Effects of foliar washing on subsequent infection of leaves of swede (Brassica napus) by Erysiphe cruciferarum

Physiol.

Pl. Path.

1, 123-132

(1971).

Effects of foliar washing on subsequent infection of leaves of Swede (Brassica napus) by Erysiphe cruciferarum T. J. PunNxx.Lt and T. F. PREECE Agricultural Botany Diuision, Department The University of Leeds, U.K. (Accepted for

publication

September

of Agricultural

Sciences,

1970)

Washing the surfaces of fully expanded leaves of swede (Brassicu napus) with distilled water for 1 h prior to inoculation with conidia of Erysiphe cruc$hwum had no effect on germination and appressorium formation, but caused a reduction in the number of conidia which produced primary and secondary hyphae. Washing also caused a reduction in the lengths of primary hyphae. When inoculation of washed leaves was delayed, the number of conidia which produced primary and secondary hyphae gradually increased up to a maximum on leaves inoculated 5 days after washing. Washed leaves inoculated after this time showed a reduction in the number of conidia producing hyphae. All leaf washings collected during the first 5 days after the initial washing stimulated spore germination and germ tube growth of Botrytti cinerea used in vitro as a test fungus, but those collected later were inhibitory. Scanning electron microscope studies showed that wax was removed from the leaf during the washing process and that little of this wax was regenerated on fully expanded leaves. -4nalyses of leaf washings using gas chromatography showed that carbohydrates were also lost from the leaf surface during washing, but within 7 days these were replaced and accumulated at the surface in excess of the amounts found on unwashed leaves. It seems unlikely, however, that the inhibition of either fungus was due to the increased carbohydrate concentration but, rather, to the accumulation of some unknown inhibitory substance.

INTRODUCTION

The prolonged persistence of water films on leaf surfaces is essential for infection by many plant pathogenic fungi. As soon as water runs off the surface, however, inorganic and organic substances [3, 14, 191 and surface waxes [IO] are lost from the leaf. The question arises as to what extent this removal of materials from potential infection sites affects disease establishment. Examination of rain-wash effects using a leaf on which water films do not normally persist might be one approach to this general problem. Water repellency, resulting in water droplets rolling off the leaf is easily observed on leaves of Swede (Brassica napus). The effects of washing Swede leaves with water prior to inoculation with conidia of Evysiphe cruciferarum Opiz ex June11 [9] are reported in this paper. MATERIALS

AND

METHODS

Seedlings of the Swede cultivar “Best of All” were grown in John Innes No. 2 compost in a glasshouse for 6 to 8 weeks, during which time 12 to 15 leaves developed on each plant. Leaves used for experimental purposes were selected by the Leaf Plastochron 1 Present

address:

ICI

Limited,

Jealott’s

Hill

Research

Station,

Bracknell,

Berkshire,

U.K.

124

T. J. Purnell

and T. F. Preece

Index (L.P.I.) method [8]. Th is method enables replicate leaves to be selected on the basis of developmental status rather than on chronological age. A low Plastochron Index denotes a young leaf. A cabinet was constructed from cast acrylic sheet (Perspex, Imperial Chemical Industries Ltd., London). A front plate, fitted with a rubber gasket allowed access, and a hole 4 cm in diameter in the centre of the front plate enabled a single leaf to be inserted and sealed into the cabinet. Either entire plants or single leaves still attached to plants were washed in this apparatus (Fig. 1). An atomizer, which was operated by a jet of air from an electromagnetic piston pump (Reciprotor, Edwards High Vacuum Ltd., Crawley, Sussex, U.K.), was supplied with glass distilled water from a Perspex reservoir on the top of the cabinet. The airflow was divided after

65cm 1 Air

+---

FIG.

