FUNGICIDAL EFFECTS OF SOME CHEMICALS ON SCLEROTINIA TRIFOLIORUM ERIKSS. Aarre Ylimäki Received August 20, 1969' Department ofPlant Pathology, Agricultural Research Centre, Tikkurila, Finland In field trials carried out by the Department of Plant Pathology it has been established that clover rot ( Sclerolinia trifoliorum Erikss.) can effectively be controlled by PCNB (quinto- zene-)-preparations (Ylimäki 1955, 1956, 1969). The effect of these fungicides and their dependence on environmental factors as well as the effectiveness of some other chemicals on S. trifoliorum has been studied. Methods Since the effect of fungicides on the growth of mycelium in S. trifoliorum is easier to study, and as fumigation of fungicides appears to be a relatively advantageous, the studies were carried out as plate tests. The chemicals were placed in weighed amounts on four filter papers of 10 mm. These were placed on the surface of agar from where at least the watersoluble chemicals could diffuse along the agar surface. The fungus was transferred into the middle of the plate (c.f. Fig. 1). In some cases the fungicides were dusted or sprayed direct on the mycelium which had already started to grow. To establish merely the fumigation effect of the chemicals, the substance under study was placed in a small plastic cup inside the cover of the Petri dish, upside downand with the fungus on the medium above it. The growth of the mycelia was measured daily and observations were made on the formation of sclerotia. Microscopical studies were made to see whether the chemicals had caused visible changes in the mycelia. From the treated dishes the mycelia and sclerotia were transferred again on the normal medium so as to established whether the treatment had caused permanent changes in the mycelia. For a study of the sporophores the sclerotia were germinated on water agar or on wet quartz sand. Results The experiments (Table 1, Figs. I—4)1 —4) showed that the chemicals had a relatively restraining effect on the mycelium of S. trifoliorum, but as many of these substances are https://www.c-info.fi/en/info/?token=oLscsMtioVDw3DcT.SYNCRlz1i_ZOt-or7BnhnA.-diNVtkwEz3EmuHI5yUFZCveCuTTKtHLjx4kEby8NIIodSZOdYFoe0hbxfBBc9Ikp1TdGiVQRinH1hzb1Zwxj3DpvrQDuyA6kgsn8WLxFRoC8xOhosO81LqbKNm5bCNv_6GxtQHfiLdOlv5uITc3vPIivcCoQNsL8_BWNLpSp_8 244 Table 1. Effect of various chemicals on Sclerolinia tri/oliorum in laboratory trials. Mycelial growth mm Preparation Active ingredients after days 2 6 12 Mercuric compounds Agrosan GN phenylmercury acetate 0.85 % + ethylmercury chloride 0.15 % 9.5 28.4 33.7 Atiran methoxyethylmercury chloride 4.6 % 16.3 29.9 44.9 Ceresan Nb. methoxymercury acetate 2.4 % 16.3 26.3 31.4 CRC mercurochloride 4 % 9.5 59.0 100 Duphar mercury spray phenylmercury cellulose ether 4.3 % 11.1 29.5 35.3 Femma phenylmercury acetate 2.0 % 10.5 43.2 51.6 •Germisan phenylmercury pyrocatechine 3.3 % 9.5 33.7 40.0 Mercadmine phenylmercury salicylate 5 % 4.8 23.2 25.3 Solusan methylethylmercury acetate (Hg 15,0 %) 7.4 18.9 20.0 Täyssato methoxyethylmercury chloride 2.2 % 7.9 29.5 66.8 Verdasan phenylmercury acetate 5 % 11.6 29.3 31.0 YF 5049 phenylmercury salicylate anilide 96 g/1 5.3 15.8 15.8 Average 10.0 30.6 41.3 Benzenes Amatin Staub hexachlorobenzene (HCB) 20 % 12.1 61.1 97.4 Avicol dust quintozene (PCNB) 20 % 0 9.4 31.8 Avicol wp. „ 50 ~ 4.8 13.2 26.1 Botrilex „ 20 „ 4.6 20.3 53.1 Brassicol dust „ 20 „ 7.5 18.9 44.5 Brassicol sup. „ 50 „ 6.0 20.2 38.8 Fartox dust „ 20 „ 9.4 22.4 24.1 Folosan tecnazene (TCNB) 5,, 1.2 5.1 11.7 Olpisan trichlorodinitrobenzene (TCDNB) 20 % 4.2 15.4 39.6 Bulbosan trichlorotrinitrobenzene (TCTNB) 7.5 % 5.3 12.6 26.8 Bulbosit rodandinitrobenzene (RDNB) 10.0 47.4 73.7 Average 5.2 40.1 38.0 Thiocarbamates Dithane Z-78 zineb 65 % 12.6 63.2 92.1 Duphar ferbam