Phytopath. Z., 78,147-151 (1973)fc) 1973 Verlag Paul Parey, Berlin und HamburgISSN 0031-9481 / ASTM-Coden: PHYZA3

Department of Botany, Randfi University, India

Variations in Amino Acids and Sugars and Their Effecton Growth and Sporulation in Fusarium oxysporum f, udum

ByMAHENDRA PRASAD and SUSHIL KUMAR CHAUDHARY

With one figure

Received October 9, 1972

Changes in amino acid and sugar contents of a growing culture of Fusariumoxysporum f. udum were studied for their effects on growth and sporulation.

Material and Methods

Fusarium oxysporum f. ucium (Butl.) Sn, et H., obtained from diseased tissues of Cajanuscajan (L.) Millsp., was grown on a sucrose-nitrate medium (sucrose 50 g, KNO, 10 g, KHgPO,5 g, MgSOj-7HgO 2.5 g, double distilled water 1000 ml). Funga! mats were harvested everyday on Whatman filter papers no. 41 and 42 and the filtrates were passed through Budincrsuction funnels. The mats were repeatedly washed with distilled water to remove all tracesof liquid media. Fungal mats obtained in three replicates on Whatman paper no. 41 were usedfor dry weight estimates (PRASAD and CHAUUHARY 1966, 1967) whereas those sieved on paperno. 42 were utilized for diromatographic studies. For this purpose, the mycelial mats werecrushed and pulverized thoroughly in a homogenizer. The extracts were centrifuged at1000—3000 r.p.m., treated with 96% ethanol and concentrated on the water bath. Knownquantities between 5 and 30 //I were diromatographically analysed by standard methods foramino acids and sugars. Identifications and quantitative gradation.s were made by comparisonwith diromatograms of pure compounds. Culture filtrates were analysed in similar fashion.Macroconidia, mieroconidia and chlamydospores were estimated according to PRASAD andCHAUDHARY (1966,1967).

Results

The pH of the medium tended slowly but steadily towards weak alkalinity;it was 6.1 on the first day and 7.4 after 30 days. In two days old mycelial extracts

10*

148 PRASAD and CHAUDHARY

four amino acids were found (;'-amino butyric acid, alanine, glutamic acid,homoserine; figure). Parallel to this, sporulation began with the formation ofminute quantities of macro- and mieroconidia. With the production of glycincand serine on the third day a considerable increase in mycelial weight occurred.

-9—<b (D

MCROCONIDIA

MACROCONiDIA

CHLAMVDOSPORE

• RY WEIGHT OF MYCELIUM

AMINQ ACID/SUGAR PRESENT IN CULTURE FILTRATE

AMINO ACID/SUGAR PRESENT IN MYCELIAL EXT«ACT

THICKNESS OF THE LINE DENOTES THE AMOUNT OF. AMINO ACID/SUGAR ASSESSED AReiTRARILY

600

500

&00

300

> 200

100

• 50

540

Z20 •

] GLUCOSE

FRUCTOSE

GLUCOSE

FRUCTOSE

SUCROSE

GLUTAMINEHOMOSERINEHOMOSERINEHISTIDINE

ARGININEASPARTJC ACID

ASPARTIC ACIDOLUTAMIC ACIDGLUTAMIC ACID

SERtNEL-CYSTINE

ASPARAQNE

TSVPTOPHANE

GLVCINE

THREONINE

DL-ALANINE

DL-ALANINE

LYSINE

METHIONINE

PRDLINETYROSINEY-AMINO BUTYRIC ACIDY-AMINO BUTYRIC ACID

VALINE

p-PHENYL-ALAKINE

L-LEUC(NE/ISOLEUC)NE

10 12 U 16 18 20 22 2 i 26 28 30

GROWTH IN DAYS

Fig. Presence of sugars and amino acids, myccliai growth, and spore formationin growing cultures of Fusarium oxysporum f. udum

, Variations in Amino Acids and Sugars 149

On the fourth day with formation of leucine/isoleucine, proline, methionine,lysine, and aspartic acid in the mediuni botb sporulation and growth invigorated.By the sixth day, 17 amino acids were detected in the mycelial extract. Formationof mieroconidia and mycelium went on unhindered but from the seventh dayonwards the population of macroconidia rapidly declined. Arginine appearedun the 9th and histidine on the 10th day; occurrence of these amino acids isconsidered to be connected with the formation of chlamydospores. As the lastamino acids, tryptophane appeared after 11 and A"phenyl alanine after 15 days.

Very few amino acids could be found in the culture filtrates during thewhole growth period. Aspartic acid, homoserine and /'-amino butyric acidappeared and disappeared again after a certain time whereas alanine and glutamicacid remained during all the time.

Sucrose was present in the filtrates for the first three days together withglucose and fructose which increased together with the decrease of sucrose.Mycelial extracts contained glucose and fructose only (figure). With increasingage of the cultures, glucose decreased steadily and fructose more rapidly inmycelia and culture filtrates.