1. Apparatus

used

30cm

to wash

------+

single

Swede

leaves.

leaving the pump, part going to the atomizer and the remainder being piped into the reservoir. This arrangement facilitated the outward flow of water to the atomizer. The volume of air passing to the atomizer was controlled by a small valve in the air line, enabling the spray passing over the leaf to be adjusted from a simulated “fine mist” to a “heavy rain”. The apparatus delivered 60 ml distilled water/mm. The length of the washing period was restricted to 1 h so that as far as possible only materials present on the leaf surface would be removed. The attached leaves were allowed to dry at room temperature for an additional hour before inoculation with conidia of E. cruciferarum. Mature, completely expanded leaves (L.P.I. range 6 to 9, 14 to 20 days old), were used in the majority of experiments. For studies which involved delaying the inoculation of leaves for increasing lengths of time, the date of washing the leaves

Effects

of foliar

washing

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of Swede

leaves

125

was arranged so that all the treatments could be inoculated at the same time. The leaves of plants grown in both summer and winter seasons were used in the experiments. Although blowing conidia from infected plants on to healthy leaves produced successful infections, these infections were not uniform in distribution. Therefore a specially designed spore settling tower was used. It was made of a transparent polyester film (Melinex, I.C.I. Ltd., London) supported by a framework of slotted, aluminium-alloy angle strips (“Dexion”, Dexion Ltd., Dexion House, Empire Way, Wembley, Middlesex). Spores were introduced by shaking infected plants above the open top of the tower. These were then distributed by a short blast of air blown upward and allowed to settle. The inoculated plants were placed on benches covered with fine wet gravel in a glasshouse. The growth of E. cruciferarum was observed after a 24 h period of incubation as follows: disks were cut from leaves, bleached in chlorine gas, stained and mounted in phenol-acetic-aniline-blue solution [17] and examined by means of a light microscope for germination, appressorium formation and the production of primary and secondary hyphae. Later, when the Stereoscan electron microscope (Cambridge Instruments S.E.M., Mark II) was used, the leaf disks were coated with 200 A of silver in a vacuum coating unit prior to examination. Each washing sample was obtained from 20 mature leaves, each washed for 1 h. The area of each leaf was determined by tracing the leaf margin on squared paper and adding the area of the squares. The washings of each collection were combined and sterilized by passing through a membrane filter (Oxoid Ltd., London). The bulked washings were concentrated from 2 1 to approximately 20 ml at 40 “C under pressure and again passed through a membrane filter and stored at 0 “C until required. In previous studies of the effect of leaf exudates on fungal development [7, 1.21 the final volume of extracts was standardized by relating them to leaf area. This procedure has been followed here and “1K” represents the concentration of solutes that would occur in a film of water 0.1 mm thick over the leaf. Thus a concentration recorded as “2K” has twice the concentration of solutes in the same volume. The biological activity of leaf washings was assayed by a sensitive test fungus [12], Botrytis cinerea. The organism was grown on dextrose peptone agar and the spores prepared for use as described by Chu-Chou & Preece [4]. The concentration of spores was adjusted densiometrically to 2.5 x lo5 spores/ml using an EEL colorimeter and confirmed by haemocytometer counts. Equal volumes of spore suspension and 2K leaf washings were mixed on cleaned, hollow-ground microscope slides. Thus the spore concentration in the tests was 1.25 x 105/ml and that of the leaf washings was 1K. The percentage of Botrytis spores which germinated and the lengths of their germ tubes were recorded by examination of 500 spores/treatment after a 4-h incubation period at 22°C. In preparation for gas chromatographic analysis of the carbohydrates contained in leaf washings, the concentrated sterile washings were passed through ion-exchange columns and the neutral fraction containing the carbohydrates was dried completely under reduced pressure. Then 0.85 ml anhydrous pyridine was added to the dry fraction which was allowed to stand for 45 min. The volatile trimethylsilyl derivatives

