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Conidial germination and germ-tube growth of Erysiphe pisi in relation to visible light and its transmission through pea leaves
[ 269 ] Trans. Br. mycol. Soc. 81 (2)
26~274
(1983) Printedin Great Britain
CONIDIAL GERMINATION AND GERM-TUBE GROWTH OF ERYSIPHE PISI IN RELATION TO VISIBLE LIGHT AND ITS TRANSMISSION THROUGH PEA LEAVES By P. G. AYRES Department of Biological Sciences, University of Lancaster, LA 1 4 YQ Germination of conidia of Erysiphe pisi, and germ-tube growth, are sensitive to low levels of irradiance at wavelengths within the visible spectrum, especially if conidia come from plants previously held in darkness for 24 h. At similar irradiances, germination is higher on the lower than on the upper epidermis of pea leaves illuminated from above. Germination, but not germ-tube growth, in vitro is stimulated by light transmitted through pea leaves. Development of conidia in vitro, in light transmitted through selected interference filters, is stimulated by bands of peak wavelengths 430 and 496 nm, inhibited by light of peak 551 nm, and insensitive to light of longer wavelengths. The significance of the responses is discussed in relation to the light transmitting properties of pea leaves which alter after mildew infection. Little is known about the effects of visible light on germination of conidia of powdery mildew fungi. Yarwood (1936) found germination of conidia of Erysiphe polygoni DC., from red clover, was stimulated by white light (probably of high intensity) if spores were collected at night but not if they were collected during the day. Germination of conidia of E. graminis DC. from barley and of other unspecified members ofthe Erysiphaceae was also stimulated by light. Drandarevski (1969) reported white light, 4700 lumen, stimulated germination of E. betae (Vafiha) Weltzien. Recently Singh & Singh (1981) reported white and green light of 150 lx stimulated germination of E. pisi DC. Reports concerning the effects of light on germ-tube growth are contradictory. Yarwood (1936) observed germ-tubes of E. polygoni were negatively phototropic to white light at low intensities and positively phototropic to a wide range of intensities of green light. Singh & Singh (1981) found exactly opposite results with E. pisi. It is difficult to compare these few reports because, apart from involving different species, either light has not been measured, or it has been measured in units (lumen or lux) to which the human eye is sensitive, rather than in units of energy. Moreover, the spectral quality of light, and its energy content, have not been specified. The spectral quality of light and its energy content are altered as it passes through a leaf. Thus, differences sometimes noted between the germination of mildew conidia on upper and lower leaf surfaces (Russell, Andrews & Bishop, 1975) may be due to differences in the light regime at the two surfaces. It was decided to make a systematic examination of the effects of visible light on the germination of
conidia of Erysiphe pisi and the development of their germ-tubes. Conidia were studied on the surface of leaves of pea (Pisum sativum L.), but the study concentrated on conidial behaviour in vitro where the effects of light could be separated from other confounding factors such as temperature, relative humidity and the chemistry and topography of the leaf surface. MA TERIALS AND METHODS
Plants and fungus Plants of garden pea (Pisum sativum L.) cv. Feltham First were grown in John Innes No.2 compost in 12 ern diam pots, 5 plants per pot. Young plants before infection, and infected stock plants used as a source of E. pisi conidia, were maintained under separate ventilated polythene covers in a glasshouse. Environmental conditions were not controlled. Investigations were carried out in the period June to September. Infected stock plants were shaken 24 h before an experiment to remove old spores and transferred to a physiological dark room at a constant 20°C. When the effect of light during this period was studied half the plants were maintained in the controlled environment room described below but were given 24 h continuous light. All subsequent operations were carried out under a photographic safelight (Ilford Ltd, London) in the dark room. Spores were dislodged from leaves by tapping the petiole. Germination on leaf surfaces Leaves were detached and their petioles passed through the side walls of water-filled black plastic
Light and conidial germination of Erysiphe pisi boxes, 5·5 x 3·5 x 2·5 em deep, so that each leaf was held horizontally. Petioles were immediately recut under water. Leaves were inoculated on either upper or lower epidermis: the lower epidermis was turned uppermost for inoculation so that spores could settle from above. Illumination from above was provided by a single metal halide lamp (400 W Kolorarc, Thorn Industries) during the following 20 h incubation period. Neutral filters were placed between the lamp and the leaf so that the final energy level was 6'0 W m- 2 at both upper and lower (light also filtered by leaf) epidermis. Reflected light was minimized by enclosing leaves in a cylinder whose inner surfaces and base were blackened. Spores were removed from the leaf surface in a dried film of Necoloidine (RD.H.) after 20 h incubation. Spores trapped in the film were stained in trypan blue in lactophenol before observation