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Effect of allopurinol treatment on biotrophic growth of some powdery mildew fungi in their specific hosts
Physiological and Molecular Plant Pathology (2001) 59, 201±211 doi:10.1006/pmpp.2001.0358, available online at http://www.idealibrary.com on
E¡ect of allopurinol treatment on biotrophic growth of some powdery mildew fungi in their speci¢c hosts M . M A R T E and P. M O N TA L B I N I * Dipartimento di Arboricoltura e Protezione delle Piante, UniversitaÁ di Perugia, Borgo XX Giugno 74, I-06121 Perugia, Italy (Accepted for publication September 2001) When supplied via roots to tobacco host plants, aqueous solutions of allopurinol, a speci®c inhibitor of xanthine oxidase and dehydrogenase, markedly reduced or completely prevented the visible development of tobacco powdery mildew fungus Erysiphe cichoracearum (E. orontii) at concentrations as low as 25±50 mM administered for periods as short as 2 days. The duration of this allopurinol-dependent protection from the pathogen was of at least 20 days after the last administration. If supplied as foliar sprays at concentrations up to 1600 mM, allopurinol was not absorbed by the leaf tissues and did not aect the fungal infection. Histological observations revealed that on tobacco plants treated with allopurinol via roots, a sharp reduction in E. cichoracearum mycelial development started only 48 h after inoculation (after formation of the ®rst haustoria) and was accompanied by a strong delay and reduction of sporulation. The proportion of haustoria partially or entirely embedded in thick translucent sheaths ( possibly callosic in nature) increased signi®cantly in allopurinol irrigated plants reaching about 41 % of the total haustoria (compared to 9 % in controls) 7 days post-inoculation. No eects of allopurinol supply was observed on Blumeria graminis and Erysiphe trifolii development on wheat and clover plants, respectively. The present results are consistent with the view that allopurinol aects pathogen growth and sporulation by inhibition c 2001 Academic Press * of xanthine dehydrogenase in the plant, the pathogen or both organisms. Keywords: allopurinol; purine catabolism; xanthine dehydrogenase; Nicotiana tabacum; Erysiphe cichoracearum; Triticum aestivum; Blumeria graminis; Trifolium pratense; Erysiphe trifolii; plant protection; histology; haustoria.
INTRODUCTION 4-hydroxypyrazolo(3,4-d)pyrimidine is a synthetic analogue of hypoxanthine commonly called allopurinol. It is a speci®c and potent inhibitor of xanthine oxidase and dehydrogenase, a property also characteristic of oxypurinol [4,6-dihydroxypyrazolo (3,4-d)pyrimidine], the principal metabolic conversion product obtained by allopurinol oxidation in vivo. When allopurinol is applied to various plant material (germinating seeds, roots and suspension-cultured plant cells) it exerts an in vivo inhibitory eect on purine catabolic metabolism with the result that xanthine, usually not detectable in leaf extracts, greatly accumulates since it is not transformed in uric acid and thereafter in ureides (allantoin and allantoic acid) [ for pertinent literature see: 2, 19]. Previous experiments have shown that allopurinol is an interfering agent against biotrophic growth of some rust fungi [10, 14, 15, 17]. Due to the absence of xanthine oxidoreductase activity in the thallus of these obligate parasites [14, 16, 17], it was initially supposed, and later also supported by * To whom all correspondence should be addressed. E-mail: [email protected]
0885-5765/01/100201+11 $35.00/00
experimental evidence [2], that the allopurinol inhibitory eect on rust fungi could result from the inhibition of host xanthine dehydrogenase which should therefore be considered a complementing factor to maintain operative purine catabolism during intercellular growth of the parasites. The concentration of allopurinol necessary to inhibit the growth of rust fungi varied in relation to the host plant considered, and ranged from 60 (wheat) to about 200±400 mM (bean and broad bean) in solutions applied by root absorption [21]. Since xanthine dehydrogenase from these plants was inhibited in vitro to almost the same extent by 400 mM allopurinol [14, 16, 17], the question arises as to whether the concentration of the inhibitor necessary to aect host xanthine dehydrogenase in vivo, and consequently the dependence of parasite growth, could be related to a dierent degree of accumulation of this compound in leaf tissue or to a dierent rate and kind of metabolic degradation product in the cell of dierent plant species. Experiments were then performed which demonstrated that both the extent of allopurinol accumulation in leaves and also its rate of metabolic conversion to riboside derivatives (inactive as xanthine dehydrogenase inhibitors) can explain why c 2001 Academic Press *
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dierent concentrations are necessary to reach the same extent of inhibition of rust fungi growth in wheat, bean and broad bean [2]. Also, in TNV-infected tobacco leaves, a strong reduction of the hypersensitive response and virus replication rate occurs following application of 400 mM. These eects correlate respectively, with in vivo xanthine dehydrogenase inhibition and interference on nucleic acid metabolism and therefore on viral replicative process by the huge amount of ribonucleoside derivative product of allopurinol and oxypurinol synthesized in treated host plants [20]. In the present work an investigation carried out on the eects of allopurinol on some powdery mildew diseases is reported. Three plant±fungal parasite interactions were considered, i.e. tobacco±Erysiphe cichoracearum DC. (E. orontii Cast.), wheat±Blumeria (Erysiphe) graminis (DC) Speer and clover±Erysiphe trifolii Grev. The results showed that allopurinol treatment strongly in¯uenced the E. cichoracearum infection on tobacco but was ineective on the other two fungal parasites in their respective host plants considered. A synthesis of some of these studies has been already presented in a meeting [22].
MATERIALS AND METHODS
true leaf stage. An aqueous suspension of freshly produced conidia of E. cichoracearum, containing 200 000 conidia per ml (unless otherwise speci®ed) was sprayed onto the upper surface of the seventh and eighth leaves, which were then allowed to dry quickly to prevent water damage to the conidial inoculum. The inoculated plants, along with non-inoculated (water sprayed) controls, either treated with allopurinol or untreated, were transferred to a climatic chamber (14 h photoperiod, 258C and 75±80 % R. H. during the illumination period, 18±208C and 90±95 % R. H. in the dark). Treated and untreated wheat and clover plants, at the three-leaf stage and with fully expanded trifoliate leaves, respectively, were inoculated by dusting them with dry conidia of B. graminis or E. trifolii, respectively and maintained in a greenhouse cell at 20±228C temperature under natural illumination. All the inoculated plants were inspected for powdery mildew symptoms every 2 days starting from 4 days after inoculation. The percentage of leaf area covered by the fungal growth, and referred to as the infection intensity, was visually estimated for every inoculated leaf. Each experiment was repeated twice. In some experiments with tobacco plants, allopurinol solutions (400 and 1600 mM) were applied as foliar spray to test the possibility that the compound may be absorbed by the cuticle and then transferred to the whole plant.
Plants and pathogens Greenhouse-grown tobacco (Nicotiana tabacum L.) cv. Havana 425, wheat (Triticum aestivum L.) cv. Mentana and clover (Trifolium pratense L.) plants, all susceptible to their speci®c powdery mildew agents, were used in the experiments. Plants were grown from seeds in pots (one tobacco plant per pot, 35±40 wheat or clover plants per pot), in a mixture of sterilized soil, compost and sand, under the following greenhouse conditions: 18±248C temperature, natural illumination supplemented with arti®cial light for a total photoperiod of 14 h. Italian isolates of E. cichoracearum, B. graminis and E. trifolii, obtained from tobacco, wheat and clover crops, respectively were routinely cultured on their speci®c (susceptible) host plants grown in the greenhouse as above, in a separate cell.
Allopurinol administration and powdery mildew inoculation Plants were supplied via roots (i.e. as a soil drench) with aqueous solutions of allopurinol at dierent concentrations in dierent experiments (see results), using 300 ml solution per pot at each application. Plants irrigated with equal volumes of water served as untreated controls in all experiments. Allopurinol-treated and untreated tobacco plants were inoculated with powdery mildew normally at the 9±10
Microscopy Tobacco plants treated with 100 mM allopurinol and water-irrigated controls were used in these investigations. Leaf samples from two allopurinol-treated and two control plants, both inoculated with E. cichoracearum, were collected at 12 and 24 h after inoculation, then every day up to the seventh. Leaf pieces, 1.0±1.5 1.5±2.0 cm, were cut from each sample and immediately subjected to a decolouration and staining procedure to obtain whole mount preparations for light microscopy. Pieces from every leaf were treated separately. In order to avoid washing o or displacement of the inoculum and infection structures not ®rmly anchored to the leaf surface, leaf pieces were processed by placing them, with the upper side up, on several layers of ®lter paper imbued with the decolourizing ¯uid. An aqueous saturated solution of chloral hydrate, which caused complete decolouration of leaf tissues within 7±10 days, was used. The decolourized pieces were washed for 2±3 h on ®lter paper drenched with distilled water, then transferred onto microscope slides where their upper surface was covered with a thin layer of agarose, following the method of Ryan and Clare [24]. The pieces were stained with one or two drops of lacto-glycerol (lactic acid, glycerol and water 1 : 1 : 1 v/v) containing 0.03 % of aniline blue, then the preparations were mounted unsectioned in the same staining solution.
