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Reprinted from PHYTOPATHOLOGY, Vol. 50, No. 12, 870-873, Dec. 1960Printed in U. S. A.

SELECTIVITY OF FUNGICIDAL MATERIALS IN AGAR CULTURES

0. Vaartaj a

SELECTIVITY OF FUNGICIDAL MATERIALS IN AGAR CULTURES

0. Vaartaja

Contribution No. 615, Forest Biology Division, ResearchBranch, Department of Agriculture, Ottawa, Canada, andNo. 41, Canada Department of Agriculture Research Sta-tion, Saskatoon, Saskatchewan.

Accepted for publication May 16, 1960.

SUMMARY

Tolerance of fungi to synthetic and natural fungici-dal materials was studied in agar cultures. Many ofthe 43 materials exerted selective action on 10 speciesrepresenting different fungal groups. Pythium de-baryanum and Phytophthora cactorum tolerated 3polyene antibiotics at concentrations inhibitory toother species tested, and were inhibited by aureomycinat rates tolerated by most others. Phytophthoracactorunt tolerated tannins and certain fungicides bet-ter than P. debaryanum. Rhizopus stolonifer was in-hibited by pentachloronitrobenzene at concentrationstolerated by all others. Trichoderma viride was in-hibited by a duramycin mixture at concentrationstolerated by all others. Pellicularia praticola toleratedmany materials better than most of the fungi, but with2 experimental fungicides (B-856 and C-272), captan,and Dyrene the situation was reversed. Russula sp.tolerated duramycin mixture, Dyrene, neomycin, andthiram at concentrations inhibitory to Mucor raman-nianus. This selectivity might be utilized in isolatingpathogens and in indirect biological disease controlwith fun gicides tolerated by antagonistic saprophytes.

In studies of fungal soil flora by the plate technique,rose bengal at about 30 ppm may be used to inhibitmany bacteria and actinomycetes. Smith and Dawson(18) found it more useful than boric acid, benzoicacid, or Chrysoidine Y. Rose bengal (18), crystalviolet (9), and oxgall (13) have the additional ad-vantage of reducing the spread of many fast-growingfungi, such as Rhizopus and Trichoderma spp. Bac-teria are further inhibited when streptomycin at 30-100 ppm is combined with some of these, usually withrose bengal. Such combinations are now widely usedin soil platings (9, 11, 12). Penicillin G (2), aureo-mycin (3, 8), and novobiocin (7) have been used inthe same way as streptomycin. In isolating basidio-mycetes from soil, Warcup (20) added either strepto-mycin or wheat straw to the media to suppress bacteria.

Selective chemicals should also be useful in isolatingroot pathogens from soil or infected plants. Maloy and

Alexander (10) thus isolated Thielaviopsis basicola(Berk. & Br.) Ferr. from soil by using carrot tissuesoaked in a streptomycin solution as "bait." Nadaka-vukaren and Homer (14) used agar containing strep-tomycin and alcohol (5500 ppm) in isolating Verticil-lium albo-atrum Reinke & Berth. from soil. Papavizasand Davey (15) isolated Rhizoctonia solani Kiihnwith pieces of Fagopyron as bait plated on media con-taining 50 ppm of aureomycin, neomycin, and strepto-mycin.

It should also be possible to suppress certain fungiand select others by using selective fungicides. Barkerand Martinson (1) used pentachloronitrobenzene toprevent Rhizopus stolonifer (Ehr. ex Fr.) Lindau fromovergrowing other fungi in soil sampling tubes. Vaar-taja (19) found that several compounds were moreeffective against Pythium debaryanum Hesse thanagainst Pellicularia praticola (Kotila) Flentje in afungicide test on corn-meal agar. Reavill (16) foundthat chloronitrobenzenes were tolerated better byPythium spp. than by species of several other fungalgenera. Schneider (17) used cycloheximide and strep-tomycin in isolating Graphium ulmi Schwarz fromwood; this fungus was suppressed less than variouscontaminants.

