Phytopath. Z., 80, 279—282 (1974)© 1974 Verlag Paul Parey, Berlin und HamburgISSN 0031-9481 / ASTM-Coden: PHYZA3

Department of Botany, Ranchi University, India

In vitro Production of Fusaric Acid and its Impacton Growth and Sporulation in Fusarium oxysporum f. udum

By

MAHENDRA PRASAO and SUSHIL KUMAR GHAUDHARY

Received October 15, 1973

The relations between growth, sporulation and toxin production werestudied in cultures of Fusarium oxysporum f. udum (Butl.) Sn. et H.

Material and Methods

The pathogen was isolated from diseased plants of Cajanus cajan (L.) Millsp. and grownon a sucrose-nitrate medium, the carbon-nitrogen ratio being 5 :1 as suggested by SANWAL(1956) for optimum production of fusaric acid (Series I). According to another report (KALY-ANASUNBARAM and SARASWATHI-DEVI 1955), series II was prepared with the addition of 7/<gZn per 25 ml. At intervals of five days the fungal mats were collected on Whatman filterpaper nr. 42, repeatedly washed with distilled water and crushed in a homogenizer. Themycelial residue was centrifuged at 1000—3000 r.p.m., extracted with 96% ethanol anddiromatographically analysed for fusaric acid (ZAHNER 1954). Culture filtrates also wereanalysed. The identity of fusaric acid was confirmed by comparison with standard diromato-grams. Dry weight estimations were made (SRINIVASA PAI 1955) and spore populations werecoilnted with a haemocytometer slide (PRASAD 1968).

Results

In series I, microconidial formation progressed rapidly from the fifth day,readied its maximum on the 25th day and then declined. Ghlamydospore forma-tion started later and reached its maximum after 35 days (table). With progres-sing incubation, length of microeonidia and diameter of dilamydospores decreas-ed. Mycelial weight increased till 30 days and decreased later. Fusaric acid wasfound in traces only in mycelial extracts but in higher and increasing amountsm culture filtrates.

280 PRASAD and CHAUDHARY

TableMycelial weight (M), spore population and fusaric acid production (in mycelial extract MEand culture filtrate CF) of Fusarium oxysporum i. udum cultured without (S I) and with Zinc(S II). Number of spores given in millions/100 ml medium. Fusaric acid shown in arbitrary

units based on spot size

s

IIIIIIIIIIIIIIIIIIIII

Days

5

10

15

20

25

30

35

Mmg

135103368

160400978500

1226541

1077695899595817

Microconidia

number

5.1

2.017.25.9

25.08.6

58.113.375.420.362.516.056.2

7.8

lengthfi.

8.36.7

8.36.78.38.36.76.76.76.76.75.06.75.0

Chlamydospores

number

—

5.714.010.623.819.145.329.758.649.274.285.697.3

diameter

—

6.76.76.76.76.76.76.75.05.03.3

3.3

1.7

Fusaric acid

ME CF

— —— 3

1 3— 73 102 94 123 115 141 148 29

— 1912 50

In series II, production of microeonidia reached its maximum after 25 daystoo, but the total number was only half to one third of that recorded in series I.Chlamydospore formation showed a linear increase again but the total numberwas higher than without Zn. Microconidia and dilamydospores decreased in sizewith progressing incubation. Production of mycelium was much higher thanwithout Zn and increased mainly between 10 and 15 days. With Zn in themedium fusaric acid was detected in larger quantities in mycelial extracts andculture filtrates. As in series I, higher amounts of the toxin were present inculture filtrates than in corresponding mycelial extracts.

Discussion

A carbon-nitrogen ratio of 5 :1 is suitable for fusaric acid production. Theaddition of Zn stimulated toxin production and formation of mycelium andchlamydospores. Formation of dilamydospores and fusaric acid proceededsimultaneously; a close association between production of microconidia andsecretion of fusaric acid could not be seen.

In cultures with or without Zn, fusaric acid is produced parallel to the logphase of mycelial growth and is therefore not an autolysis product. SANWAL(1956) reached the same conclusion with Fusarium lycopersici. Considerableamounts of fusaric acid, however, are excreted into the culture filtrates after theautolysis of the mycelium sets in; this was observed by STOLL (1954) in the

In vitro Production of Fusaric Acid and its Impact 281

cultures of Gibberella fujikuroi. In contrast to the observations of SANWAL(1956) who found exogenous secretion of fusaric acid only, the toxin was detect-ed in mycelial extract of Fusarium oxysporum f. udum. This corresponds withearlier observation on another strain of Fusarium oxysporum (STARRATT andMADHOSINGH 1967). Retention of fusaric acid in the mycelium and excretioninto the culture solution increase with the age of the cultures.

Fusaric acid has been detected in culture filtrates of several Fusaria causingwilt diseases (GAUMANN 1957, HEITEFUSS, STAHMANN and WALKER 1960, PAGE1961 and TRIONE 1960). Its production in the rhizosphere of tomato plants byFusarium lycopersici was reported by KALYANASUNDARAM (1958). Of two Fusa-rium isolates from sweet potato the strain causing wilt (but not the one causingsurface rot) produced fusaric acid (FISHER 1965). Production of fusaric acid byFusarium oxysporum f. udum is reported here for the first time, specially fromthe mycelial extract.

Summary

Fusarium oxysporum f. udum, the causal organism of wilt of Cajanus cajan(pigeon pea) liberated fusaric acid in vitro during the rapid growth phase of themycelium. This toxin was detected both in the mycelial extract as well as in theculture filtrate, in the former for the first time, showing that it was both retain-ed endogeneously and excreted extracellularly by the pathogen. Addition of Znin the culture medium stimulated the building up of fusaric acid considerablyand lessened the output of microconidia.

Zusammenfassung

In vitro-Produktion von Fusarinsaure und ihre Wirkung auf Wachstumund Sporulation von Fusarim oxysporum f. udum

Fusarium oxysporum f. udum, der Erreger einer Welkekrankheit an Caja-nus cajan (pigeon pea, Erbsenbohne) bildete in vitro Fusarinsaure in der Phasedes raschen Myzelwachstums. Das Toxin konnte in Myzelextrakten und Kultur-filtraten nachgewiesen werden. Zugabe von Zinkionen zur Nahrlosung stimu-lierte die Bildung von Fusarinsaure betrachtlich und verminderte die Bildungvon Mikrokonidien.

Part of the Ph.D. thesis of the second author submitted to Ranchi University. Theauthors are grateful to Professor J. P. SINHA, Head of the Department, for providing researdifacilities.

Literature

FPSHER, K. D., 1961: Some physiological comparisons of the Fusarium surface rot and Fusariumwilt pathogens of sweet potato. Diss. Abstr. 22, 21.

GAUMANN, E., 1957: Ober Fusarinsaure als Welketoxin. Phytopath. Z. 29,1—44.

282 PRASAD and CHAUDHARY, In vitro Production of Fusaric Acid and its Impact

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KALYANASUNDARAM, R., and L. SARASWATHI-DEVI, 1955: Zinc in the metabolism of Fusariumvasinfectum Atk. Nature 175, 945.

, 1958, Production of fusaric acid by Fusarium lycopersici Sacc. in the rhizosphere oftomato plants. Phytopath. Z. 32, 25—34.

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STARRATT, A. N., and C. MADHOSINGH, 1967: Sterol and fatty acid component of myceliumoi Fusarium oxysporum. Canad. J. Microbiol. 13, 1351—1355.

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Authors' address: Department of Botany, Ranchi University, Randii (Bihar), India.