ferbam 95 % 13.7 38.2 52.1 Average 13.2 50.7 72.1 Captan compounds Orthocide 75 captan 75 % 16.3 63.7 89.0 Orthocide 50 captan 50 % 0 57.0 76.3 Average 8.2 60.4 82.7 Copper compounds Kuprijauhe copper oxychloride 85 % 18.9 87.5 100 KT 35 „ 60 % 27.1 70.6 70.6 Average 23.0 79.1 85.3 Mycelial growth mm Preparation Active ingredients after days 2 6 12 Antibiotics Actidione cycloheximide 85—100 % 15.3 22.0 22.8 Agrimycin streptomycin 15 % + oxytetracycline 1.5 % 27.1 100 100 Griseofulvin griseofulvin 98 % 22.6 76.3 88.4 Kojic acid 5-hydroxy-2 hydroxine-ethyl-4 pyrone 30.7 100 100 Sorbistat sorbic acid 26.7 70.8 84.7 Usno usnic acid 29.2 98.8 96.5 U-4527 cycloheximide 19.4 25.9 26.8 U-7413 „ -oxime 25.2 75.9 81.6 U-7414 „ -acetate 27.6 97.5 98.7 U-7415 „ -semicarbazone 25.7 85.1 86.8 Average 25.0 75.2 78.6 Other compounds Pomarsol forte thiram 80 % 12.4 40.8 59.2 Fusarex plus tecnazene 2 % + isopropyl N-phenylcarbamate (IPG) 1 % 0 11.6 21.1 KT 6 quintozene (PCNB) 5 % + isopropyl N-phenyl- carbamate (IPC) 1 % 7.4 35.3 52.5 VP 19—40 = Brestan triphenyl stannic acetate 20 % 9.2 36.1 56.3 Bayer 4934 urbasulf 32 % (As 20 %) 11.2 21.8 23.5 Riedel B/500 oxychinolin halogen deriv. 1.0 % 2.7 44.8 100 Tuset thiram 40 % -f- zineb 20 % 11.6 31.6 91.1 Spergon chloranil 96 % 0 41.1 72.6 Phygon dichlone 50 % 0 14.7 57.9 Belvitan K methyl a-naphtylmethylether (MNME) 4 % Controls 14.3 68.8 92.6 Fig. 1. Effect of various fungicides on the growth of Sclerotinia trifoliorum mycelium. A. control, B. Brassicol super wp., C. Verdasan, D. Germisan, E. Täyssato, F. Solusan, G. CRG, H. Agrosan GN, I. Atiran, J. Duphar mercury spray, K. Femma, L. Granosan, M. Mercadmine, N. YF 5049. 245 Fig. 2. Effect of various fungicides on the growth of Sclerotinia trifoliorum mycelium. A. control, B. Folosan, C. Brassicol, D. Dithane Z-78, E. Atiran, F. Kuprijauhe, G. Verdasan, H. Ceresan Nb., I. Brassicol wp., J. Brestan, K. Duphar ferbam, L. Pomarsol forte, M. Orthocide 75. Fig. 3. Effect of various antibiotics on the growth of Sclerotinia trifoliorum mycelium. A. control, B. Actidione, G. U-4527, D. U-7413, E. U-7414, F. U-7415, G. Griseofulvin, H. Usno, I. Sorbistat, J. Brassicol wp., K. Verdasan. Fig. 4. Effect of various benzene-preparations on the growth of S. trifoliorum mycelium. A. control, B. Brassicol wp. C. Olpisan, D. Bulbosan, E. Folosan, F. Fusarex plus, G. KT 6, H. Amatin Staub, I. Bulbosit. Fig. 6. The same trial as in Fig. 5. Number of sclerotia 10, 20, 39 days after inoculation. I I light sclerotia HI black sclerotia Fig. 5. Effect of fungicides on the formation of sclerotia in petri dishes, a. control, b. MNME, c. PCNB, d. zineb, e. captan, f. TCNB. Fig. 7. Effect of some fungicides on the growth of mycelia of S. trifoliorum. a. control, b. MNME, c. PCNB, d. zineb, e. captan, f. TCNB. 246 247 Table 2. The preservation of the efficacy of TCNB and PCNB prep- arations in open dishes. Preparation Duration Growth of mycelium of trial mm rel. A. At room temperature Folosan TCNB 10 days 9.0 10 Brassicol PCNB „ 36.3 38 Control „ 95.0 100 L.S.D. 4.7»»* Folosan TCNB 10 months 45.8 48 Brassicol PCNB „ 86.3 90 Control „ 95.0 100 L.S.D. 14.4*»* B. Out of doors Folosan TCNB 16 days 15.2 17 Avicol PCNB „ 76.4 85 Control „ 90.0 100 L.S.D. 6.8»»* poisonous it is not possible to use them on grasslands. Moreover, the substances whi chin laboratory trials were rather effective, have proved to be less effective in field trials than the PCNB substances, which in field trials have effectively controlled the damage of clover rot (Ylimäki 1969). When TCNB, PCNB, or captan preparations, were sprinkled on the mycelia trans- ferred to the medium the growth of the mycelia of S. trifoliorum and the formation of sclerotia were seriously disturbed. TCNB was more effective in preventing the