126

T. J. Purnell

and

T. F. Preece

were then prepared by the addition of O-1 ml of hexamethyldisilazane, followed by 0.5 ml of trimethylchlorosilane. After a further 45 min the extract was analysed using a Pye series 104 gas chromatograph. Carbohydrates were identified by comparison with separations of known standards and by the addition of standards to the extracts. During the analyses, the a: and /I anomers of glucose were identified but the figures given for this carbohydrate are the total of both forms. The amino acids in the cationic fraction of the leaf washings were determined using the Technicon automatic amino acid analyser (Technicon Instruments, Chertsey, Surrey). The amino acids are separated in this machine by ion-exchange chromatography in columns of polystyrene resins. The 01amino groups of the amino acids interact with the sulphonic acid groups of the resin and the amino acids are eluted with acidic buffers in decreasing order of dissociation constants. The eflluent is monitored photometrically after a ninhydrin reaction. The amino acids can then be identified by their characteristic position on the elution curve. The non-volatile organic acids were separated by partition chromatography using the method described by De Kock & Morrison [5]. This separation was carried out on a silica gel column and a gradient elution technique was used. The separated fractions were collected on an automatic fraction collector. The acids in these fractions were then determined by titration with alcoholic 0.2 N sodium hydroxide. RESULTS

Germination

and appressorium formation

The number of conidia which germinated was approximately 10% higher on Swede leaves than on glass slides. On the surface of washed leaves of different ages germination and appressorium formation were little affected by the l-h period of washing with distilled water (Table 1). TABLE

Germination

1

and a@ressorium formation by conidia of E. cruciferarum ages subjected to 1 h of washing with distilled water prior

2.5 Percentage

of conidia

Percentage of appressoriaQ 5 250 conidia

Primary

examined

germinated”

conidia

in each

forming

Washed Unwashed Washed Unwashed

68 74 67 68

on swede leaves to inoculation

Leaf plastochron 4.5 6.5 67 71 66 68

67 64 64 63

of dyerent

index 9.5

11.5

66 65 61 58

66 67 60 59

case.

and secondary hyphal production

on washed leaves of daTerent ages

Primary and secondary hyphae are defined here as the first two hyphae to be produced from the conidium after appressorium formation [S]. The number of conidia producing primary and secondary hyphae was expressed as a percentage of the total spores counted whether germinated or not. On unwashed leaves of different ages there was little variation from leaf to leaf in the percentage of conidia which produced primary hyphae (Table 2). However, when these leaves were subjected

Effects

of foliar

washing

on infection

of swede

127

leaves

to a l-h period of leaf washing, there was a reduction in the number of conidia which produced these hyphae on mature and older leaves. The greatest reduction was found on mature leaves which had ceased expansion but as yet showed no signs of senescence (L.P.I. 6.5 to 9.5). Production

TABLE 2 of primary hyphae from the conidia of E. cruciferarum ages subjected to a l-h period of washing prior Percentage Leaf

plastochron

index

Washed for 1 h

2.5 4.5 6.5 9.5 11.5 a Figures

are means

from

leaves,

58 57 70 66 60

250 spores

of darerent

of conidia producing primary hyphae Unwashed Percentage control level of significance

60 53 49 40 45 three

on Swede leavep to inoculation

being

N.S. N.S. o-1 0.1 0.1

examined

in each

case.

Following the observations that the greatest effect of washing occurred on mature leaves, more detailed investigations were carried out using leaves of this age. Table 3 shows the results of four replicate experiments in which the production of primary and the later formed secondary hyphae was studied on washed and unwashed mature Swede leaves (L.P.I. 6.5 to 9.5). The experiments were conducted during both summer and winter months, and the greatest reduction in production of primary and secondary hyphae was observed in the summer. In winter a smaller effect was noted and conidia on both washed and unwashed leaves failed to produce secondary hyphae during the 24-h experimental period. Plate 1(a) and (b) illustrates typical examples of the stages of growth reached by conidia in situ on washed and unwashed mature leaves 24 h after inoculation. At this time, the appressorium on unwashed leaves was fully expanded and the primary and secondary hyphae were developing rapidly. However, on washed leaves growth was much slower, and after 24 h usually only the appressorium was formed. TABLE 3 The percentage of conidia of E. cruciferarum producing primary and secondary hyphae when inoculated onto mature Swede leaves washed for 1 h prior to inoculalion

Percentage with primary hyphae Experiment 1 2 3 4 Summer Winter Unwashed control Washed for 1 h

70 35

36 15

42 9

43 23

Percentage with secondary hyphae Experiment 1 2 3 4 Summer Winter 30 0

10 0

16 3

0 0

The effects of leaf washing on the early stages of growth of E. cruciferarum were further shown by the measurements of the length of the primary hyphae formed by

