guttation fluid NH. 10wN Germination (%) 91-8a 90-2a 96'8a 93'2a 97'8 b 88'ob
Hyphal growth (* ftm mm:";
Alternaria* Botrytis* Cladosporiumt
5487a 3333 a 9a
73143b 5472b 12a
9889bc 8234c 55 bc
::tl
Non-sterile
14190d 12565 d 61 c
t
8605 bC 9244c 47 b
Water
Glucose solution (8 p.p.m.)
90' 6a 89'0a 45-8 a
89'3 a 91'6a 64- 2b 7037b 7408b 14 b
~
Guttation fluid N03
NH.
10wN
9 1'6a
84'3 a 22·6 c
86'3 a 86'2 a 19' Oc
85' 8a 90' 0a 4' 2d
3332c 5606bc 73
52893 5108ac 7a
48353 5747 bc 10 3
ftm conidium:")
5268a 3536a lOa
Data are means of five replicates. Values in the same row within each treatment not followed by the same letter are significantly different at the 95 % level or better.
'" '"
~ ~
!:l.. ~
;:$
!:l..
~ ~
o ~ ~
-.... ....
N
...
.j::..
Fungal growth in guttation fluids
242
Table 2. Germination and hyphal growth of A. alternata, B. cinerea and C. herbarum in vitro in non-sterile guttation fluids from barley seedlings
Water
Glucose solution (8 p.p.m.)
Alternaria Botrytis Cladosporium
88"4a 97'0ab 44' 8a
87' 0a 95"4b 63-6 b
Alternaria* Botrytis* Cladosporiumt
3391a 2472a 12a
Guttation fluid N0 3
Germination (%) a 9 1"4 9 8-8" 94'4"
Hyphal growth (* {tm mm <; 4332 ab 6057" 3042a 7603 b 23 b 55"
t
{tm
NH 4
lowN
90' 8a 97'8 ab" 93'0"
90 -ga 9 8'2 ac 95-2"
conidium")
8158 d 12275" 90 d
5529bc 8234 b 82d
Data are means of five replicates. Values in the same row not followed by the same letter are significantly different at the 95 % level or better.
Sterilized guttation fluid stimulated hyphal growth of all three fungi. The response of C. herbarum was most pronounced, hyphal growth per conidium being on the average six times higher than in water. In non-sterile guttation fluid, a slight stimulation of hyphal growth occurred with B. cinerea while A. alternata and C. herbarum were not stimulated at all (Table 1). However, non-sterile guttation fluids collected from different N0 3 and NH 4 plants tested simultaneously with the above significantly inhibited the growth of A. alternata and C. herbarum. Otherwise, results were similar to those shown in Table 1, except that hyphal growth did not follow the same pattern of response towards the individual 'types' of guttation fluid. Growth of B. cinerea in sterilized fluid from NH 4 plants, for instance, was significantly lower than in fluid from N0 3 and low N plants. The pH values of the guttation fluids used varied between 6'1 and 6,6, so it seems unlikely that variation in pH could be responsible for these differences. On the whole, sterility of guttation fluids was obviously more important for conidial germination than any possible effects of plant nutrition. All non-sterile guttation fluid samples contained numerous bacteria and occasional yeasts growing around and/or between hyphae and conidia. We therefore concluded that the presence of other micro-organisms caused the general lack of stimulation of hyphal growth in non-sterile guttation fluids. However, when we tested a second collection of non-sterile guttation fluid in vitro we found that all fungi reacted in much the same way as they had done in sterilized guttation fluid in the earlier experiment. Percentage germination of C. herbarum was now stimulated and hyphal growth of all the
fungi was enhanced (Table 2) although numerous bacteria were seen again. These results suggest that not only the presence but also the level of antagonistic activity of other micro-organisms influence fungal development in guttation fluids. In order to find out whether the stimulating effect of non-sterile guttation fluids in the second experiment was perhaps due to a higher sugar content, we estimated the concentration of reducing sugars in all guttation fluids used. For the experiments in Table 1 these were equivalent to 28 flg glucose cm:" in fluid from N0 3 plants, 26'2 flg cm " in fluid from NH4 plants and 3 1'2 flg cm ? in fluid from low N plants. In the guttation fluids used in the second experiment (Table 2), we found 22'2, 29 and 44'2 flg glucose crn" respectively. The enhancement of germination in the second experiment could therefore not be merely attributed to a higher sugar content (preliminary chromatographic experiments had shown sucrose to be absent). The pH values of the guttation fluids were slightly higher in the second experiment, ranging from 6'75 to 7'45. It seems improbable, however, that this could have changed fungal behaviour to such an extent. Since leaf tissue has repeatedly been reported to contain inhibitors that affect fungal growth (Godfrey, 1976), we investigated the effects of guttation fluid on conidial germination on leaves. Again we used drops of sterile water or glucose solution as controls. The guttation fluids used were subsamples of the second collection of non-sterile fluids. Percentage germination of A. alternata and B. cinerea was high in all experiments and the few differences observed were of minor importance (Table 3). On leaves from NH 4 plants, C. herbarum germinated better in guttation fluid or glucose solution than in water. Similar results were
R. Frossard and J.J. Oertli
243
Table 3. Germination and hyphal growth of A. alternata, B. cinerea and C. herbarum in non-sterile guttation fluids on detach ed barley leaves NO. plants
NH. plants
Glucose Guttation solution fluid (8 p.p.m.) NO.
Water Alternaria Botrytis Cladosporium
93'8& 95'8& 66'2&
95'0& 96'0& 89.8&
96"4& 95'2& 9 1.8&
Alternaria" Botrytis" Cladosporiumt
6173& 4029& 16'
7167' 4860' 17'
8603 b 7044b 25'
Water
Glucose solution (8 p .p.rn.)
low N plants Guttation fluid NH.