Eect of allopurinol on some powdery mildew fungi Observations were carried out under a Zeiss Axiophot microscope equipped with bright ®eld and interference contrast optics. Two pieces per leaf from two leaves per plant ( for a total of eight pieces from treated plants and eight from control ones) were examined at each collection time. Quantitative determinations, i.e. estimate of % ensheathed haustoria and % degenerating epidermal cells (see results), were carried out by scanning at least 10 microscopy ®elds per leaf piece.
Data on the infection intensity of tobacco powdery mildew recorded when using dierent allopurinol concentrations were analysed by non-linear regression [4]. The whole data set was simultaneously ®tted into the logistic three-parameter model: Dij ÿ Cij 1 expfbij ln X ÿ ln ED50ij g
Analytical methods Xanthine dehydrogenase activity in E. cichoracearum conidia was determined after polyacrylamide gel electrophoresis according to the Mendel and MuÈller [12] procedure as previously described [17]. Xanthine and allopurinol were determined in tobacco leaf extracts by HPLC [19].
RESULTS
Statistical analysis
Y Cij
203
1
where Y is the infection intensity, X is the allopurinol concentration, D is the infection intensity in control (water treated) plants (higher asymptote), C is infection intensity in plants treated with the highest allopurinol concentration (lower asymptote), b is the slope of the curve around its in¯ection point, and ED50 is the concentration corresponding to a protection level halfway between the higher and lower asymptotes. Observation time (i) and leaf position (seventh or eighth) ( j) were introduced as categorical variables in the regression model, as shown by Streibig et al. [26]. Non-linear regression analyses were performed by using the NLIN procedure of SAS [25] and the adequacy of the model was assessed by graphical analysis of residuals and F test for lack of ®t. Possible dierences between response curves determined for each observation time and leaf position were investigated with consecutive F tests, as shown by Streibig et al. [26]. Signi®cance of quantitative data obtained from microscopy observations ( % ensheathed haustoria and % degenerating epidermal cells) was assayed by the variance analysis after arcsine transformation (but data in Fig. 4 and Table 2 are true percentages).
Powdery mildew conidial extract for xanthine dehydrogenase assay E. cichoracearum and B. graminis conidia were extracted in a mortar with a pestle and the aid of glass beads (75 mm) using potassium phosphate buer 0.1 M, containing 2 mM EDTA and 20 mM 2-mercaptoethanol. The extract was then fractionated by 80 % acetone and dialysed as previously reported for uredospore extract [17].
Eect of allopurinol on tobacco powdery mildew infection Visual evaluation. Preliminary experiments had shown that 200 mM solutions supplied via roots every day, starting either 2±5 days before or 1 day after inoculation with E. cichoracearum, prevented any visible development of the fungus (Fig. 1) without inducing phytotoxicity symptoms. To determine the eect of lower concentrations of the compound on the infection, an experiment was therefore carried out in which 10, 25, 50 and 100 mM allopurinol solutions were tested. Six plants were treated daily with each concentration, from 1 day after inoculation to the end of the experiment (18 days postinoculation). The results are shown in Table 1, where the average infection intensity recorded 12, 14, 16 and 18 days post-inoculation on each challenged leaf (seventh and eighth) is reported. Non-linear regression analysis showed that all the allopurinol concentrations used gave signi®cant protection from tobacco powdery mildew and the eect for each inoculated leaf at each observation time may be described by a sigmoidal dose-response curve. Besides a strong inhibition of the disease caused by 100 and 50 mM allopurinol solutions (about 2±5 % average infection intensity on treated plants at 18 days compared with 77 % on control plants), an important attack reduction was assured by 25 mM allopurinol (infection intensity not exceeding 16.5 %), while only a slight protective eect was recorded in plants treated with 10 mM solutions (infection intensity up to 51±65 %). In order to establish the minimum treatment duration required for an eective protection from tobacco powdery mildew, 50 mM allopurinol was, in another experiment, administered for periods of 7, 5, 3 and 2 days before inoculation to a group of six plants per period. Treatments ended the day before all the plant groups, including control (water-irrigated) plants, were inoculated with E. cichoracearum. The infection intensity averaged 72 % on the seventh leaf and 78 % on the eighth leaf in control plants 20 days post-inoculation, while only a few scattered colonies developed (infection intensity degree 1.8±2.1 %) on plants treated for 2 days (not shown). Finally, 2-days treatments with the same allopurinol concentration (50 mM) were in a third experiment carried out on three groups of plants (six plants per group), starting
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F I G . 1. Tobacco ``Havana 425'' leaves inoculated with the powdery mildew agent E. cichoracearum, 6 (a, b) and 10 (c, d) days after inoculation. (a, c) Control (water-irrigated) plants. (b, d) Plants treated via roots with 200 mM allopurinol (see text). Note the absence of visible fungal development on leaves from treated plants.
respectively (1) 1 day before inoculation; (2) the day of inoculation; and (3) 1 day after inoculation. No powdery mildew infection was normally observed on the inoculated leaves of all treated plants, irrespective of the treatment beginning, except on the seventh leaf of each plant group where rare colonies (4±6 % infection intensity) were recorded 18 days post-inoculation (67±75 % disease intensity observed in control plants) (not shown). Microscopy observations. Conidial germination and the initial stages of infection {formation of ®rst haustoria and development of infection hyphae [Fig. 2(a)]} appeared practically identical in allopurinol-treated and control plants, up to 48 h after inoculation (37±42 % germination of conidia in both plant groups). Then, starting from
the third day, the fungal growth rate decreased on treated plants, i.e. a sharp reduction in length and branching of the hyphae became obvious [Fig. 2(b±i)]. The thick mycelial plot developing on control plants began to produce conidia, born in chains of four to six elements each, from the fourth day [Fig. 2( f)], whereas a few scattered conidial chains began to dierentiate only 5±7 days post-inoculation in the poor fungal growths on allopurinol-treated plants [Fig. 2(g)]. Pathogen haustoria [Fig. 3(a±f)] were globose, elliptical or pear-shaped and often developed a number of ®nger-like protrusions [Fig. 3(b)]. Some haustoria appeared more or less severely collapsed and partially or entirely embedded in a thick sheath of translucent or pearly material with variable anity for aniline blue
Eect of allopurinol on some powdery mildew fungi
205
T A B L E 1. Infection intensity of powdery mildew on challenged leaves (seventh and eighth) of tobacco plants treated with allopurinol at dierent concentrations or water. Data recorded 12, 14, 16 and 18 days post-inoculation. Each value is the mean + S.E. of six replicates ( plants)* Infection intensity Treatment{
Leaf No.
12 days
14 days
16 days
18 days
All 10 mM
7th 8th
36.0 + 4.1 25.0 + 5.2
43.0 + 5.8 28.8 + 7.2
57.5 + 9.1 42.0 + 9.6
65.0 + 8.3 51.3 + 8.8
All 25 mM
7th 8th
12.0 + 3.4 10.5 + 3.6
13.3 + 3.8 10.8 + 3.5
14.7 + 5.7 11.0 + 3.6
16.5 + 5.4 11.0 + 3.4
All 50 mM
7th 8th
2.8 + 0.5 1.0 + 0.4
3.2 + 0.4 1.3 + 0.4
2.7 + 0.6 1.6 + 0.5
3.2 + 0.7 1.8 + 0.5
All 100 mM
7th 8th
3.6 + 1.2 4.0 + 1.9
4.0 + 1.2 4.8 + 2.5
4.0 + 1.1 4.5 + 2.1
4.6 + 1.1 5.0 + 2.4
W (control)
7th 8th
38.0 + 4.1 36.3 + 7.0
43.0 + 4.8 43.3 + 7.4
68.3 + 6.9 61.7 + 6.0
76.7 + 8.0 77.5 + 6.5
* Statistical signi®cance of data tested by non-linear regression analysis (see text). { All: allopurinol, W: water.
