EFFECTS OF METABOLITES AND ANTlMETABOLITES ON THE SPORULATION OF PERONOSPORA TABAOINA ADAM. ON TOBACCO LEAF DISKS By C. J. SHEI'HERD* and M. MANDRYK*
[Manuscript received ~arch 13, 1964]
Summary . The effects of 148 metabolites and a~tinietabolites on the sporulation or" Peronospora tabacina Adam. on leaf disks of Nieotiand tubacum cv. Virginia Gold. have been determined. (1) Normal metabolites, with the exception of flavin adenine dinucleotide, had slight althoU:gh statistically significant effects on sporulation intensIty, which suggests that inhibition-nutrition phenomena play no part in the sporulation process of P. tabacina. (2) Seven uracil analogues had an inhibitory effect on sporulation, and the reversal of inhibition by uracil suggests the active involvement of this compound in the sporulation process. (3) Canavanine at a final concentrat.ion of 120 ftgjml showed complete inhibition of sporulation. The reversal of the canavanine inhibition of sporulation by arginine, citrulline, and ornithine suggests the involvement of arginine and the functioning of an ornithine cycle in the sporulating system. (4) White, instead of the normal blue, conidia were produced in the presence of a number of sUlphur-containing compounds. It is suggested that this phenomenon depends on the chelating properties of these compounds towards copper ions, with the subsequent inactivation of tyrosinase activity in the conidia. (5) Sporulation intensities of 68 X 10'-}53 X 104 conidia per sqnare centimetre of leaf area were observed during the present study.
I. INTRODUCTION Clayton and Gaines (1933), Armstrong and Sumner (1935), and Dixon, McLean, and Wolf (1936) have shown that sporulation by Peronospora tabacina Adam. occurred only under conditions of high humidity. Cruickshank (1958), using controlled conditions, defined the relationship between moisture and the production of conidia on tobacco leaf disks. and subsequently (Cruickshank 1963) reported on the effect of light on conidial formation. The present study was concerned with the effects of exogenously applied metabolites and antimetabolites on sporulation of Peronospora tabacina Adam. on tobacco leaf disks. The techniques used made possible the study of effects on sporulation per se, whereas in previous studies (summarized in Hawker 1957) the observed effects were on fungal growth plus subsequent sporulation. The objective of this investigation was to obtain information on intermediary metabolism of the fungus-plant system during the sporulation phase, in the hope that a lead might be found towards a rational basis for a chemotherapeutic approach to disease control.
• Division of Plant Industry, CSIRO, Canberra.
Aus•. J. BioI. Sci., 1964, 17, 878-91 METABOLITES AND SPORULATION OF PERONOSPORA TABACINA 879
II. MATERIALS AND METHODS (a) Production of Infected Leaf Disks Nicotiana tabacum cv. Virginia Gold plants were grown in 6-in. fiower pots in soil mix C supplemented with fertilizer lIe as described by Matkin and Chandler (1957). When the plants were approximately 80 em high, the foliage was inoculated with a conidial suspension of P. tabacina in water (Shepherd 1962) and placed under conditions favourable for leaf infection (Cruickshank 1958). After 5 days, the sixth leaf from the base of each plant was removed, and disks (10 mm .diam.) were cut from this and floated ventral side uppermost on water or on the solution under test.