under the microscope. Spores were considered to have germinated when the germ-tube was longer than it was broad (Manners, 1966). One hundred spores and ten germ-tubes were measured for each replicate treatment. Germination in vitro Spores were collected on ethanol-washed glass covers lips after settling down a short tower 200 cm long. Spores were incubated for 20 h in 5 cm diam Petri dishes. Air was passed through the dishes at a rate of 1 em" S-1 after being adjusted to 92 % relative humidity by bubbling through dreschler bottles containing a saturated aqueous solution of K 2HP0 4 • (Preliminary experiments had shown germination was optimum at this humidity.) Petri dishes floated on a water bath held at a constant 20°. Dishes were illuminated from below, unless wrapped in silver foil to exclude light, by quartz-iodine bulbs mounted in a fan-cooled aluminium box. Bulbs were positioned at the focal point of 5 em diam bi-convex lenses fitted in the lid of the box to produce collimated light sources. A variable voltage transformer was used to regulate the light output of each bulb. The spectral composition, and hence average energy of the radiation, remained approximately constant as total irradiance increased. The water bath was constructed from Perspex which was blackened except where beams of light were transmitted. Interference filters (Barr & Stroud, Glasgow) of 5 em diam, which required a collimated light source, were mounted immediately above the lenses in the lid of the light box and below the water bath and Petri dishes. Properties of individual filters are listed in Table 2. Light inside the Petri dishes was measured with a light meter (Li Cor 185, Lambda Instruments, Lincoln, Nebraska) using a quantum
sensor which shows little variation in spectral sensitivity (Ludlow, 1982). It was, however, unlikely that the responses studied were photosynthetic, and it was considered they would depend on the energy received rather than the number of quanta, so spores were treated at fixed energy levels. These were determined for monochromatic light by multiplying the value measured with the quantum sensor by the energy constant for quanta of the peak wavelength (Nobel, 1970). For white light, and light transmitted by leaves, the energy constant of light of 550 nm (mid-point of the visible spectrum) was used. After incubation, spores were observed unstained under the microscope.
Transmission of light through leaves Third leaves were inoculated on the upper epidermis when they had fully expanded. One of each pair of leaflets was covered with a polythene bag during inoculation so that it remained as a healthy control. Plants were maintained in a controlled environment room with a temperature of 21o±2° and a relative humidity of65±5% during the light period, and a temperature of 19°±2° and a relative humidity of 75 ± 5 % during the dark period. Irradiance of 70 W m-2(40D-700 nm) at the height of the stem apex was provided by Kolorarc metal halide lamps (Thorn Industries) for 16 h per day. For measurement of light transmission, test leaves were gently clamped between a horizontal Petri dish lid (above) and the quantum sensor (below). Leaves were illuminated from above by a single metal halide lamp (400 W Kolorarc, Thorn Industries) and the distance between the light source and the leaf was held constant. Healthy and infected leaflets of the same leaf were compared. Intact leaves were too dense to allow their absorption spectrum to be measured in a spectrophotometer so pigments were extracted before spectra were determined. The extraction procedure was as follows. One gram fresh weight of tissue was macerated in 50 ern" of acetone in a pestle and mortar. Cell debris was removed by centrifugation, the supernatant decanted, and the pellet reextracted. The absorption of a sample of the combined supernatants was measured between 400 and 700 nm in a Pye Unicam SP8000 spectrophotometer.
RESULTS
White light An examination was made of the effects on germination of the light environment ofplants from which conidia were taken. Conidia were taken from
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Irradiance (W mFig. 1. Ca) Germination and Cb) germ-tube growth in vitro, of conidia of Erysiphepisi in response to irradiance by white light. Conidia taken from plants previously kept for 24 h in light CO) or dark ce). Each value is a mean of six replicates with standard error. Z)
Ca)
Cb)
Fig. 2. Appearance of typical germinating conidia of Erysiphe pisi Ca) in white light, Cb) in light filtered through a pea leaf, or (c) in dark.
plants held for 24 h in either light or darkness and germination, and germ-tube grown, in vitro, were measured in white light at irradiances from 0 to 120 W m", Germination in the dark was greater if conidia were taken from plants exposed to light
than if taken from plants exposed to darkness (Fig. 1 a). Germination in light was not affected by the light regime ofplantsfrom which conidia were taken and was not significantly affected by the level of irradiance within the range tested, 2·2 to 120 Wm- z. Each conidium produced one unbranched germtube (Fig. z c). Growth of the germ-tube was stimulated by low levels of irradiance and, in both light and darkness, was greater when conidia were taken from plants held in light than when taken from plants held in dark (Fig. 1b). Conidia for all later experiments were taken from plants held in darkness.
