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 ...