TABLE 1
EFFECTS OF VARIATIONS IN METHOD OF SHAKING DISKS ON ESTIlIlATIONS OF SPORULATION INTENSITY
10-4 X Mean No. of Time of 10-4 X Conidia per Treatment of Disks Shaking Standard Square Centimetre (min) Error of Leaf Surface
Shaken together in 50% ethanol 0·5 43 ±l!'6 69 ± 7·8 2 67 ± 8·0 Each shaken successively in 50 %ethanol 87 ±I5·6 Shaken together in 1/500 Teepol solution 66 ±23·6
(b) Sporulation Oonditions and Estimation of Oonidial Numbers Three disks were floated on 1 ml solution contained in a watchglass and three such watchglasses were contained in a petri dish lined with filter paper saturated with water. After incubation for 17 hr at 20°0 in darkness, the three disks contained in each watchghtss ,tere immersed in 1 ml of 50% ethanol contained in a small screw~capped vial. After shaking, the number of c~:midia present was estimated by haemocytom.eter counts. From each experimental treatment, eight counts were made .from each of .three replicates, each replicate consisting of three disks. The sp,orulation intensity was calculated as the number of conidia per square centimetre of ventral leaf surface. The estimation of ~porulation intensity was shown to be sensitive to slight modifications of the counting technique (Table 1). In view 'of the standard errors of the variO"us methods and the total time involved for the estimation of each sample, the technique adopted for the remainder of this study was to shake three disks together in 1 ml of 50% ethanol for 1 min. The source of the disks was also found to affect the variability of the final estimate of sporulation intensity. Disks were taken from the distal, central, and proximal thirds of a single leaf, from the central third of different leaves of the one plant, and from the central positions of similar leaves of different plants (all plants were },f ti1.e same age, hadbeell grown together, and hadreceivedidentical treatments at' all stages). The results of this study are shown in Table 2. 880 C. J. SHEPHERD AND M. MANDRYK
The disk-sampling error estimated from disks derived from the central part ouly of a single leaf was 5· 4%. During the remainder of this study, disks taken from one .leaf only of one plant were used for the comparisons of experimen~al trea~ments. It may be seen below that considerable variation of spornlation intensity of untreated (control) disks occurs between separate experiments. A mean spornlation intensity of 91 X 104 conidia per square centimetre of leaf area was observed, but individual assessments fell within the range 68 X 10'-153 X 10'.
(0) Ohemicals All sugars, vitamins, and amino acids were obtained from Messrs. L. Light and Co. Ltd., Colnbrook, England, and all purine and pyrimidine compounds from Nutritional Biochemicals Corporation, Cleveland, U.S.A. Other chemicals were of Analar quality wherever possible. The solutions of all compounds were adjusted to a pH within the range 6-7 before use.
TABLE 2 EFFECTS OF ORIGIN OF LEAF DISKS ON ESTIMATIONS OF SPORULATION INTENSITY
lO-~ X Mean No. of Coefficient Conidia per Disks taken from: of Variation Square Centimetre of Leaf Surface (%)
Different parts of same leaf 52·8 5·57
Different leaves of one plant 44·4 15·00
Similar leaves of different plants 48·4 23-82
III. EXPERIMENTAL AND RESULTS (a) Effects of Metabolites and Antimetabolites on Sporulation The technique described above was used to test the effects of 148 compounds on sporulation. In some cases, the standard method was modified to eight counts from each of two replicates of three disks in order to test all available chemicals of a particular class in one experiment. With the purines, pyrimidines, and their analogues, a series of experiments had to be conducted, owing to the impossibility of obtaining sufficient disks from a single leaf. Table 3 shows the effects of additions of purines, pyrimidines, and their analogues. All compounds were present at a final concentration of 100 fLgfml. Statistically significant stimulations of sporulation intensity were shown by guanosine and cytidylic acid. Significant decreases in sporulation intensity were caused by thymine, xanthine, 8~azaxanthine, 5-chIoroxanthine, 8-aza-adenine, theophylline, caffeine, diazouraciI, dithiouraciI, sulphaminouraoil, uracil-4~acetic acid, propylthiouracil, uridylic acid, and oxaluric acid. The presence of dithiothymine, dithiouracil, and propylthiouracil caused white conidia to be produced [see Section III(c)J. TABLE 3 EFFEOT OF PURINES, PYRIMIDINES, AND THEIR ANALOGUES ON SPORULATION All compounds used at a final concentration of 100 "g/ml