Light transmitted by leaves The effect on germination and germ-tube growth in vitro ofpassing white light through a single layer of detached pea leaves (immersed in 1 em water and overlapping at the margins) was examined. Irradiance was lower than in the previous experiment because the leaves absorbed large amounts of energy. White light (passed through 1 em of water) stimulated germination, but a greater stimulation occurred in light transmitted through leaves (Table 1). Conidia produced single unbranched, but often convoluted, germ-tubes in transmitted light (Fig. 2b), but total length was no greater than that of the even more convoluted germ-tubes of conidia
Light and conidial germination of Erysiphe pisi Table 1. Germination, andgerm-tube growth, ofconidia ofErysiphe pisi in response to irradiance of 1'0 W m:" of white light or light transmitted through pea leaves Germination (%) L.S.D. transformed values (P < 0'05) Germ-tube length C.um)
White light
Transmitted light
66'9 (54' 9)
78 .6 (62'4)
Dark control
3'5 47
24
L.S.D. (P < 0 '05 )
27
5'0
Values are mean s of eight replicates. Percentages were transformed to angles, in brackets, for analysis .
Table 2. Germination and germ-tube growth of conidia of Erysiphe pisi in response to irradiance of 6'0 W m- 2 at wavelengths seleeted by the use of wide and narrow band interference filters (a) Wide-band interference filter
Peak wavelength (nm) 45 8
Band width at half peak height (nm) Germination (%) L.S.D. transformed values (P < 0 '05 ) Germ-tube length (pm)
57 2
49 6
659
Dark control
85
109
51
95
85 '8 (67'9)
78 '6 (63' 0)
59 '8 (50' 1)
52'3 (46'4)
47 '7 (43'8)
32
26
8'0 57
46
29
L.S.D. (P < 0'05)
16
(b) Narrow band interference filter
Peak wavelength (nm )
Band at half peak height (nm) Germination (%) L.S.D. transformed values (P < 0 '05 ) Germ-tube length (pm) L.S.D. (P < 0 -05)
430
445
49 6
20
19
21
22
70 '9 (57'3)
69'6 (5 6 '5)
15'8 (23'4)
47 '1 (43'3)
24
25
79'3 (62' 9)
55 1
5'5 52
41
59 12
Values are means of eight replicates. Percentages were transformed to angles , in brackets, for analysis . Properties of filters are as specified by the manufacturers (Barr & Stroud, Glasgow ).
germinating in the dark (Fig. 2C). Single, unbranched but straighter, germ tubes were produced in white light and germ-tube length was significantly greater than in darkness. Conidial germination was greater on the lower epidermis of pea leaves in transmitted light, 60-4 % (50'3), than in white light on the upper epidermis, 49'7 % (44'9), (L.S.D., P = 0'05, was 5'0 for angularly transformed values, shown in brackets), although irradiance at both surfaces was 6'0 W m", Most conidia produced two, often branched, germ-tubes.
Light transmitted by interference filters
Germination and germ-tube growth in light whose wavebands were selected by the use of wide band interference filters were significantly stimulated by shorter wavelengths of light (filters with peakwavelengths of 458 and 496 nm) but unaffected by longer wavelengths (Table 2a). Germination in lights selected by narrow band filters was stimulated by the shorter wavebands, the highest germination occurring at the shortest wavelengths, but was inhibited in light of peak wavelength 551 nm (Table 2b). Germ-tube growth
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was stimulated by the shorter wavelengths of light, but was highest with the filter whose peak was 496 nm and was not inhibited by light whose peak was 551 nm.
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Days after inoculation Fig. 3. Percentage change in the transmission of light by pea leaves infected on the upper surface with Erysiphe pisi. Incident light 6:z'S W m", light transmitted by healthy leaf (broken line) 6·0 W m-·. Infected leaves with mycelium CO), with mycelium removed Ce ). Each value is a mean of twenty replicates with standard error.
/
Transmission properties of leaves The light transmitted by a pea leafiet infected by mildew on the upper surface was compared with that transmitted by the opposite uninfected leaf. The fungal mycelium developing on the leaf surface absorbed an increasing amount of light but if it were peeled off the leaf surface before measurements were made, it was seen that infected leaves transmitted an increasing proportion of the light incident upon them (Fig. 3). Examination of the pigments extracted from leaves in acetone showed that leaves infected for 7 days absorbed less light than healthy leaves in wavebands centred on 420 and 665 nm (Fig. 4).
