Trans . Br . mycol. Soc. 81 (4) 713-751 (1984)

[ 713 ]

Printed in Great Br itain

NOTES AND BRIEF ARTICLES

FUNGISTATIC NAPHTHOQUINONES OF VERTICILLIUM AGARICINUM

BY MOHAMED OSMAN

Department of Botany, University of Tanta , Tania, Egypt

AND L. R. G. VALADON

Department of Botany, Royal HoI/oway College, Egham Hill, Egham, Surrey TW20 oEX

Total naphthoquinones from V . agaricinum are fungistatic against four of seven fungi tested,the three not affected being Aspergillus niger, Fusarium spp., and Rh izopus nigricans. Inhibitionwas greatest in Alternaria humicola and in Penicillium notatum, much less in Aspergillus flavusand in Helminthosporium oryzae.

Extensive studies on the properties and culturerequirements of a fungal staling substance (vacuo-lation factor) produced by Fusarium oxysporumhave been carried out by Park (1964), Park &Robinson (1964) and Robinson & Park (1965).Chemical studies of this vacuolation factor estab-lished the structure of the naphthoquinone,bikaverin, as (1) (Cornforth, Ryback, Robinson &Park, 1971).

A number of fungi , including different Fusaria,Gibberella fujikuroi, Mycogone jaapii, are nowknown to contain bikaverin (Brewer, Arsenault,Wright & Vining, 1973). Valadon & Chapman(1983) were able to show by TLC that two minorpigments of Verticillium agaricinum could bebikaverin and norbikaverin while the majorpigment was agaricin, possibly related to these twopigments. Preliminary studies have shown that thenaphthoquinones from V. agaricinum grown on asolid medium either in the dark or in the light hadantifungal activities. TLC of these pigmentsshowed them to be made up of seven compoundswhich were very polar, very strongly adsorbed onto the silica gel and could not be eluted easily or atall. This note describes the antifungal activity oftotal naphthoquinones produced by V. agaricinumand presents a different bioassay from that ofRobinson & Park (1965).

MeO

Me o OH o

OMe

V. agaricinum (Link) Corda (Valadon & Mum-mery, 1971) was grown in 100 em" conical flaskscontaining 50 em" of a 2 % malt liquid medium inthe dark on a reciprocal shaker for 5 days . One em"of this suspension was transferred on to 2 % maltagar plates and grown in the dark for 8 days at24-25 0c. A deep pink colour had been producedat the end of the incubation period. The mycelium,which easily peeled off from the plates, waswashed with distilled water and the water squeezedlightly with filter paper. The mycelium was thenextracted with n-hexane in a Soxhlet apparatus for2 days and the slightly brownish liquid containingsterols was discarded. The remaining myceliumwas then extracted with ethyl acetate :glacial aceticacid (3: 1, vI v ) at 50 in a Waring blender for 1 minand filtered. This was repeated three times toextract the pigments which were now in the ethylacetate layer, and which were transferred intochloroform. The colour of the pigments concen-trated to a small volume in vacuo at 40 0 wasbrownish red .

Total naphthoquinones were estimated in at-ern" aliquot by diluting with 9 ern" glacial aceticacid, heating at 1000for 2 min, clarifying by centri-fugation and measuring the optical density at450 nm (max. A) (Brewer et al., 1973). Maximumproduction was approximately 500 mg 1-1.

The following test organisms were used: Asper-gillus niger van Tiegh., A. fiavus Link:Fr.,Alternaria humicola Nees, Fusarium spp., Helmin-thosporium ory zae Link : Fr., Penicillium notatumWestling, and Rhizopus nigricans Ehrenb. : Fr. Aspore suspension from 7-day-old dark-growncultures of each of the above fungi was used (Osman& Valadon, 1981). In each case the sporesuspension was mixed with 2 % malt agar kept at400and poured into Petri dishes. After solidification,

Notes and brief articles

observed: certain test organisms were not inhibitedwhile others were to a lesser or greater extent (T able1). This method is thus a good bioassay for thenaphthoquinones of V . agaricinum.

