Lithium chloride solutions (1 mM), supplied to cucumber plants via the root system, conferred protection against powdery mildew infection ofthe leaves by ...
Planl Pathology (1991) 40, 108-117
Lithium chloride and cucumber powdery mildew infection J, K ABOOD, D, M, LOSEL Department of Animal and Plant Sciences, University of Sheffield, Sheffield SIO 2TN, UK and P. G, AYRES Department of Biological Scienees, University of Eancaster, Lancaster LAI 4YQ, UK Lithium chloride solutions (1 mM), supplied to cucumber plants via the root system, conferred protection against powdery mildew infection ofthe leaves by Sphaerothecafuliginea. The development of infection structures was significantly inhibited by this treatment. Effective concentrations of the lithium salt increased the growth of shoots but resulted in some reduction of root growth, Tbe incorporation of litbium by different parts of plants was assessed during a 20-day treatment period, Tbe possible mode of action of lithium on this host-pathogen system is discussed. INTRODUCTION Powdery mildew caused by Sphaerotheca futiginea (Schlecht Fr) Poll, (Ballantyne, 1975) is one of the serious diseases of cucumber in different regions of the world, both on glasshouse crops and in the field. Infection has a greater effect on quality than on yield (Sitterly, 1978), Although currently available fungicides provide some control of cucumber powdery mildew, there is still a need for other control systems, not based on organochemicals, wbich avoid tbe problems of possible residues in food crops and do not interfere with the biological control of cucumber pests by natural predators (Molot & Lecoq, 1986), This requirement, as well as an increasing interest in the role of lithium in cell biology (Lazarus, 1986) has prompted a re-examination of the possibilities of control of powdery mildew by lithium salts, suggested by some earlier studies, Tbe use of lithium, a normal soil constituent which has also been extensively studied in human cell biology, may avoid some of the problems involved in the certification of organochemicals for disease control in crop plants, Tbe effects of lithium on cultivated plants were investigated by Kent (1941) and shown to include increased resistance to disease and stimulation of growth, at low concentrations, as well as phytotoxicity and inhibition of growth at higher concentrations. Smith & Blair (1950) noted a reduction in the severity of wheat powdery mildew (Erysiphe graminis) Irom 11 to 1",, of leaf area infected when they fed plants with lithium chloride (8 g/l) and cotnplete mineral solution Carter
& Wain (1964) reported that lithium sulphate, when applied to the roots of wheat seedlings, showed marked systemic activity against E. graminis. Lithium salts were also reported to reduce the susceptibility of barley (Hirata, 1959) and tobacco (Vidali & Ciferri, 1951; D'Armini, 1953) to powdery mildew, E. graminis f,sp, hordei and E. cichoracearum, respectively. It apf>eared from preliminary experiments tbat lithium salts showed some inhibitory effect on powdery mildew infections of cucumber, Tbe inbibitory influence of effective concentrations of litbium chloride on growth of 5. fuliginea was similar to that of lithium sulphate but greater than that of lithium nitrate (J, K, Abood, unpublisbed results). Lithium sulphate caused a larger increase in the starch content of leaf tissues than either lithium chloride or lithium nitrate. For these reasons lithium chloride (LiCI) was chosen for the present investigations of its possible activity as a systemic fungicide against powdery mildew infection and of its influence on tbe growth of cucumber plants, MATERIALS AND METHODS Growth of plants Seeds of cucumber {Cucumis sativus e\, Beit Alpha, susceptible to Sphaerotheca fuliginea) were grown in a mixture of equal volumes of Levington's compost and sand in a propagating ch;imber for 6 days in growth-room conditions (18 C nigbt and 20 C day temperature, 16-h
LiCl and cucumber powdery mildew infection photoperiod, light intensity 80 ^mol m"^ s ' , r,h.40-60%). Seedlings were then transplanted to 9'Cm-diameter plastic pots containing acidwashed sand and were watered daily with 30 ml nutrient solution (Rorison, 1966) which was adjusted to pH 5-5-5-8 using HCl and KOH, Equal additions of distilled water were made to all plants as required. After 10 days, half of the plants were fed with nutrient solution that contained lithium. To study the effects of LiCl on plant growth and uptake by different tissues, plants were supplied on alternate days with 30 ml of 2 mM LiCI, The dry weights of shoots and root systems of four replicate plants were determined at intervals of 4 days after the commencement of LiCl application, during a period of 20 days. To determine the effect of LiCl on powdery mildew infection, but to avoid possible phytotoxic effects noted in the first experiment (see results), plants in subsequent experiments were supplied with I mM LiCl for each of 7 days before inoculation. Estimation of lithium