DISCUSSION
The conclusions of Yarwood (1936), Drandarevski (1969) and Singh & Singh (1981) that white light stimulates germination of powdery mildew conidia
Healthy
.-- Infected
400
450
500
550
600
650
700
Wavelength (nm) Fig. 4. Absorption spectrum in acetone of extracts from pea leaves healthy or infected for 7 d with Erysiphe pisi (after removal of mycelium).
274
Light and conidial germination of Erysiphe pisi
are confirmed, and relatively low irradiances are sufficient to cause that stimulation (sunlight may reach 800 W m- 2 at the earth's surface). White light is a mixture of different wavelengths, the exact balance depending upon the nature of the source. Thus, the observed effects of white light are the net result of the individual effects of different wavelengths. These may be conflicting, for while light around 430 to 445 nm stimulated germination, light around 551 nm inhibited germination. The artificial sources used will have produced some light of wavelengths shorter than 400 nm and some of wavelengths longer than 700 nm. Any light in the ultraviolet range (350 nm and below) may also have an inhibitory effect (Yarwood, 1936). The nature of the light receptor is unknown but it is interesting that the germination response has energy requirements similar to those offungi whose sporulation is stimulated by light (Griffin, 1981). Conidia were exposed to uniform illumination so phototropic responses were not evident as in other investigations (Yarwood, 1936; Singh & Singh, 1982). White light, however, and light of short wavelengths, stimulated germ-tube growth at low irradiances. Conidia from plants held in the light were more responsive than conidia from plants held in darkness, as also found by Yarwood (1936). This could indicate the presence of higher levels of light receptor in the former. If this were true, it is to be expected that germination would have been similarly affected by the environment of the plant from which the spores came. Alternatively, it could have occurred because conidia from plants in the light had greater nutrient reserves and were, thus, potentially capable of greater growth. Since all epidermal cells of pea leaves, excepting stomatal guard cells, appear to be equally susceptible to infection, it is doubtful whether the effects of light on germ-tube growth have any importance in vivo. Effects of light on germination are probably, however, more important in vivo. Light cycles may induce some degree of diurnal rhythm in the infection cycle. The stimulatoryeffect oftransmitted light may promote development of the fungus on the lower surface of the upper leaves of the canopy and on both surfaces of the lower leaves. Thus, it
may be a factor contributing to the more luxuriant growth of mildew often observed within crops. Pea leaves transmitted increasingly more ofthe incident light as infection progressed. In particular, they absorbed less light at wavelengths corresponding to the known absorption peaks of chlorophyll a. Breakdown of chlorophyll a is known to occur more rapidly than breakdown of chlorophyll b in powdery mildew infected leaves (Paulech & Haspelova-Horvatovicova, 1970; Hewitt, 1976). Loss of the absorption peak at 420 nm would, in particular, allow more light of germinationstimulating wavelengths to pass into the vegetation.
REFERENCES
DRANDAREVSKI, C. A. (1969). Untersuchungen iiber den echten Riibenrnehltau Erysiphe betae (Vaiiha) Weltzien. II. Biologie und Klimaabhangigkeit des Pilzes. Phytopathologische Zeirschrift 65, 124-154. GRIFFIN, D. H. (1981). Fungal Physiology. Chichester: John Wiley. HEWITT, H. G. (1976). The effects of infection by Microsphaera alphitoides upon the physiology and growth of Quercus robur. Ph.D. Thesis, University of Lancaster. LUDLOW, M. M. (1982). Measurement of solar radiation, temperature and humidity. In Techniques in Bioproductioity and Photosynthesis (ed. J. Coombs & D. O. Hall), pp. 5-11. Oxford: Pergamon. MANNERS, J. G. (1966). Assessment of germination. In The Fungus Spore (ed. M. F. Madelin), pp. 165-174. London: Butterworths. NOBEL, P. S. (1970). Plant Cell Physiology. A Physiochemical Approach. San Francisco: Freeman. PAULECH, C. & HASPELOVA-HoRVATOVICOVA, A. (1970). Photosynthesis, plant pigments and transpiration in healthy barley and in barley infected by powdery mildew. Biologia (Bratislava) 25, 477-487. RUSSELL, G. E., ANDREWS, C. R. & BISHOP, C. D. (1975). Germination of Erysiphe graminis f. sp. hordei conidia on barley leaves. Annals of Applied Biology 81,161-169. SINGH, H. B. & SINGH, U. P. (1981). Reversible phototropism in germ tubes of Erysiphe polygoni. Zeitschrift fur Planzenkrankheiten und Pflanzenschutz 88, 626-630. YARWOOD, C. E. (1936). The diurnal cycle ofthe powdery mildew Erysiphe polygoni. Journal of Agricultural Research 52, 645-657.