10-4 x Mean No. of 10-4 X Mean No. of Conidia per Sporulation Conidia per Sporulation Compound Square Centimetre (% of untreated CompoUnd Square Centimetre (% of untreated of Leaf Surface control) of Leaf Surface control) Nil (oontrol) . 77-0 100-0 Nil (control) 152-9 100-0 Guanosine 97-8 127-0 Thymine 144-0 94-7 Guanylic aoid 73-2 95-0 Theobromine 160-3 104-7 Isopropylidineguanosine 90-4 117-4 Caffeine 76-4 49-9 Isoguanine sulphate 80-6 104-6 8-Aza-2,6·diaroinopurine 158-0 103-3 Cytidylic acid 90-4 123-6 Uracil 138-8 90-9 Thymine 66-9 86-9 Uracil-5-carboxylic acid 154-1 100-7 Dihyclrothymine 86-3 112-0 5-Aminouracil 144-0 94-2 Dithiothymine 69-1 89-8 Diazauracil 61-1 39-9 6-Azathymine 72-0 93-5 Thiouracil 130-0 85-0 5~Methylcytosine 67-2 87-2 Sulpharoinouracil 117-2 76-6 Deoxycytidine 64-4 83-6 Uric acid 145-3 94-9 Guanine 89-8 116-6 Uracil-4-aeetic acid 85-4 55-8 Uridine 152-9 100-0 L_S_D_ (5%) 12-27 17-4 Uramil 138-9 90-9 Dithiouraeil 67-5 44-2 Nil (control) 120-8 100-0 5-Nitrouracil 138-9 90-9 Adenine 95-6 79-2 Propylthiouracil 108-4 70-7 2-Thiocytosine 106-7 86-6 6-Azauracil 140-0 91-6 Isocytosine 112-1 92-7 6-Methyluracil 132-5 86-7 Cytosine 91-8 75-0 Uridylic acid 114-7 75-0 Xanthosine 100-0 82-8 Oxaluric acid 82-5 54-2 8~Azahypoxanthine 92-1 76-2 Inosine 156-7 102-4 Hypoxanthine 93-4 77-3 Adenosine phosphate 165-7 108-3 Xanthine 86-3 71-4 Adenosine triphosphate 139-5 91-2 8-Azaxanthine 87-3 72-4 Cytidine 176-7 115-6 S-Chloroxanthine 89-5 74-1 Adeny:lic acid 90-2 74-6 8-Aza-adenllie 87-9 72-8 Deoxyadenosine 95-6 79-2 00 00 5-Methylorotic acid 93-1 77-1 .... Theophylline 72-9 60-3
L_S_D_ (5%) 30-66 25-4 L_S_D_ (5%) 28-8 18-85 882 O. J. SHEPHERD AND M. MANDRYK
The inhibitory actions of diazouracil, uracil-4-acetic acid, and dithiouracil at concentrations of 100 f.Lg/m1 were completely reversed by the simultaneous addition of uracil to a final concentration of 400 fLgiml.
TABLE 4
EFFECT OF SUGARS AND RELATED COMPOUNDS ON SPORULATION All compounds used at a final concentration of 100 p.g/ml
10-4 x Mean No. of Sporulation Conidia per Compound I of untreated Square Centimetre (% control) of Leaf SUl'face I
Nil (control) 69·1 100·0 Glucose 56·6 81·9 Cellobiose 66·1 95·7 Xylose 72·1 104·2 Fructose 56·6 81·9 Galactose 76·8 110·5 Sucrose 60·1 87·0 Mannose 77·1 111·6 Lactose 62·3 90·2 Rhamnose 54·3 78·6 Melibiose 63·6 92·0 Maltose 60·8 87·9 Melezitose 59·7 86·4 Turanose 81·4 117·7 Fucose 57·6 83·4 Lyxose 63·4 91·7 Arabinose 79·3 114·9 Sorbose 53·3 77·1 Raffinose 59·2 85·7 Glucoheptose 55·8 80·8 Trehalose 84·2 121·9 Gluconic acid 59·7 86·4 Glucosamine 59·7 86·4 n-Acetylglucosamine 79·6 115·2. Sorbitol 54·8 79·4 Adonitol 63·4 91·8 Dulcitol 77·5 112·1 Mannitol 74·9 108·3 Erythritol 77·5 112·1 Gentiobiose 69·1 100·0 Inositol 80·9 117·0
L.S.D. (5%) 12·53 18·14
None of the sugars tested (final concentration lUO fLgiml) produced any marked effect on sporulation (Table 4), although several gave statistically siguificant effects. Of the amino acids tested (final concentration 100 "girril) glutamiue and ~-alanine ,gave ~ ~light· stimula~ion of sporul~tion., whereas a corisiqerable degree
of of
canavanine canavanine
inhibition inhibition is is reported reported in in detail detail in in Section Section III(b). III(b).
pletely pletely
reversed reversed by by
the the
simultaneous simultaneous
addition addition of of methionine methionine (400 (400 ",g/rol). ",g/rol). Reversal Reversal
The The inhibitory inhibitory
action action
of of
ethionine ethionine
(final (final concentration concentration 100 100 ",g/ml) ",g/ml) was was com
L.S.D. L.S.D.