128

T. J. Purnell

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T. F. Preece

the conidia in some experiments. From data obtained in a summer experiment the mean length of 250 primary hyphae, measured 24 h after inoculation, was 112 pm on unwashed control leaves, and only 46 pm on washed leaves. During the winter months elongation of these hyphae occurred more slowly and therefore the mean hyphal lengths recorded on unwashed and washed leaves were small, being 27 and 23 p respectively. Effect of delayed inoculation

of washed mature leaves Although the percentage of conidia which produced primary and secondary hyphae on unwashed mature leaves varied between experiments, within a single experiment leaf ages of the order L.P.I. 6.5 to 9.5 did not affect hyphal production.

Interval (days) after washing

FIG. 2. Comparison of effects observed on E. of leaf surface carbohydrates. (a) Percentage of and secondary hyphae on mature Swede leaves washine with distilled water in Tub. Unwashed secondary hyphae. O-O, P>mary hyphae; regeneration of carbohydrates on the surface of after washing with distilled water. Carbohydrate leaves = O-321 pg/ml. ”

cruciferarum with changes in the concentration conidia of E. cruciferarum producing primary when inoculated at increasing intervals after control leaf: 50% primarv hwhae and ZOO/ 0 - - - - 0, secondary hy&ae. (b) The mature Swede leaves at increasing intervals concentration in washings from control dry ,_.

I

If mature leaves of this age are allowed to stand for increasing lengths of time after washing before inoculation with conidia, then the number of conidia which produce primary and secondary hyphae is related to the time that the leaves were allowed to stand before inoculation. Figure 2(a) shows that during the summer months the greatest reduction in numbers of these hyphae was observed on leaves inoculated 0 to 3 days after washing. However, conidia on leaves inoculated 5 days after washing showed enhanced production of hyphae, being up to 20% higher than on unwashed leaves. Inoculation of washed leaves later than 5 days again revealed a reduction in hyphal production and was similar to that of the unwashed leaves. During the winter months the increase in hyphal production was not observed until 12 days after washing.

Effects

of foliar

washing

on infection

of Swede

129

leaves

Results obtained by the inoculation of washed mature Swede leaves with mildew conidia were substantiated by using the in vitro germination and germ tube growth of Botrytis cinerea to assay the biological activity of washings collected from Swede leaves at increasing intervals after an initial washing. The greatest increases in percentage germination and in germ tube growth were observed in washings collected from Swede leaves 5 days after an initial washing. Repeated experiments have shown, however, that the growth of the germ tubes is more sensitive to the concentration of leaf washings than is spore germination. Washings collected after 7 and 12 days greatly inhibited the development of germ tubes of B. cinerea. The effect of washing

on the mature leaf surface itself

The described reduction in the number of conidia which produced primary and secondary hyphae on leaves inoculated soon after washing and the enhanced hyphal production when inoculation of washed leaves was delayed for 5 days might be due TABLE 4 The quantitative loss of organic substances from the mature leaves of Swede (Brassica during summer and winter months, @ washing the foliage with distilled water for

Carbohydrates Winter Summer Glucose

o-090

Amino Winter

Fructose

0.003

0.054

Aspartic acid Serine

Inositol

Trace

0.011

Glycine

0.002

0.004

Sucrose

0.141

0.006

Lysine Histidine Arginine Ornithine Others

0.00 1 0.002 0.005 0.001

0.001 0.003 0.001 o-002 0.006

Totals

O-234

0.32 1

0.018

0.020

a Measured

O-250

acids Summer

in pg per

o-002 0.005

0.001 0.002

napus) 1 h Organic Winter

Fumaric acid Succinic acid Malonic acid

acids Summer

Trace

Trace

Trace

Trace

Trace

Trace

Trace

Trace

cma of leaf surface.