(Received for publication
22
September 1982)
26~274
(1983) Printedin Great Britain
CONIDIAL GERMINATION AND GERM-TUBE GROWTH OF ERYSIPHE PISI IN RELATION TO VISIBLE LIGHT AND ITS TRANSMISSION THROUGH PEA LEAVES By P. G. AYRES Department of Biological Sciences, University of Lancaster, LA 1 4 YQ Germination of conidia of Erysiphe pisi, and germ-tube growth, are sensitive to low levels of irradiance at wavelengths within the visible spectrum, especially if conidia come from plants previously held in darkness for 24 h. At similar irradiances, germination is higher on the lower than on the upper epidermis of pea leaves illuminated from above. Germination, but not germ-tube growth, in vitro is stimulated by light transmitted through pea leaves. Development of conidia in vitro, in light transmitted through selected interference filters, is stimulated by bands of peak wavelengths 430 and 496 nm, inhibited by light of peak 551 nm, and insensitive to light of longer wavelengths. The significance of the responses is discussed in relation to the light transmitting properties of pea leaves which alter after mildew infection. Little is known about the effects of visible light on germination of conidia of powdery mildew fungi. Yarwood (1936) found germination of conidia of Erysiphe polygoni DC., from red clover, was stimulated by white light (probably of high intensity) if spores were collected at night but not if they were collected during the day. Germination of conidia of E. graminis DC. from barley and of other unspecified members ofthe Erysiphaceae was also stimulated by light. Drandarevski (1969) reported white light, 4700 lumen, stimulated germination of E. betae (Vafiha) Weltzien. Recently Singh & Singh (1981) reported white and green light of 150 lx stimulated germination of E. pisi DC. Reports concerning the effects of light on germ-tube growth are contradictory. Yarwood (1936) observed germ-tubes of E. polygoni were negatively phototropic to white light at low intensities and positively phototropic to a wide range of intensities of green light. Singh & Singh (1981) found exactly opposite results with E. pisi. It is difficult to compare these few reports because, apart from involving different species, either light has not been measured, or it has been measured in units (lumen or lux) to which the human eye is sensitive, rather than in units of energy. Moreover, the spectral quality of light, and its energy content, have not been specified. The spectral quality of light and its energy content are altered as it passes through a leaf. Thus, differences sometimes noted between the germination of mildew conidia on upper and lower leaf surfaces (Russell, Andrews & Bishop, 1975) may be due to differences in the light regime at the two surfaces. It was decided to make a systematic examination of the effects of visible light on the germination of
conidia of Erysiphe pisi and the development of their germ-tubes. Conidia were studied on the surface of leaves of pea (Pisum sativum L.), but the study concentrated on conidial behaviour in vitro where the effects of light could be separated from other confounding factors such as temperature, relative humidity and the chemistry and topography of the leaf surface. MA TERIALS AND METHODS
Plants and fungus Plants of garden pea (Pisum sativum L.) cv. Feltham First were grown in John Innes No.2 compost in 12 ern diam pots, 5 plants per pot. Young plants before infection, and infected stock plants used as a source of E. pisi conidia, were maintained under separate ventilated polythene covers in a glasshouse. Environmental conditions were not controlled. Investigations were carried out in the period June to September. Infected stock plants were shaken 24 h before an experiment to remove old spores and transferred to a physiological dark room at a constant 20°C. When the effect of light during this period was studied half the plants were maintained in the controlled environment room described below but were given 24 h continuous light. All subsequent operations were carried out under a photographic safelight (Ilford Ltd, London) in the dark room. Spores were dislodged from leaves by tapping the petiole. Germination on leaf surfaces Leaves were detached and their petioles passed through the side walls of water-filled black plastic
Light and conidial germination of Erysiphe pisi boxes, 5·5 x 3·5 x 2·5 em deep, so that each leaf was held horizontally. Petioles were immediately recut under water. Leaves were inoculated on either upper or lower epidermis: the lower epidermis was turned uppermost for inoculation so that spores could settle from above. Illumination from above was provided by a single metal halide lamp (400 W Kolorarc, Thorn Industries) during the following 20 h incubation period. Neutral filters were placed between the lamp and the leaf so that the final energy level was 6'0 W m- 2 at both upper and lower (light also filtered by leaf) epidermis. Reflected light was minimized by enclosing leaves in a cylinder whose inner surfaces and base were blackened. Spores were removed from the leaf surface in a dried film of Necoloidine (RD.H.) after 20 h incubation. Spores trapped in the film were stained in trypan blue in lactophenol before observation under the microscope. Spores were considered to have germinated when the germ-tube was