(5%) (5%)
18·2 18·2 20·9 20·9
Canavanine Canavanine
1·2 1·2 1·4 1·4
,a- Furylalanine Furylalanine
51·7 51·7 59·1 59·1
Ethionine Ethionine
8·6 8·6 10·9 10·9
Norleucine Norleucine
100·0 100·0 114·5 114·5
Norvaline Norvaline
88·8 88·8 101·8 101·8
Methionine Methionine sulphoxide sulphoxide
71·0 71·0 81·4 81·4
Cysteic Cysteic acid acid
73·6 73·6 84·6 84·6
Homocysteine Homocysteine
94·8 94·8 108·6 108·6
Homoserine Homoserine
100·0 100·0 114·5 114·5
Valine Valine
76'4 76'4 87·5 87·5
Glycine Glycine
75·0 75·0 86·0 86·0
Citrulline Citrulline
70·1 70·1 80·4 80·4
a.-Amino a.-Amino butyric butyric acid acid
72'9 72'9 83·6 83·6
Tryptophan Tryptophan
79·6 79·6 91·3 91·3
Tyrosine Tyrosine
91'0 91'0 104·2 104·2
Threonine Threonine
75'5 75'5 86·6 86·6
Serine Serine
104'4 104'4 119·6 119·6
Phenylalanine Phenylalanine
102'2 102'2 117·2 117·2
Proline Proline
85'8 85'8 98·4 98·4
Ornithine Ornithine
99·0 99·0 113'5 113'5
Methionine Methionine
86'9 86'9 100·3 100·3
,a-Alanine ,a-Alanine
115·0 115·0 131·9 131·9
Lysine Lysine
102'6 102'6 117 117
·6 ·6
Cysteine Cysteine
88·4 88·4 101·6 101·6
Leucine Leucine
82·3 82·3 94·4 94·4
Isoleucine Isoleucine
76·6 76·6 87·8 87·8
Asparagine Asparagine
73·8 73·8 84·7 84·7
Aspartic Aspartic acid acid
71'9 71'9 87'4 87'4
y-Aroinobutyric y-Aroinobutyric acid acid
104·6 104·6 112·0 112·0
Glutamine Glutamine
110·5 110·5 126·6 126·6
Glutamic Glutamic acid acid
103·5 103·5 118+ 118+
Alanine Alanine
69·8 69·8 80·0 80·0
Hydroxyproline Hydroxyproline
82·8 82·8 94·9 94·9
Histidine Histidine
92·8 92·8 106·4 106·4
Arginine Arginine
103·4 103·4 118·5 118·5
Nil Nil '(control) '(control)
87·2 87·2 100·0 100·0
of of Leaf Leaf Surface Surface
control) control)
Square Square
Centimetre Centimetre
Compound Compound
(% (% of of untreated untreated
Conidia Conidia per per
Sporulation Sporulation
10- x x Mean Mean No. No. of of 4 4
All All
compounds compounds
used used at at
a a final final concentration concentration of of 100 100 p..g/ml p..g/ml
EFFECT EFFECT
OF OF AMINO AMINO
ACIDS ACIDS AND AND ANALOGUES ANALOGUES ON ON SPORULATION SPORULATION
TABLE TABLE 5 5
(Table (Table 5). 5).
of of inhibition inhibition
was was seen seen in in
the the presence presence of of
ethionine, ethionine, tB-furylalanine, tB-furylalanine, and and canavanine canavanine
METABOLITES METABOLITES
AND AND SPORULATION SPORULATION OF OF PERONOSPORA PERONOSPORA TABACINA TABACINA 883 883 884 C. J, SHEPHERD AND M. :MA.N'DRYK
Table 6 shows the effects of additions of various vitamins at a final concen tration of 20 fLg/m!. Statistically significant stimulations of sporulation were shown by riboflavin, nicotinic acid, and p-aminobenzoic acid and inhibitions by choline,
TABLE 6
EFFEOT OF VITAMINS ON SPORULATION All compounds used at a final concentration of 20 jLgjrnl
10-" x Mean No. of Sporulation Compound Conidia per Square Centimetre (% of untreated of Leaf Surface control)
Nil (control) 84·7 100·0 Nicotinic acid 109·0 127·4 p·A.minobenzoic acid 118·4 139·6 Thiamine 84·0 99·2 Choline 72·6 85·7 Pyridoxin 75·9 89·6 Pantothenic acid 72·2 85·3 Biotin 91·4 108·0 Riboflavin 98·5 116·4
L.S.D. (5%) 8·4 9·9
pyridoxin, and pantothenic acid. The effects of ribofiavin-5-phosphate (R-5-P) and fiavin adenine dinucleotide (FAD) are shown separately in Table 7.