[Fig. 3(c±f)]. The proportion of ensheathed haustoria in control plants was about 1 % of the total haustoria 24 h after inoculation, 3±5 % from the second to the ®fth day and rose to nearly 9 % at the seventh day. This proportion in treated plants was also 1 % 1 day post-inoculation but increased rapidly thereafter to reach about 41 % at the last collection times (Fig. 4), dierences from control values being statistically signi®cant. Starting from 24 to 48 h after inoculation, single epidermal cells located under the fungal colonies showed degeneration signs, i.e. coagulation of the cell content and increase in its anity for the stain [Fig. 3(g) and (h)]. The average percentage of degenerated cells in colonized leaf areas was initially very low but tended to rise in all plants, particularly in treated ones where it attained about 2 % at the seventh day post-inoculation, compared to 0.7 % recorded in controls at the same observation time (Table 2).
Duration of allopurinol eect in tobacco Tobacco plants treated with 50 mM allopurinol for 2 days as above (i.e. starting 1 day before inoculation, the day of inoculation, or 1 day after inoculation), and challenged with powdery mildew on the seventh and eighth leaves, were used to estimate the duration of the allopurinoldependent protection against the pathogen. To this purpose, a second inoculation with a conidial suspension of E. cichoracearum was carried out on the ninth and 10th leaves of these plants 10, 20 or 30 days after the treatment end, using four treated and four control plants per each treatment timing and inoculation date. The results showed that a good protection was obtained in all cases against the second inoculation carried out in 10 and 20 days after the last allopurinol supply (0.6±1.4 % average
infection intensity compared to 55±58 % of control plants), while the protective eect sharply decreased when the second inoculation was at 30 days (about 34 % infection intensity in treated plants, 57±61 % in controls) (not shown).
Eect of allopurinol on wheat and clover powdery mildew infection Allopurinol solutions 60 and 100 mM were experimented on wheat, while 100, 200 and 400 mM concentrations were tested on clover plants. In both cases four pots were irrigated daily with each solution, from 5 days before inoculation to the experiment end (18 days after powdery mildew inoculation). No allopurinol-dependent protection from B. graminis was recorded with both dosages used, the average powdery mildew severity reaching 65±75 % in treated and control wheat plants. Similarly, clover powdery mildew infection was never reduced by allopurinol treatment at all the concentrations experimented. Although somewhat variable from one pot to another, the infection intensity on clover also attained considerable average levels (53±68 %) in all plant groups. Mild toxicity eects (chlorosis and growth reduction in both plant species, along with small necrotic spots in clover) were sometimes observed on treated plants, especially those irrigated with the highest allopurinol concentrations (100 mM in wheat, 400 mM in clover).
Allopurinol is not absorbed when applied on tobacco leaf surface Leaf extracts obtained from tobacco leaves which were sprayed onto the upper surface with 400 and 1600 mM
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F I G . 2. Micrographs illustrating the development of E. cichoracearum on leaves of tobacco ``Havana 425'' plants treated via roots with 100 mM allopurinol (see text) and of water-irrigated controls. (a) Conidial germination and initial growth of germ tubes, as usually observed in both control and treated plants 6±12 h post-inoculation (at 6 h on allopurinol-irrigated plant in this picture). (b±i) Examples of mycelial development and sporulation in treated plants (b, d, g) and control ones (c, e, f, h, i) at 3 (compare b and c), 4 (d, e, f) and 7 days (g, h, i). Note the growth reduction of mycelium in treated plants from the third day (the mycelial plots in e and h exemplify non-sporulating areas of pathogen colonies on control plants). Sporulation starts at 4 days in control plants ( f), conidial production attaining high densities in sporulating areas at 7 days (i), when rare conidial chains begin to develop on treated plants (g). Interference contrast microscopy of whole mount preparations from leaf samples decolourized with chloral hydrate and stained with aniline blue; c, germinating conidium (inoculum); ch, conidial chain (sporulation). Bars 50 mm.
Eect of allopurinol on some powdery mildew fungi
207
T A B L E 2. Percentage degenerated epidermal cells in powdery mildew invaded leaf areas of tobacco ``Havana 425'' treated with allopurinol 100 mM or water. Data recorded 2, 3, 5 and 7 days post-inoculation. Each value is the mean + SE of at least 80 replicates (microscopy ®elds)* Days after inoculation Treatment{
2
3
5
7
All 100 mM W (control)
0.65 + 0.10 0.13 + 0.06
0.82 + 0.13 0.24 + 0.08
1.20 + 0.16 0.35 + 0.06
1.93 + 0.31 0.68 + 0.15
* Statistical signi®cance of data tested at each observation date by the variance analysis after arcsine transformation. Dierences between allopurinol-treated and control plants were always statistically signi®cant (P 4 0.01). { All: allopurinol, W: water.
allopurinol solutions were analysed for the presence of xanthine at intervals after the treatment beginning. The HPLC analysis of puri®ed extracts from all treated leaves showed the presence of allopurinol but not xanthine. This ®nding indicates that the compound was not absorbed through the cuticle and not translocated in the plant cells where inhibition of xanthine dehydrogenase and consequent xanthine accumulation would take place, as experienced when allopurinol is applied via roots [19].
Eect of foliar spray application of allopurinol on conidial germination and infection process of tobacco powdery mildew In order to test possible direct eects of allopurinol on E. cichoracearum germination and infection capability, the compound was administered as a foliar spray either before or during the inoculation. Allopurinol solutions 400 and 1600 mM (concentrations much higher than those inhibiting fungal development when administered by roots) were either sprayed onto the leaves of tobacco ``Havana 425'' plants 3, 2 and 1 day before inoculation of the same leaves or used as liquid medium for the fungal inoculum (treatment simultaneous with the inoculation). Four plants were used for each treatment along with the same number of untreated controls. The inoculum density in this experiment was 1 500 000 conidia per ml. Powdery mildew colonies developing on inoculated leaves were counted under a stereomicroscope 8 and 12 days after inoculation. The results showed that the number of fungal colonies was not aected by the treatment, ranging in all plants from 2.2 to 3.0 colonies cm ÿ2 leaf surface.
Xanthine dehydrogenase activity in conidial extract from E. cichoracearum and B. graminis To establish whether the inhibitory eect exerted by allopurinol on E. cichoracearum growth in tobacco could be due to an eect on the host only or also on the fungal enzyme, the presence of xanthine dehydrogenase in fungal conidia was investigated. Using an electrophoretic method the presence of a xanthine dehydrogenase band
was demonstrated in conidial extracts of E. cichoracearum (Fig. 5) and B. graminis (not shown), indicating that this fungal obligate parasite contains an active enzyme.
DISCUSSION The present research shows that powdery mildew fungus growth is strongly reduced in tobacco plants by allopurinol supplied by root absorption. Allopurinol concentration needed to reach a considerable eect was relatively low (25±50 mM) compared with that used to inhibit rust fungi in bean and broad bean (up to 200 mM) [21]. Moreover, the duration of treatment required to reach the highest eect was very short (2 days) irrespective of whether it started before inoculation or 1 day after inoculation when fungal haustoria were already dierentiating and the consequent host±parasite biotrophic exchange was initiated. No eect was obtained if allopurinol was applied on the leaf surface, probably because no allopurinol absorption occurred through the cuticle layer. This was demonstrated by spraying allopurinol onto the leaves at concentrations up to 32 times higher than those used by root absorption to inhibit powdery mildew growth. The foliar treatments were in fact ineective on conidial germination and penetration process thus allowing for a full development of the infection. These strongly suggest that the marked growth reduction by root-supplied allopurinol is exerted at the haustorium level during the metabolic exchange between the host and the pathogen. Such a view was also supported by the light microscopy observations carried out on tobacco leaves which essentially con®rmed that allopurinol treatment did not aect the pre-penetration development of E. cichoracearum and showed that an appreciable fungal growth reduction only started after 48 h post-inoculation (after the ®rst haustoria were established). The allopurinol-dependent decrease in pathogen development and sporulation was associated with a considerable rise in the percentage of ensheathed haustoria compared with controls. A similar increase was
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F I G . 3. Haustoria of E. cichoracearum and degenerating epidermal cells in powdery mildew infected leaf areas of tobacco ``Havana 425'' plants treated via roots with 100 mM allopurinol (see text) and of water-irrigated controls. (a, b) A young, round-shaped haustorium (a, 1 day post-inoculation) and mature haustoria showing ®nger-like lobes (b, 4 days) in epidermal cells of control plants. (c±f) Examples of haustoria partially (c) or entirely covered (d±f) by a thick sheath, displaying a variable anity for aniline blue and sometimes appearing continuous with a thickening of the host cell wall (e), in allopurinol-treated plants (c and d, 2 days post-inoculation; e, 3 days; f, 5 days). (g, h) Epidermal cells showing degeneration signs, i.e. coagulation and increased anity for the stain of their content, in a control plant (g, 3 days after inoculation) and in an allopurinol-treated plant (h, 5 days). Interference contrast microscopy of whole mount preparations from leaf samples decolourized with chloral hydrate and stained with aniline blue; c, germinating conidium (inoculum); cg, coagulated cell content; dc, degenerated host cell; ha, haustorium; hb, haustorium body; hy, hyphae; f, haustorial ®nger-like lobes; nu, host cell nucleus; pa, cell wall papilla; sh, haustorial sheath; th, host cell wall thickening. Bars: a±f 10 mm; g, h 25 mm.