Discussion

In conformity with the observations of GHOSH et al. (1965), HASIJA (1968)and SRIVASTAVA and TANDON (1969), hydrolysis of sucrose into glucose andfructose takes place. Mycelial growth and spore formation were influenced bythe utilization of fructose and glucose and not for sucrose which could not bedetected in the mycelium. Utilization of fructose and glucose begins slowly andbecomes more rapid with the bulk formation of fungal mats and profusesporulation.

Amino acids have been investigated in cultures of various Fusaria (NATA-

KAjAN 1958, 2AKORDON£TS 1965, VENKATA RAM 1956). Similar to our observa-tions, BiLAi and ZAKORDONETS (1965) found fewer amino acids occurring insmaller quantities in culture filtrates than in mycelial extracts. MAHADEVAN

and CAROSELLI (1966), on the other hand, found a larger ammo acid pool inyounger culture filtrates than in older ones. In our cultures, alanine decreasedafter about 25 days. In unpublished studies we found that fusaric acid produc-tion increased many times after 25 days of incubation. It can be inferred thatalanine depletion has a bearing on toxin production. This confirms earlierobservations of KERN and SANWAL (1954), FLUCK and RICHLE (1955) andSANWAL (1956). The decline in alanine is in variance to the findings of VENKATARAM (1956).

Our observations suggest that cohesive relationships exist between theappearance of amino acids in the mycelium and the formation of various sporeforms. 7-amino butyric acid, alanine, glutamic acid, and homoserine couldinitiate formation of macro- and mieroconidia on the second day whereashistidine and arginine could induce chlamydospore production on the tenth day.A similar role of these two amino acids was reported by SEMPIO and RAGGI

(1966) in uredospore differentiation by Vromyces fahae. With the appearance

150 PRASAD and CHAUDHARY

of glycine and serine, rapid mycelial growth began. The linear increase inmycelia] weight and formation of microconidia and chlamydospores is notaffected by the presence or absence of amino acids during the whole growthperiod. Formation of macroconidia, however, decreased suddenly with thcappearance of asparagine and cystine and steadily declined thereafter.

Summary

Sugars and amino acids were analysed in mycelial extracts and culturefiltrates of Fusarium oxysporum f. udum in relation to growth and spore forma-tion. Sucrose is not assimilated as such and hydrolysis into glucose and fructose-is essential to initiate formation of micro- and macroconidia. Utilization ofglucose and fructose is slow at the beginning but increases with rapid growthand sporulation. Alanine, glutamic acid, ;.'-amino butyric acid and homoserineinitiate formation of macro- and microconidia on the second day. Arginine andhistidine stimulate chlamydospore formation on the 9th and 10th day. With theappearance of asparagine and cystine in the mycelial extract the number ofmacroconidia decreases whereas mycelial growth and formation of the otherspore forms continues steadily. The appearance of glycine and .serine correspondsto a rapid increase in mycelial weight. Larger numbers and amounts of aminoacids were found in mycelial extracts than in culture filtrates.

Zusa mmenf assung

Veranderungen im Gehalt an Zuckern und AminosSuren in Kulturen vonFusariHtn oxysporum f. udum im Zusammenhang mit Wachstum und Sporenbildung

Die in der Nahrlosung enthaltene Saccharose wird erst nach Spaltung inGlucose und Fructose vom Pilz verwertet. Die Verwertung beginnt langsam undnimmt mit intensiverer Myzel- und Sporenbildung zu. Mit dem Auftreten vonAlanm, Glutaminsaure, r-Aminobuttersaure und Homoserin im Myzel {nachzwei Tagen) setzt die Bildung von Makro- und Mikrokonidien ein, mit demAuftreten von Arginin und Histidin (nach neun und zehn Tagen) die Chlamydo-sporenbildung. Die Makrosporenbildung geht beim Auftreten von Asparaginund Cystin im Myzelextrakt zuruck, wahrend Myzelwachstum und Mikro-konidienbildung gleichmafiig weitergehen. Im Myzelextrakt wurden Amino-sauren in groRerer Zahl und Menge gefunden als im Kulturfiltrat.

Part of the Ph.D. thesis of the second author submitted to Randii University. We areIndebted to Prof. J. P. SINHA, Head of thc Department, for providing facilities. The secondauthor thanks the Government of India, Ministry of Education, for the financial assistanceaccorded under the Researdi Training Sdieme, We are grateful to Dr. P. N. SRIVASTAVA (atpresent at the University of Montreal, Canada) and Mr. R. K. VARSHNFY (Zoological Surveyof India, Patna) for technical guidance in diromatographic work and to Mr. J. N. DTIVEDYfor the preparation of the diagram. | ;

Variations in Amino Acids and Sugars 151

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SEMPIO, C , e V. RAGGI, 1966: Aminoacidi (iberi e combinate in piante di Fava (Vicia fabamajor) infectate con Uromyces fabae. Phytopath, Z. 55, 117—171,

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Address of the authors; Department of Botany, Ranchi University, Randii (Bihar),India.