TABLE 7 EFFECTS OF RlBOFLAV!N·5·FROSPRATE (R.5.P) AND FLAVIN ADENINE DINUCLEOTIDE (FAD) ON SPORULATION UNDER LIGHT AND DARK CONDITIONS All compounds used at a final concentration of 50 p.g/ml
10-4 x Mean No. of Incubation Sporulation Compound Conidia per Conditions Square Centimetre (% of dark of Leaf Surface control)
Light Nil (control) 0 0 R·5-P 2·04 2·4 FAD 2·50 3·0
Dark Nil (control) 82·1 100·0 R-5·P 121'8 148'4 FAD 142'8 173·9
L.S.D. (5%) 21·0 25·6
Replicate leaf disk samples were placed in complete darkness and under continuous fluorescent illumination (800 f.c.), which is sufficient to inhibit sporulation completely (Crnickshank 1963). In darkness, both compounds produce a marked METABOLITES AND SPORULATION OF PERONOSPORA TABACINA 885 increase in sporulation intensity. Under continuous light and in the absence of R-5-P and FAD neither conidia nor conidiophores are produced. In the presence of these compounds under, continuous light, conidiophores are produced in large numbers (approximately 50% of the number found on untreated, dark-incubated disks) and a small number of conidia are found.
TABLE 8 EFFECT OF INORGANIC PHOSPHATES ON SPORULATION All compounds used at a final concentration of 12·5 ftg phosphorus/ml
10-4 x Mean No. of Sporulation Conidia per Compound (% of untreated Square Centimetre control) of Leaf Surface - Nil (control) 68·2 100·0 Sodium orthophosphate 75·2 1l0'3 Sodium pyrophosphate 74·5 109·3 Sodium hexametaphosphate 60·3 88·4
L.S.D. (5%) 5·7 16·8
Tables 8, 9, and 10 show .respectively that various inorganic phosphates, inorganic nitrogen compounds, and sodium salts of fatty acids have no significant effects on sporulation. Sodium oxalate, however, showed a significant inhibition of sporulation.
TABLE 9 EFFECT OF INORGANIC NITROGEN COMPOUNDS ON SPORULATION All compounds used at a final concentration of 25 p.g nitrogen/mJ
lO-llxMeanNo.of Sporulation Conidia per Compound (% of untreated Square. Centimetre control) of Leaf Surface
Nil (control) 71·6 100·0 Sodium nitrate 71·1 99·3 Sodium nitrite 73·7 103·0 Ammonium sulphate 69·4 98·3
L.S.D. (5%) 9·6 13·4
The addition of iron, copper, manganese, zinc, cobalt, molybdenum, and boron ions, each at 10 flog/ml, and of magnesium and calcium ions (50 flog/ml) produced no detectable e/fects on sporulation intensity. A mixture of all ions together showed a slight inhibition of sporulation. Additional tests with the sugars, natural amino acids, and natural purines and pyrimidines at final concentrations of 50 and 200 flog/ml gave results in accord with those reported above. 886 C. J. SHEPHERD AND M. MANDRYK
(b ) Inhibition of Sporulation by Oanavanine In view of the strongly inhibitory effect of canavanine on sporulation (Table 5), and its reversal by arginine in fungi (Horowitz and Srb 1948), it was decided to inve"stigate the action of this compound in detail.
TABLE 10
EFFECT OF SODIUII-[ SALTS OF FATTY ACIDS ON SPORULATION All compounds used at a final concentration of 100 p,g/mJ.
10-4 x Mean No. of Sporulation Conidia per Compound (% of untreated Square Centimetre control) of Leaf Surface
Nil (control) 68·2 100·0 Sodium fumarate 73·2 107·3 Sodium lactate 63·7 93·4 Sodium succinate 63·8 93·5 Sodium acetate 62·4 91·5 Sodium citrate 64·7 94·8 Sodium oxalate 53·7 78·7 Sodium propionate 62·0 90·9 Sodium tartrate 58·7 86·1 Sodium malate 66·1 96·8 . . L.S.D. (5%) 5·7 16·8
Figure 1 shows the effect of canavanine on sporulation intensity. The LDso is approximately 64 p.g/ml and inhibition is complete at approximately 120 p.g/ml.
~ 100, ___
5 80
Q~ 60 ~ 15• 46 "~ z o 20 5 ii!: 0' , \ 2 1 10 100 1000 1/1 CANAVANINE r",_G/ML) Fig. I.-Effect of canavanine on sporulation.