Finally, the naphthoquinones of V. agaricinumare more in number (Valadon & Chapman, 1983)compared to those of F. oxysporum where it ispossible to obtain bikaverin in a pure form. It is notpossible so far to do the same with the pigments ofV . agaricinum which are very polar and difficult toelute. However, the total naphthoquinones of V.agaricinum are very effective in inhibiting thegrowth of certain fungi .

o7'5±O'3*

17'O±o'8o

9'5 ±0'514'O±1 'O

o

Test organism

Aspergillus nigerA ·fiavusAlternaria humicolaFusarium spp.Helminthosporium oryzaePenicillium notatumRhizopus nigricans

Table 1. Inhibitory effect of total naphthoquinones(0 ' 1 pg em- a) from V. agaricinum on some test

organisms

(T he controls (discs soaked in CHCl.) had noeffect on any of the test organisms.)

Mean diam ofinhibition zone (mm)

* S.E.M.

four filter paper discs (6 rom diam) contammg0'1 pg total naphthoquinones em-a in CHCla orcontrols (discs soaked in CHCla)were placed on theagar surface of each plate and then incubated in thedark at 25 ±1

0 for 48 h. At least 2 plates of eachtest organism were used and the results are givenas mean of at least 8 replicates ± standard error ofthe mean (S.E,M.) (Table 1).

The results (Table 1) show that only four of theseven organisms are inhibited by the naphtho-qui nones of V. agaricinum while Alternaria humicolaand Penicillium notatum are the most inhibited. Theresults are different from those of Park (1964) whofound that leader hyphae of all 20 fungi testedagainst the staled solution (= bikaverin+norbi-kaverin) from Fusarium oxysporum developedpermanent inhibition. The difference may beaccounted for partly by the fact that the staledsolution of F . oxysporum contained othermetabolites besides bikaverin. Also, the assay in thepresent investigation was based on spore germina-tion. In the present study, 0'1 pg cm ? naphtho-Quinones were used and a differential response was

REFERENCES

BREWER, D., ARsENAULT, G. P., WRIGHT, J. L. C. &VINING, L. C. (1973). Production of bikaverin byFusarium oxysporum and its identity with lycopersin.The Journal of Antibiotics 26, 778-781.

CORNFORTH, J. W., RYBACK, G ., ROBINSON, P. M. &PARK, D. (1971). Isolation and characterisation of afungal vacuolation factor (bikaverin). Journal of theChemical Society (C.) 2786-2788.

OSMAN, M. & VALADON, L. R. G . (1981). Effect of light(especially near-U'V) on spore germination and ultra-structure of Verticillium agaricinum. Transactions of theBritish Mycological Society 77, 187-189.

PARK, D. (1964) . Some properties of a staling substancefrom Fusarium oxysporum. Transactions of the BritishMycological Society 47, 541-546.

PARK, D. & ROBINSON, P. M. (1964). Isolation andbioassay of a fungal morphogen. Nature 203, 988-989.

ROBINSON, P. M . & PARK, D. (1965 ). The production andquantitative estimation of a fungal morphogen, Trans-actions of the British Mycological Society 48, 561-571,

VALADON, L. R. G . & MUMMERY, R. S. (1971). Effect oflight on nucleic acids, proteins and carotenoids ofVerticillium agaricinum. Microbios 4, 227-240.

VALDON, L. R. G. & CHAPMAN, D. J. (1983). Thenaphthoquinones (fun gal vacuolation factor) of Verti-cillium agaricinum. Microbios Letters 24, 115-119.

PESTICIDE EFFECTS ON GERMINATION OF BEA UVERIA BASSIANA

BY B. A. WATT, R. A. LEBRUN AND P . A. LOGAN

Department of Plant Pathology and Entomology, University of Rhode Island,Kingston RI 02881 , U.S.A.

Four fungicides and four insecticides significantly inhibited germination of B. bassianacompared with controls. The chemicals can be classed according to four levels of inhibition.Acylalanine and fenvalarate were least inhibitory, permethrin was slightly more inhibitory,azinphos-methyl and chlorothalonil were strongly inhibitory while metiram, carbofuran andmancozeb were the most inhibitory.

The effects of pesticides on entomogenous fungihave been extensively reviewed by Roberts &

Campbell (1977). Tedders (1981) has reportedinhibition of Beauveria bassiana (Bals.) Vuill.

Trans . Br , mycol. Soc. 82 (4), (1984) Printed in Grear Britain