This study describes a systematic effort to demon-strate the selective effects of a variety of chemicals onisolates representing different fungal groups.

Methods.-Antibiotic materials and synthetic com-pounds of chemically different types were selected.The following 43 materials were tested, each at 4 con-centrations:

Acti-dione, cycloheximide (3- [2- (3,5-dimethy1-2-oxocyclohexyl)-2-hydroxyethyl]glutarimidel at 4, 20,100, 500 ppm (provided by Upjohn Company) ; Alba-mycin (novobiocin), antibiotic at 4, 20, 100, 500 ppm(Upjohn Company) ; Anisomycin, antibiotic at 4. 20,100, 500 ppm (Chas. F. Pfizer Company) ; aureomy-cin, chlortetracycline (7-chloro-4-dimethylamino-1.4.4a,5, 5a, 6, 11, 12a - octahydro - 3,6,10,12,12a-pentallydroxy-6-methy1-1,11-dioxo-2-naphthacenecarboxamide) at 0.2,1, 5, 25 ppm B-856, 1,3-dichloro-5,5-diphenylhydantoin

10, 50, 250 ppm (Ethyl Corporation) ; B(ayer)22555, p-dimethylaminobenzendiazo sodium sulfonate

14, 70, 350 ppm (Chemagro Corporation) ;

December, 1960] VAARTAJA: SELECTIVITY OF FUNGICIDES IN AGAR

Table 1.-Effects of 2 antibiotics on growth of the test fungi in corn-meal agar.

871

Daily radial growth rate (mm)Control With Mycostatin 4 With Acti-dione

Fungus medium VL L H VH VL L H VH

Rhizopus stoloniler 21 4.5 1 0 0 14 9.3 1 0Trichoderma viride 17 1.5 0 0 0 3 2 0 0Fusarium solaniRhizoctonia

3.58

2.70

1.7 0 3 .57.3

2.10 0 3.3 00

0 00

Streptom yces sp. .2 .2 .2 .2 .2 .2 .2 .2 .2Pythium debaryarum 14 14 14 12 10 0 0 0 0Ph ytophthora cactorum 3.8 3.8 3.3 3.6 1.7 0 0 0 0Thielaviopsis basicola 1.1 1.1 .7 0 0 0 0 0 0Mucor ramannianus 2.3 1.3 0 0 0 2.0 1.4 .6 0Russula sp. 1.1 1 0 0 0 0 0 0 0Boletus granulatus 0.9 0.3 0 0 0 0 0 0 0

'Concentration of these antibiotics: VI, (very low) = 4 ppm; L (low) = 20 ppm; H (high) = 100 ppm; VH (veryhigh) = 500 ppm.