The inhibitory effect of canavanine may be reversed by the simultaneous addition of a number of amino acids and other nitrogenous compounds (Fig. 2). Complete reversal of the inhibition due to canavanine (concn. 0·5 p.mole/ml) is effected by arginine at a concentration of 2 ,lLmoles/ml. Citrulline and ornithine give a 90% METABOLITES AND SPORULATION OF PERONOSPORA TABACINA 887
l'eversal of the inhibition at a concentration of 6 ,umolesjml, while aspartic acid, glutamic acid, glycine, lysine, leucine, proline, creatine, urea, and ammonium carbonate give reversals of 32-55% at concentrations of 4-6 ,umolesjml. Canavanine has been described as an efficient inhibitor of conidial germination (Shepherd 1962) and would be a useful therapeutic agent for use in disease control, except for its strongly phytotoxic properties.
F>------ a ____ l:J..-- -- !='TRULL'NE a a ORNITHINE / ti.. 8 ii g" ~ 8 PROLINE ASPARTIC ACID I2- GLUTAMIC ACID GLYCINE LYSINE I CREATINE LEUCINE ~ IONS iii ~ 0 1 I I ! o 2 3 4 !3 6 COMPOUND (,....MOLES]Mt.) Fig. 2.-Reversal of canavanine inhibition by various amino compounds and ammonium ions. Canavanine was present in all experiments at a concentration of 0·5 fLmolefml. (c) Production of White Spores by P. tabacina While the spores of P. tabacina are noticeably violet-blue in mass, white spores were seen to be produced in the presence of dithiothymine, dithiouracil, and propylthiouracil [see Section III(a)]. White spores have also been observed previously by the present authors in the greenhouse after spraying P. tabacina infected plants with zinc dimethyldithiocarbamate. 888 C. J. SHEPHERD AND M. MANDRYK Leaf disks produced white spores when floated on aqueous suspensions of tetramethyl thiuram disulphide, sodium diethyldithiocarbamate, and zinc and manganese dimethyldithiocarbamates (cancns. 400 fLgfml). However, blue spores are produced in the presence of a suspension of copper dimethyldithiocarbamate (collen. 400 "g/ml), or in the presence of other dithiocarbamates to which had been added an equimolar amount of cupric ions. Tetramethyl thiuram disulphide and the dithiocarbamates, at concentrations of 400 fLg/m1, caused no diminution of the sporulation intensity: the white spores are fully viable, as judged by germination on agar (Shepherd 1962) or on the leaf surface (Shepherd and Mandryk 1963), and are pathogenic. Mter infection of N. tabacum by white spores, the subsequent spore crop is the normal blue colour, which indicates a phenotypic change only. White spores may also occasionally be produced when the temperature during the sporulation period is held at 27-28°0 and the humidity is near satura.tion. Such spores are non-viable. White spores are morphologically indistinguishable from the normal coloured ones. IV. DISCUSSION It may be seen from the above data that no normal metabolite was found to be strongly inhibitory to spore formation, although statistically significant effects may be seen in several cases. These results are in marked contrast to those reported by Samborski and Forsyth (1960) for the effects of metabolites on rust development on detached wheat leaves. These authors found that a metabolite, e.g. methionine, might stimulate growth at one concentration but be strongly inhibitory at another. Even though the majority of metabolites were only tested at three levels of concen tration, it may be inferred from the results reported above that inhibition-nutrition phenomena, as suggested by Garber (1956), play no part in the sporulation processes of P. tabacina. Conversely, no very marked stimulations ofsporuIation intensity by exogenously supplied nutrients couId be demonstrated, except in the case of FAD, which perhaps indicated that the experimental system used provided near-optimal nutrition. An alternative explanation of the apparent lack of activity of normal metabolites could be sought in difficulties of permeability to these compounds in the dual plant-fungus system. However, the strong inhibitions shown by some compounds, such as diazauracil and canavanine, and the reversal of such inhibitions by uracil and arginine respectively, suggest that this latter explanation probably does not hold. No clear explanation can be seen for the stimulating effects observed, except in the cases of nicotinic and p-aminobenzoic acids where Shepherd, Stuart, and lVIandryk (1963) observed that these compounds lead to the production of an increased number of conidiophores per unit area of leaf surface. The inhibition of sporulation by