Eect of allopurinol on some powdery mildew fungi
209
50
Ensheathed hustoria (%)
40
30
20
10
0
1
2 3 4 Days after inoculation
5
7
F I G . 4. Percentage ensheathed haustoria of E. cichoracearum, recorded at intervals (days) after inoculation, in infected leaves of tobacco ``Havana 425'' plants treated via roots with 100 mM allopurinol (ÐsÐ) (see text) and of water-irrigated controls (ÐhÐ). Each value is the mean + SE of 80 replicates (microscopy ®elds). Dierences between allopurinol-treated plants and controls were statistically signi®cant (P 4 0.01) at every collection date, except 1 day post-inoculation (variance analysis after arcsine transformation of data).
previously observed, although to a somewhat lesser extent, in tobacco resistance and induced resistance to powdery mildew [23] as well as in wheat and bean plants protected from rusts by allopurinol [10]. Since haustorial sheath, mostly callosic in nature, is considered as a structural plant response to impaired viability and functionality of the haustoria [6, 9], the increased frequency of sheaths recorded in the present work should re¯ect pathogen metabolic disorders caused by the allopurinol supply. The proportion of degenerating epidermal cells in leaf areas invaded by the fungus, although generally low, became signi®cantly higher in treated than in untreated plants during the disease development. If this cell death was a consequence of the fungal parasitic action, it may be hypothesized that the natural sensitivity of susceptible host cells to the infection damage is considerably increased by the eects of treatment on plant metabolism. Owing to the speci®c allopurinol-mediated in vivo inhibition of xanthine dehydrogenase, ®rst enzyme of the oxidative purine pathway, it seems obvious that oxidative purine degradation is a basic process to keep the host±parasite metabolic exchange operative. Previous
F I G . 5. Polyacrylamide disc-gel electrophoresis of dialysed extracts (80 % acetone precipitates) of E. cichoracearum conidia (the same results were obtained with conidia of B. graminis). After electrophoresis gel 2 was incubated for 1 h in 0.1 mM pyrophosphate buer, pH 8.0, containing 2 mM substrate (hypoxanthine) and 0.1 mM phenazine methosulphate/1 mM p-nitrobluetetrazolium (electron acceptors) solution. A speci®c violet formazan band was visualized (arrow). Gel 1 was incubated as gel 2 without the substrate and represents the blank showing unspeci®c background staining and unstained areas.
studies demonstrated that germinating uredospore extract of Uromyces fabae [14], Uromyces phaseoli [16] and Puccinia recondita f. sp. tritici [17] did not exhibit an active xanthine dehydrogenase. This may be connected with the presence of a defective structural gene and/or the absence of a functional genes coding for the enzyme prosthetic group, particularly the molybdenum cofactor [7, 13, 27] or other cofactors the occurrence of which is shown by the wellknown desulfo defective forms of the enzyme [8, 11]. This constitutive de®ciency may be complemented by host factors during the biotrophic growth of the pathogen. In short, the parasite may keep its own oxidative purine catabolism operative using uric acid synthesized in the leaf cells or using the host synthesizing machinery to build up an active xanthine dehydrogenase holoenzyme [18]. Unlike rusts, powdery mildew fungi instead have their own active xanthine dehydrogenase (Fig. 5). Therefore, allopurinol-mediated powdery mildew growth inhibition in tobacco is presumably connected with xanthine
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dehydrogenase inhibition in both host and parasite cells. This two-sites inhibition may explain the higher ecacy of allopurinol against E. cichoracearum obtained with very low allopurinol concentrations and limited treatment durations, starting either before the infection process is initiated or when it is already established. In contrast with these results is the complete ineectiveness of allopurinol, even at high concentrations, against wheat and clover powdery mildew fungi (B. graminis and E. trifolii). The lack of eect against B. graminis was ®rst observed some years ago when powdery mildew colonies due to natural infections could be observed in allopurinol-treated wheat plants (Montalbini, unpublished work). Recently similar results have also been reported by other authors [1]. The insensitivity of B. graminis and E. trifolii to allopurinol is dicult to explain. Since powdery mildew fungi establish their parasitic relationship with the epidermal cells, it may be that in wheat and clover the allopurinol supplied by roots does not reach the epidermal layer or at least its concentration in this tissue is very low and not sucient to cause any appreciable reduction of powdery mildew infection. It is known that speci®c transporter proteins are involved in the transfer of dierent nitrogenous compounds across the cell membranes [5]. A signi®cant property of these proteins is their substrate speci®city, as is the case of purine transporters [3]. It may therefore be supposed that speci®c transporter proteins exist in plants which distribute purines in the epidermal tissue. Possibly, these proteins are able to transport allopurinol in tobacco but not in wheat and clover due to a lack of speci®city in the latter plant species for allopurinol which is a synthetic purine analogue. In view of the high eectiveness of allopurinol against E. cichoracearum, as indicated by the low dosages (much lower than those causing phytotoxic eects in tobacco) and the short periods of treatment (2 days) required for a high and relatively long-lasting (at least 20 days) protection from the parasite, a practical application of this purine catabolism inhibitor in tobacco powdery mildew control may be hypothesized.
The authors thank Dr A. Onofri, Dipartimento di Scienze Agro-ambientali e della Produzione vegetale, University of Perugia, for assistance in statistical analysis.
REFERENCES Â daÂm AL, Galal AA, Manninger K, Barna B. 2000. 1. A Inhibition of the development of leaf rust (Puccinia recondita) by treatment of wheat with allopurinol and production of a hypersensitive-like reaction in a compatible host. Plant Pathology 49: 317±323.
2. Della Torre G, Montalbini P. 1995. Allopurinol metabolic conversion products and xanthine accumulation in allopurinol-treated plants. Plant Science 111: 187±198. 3. Diallinas G, Valdez J, Sophianopoulou V, Rosa A, Scazzocchio C. 1998. Chimeric purine transporters of Aspergillus nidulans de®ne a domain critical for function and speci®city conserved in bacterial, plant and metazoan homologues. The EMBO Journal 17: 3827±3837. 4. Finney DJ. 1979. Bioassay and the practice of statistical inference. International Statistical Review 47: 1±12. 5. Frommer WB, Kwart M, Hirner B, Fisher WN, Hummel S, Ninnemann O. 1994. Transporters for nitrogenous compounds in plants. Plant Molecular Biology 26: 1651±1670. 6. Heath MC. 1995. Signal exchange between higher plants and rust fungi. Canadian Journal of Botany 73(Suppl. 1): S616±S623. 7. Kuper J, Palmer T, Mendel RR, Scwarz G. 2000. Mutations in the molybdenum cofactor biosynthetic protein Cnx1G from Arabidopsis thaliana de®ne functions for molybdopterin binding, molybdenum insertion, and molybdenum cofactor stabilization. Proceedings of the National Academy of Sciences, USA 97: 6475±6480. 8. Ikegami T, Nishino T. 1986. The presence of desulfo xanthine dehydrogenase in puri®ed and crude enzyme preparations from rat liver. Archives of Biochemistry and Biophysics 247: 254±260. 9. Little®eld LJ, Heath MC. 1979. Ultrastructure of Rust Fungi. New York, NY, U.S.A.: Academic Press. 10. Marte M, Montalbini P. 1999. Histological observations on Uromyces phaseoli and Puccinia recondita infection in allopurinol-treated susceptible plants. Journal of Phytopathology 147: 163±168. 11. Massey V, Komai H, Palmer G, Elion G. 1970. The existence of nonfunctional active sites in milk xanthine oxidase: reaction with functional active inhibitors. Vitamins and Hormones (New York) 28: 505±531. 12. Mendel RR, MuÈller AJ. 1976. A common genetic determinant of xanthine dehydrogenase and nitrate reductase in Nicotiana tabacum. Biochemie und Physiologie der P¯anzen 170S: 538±541. 13. Mendel RR, Scwarz G. 1999. Molybdoenzymes and molybdenum cofactor in plants. Critical Reviews in Plant Sciences 18: 33±69. 14. Montalbini P. 1991. Levels of ureides and enzymes of ureide synthesis in Vicia faba leaves infected by Uromyces fabae and eect of allopurinol on biotrophic fungal growth. Phytopathologia Mediterranea 30: 83±92. 15. Montalbini P. 1992. The inhibition of rust fungus growth in allopurinol-treated bean plants: indirect evidence for the involvement of xanthine oxidoreductase in the biotrophism of Uromyces phaseoli. Journal of Phytopathology 135: 207±216. 16. Montalbini P. 1992. Changes in xanthine oxidase activity in bean leaves induced by Uromyces phaseoli infection. Journal of Phytopathology 134: 63±74. 17. Montalbini P. 1992. Ureides and enzymes of ureide synthesis in wheat seeds and leaves and eect of allopurinol on Puccinia recondita f. sp. tritici infection. Plant Science 87: 225±231. 18. Montalbini P. 1995. Eect of rust infection on purine catabolism enzyme levels in wheat leaves. Physiological and Molecular Plant Pathology 46: 275±292. 19. Montalbini P, Della Torre G. 1995. Allopurinol metabolites and xanthine accumulation in allopurinol-treated tobacco. Journal of Plant Physiology 147: 321±327.