captan, N-(trichloromethylthio)-4-cyclohexene-1,2- di-carboximide at 2, 10, 50, 250 ppm; catechol at 4, 20,100, 500 ppm; cinnamic acid at 4, 20, 100, 500 ppm;coumarin at 4, 20, 100, 500 ppm; C-272, trans-1,2-bis(ethylsulfonylethylene) at 0.8, 4, 20. 100 ppm(Chemagro Corporation) ; dimethyl formamide at 4,20, 100, 500 ppm; d-sorbitol at 4, 20, 100, 500 ppm;duramycin, a mixture of antibiotics called F-17 andlargely containing duramycin, at 4, 20, 100, 500 pm(Northern Utilization Research Branch, U. S. Depart-ment of Agriculture, Peoria, Ill.) ; Dyrene, 2,4-dichloro-6-(o-chloroanilino)-2-triazine at 2, 10, 50, 250ppm (Chemagro Corporation) ; ethyl alcohol at 8, 40,200. 1.000 ppm; gallic acid at 4, 20. 100, 500 ppm;gladiolic acid, antibiotic at 2, 10, 50, 250 ppm; griseo-fulvin, antibiotic at 4, 20, 100, 500 ppm; Karathane,2- (1 - methy lheptyl) - 4,6 - dinit rophenyl crotonate andisomers, at 0.9, 4.5, 23, 46 ppni; Mycostatin (nystatin),polyene antibiotic at 4, 20. 100, 500 ppm; neomycin,antibiotic at 3, 15, 75, 350 ppm; nootkatin, antibioticat 4, 20, 100, 500 ppm (Forest Products Laboratory,Vancouver, B. C.) ; oligomycin, antibiotic at 4, 20. 100,500 ppm (Chas. F. Pfizer Company) ; PCNB, penta-chloronitrobenzene at 3, 15, 75, 350 ppm; phytoactin,polypeptide antibiotic at 1, 5, 25, 125 ppm (Pabst Lab-oratories) ; pimacrin, polyene antibiotic at 4, 20, 100.500 ppm; pinosylvin, antibiotic at 4, 20, 100, 500 (Dr.Erdtman, Stockholm, Sweden) ; pinosylvin mono-methyl ether (as pinosylvin) ; pyrogallol at 4, 20, 100,500 ppm; Rimocidin, polyene antibiotic at 4, 20, 100,500 ppm (Chas. F. Pfizer Company) ; rose bengal at4. 20, 100, 500 ppm; sodium metabisul fate at 4, 20,100, 500 ppm; sodium propionate at 4, 20, 100, 500ppm; sodium taurochocolate at 4, 20, 100. 500 ppm;spruce bark (powder from Picea glauca (Moench)Voss, at 8, 40, 200, 1000 ppm; streptomycin sulfate at4, 20, 100, 500 ppm; tannic acid at 4. 20, 100, 500ppm; taxifolin, antibiotic at 4, 20. 100 500 ppm (For-est Products Laboratory, Vancouver, B. C.) ; thiram,bis(dimethylthiocarbamoyl) disulfide at 3, 15, 75, 350ppm; thujaplicin, antibiotic at 4, 20, 100, 500 ppm(Forest Products Laboratory, Vancouver, B. C.) ;ustilagic acid, antibiotic at 4, 20, 100, 500 ppm (Dr.R. Haskins, National Research Council, Prairie Re-

gional Laboratory, Saskatoon, Sask.), zineb, zincethylenebis[dithiocarbamate] at 2.6, 13, 65, 325 ppm.

The materials were mixed in melted corn-meal agarcooled to about 60°C and immediately poured intoPetri dishes. The dishes were seeded with the follow-ing species.

1) Rhizopus stolonifer, saprophyte, common aerialcontaminant; 2) Trichoderma viride Pers. ex Fr.,saprophyte, very common in most soils; 3) Fusariumsolani (Mart.) App. & Wr., saprophyte, very commonin most agricultural soils; 4) Rhizoctonia solani (per-fect stage: Pellicularia praticola), virulent seedlingpathogen, common in many forest nurseries and per-haps in many agricultural soils; 5) Strepromyces sp.,saprophyte with strongly antagonistic ability againsta large number of other fungi, common in neutral andalkaline soils; 6) Pythium debaryanum, virulent un-specialized seedling pathogen, very common in mostagricultural soils; 7) Phytopthora cactorum (Leb. &Cohn) Schroet.. unspecialized pathogen, common incertain agricultural soils; 8) Thielaviopsis basicola,unspecialized pathogen, common in certain agri-cultural soils; 9) Mucor ramannianus Miiller, sapro-phyte and epiphyte, very common in forest soils; 10)Russula sp., possibly a mycorrhizal fungus, common inforest soils.

These isolates originated in Saskatchewan or Mani-toba from diseased tree seedlings in forest nurserysoils, except that no. 1 was from a pine seed, no. 2from a orchid root, and no. 10 from a fruiting body.