longer than it was broad (Manners, 1966). One hundred spores and ten germ-tubes were measured for each replicate treatment. Germination in vitro Spores were collected on ethanol-washed glass covers lips after settling down a short tower 200 cm long. Spores were incubated for 20 h in 5 cm diam Petri dishes. Air was passed through the dishes at a rate of 1 em" S-1 after being adjusted to 92 % relative humidity by bubbling through dreschler bottles containing a saturated aqueous solution of K 2HP0 4 • (Preliminary experiments had shown germination was optimum at this humidity.) Petri dishes floated on a water bath held at a constant 20°. Dishes were illuminated from below, unless wrapped in silver foil to exclude light, by quartz-iodine bulbs mounted in a fan-cooled aluminium box. Bulbs were positioned at the focal point of 5 em diam bi-convex lenses fitted in the lid of the box to produce collimated light sources. A variable voltage transformer was used to regulate the light output of each bulb. The spectral composition, and hence average energy of the radiation, remained approximately constant as total irradiance increased. The water bath was constructed from Perspex which was blackened except where beams of light were transmitted. Interference filters (Barr & Stroud, Glasgow) of 5 em diam, which required a collimated light source, were mounted immediately above the lenses in the lid of the light box and below the water bath and Petri dishes. Properties of individual filters are listed in Table 2. Light inside the Petri dishes was measured with a light meter (Li Cor 185, Lambda Instruments, Lincoln, Nebraska) using a quantum
sensor which shows little variation in spectral sensitivity (Ludlow, 1982). It was, however, unlikely that the responses studied were photosynthetic, and it was considered they would depend on the energy received rather than the number of quanta, so spores were treated at fixed energy levels. These were determined for monochromatic light by multiplying the value measured with the quantum sensor by the energy constant for quanta of the peak wavelength (Nobel, 1970). For white light, and light transmitted by leaves, the energy constant of light of 550 nm (mid-point of the visible spectrum) was used. After incubation, spores were observed unstained under the microscope.
Transmission of light through leaves Third leaves were inoculated on the upper epidermis when they had fully expanded. One of each pair of leaflets was covered with a polythene bag during inoculation so that it remained as a healthy control. Plants were maintained in a controlled environment room with a temperature of 21o±2° and a relative humidity of65±5% during the light period, and a temperature of 19°±2° and a relative humidity of 75 ± 5 % during the dark period. Irradiance of 70 W m-2(40D-700 nm) at the height of the stem apex was provided by Kolorarc metal halide lamps (Thorn Industries) for 16 h per day. For measurement of light transmission, test leaves were gently clamped between a horizontal Petri dish lid (above) and the quantum sensor (below). Leaves were illuminated from above by a single metal halide lamp (400 W Kolorarc, Thorn Industries) and the distance between the light source and the leaf was held constant. Healthy and infected leaflets of the same leaf were compared. Intact leaves were too dense to allow their absorption spectrum to be measured in a spectrophotometer so pigments were extracted before spectra were determined. The extraction procedure was as follows. One gram fresh weight of tissue was macerated in 50 ern" of acetone in a pestle and mortar. Cell debris was removed by centrifugation, the supernatant decanted, and the pellet reextracted. The absorption of a sample of the combined supernatants was measured between 400 and 700 nm in a Pye Unicam SP8000 spectrophotometer.
RESULTS
White light An examination was made of the effects on germination of the light environment ofplants from which conidia were taken. Conidia were taken from
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Irradiance (W mFig. 1. Ca) Germination and Cb) germ-tube growth in vitro, of conidia of Erysiphepisi in response to irradiance by white light. Conidia taken from plants previously kept for 24 h in light CO) or dark ce). Each value is a mean of six replicates with standard error. Z)
Ca)
Cb)
Fig. 2. Appearance of typical germinating conidia of Erysiphe pisi Ca) in white light, Cb) in light filtered through a pea leaf, or (c) in dark.
plants held for 24 h in either light or darkness and germination, and germ-tube grown, in vitro, were measured in white light at irradiances from 0 to 120 W m", Germination in the dark was greater if conidia were taken from plants exposed to light
than if taken from plants exposed to darkness (Fig. 1 a). Germination in light was not affected by the light regime ofplantsfrom which conidia were taken and was not significantly affected by the level of irradiance within the range tested, 2·2 to 120 Wm- z. Each conidium produced one unbranched germtube (Fig. z c). Growth of the germ-tube was stimulated by low levels of irradiance and, in both light and darkness, was greater when conidia were taken from plants held in light than when taken from plants held in dark (Fig. 1b). Conidia for all later experiments were taken from plants held in darkness.