Eect of allopurinol on some powdery mildew fungi 20. Montalbini P, Della Torre G. 1996. Evidence of a two-fold mechanism responsible for the inhibition by allopurinol of the hypersensitive response induced in tobacco by tobacco necrosis virus. Physiological and Molecular Plant Pathology 48: 273±287. 21. Montalbini P, Della Torre G, Umesh Kumar NN. 1994. Allopurinol [4-hydroxypyrazolo (3,4-d) pyrimidine] a xanthine oxidoreductase inhibitor and interfering agent against biotrophic growth of rust fungi: modalities of application to host plants. Phytopathologia Mediterranea 33: 41±50. 22. Montalbini P, Marte M, Guiderdone SM. 2001. The oxidative purine catabolism is involved in compatible and incompatible host±pathogen relationships. Proceedings of 5th International Congress of the European Foundation for Plant Pathology on Biodiversity in Plant Pathology. Taormina, Italy, 18±22 September, 2000 (in press).
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23. Polverari A, Guiderdone SM, Marte M. 2000. Aspetti istologici della resistenza e della resistenza indotta contro l'oidio del tabacco (Erysiphe cichoracearum DC.). In: Atti Giornate Fitopatologiche, vol. 2. Perugia, Italy, 16±20 April 2000, 291±292. 24. Ryan CC, Clare BG. 1974. Coating of leaf surfaces with agarose to retain fungal inoculum in situ for staining. Stain Technology 49: 15±18. 25. SAS Institute Inc. 1988. SAS. Introductory Guide for Personal Computers, release 6.03. Cary, NC, U.S.A.: SAS Institute Inc. 26. Streibig JC, Rudemo M, Jensen JE. 1993. Dose-response curves and statistical methods. In: Streibig JC, Kudsk P, eds. Herbicide Bioassays, vol. 3. Boca Raton, FL, U.S.A.: CRC Press, 29±55. 27. Venton AM, Deistung J, Bray RC. 1988. The isolation of demolybdo xanthine oxidase from bovine milk. Biochemistry Journal 255: 949±956.
E¡ect of allopurinol treatment on biotrophic growth of some powdery mildew fungi in their speci¢c hosts M . M A R T E and P. M O N TA L B I N I * Dipartimento di Arboricoltura e Protezione delle Piante, UniversitaÁ di Perugia, Borgo XX Giugno 74, I-06121 Perugia, Italy (Accepted for publication September 2001) When supplied via roots to tobacco host plants, aqueous solutions of allopurinol, a speci®c inhibitor of xanthine oxidase and dehydrogenase, markedly reduced or completely prevented the visible development of tobacco powdery mildew fungus Erysiphe cichoracearum (E. orontii) at concentrations as low as 25±50 mM administered for periods as short as 2 days. The duration of this allopurinol-dependent protection from the pathogen was of at least 20 days after the last administration. If supplied as foliar sprays at concentrations up to 1600 mM, allopurinol was not absorbed by the leaf tissues and did not aect the fungal infection. Histological observations revealed that on tobacco plants treated with allopurinol via roots, a sharp reduction in E. cichoracearum mycelial development started only 48 h after inoculation (after formation of the ®rst haustoria) and was accompanied by a strong delay and reduction of sporulation. The proportion of haustoria partially or entirely embedded in thick translucent sheaths ( possibly callosic in nature) increased signi®cantly in allopurinol irrigated plants reaching about 41 % of the total haustoria (compared to 9 % in controls) 7 days post-inoculation. No eects of allopurinol supply was observed on Blumeria graminis and Erysiphe trifolii development on wheat and clover plants, respectively. The present results are consistent with the view that allopurinol aects pathogen growth and sporulation by inhibition c 2001 Academic Press * of xanthine dehydrogenase in the plant, the pathogen or both organisms. Keywords: allopurinol; purine catabolism; xanthine dehydrogenase; Nicotiana tabacum; Erysiphe cichoracearum; Triticum aestivum; Blumeria graminis; Trifolium pratense; Erysiphe trifolii; plant protection; histology; haustoria.
INTRODUCTION 4-hydroxypyrazolo(3,4-d)pyrimidine is a synthetic analogue of hypoxanthine commonly called allopurinol. It is a speci®c and potent inhibitor of xanthine oxidase and dehydrogenase, a property also characteristic of oxypurinol [4,6-dihydroxypyrazolo (3,4-d)pyrimidine], the principal metabolic conversion product obtained by allopurinol oxidation in vivo. When allopurinol is applied to various plant material (germinating seeds, roots and suspension-cultured plant cells) it exerts an in vivo inhibitory eect on purine catabolic metabolism with the result that xanthine, usually not detectable in leaf extracts, greatly accumulates since it is not transformed in uric acid and thereafter in ureides (allantoin and allantoic acid) [ for pertinent literature see: 2, 19]. Previous experiments have shown that allopurinol is an interfering agent against biotrophic growth of some rust fungi [10, 14, 15, 17]. Due to the absence of xanthine oxidoreductase activity in the thallus of these obligate parasites [14, 16, 17], it was initially supposed, and later also supported by * To whom all correspondence should be addressed. E-mail: [email protected]
0885-5765/01/100201+11 $35.00/00
experimental evidence [2], that the allopurinol inhibitory eect on rust fungi could result from the inhibition of host xanthine dehydrogenase which should therefore be considered a complementing factor to maintain operative purine catabolism during intercellular growth of the parasites. The concentration of allopurinol necessary to inhibit the growth of rust fungi varied in relation to the host plant considered, and ranged from 60 (wheat) to about 200±400 mM (bean and broad bean) in solutions applied by root absorption [21]. Since xanthine dehydrogenase from these plants was inhibited in vitro to almost the same extent by 400 mM allopurinol [14, 16, 17], the question arises as to whether the concentration of the inhibitor necessary to aect host xanthine dehydrogenase in vivo, and consequently the dependence of parasite growth, could be related to a dierent degree of accumulation of this compound in leaf tissue or to a dierent rate and kind of metabolic degradation product in the cell of dierent plant species. Experiments were then performed which demonstrated that both the extent of allopurinol accumulation in leaves and also its rate of metabolic conversion to riboside derivatives (inactive as xanthine dehydrogenase inhibitors) can explain why c 2001 Academic Press *
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dierent concentrations are necessary to reach the same extent of inhibition of rust fungi growth in wheat, bean and broad bean [2]. Also, in TNV-infected tobacco leaves, a strong reduction of the hypersensitive response and virus replication rate occurs following application of 400 mM. These eects correlate respectively, with in vivo xanthine dehydrogenase inhibition and interference on nucleic acid metabolism and therefore on viral replicative process by the huge amount of ribonucleoside derivative product of allopurinol and oxypurinol synthesized in treated host plants [20]. In the present work an investigation carried out on the eects of allopurinol on some powdery mildew diseases is reported. Three plant±fungal parasite interactions were considered, i.e. tobacco±Erysiphe cichoracearum DC. (E. orontii Cast.), wheat±Blumeria (Erysiphe) graminis (DC) Speer and clover±Erysiphe trifolii Grev. The results showed that allopurinol treatment strongly in¯uenced the E. cichoracearum infection on tobacco but was ineective on the other two fungal parasites in their respective host plants considered. A synthesis of some of these studies has been already presented in a meeting [22].