All the chemicals were tested once with these 10isolates. The most interesting findings were retestedin at least 5 replications. Some of the chemicals werealso tested with further isolates of these and otherfungal species, as reported in more detail in the re-sults. Among these was Boletus granulatus (L.) Fr.(Table 1), a common mycorrhizal fungus. The iso-late was obtained from a fruiting body in Saskatche-wan.

The cultures were incubated at room temperature.When the control colonies of an isolate reached abouthalf the diameter of the plate, the radial growth wasmeasured for all tests of the isolate. Records of thelowest concentrations totally inhibitory and of those

872 1'115 TOPATHOLOGY i Vol. 50

Table 2.—Concentration' of antibiotics and fungicides required to inhibit various fungi.

MaterialRhizopusstolonifer

Tricho-dermaviride

Fusariumsolani

Rhizoc-toniasolani

Strepto-myces

sp.

Pythiumdebar-yanum

Phytoph-thora

cactorum

Thiela-viopsisbasicola

Mucorraman-nianus

Russulasp.

PCNB VI? VH"" bp VII"DuramycinRiniocidin

VH"VL"

L"VU' VL"

H"L

VH' P_o

VH"_o

HP

LH"L

VII"L

Pimacrin LhP H" L"P --e L"' H"Mycostatin H" VL" H" L L"B-856 VH VL" H LP H" H" VH VII"Captan H H" H HDyrene H', VH" _p L" —" VH" H" H"Albamycin VL'' VH"Rose bengal _P _P L" VH" VH" VH"Streptomycin VH"Neomycin P VH" P \ H"Aureomycin H"P VIP " VI-I''"C-272 _p VII L" II VHThiram L H" L 12 L VL" I. VL" H"Catechol VII VH" VH" II" VII VH" VII I-I"Tannic acid VH VH" - VII VH"Gallic acid VII VH" - VH"Pyrogallol -" VII" L" I-I" L" H HSodium meta-

bisulfate VII" VW" H H" 11" H"Act idione VH" H" VH" Fl VLb VL" VH" VHB. 22555 VH" _c _op VH H H" VII —P

Zineb VH VII VH VII" VH VH H" H" VH VH"Ustilagic acid H VH" VH" 11 VH H L" VH 11Thujaplicin L" HP L VL L LP VL L VLKarathene —" VHP VII HP VH"

" Inhibition was complete at concentrations given. The concentrations VH (very high), 11 (high), L (low), and VL(very low) varied for each material as listed in the text.

b Particularly effective inhibition.Unusual tolerance.

' Partial inhibition (growth roe reduction > 75%) at any lower concentration.indicates no inhibition at VH.

allowing only limited I < 25%) growth were tabulatedfor all materials.

Results and (Iiseussion.—Table 1 gives examples ofactual growth rates and types of selectivity. Table 2compares most of the materials, excluding those withlittle or no selectivity.

The selective materials in Table 2 are arranged asclosely as possible so that those mainly inhibitory tothe isolates on the left are given first. Thus, the firstmaterial. PCNB, inhibited the first isolate. R. stoloni-fer at the lowest concentration tested but had no effecton most of the other isolates; the second material,duramycin mixture, was especially effective on thesecond isolate, Trichoderma viride; and so on.

Itimocidin, pimacrin. and Mycostatin. which weretolerated by Pythium debaryanum and Phytophthoracactorum, belong to the polyene group Of antibiotics.The results suggest that these may be used in isolatingfungi of this group. Further test s were made with 6more isolates of Pythium spp. and P. cactoram andmiscellaneous other fungi. The results indicate thatthe tolerance is common in. and mainly limited to. thisgroup of fungi. Only one of the other isolates, Mucorsp., tolerated these antibiotics almost as well asPythium.

It was found that heat destroys polyene antibiotics.According to Dekker and Ark (4) these compoundsmay be oxidized in the presence of water and oxygen.

but the oxidation is counteracted by ascorbic acid,gallic acid, and other compounds. In consideration ofthe risk of oxidization, a high concentration shouldbe used in isolations. Most Pythiaceae tolerated con-centrations of about 500 ppm.