Light transmitted by leaves The effect on germination and germ-tube growth in vitro ofpassing white light through a single layer of detached pea leaves (immersed in 1 em water and overlapping at the margins) was examined. Irradiance was lower than in the previous experiment because the leaves absorbed large amounts of energy. White light (passed through 1 em of water) stimulated germination, but a greater stimulation occurred in light transmitted through leaves (Table 1). Conidia produced single unbranched, but often convoluted, germ-tubes in transmitted light (Fig. 2b), but total length was no greater than that of the even more convoluted germ-tubes of conidia
Light and conidial germination of Erysiphe pisi Table 1. Germination, andgerm-tube growth, ofconidia ofErysiphe pisi in response to irradiance of 1'0 W m:" of white light or light transmitted through pea leaves Germination (%) L.S.D. transformed values (P < 0'05) Germ-tube length C.um)
White light
Transmitted light
66'9 (54' 9)
78 .6 (62'4)
Dark control
3'5 47
24
L.S.D. (P < 0 '05 )
27
5'0
Values are mean s of eight replicates. Percentages were transformed to angles, in brackets, for analysis .
Table 2. Germination and germ-tube growth of conidia of Erysiphe pisi in response to irradiance of 6'0 W m- 2 at wavelengths seleeted by the use of wide and narrow band interference filters (a) Wide-band interference filter
Peak wavelength (nm) 45 8
Band width at half peak height (nm) Germination (%) L.S.D. transformed values (P < 0 '05 ) Germ-tube length (pm)
57 2
49 6
659
Dark control
85
109
51
95
85 '8 (67'9)
78 '6 (63' 0)
59 '8 (50' 1)
52'3 (46'4)
47 '7 (43'8)
32
26
8'0 57
46
29
L.S.D. (P < 0'05)
16
(b) Narrow band interference filter
Peak wavelength (nm )
Band at half peak height (nm) Germination (%) L.S.D. transformed values (P < 0 '05 ) Germ-tube length (pm) L.S.D. (P < 0 -05)
430
445
49 6
20
19
21
22
70 '9 (57'3)
69'6 (5 6 '5)
15'8 (23'4)
47 '1 (43'3)
24
25
79'3 (62' 9)
55 1
5'5 52
41
59 12
Values are means of eight replicates. Percentages were transformed to angles , in brackets, for analysis . Properties of filters are as specified by the manufacturers (Barr & Stroud, Glasgow ).
germinating in the dark (Fig. 2C). Single, unbranched but straighter, germ tubes were produced in white light and germ-tube length was significantly greater than in darkness. Conidial germination was greater on the lower epidermis of pea leaves in transmitted light, 60-4 % (50'3), than in white light on the upper epidermis, 49'7 % (44'9), (L.S.D., P = 0'05, was 5'0 for angularly transformed values, shown in brackets), although irradiance at both surfaces was 6'0 W m", Most conidia produced two, often branched, germ-tubes.
Light transmitted by interference filters
Germination and germ-tube growth in light whose wavebands were selected by the use of wide band interference filters were significantly stimulated by shorter wavelengths of light (filters with peakwavelengths of 458 and 496 nm) but unaffected by longer wavelengths (Table 2a). Germination in lights selected by narrow band filters was stimulated by the shorter wavebands, the highest germination occurring at the shortest wavelengths, but was inhibited in light of peak wavelength 551 nm (Table 2b). Germ-tube growth
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273
was stimulated by the shorter wavelengths of light, but was highest with the filter whose peak was 496 nm and was not inhibited by light whose peak was 551 nm.
I 0
3
8
5
10
Days after inoculation Fig. 3. Percentage change in the transmission of light by pea leaves infected on the upper surface with Erysiphe pisi. Incident light 6:z'S W m", light transmitted by healthy leaf (broken line) 6·0 W m-·. Infected leaves with mycelium CO), with mycelium removed Ce ). Each value is a mean of twenty replicates with standard error.
/
Transmission properties of leaves The light transmitted by a pea leafiet infected by mildew on the upper surface was compared with that transmitted by the opposite uninfected leaf. The fungal mycelium developing on the leaf surface absorbed an increasing amount of light but if it were peeled off the leaf surface before measurements were made, it was seen that infected leaves transmitted an increasing proportion of the light incident upon them (Fig. 3). Examination of the pigments extracted from leaves in acetone showed that leaves infected for 7 days absorbed less light than healthy leaves in wavebands centred on 420 and 665 nm (Fig. 4).