growth of mycelia than PCNB (Fig. 4), whereas the preventive effect on the formation of sclerotia was very similar in both (Figs. 5 and 6). Fig. 8. Effect of PCNB preparations on the growth of mycelia and formation of sclerotia of S. trifoliorum A. control, B. Avicol wp. 248 Table 3. Preservation of TCNB and PCNB preparations in different conditions Way of storage Preparation Growth of mycelium mm rel. Trial I In a room, closed dish Brassicol sup. PCNB 32.5 34 ~ Brassicol dust ~ 44.5 47 „ Avicol wp. ~ 23.5 25 „ Folosan TCNB 7.0 7 In a room, open dish, dry Brassicol sup. PCNB 39.0 41 ~ Brassicol dust ~ 43.5 46 ~ Avicol wp. „ 25.5 27 „ Folosan TCNB 8.5 9 In a room, open dish, wet Brassicol sup. PCNB 31.5 33 „ Avicol wp. „ 44.5 47 Out of doors, open dish, wet Brassicol sup. PCNB 45.0 47 ~ Brassicol dust ~ 51.0 54 ~ Avicol wp. ~ 26.5 28 „ Folosan TCNB 12.5 13 Control 95.0 100 L.S.D. 4.4»»» Trial II In a room, closed dish Folosan, an old amount TCNB 12.5 14 „ Folosan, an new amount ~ 18.0 20 „ Botrilex, an old amount PCNB 57.5 63 ~ Botrilex, an new amount „ 69.3 76 In a room, open dish Folosan TCNB 41.0 45 „ Botrilex PCNB 79.5 88 In a cool place, open dish Folosan TCNB 19.8 22 „ Botrilex PCNB 32.3 36 Control 90.7 100 L.S.D. 12.6»** Fig. 9. Evaporation trial with benzene prepara- tions. A. control, B. Brassicol wp., C. Avicol wp., D. Brassicol dust, E. Folosan; storage 1. in closed parcel 2. in open dish out of doors, wet 3. in open dish in a room, dry 4. open dish in a room, wet. 249 Table 4. Effect of temperature on the preservation of PCNB preparations. Substances preserved in open dishes during one month. Growth of mycelium Temperature Preparation (15 —37°C) (4—18°C) mm rel. mm rel. Brassicol super wp. 31.0 41 24.0 32 Avicol wp. 23.5 31 27.0 36 Avicol dust 24.0 32 24.0 32 Botrilex dust 26.5 35 25.0 33 Brassicol dust 25.0 33 25.0 33 Control 75.0 100 L.S.D. 9.o*** Observed microscopically, the mycelia which had interrupted their growth, seemed alive, even if abnormally thick and crooked (Fig. 7). The formation of appressors was more abundant in dishes treated with fungicides than in untreated dishes. The sclerotia formed more rapidly in control dishes. They were mainly on the edges of the dishes, whereas in the dishes treated with chemicals their occurrence was haphazard and they appeared mostly in groups joined to each other (Fig. 5). When transferred to an untreated medium, the abnormal mycelia and sclerotia were able again to form mycelia and sclerotia that seemed to be totally healthy. The sclerotia from the mycelia treatedwith TCNB as well as with PCNB preparations formed numerous sporophores, they developed no apothecia, however, whereas in untreated sclerotia they developed normally. Although pieces taken from the mycelia treated with both substances and placed on nonpoisonous medium formed normal kind of mycelium and sclerotia, these sclerotia did not develop normal sporophores as did the sclerotia of the control. It seems possible that TCNB and PCNB substances may have effects of more lasting nature on the S. trifoliorum fungus. Preventing the growth of the mycelia of S. trifoliorum and the formation of sclerotia is clearly a fungistatic process (cf. Strecker 1957). The activity of these substances is depen- dent on the temperature: in the same temperature TCNB is more fungistatic than PCNB. Since the vapor pressure of the TCNB is 4—5 times greater in the same temperature as the vapor pressure of PCNB (Reavill 1954), the difference in the efficacy of the substances may depend mainly on the difference in fumigation. In all trials wheretheTCNB preparation was used, it was more effective on S. trifoliorum than the PCNB preparations (Tables 2—3, Figs. 2, 4 and 9). As the preventive treatments in the control of clover rot should be started