Germination (%) 96'0&b 96'8& 94'0b 96"4& 95'2& 96'4& 77'0& 92·8b 97'0 b
Water 95'0& 95'4& 88'2&
Glucose Guttation solution fluid (8 p .p.m.) 10wN 96'2& 96'0& 88'4&
98'6 b 94'4& 90'2&
7933' 4144' 16b
6900' 6784b 19'
H yphal growth (* pm mm" ; t I'm conidium" ) 7702' 4297' 11 a
8485& 4588' 18b
11680& 8230b 47c
7900' 3895' 17· b
Data are means of five replicates. Values within each nitrogen treatment not followed by the same letter are significantly different at the 95 % level or better.
obtained on leaves from N0 3 plants; the differences, however, were not significant due to high variation between replicates in the water control. Similarly, variability of hyphaI growth of A . alternata and C. herbarum was high between replicates on leaves . A significant stimulation of A. alternata by guttation fluid was found, however, on leaves from NO. plants and of C. herbarum on leaves from NH. plants. B. cinerea, on the other hand, was stimulated by guttation fluid in all three treatments. No consistent correlation between nitrogen nutrition of the plants and conidial germination could be detected. Both water controls and glucose solutions supported some bacterial growth on leaf surfaces; when compared with guttation fluids, however, it was judged as low . DISCUSSION
Fertilization of grasses and other herbaceous plants with ammonium salts may enhance exudation of glutamine, and probably other amino acids, either through bleeding from cut surfaces or through guttation (Greenhill & Chibnall, 1934; Curtis, 1944; Raleigh, 1946). Starting from the hypothesis that amino acids may be limiting nutrients restricting conidial germination (cf. Blakeman, 1978), a marked increase of hyphal growth in guttation fluid from NH. plants was to be expected. Nevertheless, we found no unequivocal correlation between the type or amount of nitrogen supplied to barley seedlings and the stimulation of growth and germination of A. alt ernata, B. cinerea and C. herbarum conidia in guttation fluid. The stimulation of hyphaI growth of A. alternata and B . cinerea by sterilized guttation fluid from NH. plants could not be confirmed when the experiment
was repeated with another series of fluids (results not reported here). Yet in our second experiment, with non-sterile fluids, we observed this tendency again. Apparently the chemical composition of guttation fluids is subject to considerable variation, which in turn affects fungal growth. This observation is supported by results obtained with Helminthosporium sorokinianum (Endo & Amacher, 1964) and Saccharomyces cerevisiae (Perrin, 1972) in guttation fluids . Different fungi will not necessarily respond alike towards the same sample of guttation fluid as is seen when one compares hyphal growth of C. herbarum with that of the other fungi tested here. C. herbarum grew equally well in fluid from low N plants as in fluid from NH. plants (T able 2). This may have been due to the high sugar content of the guttation fluid of the low N plants. C. herbarum has been reported to show increasing germination with increasing sugar concentrations (M cBride & Hayes, 1979). The presence of a natural microflora clearly lowered percentage germination or hyphal growth (T able 1). The inhibitory effect of bacteria and yeasts on fungal germination in media with a low nutrient supply is well documented (Blakeman & Fraser, 1971; Brodie & Blakeman, 1976; Blakeman & Brodie, 1977; McBride & Hayes, 1979 ; Sharman & Heale, 1979). It is generally assumed that competition for nutrients is the most important cause of inhibition, although other mechanisms, for example the production of antibiot ic substances, cannot be ruled out (Blakeman & Brodie, 1976; Fokkema, 1976; Sharman & Heale, 1979). In our second experiment non-sterile guttation fluids failed to inhibit germination of the test fungi
244
Fungal growth in guttation fluids
(Table 2) although microscopic observation clearly REFERENCES showed large numbers of bacteria and some yeasts. Unfortunately, sterile subsamples of the same BLAKEMAN, J. P. (1978). Microbial competition for nutrients and germination of fungal spores. Annals of guttation fluids could not be tested due to the Applied Biology 89, 151-155. amount available. It may therefore be possible that hypha I growth would have been stimulated even BLAKEMAN, J. P. & ATKINSON, P. (1976). Evidence for a spore germination inhibitor co-extracted with wax from more had the fluids been sterilized. Nevertheless, leaves. In Microbiology of Aerial Plant Surfaces (ed. the microflora present in these specific samples was e. H. Dickinson & T. F. Preece), pp. 441-449. less effective in inhibiting conidial germination. London: Academic Press. Previous investigations on the effects of guttation BLAKEMAN, J. P. & BRODIE, I. D. S. (1976). Inhibition of fluids on fungi have almost exclusively been made pathogens by epiphytic bacteria on aerial plant with sterilized guttation fluids (Lewis, 1962; Endo surfaces. In Microbiology of Aerial Plant Surfaces (ed. C. H. Dickinson & T. F. Preece), pp. 529-557. & Amacher, 1964; Endo & Oertli, 1964; Healy & London: Academic Press. Britton, 1968; Potbury & Drysdale, 1969; Perrin, 1972). Our results demonstrate that conclusions BLAKEMAN, J. P. & BRODIE, I. D. S. (1977). Competition for nutrients between epiphytic micro-organisms and obtained with sterilized samples will not necessarily germination of spores of plant pathogens on beetroot apply to the actual situation in the field. leaves. Physiological Plant Pathology 10, 29-42. On detached barley leaves, C. herbarum had BLAKEMAN, J. P. & FRASER, A. K. (1971). 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(Received for publication 19 February 1981)