continuous light has been discussed by Cruickshank (1963), and a similar effect has been noted to occur with Alternaria solani (E. & M.) Jones & Grantly Vickers (Lukens 1963). With the latter organism the light inhibition could be reversed by the addition of R·5·P and the action spectrum of the light inhibition further suggested a key role for this flavin in the sporulation METABOLITES AND Sl'ORULATION OF l'ERONOSPORA TABACINA 889 process. In the present study, the small degree of reversal shown with both R-5-P and FAD, together with the characteristics of the action spectrum of light determined by Cruickshank (1963), suggest that the mechanism of the inhibition by light in P. tabacina differs from that postulated for A. solani. In view of the effect of riboflavin on conidial germination shown by Shepherd (1962) it is interesting to note that in the present study ribofiavin itself has a significant stimulating effect on sporulation intensity (116·4% of the untreated control), the mononucleotide a greater effect (148·4%), and FAD the largest effect (173· 9%), which suggests the possible conversion of ribofiavin to FAD in the sporulating system. The inhibitions demonstrated in the case of the seven uracil analogues and their reversal by the addition of uracil suggests the active involvement of this compound in the sporulation process. Previously Shepherd (1962) suggested that uracil synthesis is limiting during the phase of germ-tube elongation. Xanthine, its analogues, and the related compounds caffeine and theophylline all show some degree of inhibition of sporulation, but the mechanism of this inhibition is unknown. 'Theobromine was not inhibitory, but differs from theophylline and caffeine by being unsubstituted in position 1 of the purine skeleton. Samborski and Forsyth (1960) showed that sorbose, lyxose, and the sugar alcohols were ~ffect~ve inhibitors of rust development in wheat. In contrast to these results, it may be seen from the data reported above that no marked effects on sporulation were shown after the addition of sugars to the suspending medium, although sorbose and sorbitol did give a statistically significant degree of inhibition. While none of the natural amino acids was inhibitory, of the amino acid analogues tested canavanine was the most effective for inhibiting sporulation. In accordance with the report by Samborski and Forsyt,h (1960), it was found that the action of ethionine could be reversed by added methionine. Canavanine inhibition of growth of bacteria and its reversal by arginine, citrulline, and ornithine was first demonstrated by Volcani and Snell (1948). A similar growth inhibition of the fungus Neurospora by canavanine was demonstrated by Horowitz and Srb (1948). The latter authors showed that the inhibition could be reversed by arginine and also by lysine and methionine in some cases, and that other amino acids gave a partial reversal of the inhibition. In the, present study, complete reversal of canavanine inhibition was only 0 effected by arginine, citrulline, and ornithine (all of which gave about 90 / 0 reversal) while other amino acids gave about 30-50% reversal. It would appear from the degree of reversal given by ammonium ions that this latter group of aminC? acids, including lysine and methionine, might he contributing to arginine synthesis via transamination and other synthetic pathways. This action of lysine and methionine is similar to that shown for these compounds by Volcani and Snell (1948), whereas their effects on the reversal of canavanine inhibition in Neurospora would appear more specific (Horowitz and Srb 1948). The latter authors report the ratio of canavanine concentration to arginine concentration to be 0·3 for complete reversal of Neurospora, which is comparable with the value of 0·25 found in the present study. The ratio for complete reversal and the sensitivity to canavanine inhibition 890 C. J. SHEPHERD AND M. MANDRYK was shown ,to vary with different strains of Neurospora, but the Canberra strain and the SOl-strain (Hill 1963) of P. tabacina exhibited identical sensitivities to canavanine inhibition of both conidial germination and sporulation. It is postulated that the production of white spores in the presence of thio pyrimidines, tetramethyl thiuram disulphide, and the dithiocarbamates depends on the chelating properties of these compounds towards copper ions. This hypothesis is supported by the fact that blue spores are produced in the presence of copper dimethyldithiocarbamate or when