MATERIALS AND METHODS
true leaf stage. An aqueous suspension of freshly produced conidia of E. cichoracearum, containing 200 000 conidia per ml (unless otherwise speci®ed) was sprayed onto the upper surface of the seventh and eighth leaves, which were then allowed to dry quickly to prevent water damage to the conidial inoculum. The inoculated plants, along with non-inoculated (water sprayed) controls, either treated with allopurinol or untreated, were transferred to a climatic chamber (14 h photoperiod, 258C and 75±80 % R. H. during the illumination period, 18±208C and 90±95 % R. H. in the dark). Treated and untreated wheat and clover plants, at the three-leaf stage and with fully expanded trifoliate leaves, respectively, were inoculated by dusting them with dry conidia of B. graminis or E. trifolii, respectively and maintained in a greenhouse cell at 20±228C temperature under natural illumination. All the inoculated plants were inspected for powdery mildew symptoms every 2 days starting from 4 days after inoculation. The percentage of leaf area covered by the fungal growth, and referred to as the infection intensity, was visually estimated for every inoculated leaf. Each experiment was repeated twice. In some experiments with tobacco plants, allopurinol solutions (400 and 1600 mM) were applied as foliar spray to test the possibility that the compound may be absorbed by the cuticle and then transferred to the whole plant.
Plants and pathogens Greenhouse-grown tobacco (Nicotiana tabacum L.) cv. Havana 425, wheat (Triticum aestivum L.) cv. Mentana and clover (Trifolium pratense L.) plants, all susceptible to their speci®c powdery mildew agents, were used in the experiments. Plants were grown from seeds in pots (one tobacco plant per pot, 35±40 wheat or clover plants per pot), in a mixture of sterilized soil, compost and sand, under the following greenhouse conditions: 18±248C temperature, natural illumination supplemented with arti®cial light for a total photoperiod of 14 h. Italian isolates of E. cichoracearum, B. graminis and E. trifolii, obtained from tobacco, wheat and clover crops, respectively were routinely cultured on their speci®c (susceptible) host plants grown in the greenhouse as above, in a separate cell.
Allopurinol administration and powdery mildew inoculation Plants were supplied via roots (i.e. as a soil drench) with aqueous solutions of allopurinol at dierent concentrations in dierent experiments (see results), using 300 ml solution per pot at each application. Plants irrigated with equal volumes of water served as untreated controls in all experiments. Allopurinol-treated and untreated tobacco plants were inoculated with powdery mildew normally at the 9±10
Microscopy Tobacco plants treated with 100 mM allopurinol and water-irrigated controls were used in these investigations. Leaf samples from two allopurinol-treated and two control plants, both inoculated with E. cichoracearum, were collected at 12 and 24 h after inoculation, then every day up to the seventh. Leaf pieces, 1.0±1.5 1.5±2.0 cm, were cut from each sample and immediately subjected to a decolouration and staining procedure to obtain whole mount preparations for light microscopy. Pieces from every leaf were treated separately. In order to avoid washing o or displacement of the inoculum and infection structures not ®rmly anchored to the leaf surface, leaf pieces were processed by placing them, with the upper side up, on several layers of ®lter paper imbued with the decolourizing ¯uid. An aqueous saturated solution of chloral hydrate, which caused complete decolouration of leaf tissues within 7±10 days, was used. The decolourized pieces were washed for 2±3 h on ®lter paper drenched with distilled water, then transferred onto microscope slides where their upper surface was covered with a thin layer of agarose, following the method of Ryan and Clare [24]. The pieces were stained with one or two drops of lacto-glycerol (lactic acid, glycerol and water 1 : 1 : 1 v/v) containing 0.03 % of aniline blue, then the preparations were mounted unsectioned in the same staining solution.
Eect of allopurinol on some powdery mildew fungi Observations were carried out under a Zeiss Axiophot microscope equipped with bright ®eld and interference contrast optics. Two pieces per leaf from two leaves per plant ( for a total of eight pieces from treated plants and eight from control ones) were examined at each collection time. Quantitative determinations, i.e. estimate of % ensheathed haustoria and % degenerating epidermal cells (see results), were carried out by scanning at least 10 microscopy ®elds per leaf piece.
Data on the infection intensity of tobacco powdery mildew recorded when using dierent allopurinol concentrations were analysed by non-linear regression [4]. The whole data set was simultaneously ®tted into the logistic three-parameter model: Dij ÿ Cij 1 expfbij ln X ÿ ln ED50ij g
Analytical methods Xanthine dehydrogenase activity in E. cichoracearum conidia was determined after polyacrylamide gel electrophoresis according to the Mendel and MuÈller [12] procedure as previously described [17]. Xanthine and allopurinol were determined in tobacco leaf extracts by HPLC [19].
RESULTS
Statistical analysis
Y Cij
203
1
where Y is the infection intensity, X is the allopurinol concentration, D is the infection intensity in control (water treated) plants (higher asymptote), C is infection intensity in plants treated with the highest allopurinol concentration (lower asymptote), b is the slope of the curve around its in¯ection point, and ED50 is the concentration corresponding to a protection level halfway between the higher and lower asymptotes. Observation time (i) and leaf position (seventh or eighth) ( j) were introduced as categorical variables in the regression model, as shown by Streibig et al. [26]. Non-linear regression analyses were performed by using the NLIN procedure of SAS [25] and the adequacy of the model was assessed by graphical analysis of residuals and F test for lack of ®t. Possible dierences between response curves determined for each observation time and leaf position were investigated with consecutive F tests, as shown by Streibig et al. [26]. Signi®cance of quantitative data obtained from microscopy observations ( % ensheathed haustoria and % degenerating epidermal cells) was assayed by the variance analysis after arcsine transformation (but data in Fig. 4 and Table 2 are true percentages).
Powdery mildew conidial extract for xanthine dehydrogenase assay E. cichoracearum and B. graminis conidia were extracted in a mortar with a pestle and the aid of glass beads (75 mm) using potassium phosphate buer 0.1 M, containing 2 mM EDTA and 20 mM 2-mercaptoethanol. The extract was then fractionated by 80 % acetone and dialysed as previously reported for uredospore extract [17].
Eect of allopurinol on tobacco powdery mildew infection Visual evaluation. Preliminary experiments had shown that 200 mM solutions supplied via roots every day, starting either 2±5 days before or 1 day after inoculation with E. cichoracearum, prevented any visible development of the fungus (Fig. 1) without inducing phytotoxicity symptoms. To determine the eect of lower concentrations of the compound on the infection, an experiment was therefore carried out in which 10, 25, 50 and 100 mM allopurinol solutions were tested. Six plants were treated daily with each concentration, from 1 day after inoculation to the end of the experiment (18 days postinoculation). The results are shown in Table 1, where the average infection intensity recorded 12, 14, 16 and 18 days post-inoculation on each challenged leaf (seventh and eighth) is reported. Non-linear regression analysis showed that all the allopurinol concentrations used gave signi®cant protection from tobacco powdery mildew and the eect for each inoculated leaf at each observation time may be described by a sigmoidal dose-response curve. Besides a strong inhibition of the disease caused by 100 and 50 mM allopurinol solutions (about 2±5 % average infection intensity on treated plants at 18 days compared with 77 % on control plants), an important attack reduction was assured by 25 mM allopurinol (infection intensity not exceeding 16.5 %), while only a slight protective eect was recorded in plants treated with 10 mM solutions (infection intensity up to 51±65 %). In order to establish the minimum treatment duration required for an eective protection from tobacco powdery mildew, 50 mM allopurinol was, in another experiment, administered for periods of 7, 5, 3 and 2 days before inoculation to a group of six plants per period. Treatments ended the day before all the plant groups, including control (water-irrigated) plants, were inoculated with E. cichoracearum. The infection intensity averaged 72 % on the seventh leaf and 78 % on the eighth leaf in control plants 20 days post-inoculation, while only a few scattered colonies developed (infection intensity degree 1.8±2.1 %) on plants treated for 2 days (not shown). Finally, 2-days treatments with the same allopurinol concentration (50 mM) were in a third experiment carried out on three groups of plants (six plants per group), starting
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F I G . 1. Tobacco ``Havana 425'' leaves inoculated with the powdery mildew agent E. cichoracearum, 6 (a, b) and 10 (c, d) days after inoculation. (a, c) Control (water-irrigated) plants. (b, d) Plants treated via roots with 200 mM allopurinol (see text). Note the absence of visible fungal development on leaves from treated plants.