The lithium content in various parts of plants was estimated by flame photometry (410 Croning) using the procedure of Gloterman et ai (1978), After drying plant samples in an oven at 70-80°C for 24 h, 0-1 g of tissue was accurately weighed, partially ground and transferred to thick-walled test tubes. Concentrated H1SO4 (2 ml) and H2O2 (1 ml) were added and the test tubes were heated on a heating block at 30035OX for 3-5 h, then cooled and diluted with distilled water to 25 ml. A calibration curve, prepared from standard solutions at lithium concentrations from 0 (reagent blank) to 20 p,p,m., was used to calculate the amount of lithium in each tissue sample. The lithium content of conidia was estimated by a similar procedure from conidia collected, 9 days after inoculation, by tapping the leaves over aluminium foil, Tbe conidia were then transferred into weighed 20 ml beakers and dried at 80 C, After dissolving in 2 ml H2SO4, the fungal tissue was transferred to thick-walled test tubes and lithium digestion and estimation were carried out as described above.
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Fig. 1. Diagram of cucumber leaf showing positions of sampled segments. an electric fan. The inoculated plants were maintained in the same growth conditions as before until sporulation was abundant. Older conidia were tapped off stock plants and discarded. 6 h prior to each experiment, so that only freshly developed conidia were used as inoculum. Plants used in experiments were inoculated at the end of a photoperiod by cutting I-cm- segments from leaves of stock plants and touching each on a marked area in one of four positions on a healthy second leaf (Fig, 1), Preliminary investigations of powdery mildew infection of uniformly inoculated leaves of cucumber plants treated with 2 mM LiCl had shown more fungal growth in tbe vicinity of major veins than over interveinal areas (Fig, 2). whereas on similarly inoculated control plants fungal growth was evenly distributed over tbe leaf surface. It -^vas tberefore of interest to compare litbium uptake and fungal development in tbese two regions. After inoculation, the plants were covered with plastic bags for 24 h to increase the relative humidity around the spores, and were held in the growth room conditions alread> specified. Leaf segments from control and lithium-treated plants were sampled 6. 12, 18, 24, 36. 48, 96, and 144 h after inoculation. Assessment of fungal development
Maintenance and preparation of inoculum
Stock plants at tbe third-leaf stage were inoculated with powdery mildew spores by exposing them for 30 min to heavily infected plants within a chamber in which air was circulated by means of
To observe tbe early stages of development, segments taken after 6, 12 and 18 b were mounted in 0-5% trypan blue in lactophenol and heated gently at 60 C for 1 min, (Russell et ai, 1975) Although some displacement ol spores ma\ occur
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Fig, 2, (a) C ontrol and (b) lithium-treatcd lea\es showing the development of Sphaerothecu fuliginea. 7 days after inoculalion in this process, care was taken to standardize the mounting procedure so that valid comparisons between treatments could be made. The segments taken at 24, 36. 4!^. 96 and 144 h were dipped in a mixture of (he three \olumes of absolute alcohol and one volume of glacial aceiic atid to bleach ihc tissue, cleared with lactophenol for 8 h at 60 C and stained vMth 0 ?"i. trypan blue in lactophenol tor 30 mm according to the procedure of Carver & Carr (1977), but with reduced time of staining and omitting the destaming procedure. At each sampling time, a transect ol approximately 10 fields across each segment was examined (1 50 200 spores per replicate) using the x 40 ob|ccti\c, except in Ihc case of measurements of conidiophorc formation vshen the » 10 objectuc was used Two similar segments from a leal represented one replicate and each experiment was replicated tour times. The proportion of conidia which had tbrmed a germ tube longer ihan the width ol spores was assessed Earlier stages of germination were not casiK detected as germ tubes \cry quickK reached this length (icrmination percentages were determined d, 12 anil IK h .liter inoculation Prclimmar> experiments had shown no liirthcr increase in germination alter IX h Kccausc the germination hchaMoiir o l S liitii;iiifd spores IS dilVcrcnl Irom tli.it otHihcr powilcrv niiklcw spores, e g hry^iphc \;rnu\t
nolog\ gi\cn in Fig, 3 was adopted. The hypha cut olf by a septum at the apical end of an appressorium. after the appearance of the first haustorium. is referred to as the primary hypha; the h>pha developed after the appearance of a septum on the secondary germ tube is termed the secondary hyphii The lengths of the primary
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Fig. 5. Changes in lithium content of different parts of cucumber plants during treatment with 2 n-i,M lithium chloride. Vertical bars indicate standard errors, —D—, first leaf; • . second leaf; O. stem and apical shoot; • . root.