to an initial increase and subsequent decrease of leaf turgidity. This seems unlikely, however, because numerous leaves held experimentally in different states of turgidity and inoculated with conidia showed no differences in the early stages of infection. Another possibility is that the removal of surface waxes and their regeneration might be involved. Wax is removed, and this process is easily revealed by using the Stereoscan electron microscope [Plate l(c) and (d)]. However, there is very little regeneration of wax at the surface of mature leaves after washing [Plate 1 (d) and (e)]. Various other leaf surface substances including carbohydrates, amino acids, organic acids and inorganic salts may be removed also (Table 4). The amounts of amino acids and organic acids lost from the leaf during washing were relatively small, but the loss of carbohydrates was considerably higher. Many workers have shown that carbohydrates, particularly glucose and fructose, present in the washings of various plant parts are important in the stimulation of the germination and growth of fungal pathogens [11, 151. For this reason further studies were restricted to the

130

T. J. Purnell

and

T. F. Preece

analyses of carbohydrates. Such analyses of leaf washings collected at increasing intervals after washing with distilled water indicated a regeneration of carbohydrates at the surface [Fig. 2(b)]. DISCUSSION

Germination of powdery mildew conidia has often been found to be greater on leaves of the host than on glass slides, under the same conditions of relative humidity [18]. Yarwood & Hazen [21] suggested that this stimulation of germination was due to the presence of chemical stimulators on the leaf surface. In the case of S’haerotlzeca macularis the surface waxes of several strawberry varieties have been shown to stimulate germination of conidia in vitro [1&j. In the present investigations, however, no evidence of such a stimulator of germination was found in the washings or surface wax obtained from mature Swede leaves. The increased germination on Swede leaves over that on glass slides may however, be due to the existence of a favourable microclimate at the surface [13], the presence of a volatile stimulator [2] or to the absorption of moisture by conidia through the epidermis as suggested by Weinhold [20]. The production and elongation of primary and secondary hyphae was inhibited when mature, fully expanded Swede leaves were washed with water for 1 h and inoculated with conidia of E. cruciferarum. The development of these hyphae was, however, markedly stimulated when inoculation of the washed leaves was delayed for 5 days. Conidia inoculated on to leaves 12 days after washing again showed inhibition of primary and secondary hyphae. Experiments carried out during the winter months revealed effects similar to those observed on washed leaves in the summer. A longer period elapsed, however, before enhanced growth was observed, and this is thought to be due to the slow growth of the fungus and the slower regeneration of leaf surface materials in the winter. There was little reduction in the number of conidia which produced primary and secondary hyphae on washed immature leaves. As yet no explanation for this difference from mature leaves has been found, but it may be that, because of the large amounts of wax on young leaves, the surface is little affected by a l-h period of washing with water. In the washing water, wax is removed but very little is regenerated on mature fully expanded leaves. Conversely, carbohydrates, and possibly other water-soluble substances, are not only replaced, but later accumulate in excess of the amounts found on unwashed leaves. Spore germination and the early stages of growth of many leaf-infecting fungi have often been reported to be influenced by organic substances present at the plant surface [I, II, 123. Whether the effect is one of stimulation or inhibition may well depend on the concentration of these substances at the surface [12]. It is unlikely that the increased amounts of carbohydrates or amino acids on rain-washed Swede leaves can account for the reduction in hyphal production by conidia of E. cruciferarum or the inhibition of germ tube growth of B. cinerea. Results of experiments not reported here have shown that the addition of carbohydrates and amino acids (in concentrations up to five times that found at the leaf surface) to Swede leaf washings further enhanced the germination and germ tube growth of B. cinerea.

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19. TUKEY, H. B., JR, TUKEY, H. B. & WITTWER, S. H. (1958). Loss of nutrients by foliar leaching as determined by radioisotopes. Proc. Am. Sot. hart. Sci. 71, 496-506. 20. WEINHOLD, A. R. (1958). Ecological and physiological studies on the development of pealch powdery mildew. Ph.D. thesis, University of California. 21. YARWOOD, C. E. & HAZEN, W. E. (1944). The relative humidity at leaf surfaces. Am. 3. Bat. 31, 129-135.