DISCUSSION
The conclusions of Yarwood (1936), Drandarevski (1969) and Singh & Singh (1981) that white light stimulates germination of powdery mildew conidia
Healthy
.-- Infected
400
450
500
550
600
650
700
Wavelength (nm) Fig. 4. Absorption spectrum in acetone of extracts from pea leaves healthy or infected for 7 d with Erysiphe pisi (after removal of mycelium).
274
Light and conidial germination of Erysiphe pisi
are confirmed, and relatively low irradiances are sufficient to cause that stimulation (sunlight may reach 800 W m- 2 at the earth's surface). White light is a mixture of different wavelengths, the exact balance depending upon the nature of the source. Thus, the observed effects of white light are the net result of the individual effects of different wavelengths. These may be conflicting, for while light around 430 to 445 nm stimulated germination, light around 551 nm inhibited germination. The artificial sources used will have produced some light of wavelengths shorter than 400 nm and some of wavelengths longer than 700 nm. Any light in the ultraviolet range (350 nm and below) may also have an inhibitory effect (Yarwood, 1936). The nature of the light receptor is unknown but it is interesting that the germination response has energy requirements similar to those offungi whose sporulation is stimulated by light (Griffin, 1981). Conidia were exposed to uniform illumination so phototropic responses were not evident as in other investigations (Yarwood, 1936; Singh & Singh, 1982). White light, however, and light of short wavelengths, stimulated germ-tube growth at low irradiances. Conidia from plants held in the light were more responsive than conidia from plants held in darkness, as also found by Yarwood (1936). This could indicate the presence of higher levels of light receptor in the former. If this were true, it is to be expected that germination would have been similarly affected by the environment of the plant from which the spores came. Alternatively, it could have occurred because conidia from plants in the light had greater nutrient reserves and were, thus, potentially capable of greater growth. Since all epidermal cells of pea leaves, excepting stomatal guard cells, appear to be equally susceptible to infection, it is doubtful whether the effects of light on germ-tube growth have any importance in vivo. Effects of light on germination are probably, however, more important in vivo. Light cycles may induce some degree of diurnal rhythm in the infection cycle. The stimulatoryeffect oftransmitted light may promote development of the fungus on the lower surface of the upper leaves of the canopy and on both surfaces of the lower leaves. Thus, it
may be a factor contributing to the more luxuriant growth of mildew often observed within crops. Pea leaves transmitted increasingly more ofthe incident light as infection progressed. In particular, they absorbed less light at wavelengths corresponding to the known absorption peaks of chlorophyll a. Breakdown of chlorophyll a is known to occur more rapidly than breakdown of chlorophyll b in powdery mildew infected leaves (Paulech & Haspelova-Horvatovicova, 1970; Hewitt, 1976). Loss of the absorption peak at 420 nm would, in particular, allow more light of germinationstimulating wavelengths to pass into the vegetation.
REFERENCES
DRANDAREVSKI, C. A. (1969). Untersuchungen iiber den echten Riibenrnehltau Erysiphe betae (Vaiiha) Weltzien. II. Biologie und Klimaabhangigkeit des Pilzes. Phytopathologische Zeirschrift 65, 124-154. GRIFFIN, D. H. (1981). Fungal Physiology. Chichester: John Wiley. HEWITT, H. G. (1976). The effects of infection by Microsphaera alphitoides upon the physiology and growth of Quercus robur. Ph.D. Thesis, University of Lancaster. LUDLOW, M. M. (1982). Measurement of solar radiation, temperature and humidity. In Techniques in Bioproductioity and Photosynthesis (ed. J. Coombs & D. O. Hall), pp. 5-11. Oxford: Pergamon. MANNERS, J. G. (1966). Assessment of germination. In The Fungus Spore (ed. M. F. Madelin), pp. 165-174. London: Butterworths. NOBEL, P. S. (1970). Plant Cell Physiology. A Physiochemical Approach. San Francisco: Freeman. PAULECH, C. & HASPELOVA-HoRVATOVICOVA, A. (1970). Photosynthesis, plant pigments and transpiration in healthy barley and in barley infected by powdery mildew. Biologia (Bratislava) 25, 477-487. RUSSELL, G. E., ANDREWS, C. R. & BISHOP, C. D. (1975). Germination of Erysiphe graminis f. sp. hordei conidia on barley leaves. Annals of Applied Biology 81,161-169. SINGH, H. B. & SINGH, U. P. (1981). Reversible phototropism in germ tubes of Erysiphe polygoni. Zeitschrift fur Planzenkrankheiten und Pflanzenschutz 88, 626-630. YARWOOD, C. E. (1936). The diurnal cycle ofthe powdery mildew Erysiphe polygoni. Journal of Agricultural Research 52, 645-657.
(Received for publication
22
September 1982)