respectively (1) 1 day before inoculation; (2) the day of inoculation; and (3) 1 day after inoculation. No powdery mildew infection was normally observed on the inoculated leaves of all treated plants, irrespective of the treatment beginning, except on the seventh leaf of each plant group where rare colonies (4±6 % infection intensity) were recorded 18 days post-inoculation (67±75 % disease intensity observed in control plants) (not shown). Microscopy observations. Conidial germination and the initial stages of infection {formation of ®rst haustoria and development of infection hyphae [Fig. 2(a)]} appeared practically identical in allopurinol-treated and control plants, up to 48 h after inoculation (37±42 % germination of conidia in both plant groups). Then, starting from
the third day, the fungal growth rate decreased on treated plants, i.e. a sharp reduction in length and branching of the hyphae became obvious [Fig. 2(b±i)]. The thick mycelial plot developing on control plants began to produce conidia, born in chains of four to six elements each, from the fourth day [Fig. 2( f)], whereas a few scattered conidial chains began to dierentiate only 5±7 days post-inoculation in the poor fungal growths on allopurinol-treated plants [Fig. 2(g)]. Pathogen haustoria [Fig. 3(a±f)] were globose, elliptical or pear-shaped and often developed a number of ®nger-like protrusions [Fig. 3(b)]. Some haustoria appeared more or less severely collapsed and partially or entirely embedded in a thick sheath of translucent or pearly material with variable anity for aniline blue
Eect of allopurinol on some powdery mildew fungi
205
T A B L E 1. Infection intensity of powdery mildew on challenged leaves (seventh and eighth) of tobacco plants treated with allopurinol at dierent concentrations or water. Data recorded 12, 14, 16 and 18 days post-inoculation. Each value is the mean + S.E. of six replicates ( plants)* Infection intensity Treatment{
Leaf No.
12 days
14 days
16 days
18 days
All 10 mM
7th 8th
36.0 + 4.1 25.0 + 5.2
43.0 + 5.8 28.8 + 7.2
57.5 + 9.1 42.0 + 9.6
65.0 + 8.3 51.3 + 8.8
All 25 mM
7th 8th
12.0 + 3.4 10.5 + 3.6
13.3 + 3.8 10.8 + 3.5
14.7 + 5.7 11.0 + 3.6
16.5 + 5.4 11.0 + 3.4
All 50 mM
7th 8th
2.8 + 0.5 1.0 + 0.4
3.2 + 0.4 1.3 + 0.4
2.7 + 0.6 1.6 + 0.5
3.2 + 0.7 1.8 + 0.5
All 100 mM
7th 8th
3.6 + 1.2 4.0 + 1.9
4.0 + 1.2 4.8 + 2.5
4.0 + 1.1 4.5 + 2.1
4.6 + 1.1 5.0 + 2.4
W (control)
7th 8th
38.0 + 4.1 36.3 + 7.0
43.0 + 4.8 43.3 + 7.4
68.3 + 6.9 61.7 + 6.0
76.7 + 8.0 77.5 + 6.5
* Statistical signi®cance of data tested by non-linear regression analysis (see text). { All: allopurinol, W: water.
[Fig. 3(c±f)]. The proportion of ensheathed haustoria in control plants was about 1 % of the total haustoria 24 h after inoculation, 3±5 % from the second to the ®fth day and rose to nearly 9 % at the seventh day. This proportion in treated plants was also 1 % 1 day post-inoculation but increased rapidly thereafter to reach about 41 % at the last collection times (Fig. 4), dierences from control values being statistically signi®cant. Starting from 24 to 48 h after inoculation, single epidermal cells located under the fungal colonies showed degeneration signs, i.e. coagulation of the cell content and increase in its anity for the stain [Fig. 3(g) and (h)]. The average percentage of degenerated cells in colonized leaf areas was initially very low but tended to rise in all plants, particularly in treated ones where it attained about 2 % at the seventh day post-inoculation, compared to 0.7 % recorded in controls at the same observation time (Table 2).
Duration of allopurinol eect in tobacco Tobacco plants treated with 50 mM allopurinol for 2 days as above (i.e. starting 1 day before inoculation, the day of inoculation, or 1 day after inoculation), and challenged with powdery mildew on the seventh and eighth leaves, were used to estimate the duration of the allopurinoldependent protection against the pathogen. To this purpose, a second inoculation with a conidial suspension of E. cichoracearum was carried out on the ninth and 10th leaves of these plants 10, 20 or 30 days after the treatment end, using four treated and four control plants per each treatment timing and inoculation date. The results showed that a good protection was obtained in all cases against the second inoculation carried out in 10 and 20 days after the last allopurinol supply (0.6±1.4 % average
infection intensity compared to 55±58 % of control plants), while the protective eect sharply decreased when the second inoculation was at 30 days (about 34 % infection intensity in treated plants, 57±61 % in controls) (not shown).
Eect of allopurinol on wheat and clover powdery mildew infection Allopurinol solutions 60 and 100 mM were experimented on wheat, while 100, 200 and 400 mM concentrations were tested on clover plants. In both cases four pots were irrigated daily with each solution, from 5 days before inoculation to the experiment end (18 days after powdery mildew inoculation). No allopurinol-dependent protection from B. graminis was recorded with both dosages used, the average powdery mildew severity reaching 65±75 % in treated and control wheat plants. Similarly, clover powdery mildew infection was never reduced by allopurinol treatment at all the concentrations experimented. Although somewhat variable from one pot to another, the infection intensity on clover also attained considerable average levels (53±68 %) in all plant groups. Mild toxicity eects (chlorosis and growth reduction in both plant species, along with small necrotic spots in clover) were sometimes observed on treated plants, especially those irrigated with the highest allopurinol concentrations (100 mM in wheat, 400 mM in clover).
Allopurinol is not absorbed when applied on tobacco leaf surface Leaf extracts obtained from tobacco leaves which were sprayed onto the upper surface with 400 and 1600 mM
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F I G . 2. Micrographs illustrating the development of E. cichoracearum on leaves of tobacco ``Havana 425'' plants treated via roots with 100 mM allopurinol (see text) and of water-irrigated controls. (a) Conidial germination and initial growth of germ tubes, as usually observed in both control and treated plants 6±12 h post-inoculation (at 6 h on allopurinol-irrigated plant in this picture). (b±i) Examples of mycelial development and sporulation in treated plants (b, d, g) and control ones (c, e, f, h, i) at 3 (compare b and c), 4 (d, e, f) and 7 days (g, h, i). Note the growth reduction of mycelium in treated plants from the third day (the mycelial plots in e and h exemplify non-sporulating areas of pathogen colonies on control plants). Sporulation starts at 4 days in control plants ( f), conidial production attaining high densities in sporulating areas at 7 days (i), when rare conidial chains begin to develop on treated plants (g). Interference contrast microscopy of whole mount preparations from leaf samples decolourized with chloral hydrate and stained with aniline blue; c, germinating conidium (inoculum); ch, conidial chain (sporulation). Bars 50 mm.
Eect of allopurinol on some powdery mildew fungi
207
T A B L E 2. Percentage degenerated epidermal cells in powdery mildew invaded leaf areas of tobacco ``Havana 425'' treated with allopurinol 100 mM or water. Data recorded 2, 3, 5 and 7 days post-inoculation. Each value is the mean + SE of at least 80 replicates (microscopy ®elds)* Days after inoculation Treatment{
2
3
5
7
All 100 mM W (control)
0.65 + 0.10 0.13 + 0.06
0.82 + 0.13 0.24 + 0.08
1.20 + 0.16 0.35 + 0.06
1.93 + 0.31 0.68 + 0.15
* Statistical signi®cance of data tested at each observation date by the variance analysis after arcsine transformation. Dierences between allopurinol-treated and control plants were always statistically signi®cant (P 4 0.01). { All: allopurinol, W: water.
allopurinol solutions were analysed for the presence of xanthine at intervals after the treatment beginning. The HPLC analysis of puri®ed extracts from all treated leaves showed the presence of allopurinol but not xanthine. This ®nding indicates that the compound was not absorbed through the cuticle and not translocated in the plant cells where inhibition of xanthine dehydrogenase and consequent xanthine accumulation would take place, as experienced when allopurinol is applied via roots [19].
Eect of foliar spray application of allopurinol on conidial germination and infection process of tobacco powdery mildew In order to test possible direct eects of allopurinol on E. cichoracearum germination and infection capability, the compound was administered as a foliar spray either before or during the inoculation. Allopurinol solutions 400 and 1600 mM (concentrations much higher than those inhibiting fungal development when administered by roots) were either sprayed onto the leaves of tobacco ``Havana 425'' plants 3, 2 and 1 day before inoculation of the same leaves or used as liquid medium for the fungal inoculum (treatment simultaneous with the inoculation). Four plants were used for each treatment along with the same number of untreated controls. The inoculum density in this experiment was 1 500 000 conidia per ml. Powdery mildew colonies developing on inoculated leaves were counted under a stereomicroscope 8 and 12 days after inoculation. The results showed that the number of fungal colonies was not aected by the treatment, ranging in all plants from 2.2 to 3.0 colonies cm ÿ2 leaf surface.