rather early (Ylimäki 1969), it is important to know how long the fungicide remains effective in field conditions. The length of the time the substances preserve their effectiveness in storage should also be known. To study these qualities, the TCNB and PCNB preparations were kept in different temperature and humidity conditions. In the trials the effect of the temperature, the humidity and the air current on the effectiveness of TCNB and PCNB was so small that the above factors cannot have a 250 perceptibly lowering effect on the usability of TCNB or PCNB on the fields in autumn conditions (Table 4). In fact, the substances may loose their effectiveness when stored at higher temperatures in open or badly closed covers. In the trials this applied in particular to the PCNB preparations (Tables 2 and 3). Summary In efficiency tests carried out in laboratory (Table 1, Figs. I—4) it was established that on media many chemicals had a restraining effect on the growth of the mycelia ofSclerotinia trifoliorum. In addition to PCNB preparations, TCNB substances very severely restrained the growth of mycelia and the formation of sclerotia of the fungus (Figs. s—B).5—8). Sclerotia formed of mycelia treated with TCNB as well as with PCNB substances developed numerous sporophores which were not fertile however. The effect of both PCNB and TCNB on the mycelium of S. trifoliorum proved only restricting on the growth. It did not destroy the mycelia. The activity of the substances is dependent on the temperature and the higher effectivity of TCNB is based on its greater degree of vaporization (Tables 2—3, Figs. 2,4 and 9). In autumn and in outdoor conditions on the field the vaporization of the substances is so small that it does not lower the effectiveness to any great extent (Tables 2-3, Fig. 9). REFERENCES Reavill, M. 1954. Effect of certain chloronitrobenzenes on germination, growth and sporulation of some fungi. Ann. Appi. Biol. 41; 448—460. Strecker, B. 1957. Untersuchungeniiber die Einwirkung von organischen Fungiziden auf Bodenpilze. Z. Pfl.krankh. (Pfl.path.) und Pfl.schutz 64: 9—35. Ylimäki, A. 1955. On the effectiveness of penta- and tetrachloronitrobenzenes on clover rot ( Sclerotinia trifoliorum Erikss.) Acta Agr. Fenn. 83: 147—158. —»— 1956. Additional experiments on the chemical control ofclover rot. Selostus: Lisäkokemuksia apila- mädän torjumisesta kemiallisilla aineilla. Valt. Maatal.koetoim. Julk. 148: 31 —49. —»— 1969. Apilamätä apilan talvehtimisen heikentäjänä Suomessa. Summary: Clover rot as a cause of poor overwintering of clover in Finland. J. Sei. Agric. Soc. Finl. 41: SELOSTUS ERÄIDEN KEMIKAALIEN VAIKUTUS SCLEROTINIA TRIFOLIORUM SIENEEN Aarre Ylimäki Kasvitautien tutkimuslaitos , Maatalouden tutkimuskeskus, Tikkurila Laboratoriossa suoritetuissa tehokokeissa (taulukko 1, kuvat I—4)1 —4) ilmeni, että monilla kemikaaleilla oli ravintoalustoilla S. trifoliorum'in rihmaston kasvua ehkäisevä vaikutus, mutta useat niistä eivät kuiten- kaan ole käyttökelpoisia nurmilla myrkyllisyytensä takia. PCNB-valmisteiden ohella myös TCNB-aineella oli erittäin voimakas sienen rihmaston kasvua ja rihmastopahkojen muodostumista ehkäisevä vaikutus (kuvat s—B). Sekä TCNB- että PCNB- aineilla käsitellyistä rihmastoista muodostuneet rihmastopahkat kehittivät lukuisia itiöemien aiheita, jotka kuitenkaan eivät olleet fertiilejä. Sekä PCNB;n että TCNB:n vaikutus S. trifoliorum'in rihmastoon oli vain kasvua ehkäisevä, ei tappava. Aineiden aktiivisuus on riippuvainen lämpötilasta ja perustuu TCNBm suurempi tehokkuus sen suurem- paan kaasuuntuvuuteen (taulukot 2—5, kuvat 2,4, 9). Syksyisin ulkona pellolla vallitsevissa sääoloissa on aineiden kaasuuntuvuus kuitenkin siksi vähäinen, ettei sillä ole mainittavasti tehoa heikentävää vaikutusta (taulukot 2—5, kuva 9).