Effects

of foliar

washing

on infection

of Swede

leaves

131

Thus there appear to be two significant interacting substances or types of substances affecting infection which are washed off mature Swede leaves by rain. The first is possibly carbohydrate in nature, and within 5 days of washing, is replaced from within the leaves resulting in an increase in the number of conidia which produce primary and secondary hyphae results. The second substance is an inhibitor of hyphal growth. It is replaced gradually on the leaf surface after washing. Not until 12 days after washing does it build up to a degree that can be detected by reduced hyphal production. Thus, the hyphal growth curves are the result of two interacting factors. These results suggest that further studies and parallel investigations of other host/parasite combinations are now needed. We wish to thank Professor J. H. Western for his advice during the course of this work, Professor H, W. Woolhouse for the use of the Pye 104 Chromatograph, Professor R. D. Preston for the use of the Technicon automatic amino acid analyzer and Dr. J. Sikorski for Stereoscan electron microscope facilities. We are also grateful to the Ministry of Agriculture, Fisheries and Food for the provision of a postgraduate studentship to one of us (T. J. P.). REFERENCES 1. BLAKEMAN, J. P. (1968). Studies on the influence of leaf washings on infection by Mycosphaerella ligulicola. Ann. appl. Biol. 61, 77-88. Studies in the physiology of parasitism. IX. The effect on germination of 2. BROWN, W. (1922). fungal spores of volatile substances arising from plant tissues. Ann. Bot. 36, 285-300. 3. CARLISLE, A., BROWN, A. H. F. & WHITE, L. J. (1966). The organic matter and nutrient elements in the precipitation beneath a sessile oak (Q. petraea) canopy. 3. Ecol. 54, 87-89. 4. CHU CHOW, M. M. L. & PREECE, T. F. (1968). The effects of _pollen -grains on infections caused by Bot& cinerea Fr. Ann. apP1. Biol. i2, 1 i-22. 5. DE KOCK. P. C. & MORRISON. R. I. (1958). The metabolism of chlorotic leaves. 2. Organic acids. Biochek. 3. 70, 272-277. . ' ' 6. DELP, C. J. (1954). Effect of temperature and humidity on the grape powdery mildew fungus. Phytopathology 44, 6 15-625. 7. DUNN, C. L., BROWN, K. F. & MONTAGNE, J. TH. W. (1969). Antagonism between fungicides and water soluble exudates from leaves of plants. Phytopath. <. 64, 112-l 18. a. ERICKSON, R. C. & MICHELINI, F. J. (1957). The plastochron index. Am. 3. Bot. 44,297-305. 9. JUNELL, L. (1967). A revision of Erysiphz communis (Wallr.) Fr. semu Blumer. Suet& bot. lid&.

61, 209-229. 10. JUNIPER, B. E. (1959). The surface of plants. Endeavour 18, 20-25. 11. KOSUGE, T. & HEWITT, W. B. (1964). Exudates of grape berries and their effects on germination of conidia of Bohytis c-inerea. Phytopathology 54, 167-173. 12. KOVACS, A. & SZEOKE, E. (1956). Die phytopathologische Bedeutung der kutikularen Exkretion. Phytopath. <. 27, 335-349. 13. LONGTREE, K. (1939). The effect of temperature and relative humidity on the powdery mildew of roses. Mem. Cornell Univ. ag& Exp. Stn 223. 14. MORCXN, J. V. & TUKEY, H. B., JR (1964). Characterization of leachate from plant foliage. Pl. Physiol., Lancaster 39, 590-593. 15. ORELLANA, R. G. & THOMAS, C. A. (1962). Nature and predisposition of castor beans to Botrytk I. Relation of leachable sugars and certain other biochemical constituents of the capsule to varietal susceptibility. Phytopathology 52, 533-538. 16. PERIES, 0. S. (1962). Studies on strawberry mildew caused by Sphaerotheca macularis. II. Host parasite relationships on foliage of strawberry varieties. Ann. appl. Biol. 50, 225-233. 17. PREECE, T. F., BARNES, G. & BAILEY, J. M. (1967). Junctions between epidermal cells as sites of appressorium formation by plant pathogenic fungi. Pl. Path. 16, 117-l 18. 18. ROGERS, M. H. (1959). Some effects of moisture and host plant susceptibility on the development of powdery mildew of roses caused by Sphaerotheca pannosa var. rosae. Mem. Cornell Univ. Agti. Exp. Stn 363.