The results in Table 2 as well as in tests with 6other isolates suggest that Phytophthora cactorum canbe separated from the usually faster-growing Pythiumspp. through its better tolerance of C-272, thiram, andtannins (tannic acid. gallic acid. catechol, pyrogallol).It remains to be seen whether this is true with allisolates and species of these genera.

The high activity of PCNB on R. stolonifer, referredto in the introduction, was confirmed. Unfortunately,an isolate of Mucor sp. tolerated PCNB somewhatbetter. This chemical reduced but did not inhibit thegrowth of many fungi. The reduction was especiallypronounced with Trichoderma viride

These results, as well as those published earlier (5,19), show that Rhizoctonia solani can be selected bysuppressing other fungi with various chemicals. Onepossibility, for instance, is to use a medium contain-ing aureomycin (at 500 ppm) against bacteria. Strep-tomyces, and Pythiaceae, and PCNB (20 ppm) againstR. stolonifer and Trichoderma spp. An isolate ofRhizoctonia repens Bernard from roots of Goodycarasp. behaved similarly to the R. solani (P. praticola)in the tables.

December, 19601 VAARTAJ A : SELECTIVITY OF FUNGICIDES IN AGAR 873

Selective isolation of such slow-growing fungi asThielaviopsis basicola is especially difficult. It mightbe done, however, with a medium containing variouscombinations of several materials. Further testing isneeded to determine the best concentrations. The re-sults in Table 2 suggest a combination of Mycostatin,Dyrene, or B-856 (against R. solani), PCNB (againstR. stolonifer and T. viride). and neomycin (againstPythiaceae and Fusarium solani).

The few materials selectively effective (but only toa limited degree) against Fusarium solani were neo-mycin at 500 ppm, PCNB at 250 ppm, and sodiummetabisulfate (to select Rhizoctonia solani at 100ppm).

The few tests for materials selective against Glio-cladium sp. and Penicillium spp. were mostly unsuc-cessful. Some promise was shown by PCNB, duramy-cin. zineb, and Karathane. The duramycin prepara-tion used contained other antibiotics as well. Thepromising results obtained should be retested withpurer materials. Hampton and Buckholtz (6) reportedthat coumarin stimulates sporulation of Pythiumgraminicolum Subramaniam. That was not so with P.debaryanum in my tests.

. •Mucor ranumnianus was Included to test for selec-tive inhibition in isolations from mycorrhizae of foresttrees. M. ranzannianus is common on tree roots and

grows faster than mycorrhizal fungi. The results sug-gest that thiram, neomycin, Dyrene, pirnacrin, andduramycin may be selective if used at exactly the rightconcentrations. Further tests with various isolates ofbasidiomycetes showed, however. that the only (possi-bly) mycorrhizal isolate (Russula sp.) used with allthe chemicals cannot represent all the variety of re-sponses in these fungi. Differing from Russula sp.,isolates of Boletus granulatus and B. luteus L., for in-stance, were greatly inhibited by 10 ppm of pimacrinbut tolerated 10 ppm of Rimocidin well. These werestimulated by 8 and 40 ppm of captan, which containsplenty of nitrogen. This suggests that further testsshould be made with a richer medium.

The results in the tables also suggest useful selec-tivity in disease control. If a soil fungicide suppressedthe main pathogen but not the antagonistic sapro-phytes. it may exert an indirect biological control inaddition to the direct one (19). Such appears possibleespecially with Mycostatin. B-856, captan, and Dyrene(against Rhizoctonia), thiram, the tannins, Albamycin(novobiocin), Acti-dione, B-22555 (against Pythia-ceae), and zineb, ustilagic acid. thujaplicin, and Kara-thane (against T. basicola). Albamycin (novobiocin)may be especially useful against Streptonzyces scabies(Thaxter) Waksm.-Forest Biology Laboratory, Cana-da Agriculture Research Station, Saskatoon, Sas-katchewan, Canada.

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