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Time (days) Fig. 4. Changes in dry weight of (a) shoot and (b) root of cucumber plants in control conditions and under treatment with 2 mM lithium chloride. Vertical bars indicate standard errors, —•—. LiCl; —D—. control.
germ tube 6 and 12bafter inoculation, and ofthe secondary germ tube 18 h after inoculation, were measured by means of a calibrated eyepiece graticule. Tbe lengths of tbe primary and secondary byphae were measured after 24 and 36 h. Only single, well separated fungal units were counted at each observation to eliminate the possibility of errors resulting from inhibition due to crowding, Mycelial density was measured 48 and 96 h after inoculation by counting the number of hyphae crossing the line on the eyepiece graticule. Numbers of conidiophores were determined in the same way as mycelial density, 144 h after inoculation,
ment. After this time root growth was significantly inhibited and there was no further increase in root dry weight (Fig, 4b), After 20 days of treatment, root systems were significantly lower in dry weight than those of control plants (P^OQX). The lithium content of tissues increased significantly with time in treated plants (Fig, 5). but after 12 days the ascending lithium stream appeared to have been diverted from the first leaf. which had by then reached final expansion, thereafter maintaining a constant concentration of lithium, to the second leaf which accumulated higher concentrations of lithium. After 16 days there was no further increase in lithium content in the second leaf while that of the root continued to rise. In both first and second lea\es. the amount of lithium detected after 8 and 12 days of treatment in the veins and adjacent tissues was not significantly different from that in tissues of interveinal areas (Fig, 6), Small amounts of lithium were deteeted in untreated, eontrol plants, ranging from 0-025 to 0-027 mg g ' dry weight in the second leaf and from 0-025 to 0-032 mg g ' in the root, after 8 and 20 da\ s of growth.
RESULTS
Germination of conidia and development of infection structures
Effects of lithium treatment on growth of cucumber plants
The normal pattern of conidial germination and subsequent development of infection structures is illustrated in Fig, 7, The primary gcrn-| tube, which formed within ,^ 6 h after inoculation, was sometimes forked (Fig, 7a), It functioned as an appressorium from whieh penetration of an epi-
Lithium chloride markedly increased the dry weight of shoots throughout the period studied (Fig. 4a). In contrast, roots showed no response to lithium during the first 12 days of the treat-
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Fig. 6. Distribution of lithium in leaves of cucumber plants treated with 2 mM lithium chloride. Vertical bars indicate standard errors, • . first leaf vein segment; a, second leaf interveinal segment; O, second leaf vein segment; • . second leaf interveinal segment. dermal cell occurred and, in the conditions ofthe present study, gave rise to a haustorium within the first 12 h after inoculation (Fig, 7b), During the following 6 h, a secondary germ tube was formed (Fig, 7c), Primary and secondary hyphae, separated from the primary and secondary germ tubes by septa, were observed within 24 h of inoculation (Fig, 7d) and after 5-6 days conidiophores began to develop from the superficial mycelium. Effect of lithium treatment on conidial germination and the development of infection structures Lithium treatment of host plants decreased the rate of germ-tube formation by S. fuliginea conidia (Fig, 8a), Analysis of variance showed these differences to be significant after 12 and 18 h but a similar tendency at 6 h after inoculation was not significant. Leaves of both control and lithium-treated plants showed more germination in the vicinity of veins than between them after 12 h (P