equimolar amounts of copper are added together with the other compounds. The behaviour of the blue pigment in spore fractionation studies suggests it to be a melanin-like compound (authors, unpublished data), in which case poly phenol oxidase, which has a copper prosthetic group, would be expected to be involved in its formation. Thus the action of the chelating compounds may be explained on the basis of removal of the metal prosthetic group and the subsequent inactivation of the enzyme and non-production of pigment. On adding copper to the chelating compounds, the prosthetic group is not removed and blue spores are produced. In the above study, sporulation 4 4 intensities of 68 X 10 -153 X 10 (mean 91 X 104) spores per square centimetre of leaf surface were observed on untreated leaf disks. These values are comparable with 22·0 X 10'-144·5 X 10' spores per square centimetre reported by Corbaz (1961) on leaves of three tobaoco varieties in Europe. However, Rider, Cruiokshank, and Bradley (1961) have reported sporulation intensities in the range 3·5 X 104-22,5 X 4 10 spores per square centimetre for field~grown plants. The differenoes between these latter intensities and those reported by Corbaz (1961) and th~ present authors could wen be due to the differing degrees of colonization of the leaf by fungus under differing environmental conditions prior to the induction of sporulation. V. ACKNOWLEDGMENTS The authors wish to thank Mr. W. Schmid and Mr. H. Tantala for their technical assistance and Mr. G. A. McIntyre, Division of Mathematical Statistics, CSIRO, for his advice on statistical matters. They are indebted to Professor B. Dempsey, Royal Military College, Duntroon, A.C.T., for a gift of various dithio carbamates. VI. REFERENCES ARMSTRONG, G. H., and SUMNER, C. B. (1935).-Investigations on downy mildew of tobacco. Bull. S.C. Agric. Exp. Sta. No. 303. CLAYTON, E. C., and GAINES, J. G. (1933).-Control of downy mildew disease of tobacco through temperature regulation. Science 78: 609-10. CORBAI!';, R. (1961).-Considerations sur l'epidemic de mildiou du tabac (PeronDspora tabacina Adam.) en Europe. Phytopath. Z. 42: 39-44. CRUICKSHANK, I. A. M. (1958),-Environment and sporulation in phytopathogenic fungi. 1. Moisture in relation to the produotion and discharge of conidia of PeroMspora tabacina Adam. Aust. J. Biol. Sci. 11: 162-70. CRUIOKSHANK, 1. A. M. (1963).-Environment and sporulation in phytopathogenic fungi. IV. The effect of light and the formation of conidia of Peronospora tabacina Adam. Ausl. J. Bioi. Sci. 16: 88-98. METABOLITES AND SPORULATION OF PERONOSPORA TABACINA 891 DIXON, L. F., McLEAN, R. A., and WOLF, F. A. (1936).-Relationship of climatological conditions to the tobacco downy mildew. Phytopathology 26: 735-59. GARBER, E. D. (1956).-A nutrition-inhibition hypothesis of pathogenicity. Amer. Nat. 90: 183-94. HAWKER, L. E. (1957).-"The Physiology of Reproduction in Fungi." Cambridge Monogr. Exp. BioI. No.6. (Cambridge Univ. Press.) HILL, A. V. (1963).-A strain of Peronospora tabacina pathogenic to tobacco lines with resistance derived from Nicotiana debneyi and N. goodspeedii. Nature 199: 396. HOROWITZ, N. B., and SRB, A. M. (194S).-Growth inhibition of Neurospora by canavanine and its-reversal. J. Biol. Ohem. 174: 371-S. LmCENs, R. J. (1963).--Photo·inhibition of sporulation in Alternaria salam. Amer. J. Bot. 50: 720-4. MATKIN, O. H., and CHANDLER, P. A. (1957).-U.C. type soil mixes for container-grown plants. Ext. Servo LeaH. Calif. Agric. Exp. Sta. No. 89. RlDER, N. E., CRUICKSHANK, 1. A. M., and BRADLEY, E. F. (1961).-Environment and sporulation in phytopathogenic fungi. lIT. Peronospora tabacina Adam.-field environment, sporulation, and forecasting. Aust. J. Agric. Res. 12: 1119-25. SAMBORSKI, D. J., and FORSYTH, F. R. (1960).-Inhibition of rust development on detached wheat leaves by metabolites, antimetabolites and enzyme poisons. Ganad. J. Bot. 38: 467-76. SHEPHERD, C. J. (1962).-Germination of conidia of Peronospora tabacina Adam. I. Germination in vitro. Aust. J. Biol. Sci. 15: 483-508. SHEPHERD, C. J., and MANDRYK, M. (1963).-Germination of conidia of Peronospora tabacina Adam. II. Germination in vivo. Aust. J. Biol. Sci. 16: 77-87. SHEPHERD, C. J., STUART, F., and MANDRYK, M. (l963).-Isolation and maintenance of single spore lines of Peronospora tabacina (Adam.). Nature 197: 515. VOLVANI, B. E., and SNELL; E. E. (194S).-The effect of canavanine, arginine and related compounds on the growth of bacteria. J. Biol. Ohem. 174: 893-902.