Xanthine dehydrogenase activity in conidial extract from E. cichoracearum and B. graminis To establish whether the inhibitory eect exerted by allopurinol on E. cichoracearum growth in tobacco could be due to an eect on the host only or also on the fungal enzyme, the presence of xanthine dehydrogenase in fungal conidia was investigated. Using an electrophoretic method the presence of a xanthine dehydrogenase band
was demonstrated in conidial extracts of E. cichoracearum (Fig. 5) and B. graminis (not shown), indicating that this fungal obligate parasite contains an active enzyme.
DISCUSSION The present research shows that powdery mildew fungus growth is strongly reduced in tobacco plants by allopurinol supplied by root absorption. Allopurinol concentration needed to reach a considerable eect was relatively low (25±50 mM) compared with that used to inhibit rust fungi in bean and broad bean (up to 200 mM) [21]. Moreover, the duration of treatment required to reach the highest eect was very short (2 days) irrespective of whether it started before inoculation or 1 day after inoculation when fungal haustoria were already dierentiating and the consequent host±parasite biotrophic exchange was initiated. No eect was obtained if allopurinol was applied on the leaf surface, probably because no allopurinol absorption occurred through the cuticle layer. This was demonstrated by spraying allopurinol onto the leaves at concentrations up to 32 times higher than those used by root absorption to inhibit powdery mildew growth. The foliar treatments were in fact ineective on conidial germination and penetration process thus allowing for a full development of the infection. These strongly suggest that the marked growth reduction by root-supplied allopurinol is exerted at the haustorium level during the metabolic exchange between the host and the pathogen. Such a view was also supported by the light microscopy observations carried out on tobacco leaves which essentially con®rmed that allopurinol treatment did not aect the pre-penetration development of E. cichoracearum and showed that an appreciable fungal growth reduction only started after 48 h post-inoculation (after the ®rst haustoria were established). The allopurinol-dependent decrease in pathogen development and sporulation was associated with a considerable rise in the percentage of ensheathed haustoria compared with controls. A similar increase was
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F I G . 3. Haustoria of E. cichoracearum and degenerating epidermal cells in powdery mildew infected leaf areas of tobacco ``Havana 425'' plants treated via roots with 100 mM allopurinol (see text) and of water-irrigated controls. (a, b) A young, round-shaped haustorium (a, 1 day post-inoculation) and mature haustoria showing ®nger-like lobes (b, 4 days) in epidermal cells of control plants. (c±f) Examples of haustoria partially (c) or entirely covered (d±f) by a thick sheath, displaying a variable anity for aniline blue and sometimes appearing continuous with a thickening of the host cell wall (e), in allopurinol-treated plants (c and d, 2 days post-inoculation; e, 3 days; f, 5 days). (g, h) Epidermal cells showing degeneration signs, i.e. coagulation and increased anity for the stain of their content, in a control plant (g, 3 days after inoculation) and in an allopurinol-treated plant (h, 5 days). Interference contrast microscopy of whole mount preparations from leaf samples decolourized with chloral hydrate and stained with aniline blue; c, germinating conidium (inoculum); cg, coagulated cell content; dc, degenerated host cell; ha, haustorium; hb, haustorium body; hy, hyphae; f, haustorial ®nger-like lobes; nu, host cell nucleus; pa, cell wall papilla; sh, haustorial sheath; th, host cell wall thickening. Bars: a±f 10 mm; g, h 25 mm.
Eect of allopurinol on some powdery mildew fungi
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50
Ensheathed hustoria (%)
40
30
20
10
0
1
2 3 4 Days after inoculation
5
7
F I G . 4. Percentage ensheathed haustoria of E. cichoracearum, recorded at intervals (days) after inoculation, in infected leaves of tobacco ``Havana 425'' plants treated via roots with 100 mM allopurinol (ÐsÐ) (see text) and of water-irrigated controls (ÐhÐ). Each value is the mean + SE of 80 replicates (microscopy ®elds). Dierences between allopurinol-treated plants and controls were statistically signi®cant (P 4 0.01) at every collection date, except 1 day post-inoculation (variance analysis after arcsine transformation of data).
previously observed, although to a somewhat lesser extent, in tobacco resistance and induced resistance to powdery mildew [23] as well as in wheat and bean plants protected from rusts by allopurinol [10]. Since haustorial sheath, mostly callosic in nature, is considered as a structural plant response to impaired viability and functionality of the haustoria [6, 9], the increased frequency of sheaths recorded in the present work should re¯ect pathogen metabolic disorders caused by the allopurinol supply. The proportion of degenerating epidermal cells in leaf areas invaded by the fungus, although generally low, became signi®cantly higher in treated than in untreated plants during the disease development. If this cell death was a consequence of the fungal parasitic action, it may be hypothesized that the natural sensitivity of susceptible host cells to the infection damage is considerably increased by the eects of treatment on plant metabolism. Owing to the speci®c allopurinol-mediated in vivo inhibition of xanthine dehydrogenase, ®rst enzyme of the oxidative purine pathway, it seems obvious that oxidative purine degradation is a basic process to keep the host±parasite metabolic exchange operative. Previous
F I G . 5. Polyacrylamide disc-gel electrophoresis of dialysed extracts (80 % acetone precipitates) of E. cichoracearum conidia (the same results were obtained with conidia of B. graminis). After electrophoresis gel 2 was incubated for 1 h in 0.1 mM pyrophosphate buer, pH 8.0, containing 2 mM substrate (hypoxanthine) and 0.1 mM phenazine methosulphate/1 mM p-nitrobluetetrazolium (electron acceptors) solution. A speci®c violet formazan band was visualized (arrow). Gel 1 was incubated as gel 2 without the substrate and represents the blank showing unspeci®c background staining and unstained areas.
studies demonstrated that germinating uredospore extract of Uromyces fabae [14], Uromyces phaseoli [16] and Puccinia recondita f. sp. tritici [17] did not exhibit an active xanthine dehydrogenase. This may be connected with the presence of a defective structural gene and/or the absence of a functional genes coding for the enzyme prosthetic group, particularly the molybdenum cofactor [7, 13, 27] or other cofactors the occurrence of which is shown by the wellknown desulfo defective forms of the enzyme [8, 11]. This constitutive de®ciency may be complemented by host factors during the biotrophic growth of the pathogen. In short, the parasite may keep its own oxidative purine catabolism operative using uric acid synthesized in the leaf cells or using the host synthesizing machinery to build up an active xanthine dehydrogenase holoenzyme [18]. Unlike rusts, powdery mildew fungi instead have their own active xanthine dehydrogenase (Fig. 5). Therefore, allopurinol-mediated powdery mildew growth inhibition in tobacco is presumably connected with xanthine
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dehydrogenase inhibition in both host and parasite cells. This two-sites inhibition may explain the higher ecacy of allopurinol against E. cichoracearum obtained with very low allopurinol concentrations and limited treatment durations, starting either before the infection process is initiated or when it is already established. In contrast with these results is the complete ineectiveness of allopurinol, even at high concentrations, against wheat and clover powdery mildew fungi (B. graminis and E. trifolii). The lack of eect against B. graminis was ®rst observed some years ago when powdery mildew colonies due to natural infections could be observed in allopurinol-treated wheat plants (Montalbini, unpublished work). Recently similar results have also been reported by other authors [1]. The insensitivity of B. graminis and E. trifolii to allopurinol is dicult to explain. Since powdery mildew fungi establish their parasitic relationship with the epidermal cells, it may be that in wheat and clover the allopurinol supplied by roots does not reach the epidermal layer or at least its concentration in this tissue is very low and not sucient to cause any appreciable reduction of powdery mildew infection. It is known that speci®c transporter proteins are involved in the transfer of dierent nitrogenous compounds across the cell membranes [5]. A signi®cant property of these proteins is their substrate speci®city, as is the case of purine transporters [3]. It may therefore be supposed that speci®c transporter proteins exist in plants which distribute purines in the epidermal tissue. Possibly, these proteins are able to transport allopurinol in tobacco but not in wheat and clover due to a lack of speci®city in the latter plant species for allopurinol which is a synthetic purine analogue. In view of the high eectiveness of allopurinol against E. cichoracearum, as indicated by the low dosages (much lower than those causing phytotoxic eects in tobacco) and the short periods of treatment (2 days) required for a high and relatively long-lasting (at least 20 days) protection from the parasite, a practical application of this purine catabolism inhibitor in tobacco powdery mildew control may be hypothesized.
The authors thank Dr A. Onofri, Dipartimento di Scienze Agro-ambientali e della Produzione vegetale, University of Perugia, for assistance in statistical analysis.
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