C E R T A I N CHEMICAL R E Q U I R E M E N T S F O R G R O W T H AND S P O R U L A T I O N OF A L T E R N A R I A S P E C I E S by N. R. RAJDERKAR

Department o/Botany, Marathwada University, Aurangabad (18.V.1965) INTRODUCTION Studies on mineral nutrition of m a n y fungi have been made (see references) following pioneering work by STEINBERG (1935) on Aspergillus niger V. TIEGH., upon which much of our present information is based. LEWIS (1952) and NEWTON (1946) have shown that nutritional requirements for the growth of Alternaria sotani (ELL. & MARTIn) JONES & GROUT. is met by simple mineral solutions with glucose as an energy source. Deficiencies or excesses of mineral elements reduce to inhibit fungal sporulation, but there is little evidence that sporulation requires an element non essential to growth. With limiting amounts of certain elements, however, sporulation is drastically reduced, even though there is little or no effect oi1 growth. Other nutritional factors important to sporulation are concentrations of nutrients, carbon and nitrogen sources, C/N ratio, specific reproductive factors and vitamins. In general, disaccharides and polysaccharides support more fruiting than do monosaccharides: and within limits, low concentrations of sugar promote a more rapid onset of sporulation than high concentrations do. The ability to utilize nitrate, ammonium or organic nitrogen sources determines the extent of vegetative growth, and consequently, the reproductive capacity of a fungus. Some specific nitrogen compounds at high concentrations inhibit sporulation; however, the role of nitrogen in sporulation is more general. In recent years, the use of vegetable juice media has come into wider acceptance because of its property of stimulating sporulation of m a n y fungi; moreover, fungi such as Stemphylium solan( and Fusarium spp. which require light for sporulation, sporulated on vegetable juice agar without illumination. MILLER (1956) concluded that the capacity of the juice to stimulate sporulation did not lay in its mineral constituents.

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OF ALTERNARIA

Preliminary observations indicated t h a t vegetable juice agar was markedly superior to potato-dextrose-agar (PDA) in stimulating sporulation of A. nyctanthi and A. tropaeoli. The purpose of the present study was to devise a chemically defined medium on which the fungus would grow; constituents of vegetable juice or other metabolites could then be assayed on such a medium for stimulation of sporulation. MATERIAL AND METHOD

Single-spore cultures of Alternaria nyctanthi and A. tropaeoli spec. nov. were used throughout this study. Cultures were grown on vegetable juice agar (2% V-8 juice, 0.1% CaCO3 and 2% agar) and potato-dextrose-agar for 7 days at 24 C° in the dark, exposed to illumination (200 ft.C 0 for 2, 4, 6, and 8 hr; then returned to the dark for sporulation. TABLE I QuMitc~tive composition o/ 4 chemically de/i~ed media o~ which sporulation of A. nyctanthi and A. tropaeoli was compared.

Synthetic

White's

Czapek's

Vegetable juice

Glucose Glycir~e Nicotinic acid Thiamine Pyridoxine NH4NO ~ CaSO~

Sucrose Glycine Nicotinic acid Thiamine Pyridoxine Ca (NO3)2 Na~SO4 I~CI, K1. Na~HPO 4 MgSO4-7H20 Fe, Mn, Zn.

Sucrose

Glucose

NaNO~

NH~NO,~ CaC12 KC1 NH4H~PO 4 MgSO4.7H20 Fe, Mn, Zn.

KH~PO t MgSO4"7H~O Fe, tvln, Zn, Cu-A1

KC1 KH~HPO~ MgSO4-7H~P Fe

To test the hypothesis that vegetable juice does contain metabolites necessary for sporulation, particularly in the absence of illumination, a chemically defined medium, which permitted vegetable growth but not spornlation had to be devised, for bioassay for these specific sporulation metabolities using fractions of vegetable juice. Cultures were grown in the light and dark on several chemically defined media (Table I), with numerous modifications. The first medium tested was synthetic, the second White's medium subsequently modified by adding boron. The third mineral medium tested was one based on a crude estimate of vegetable juice, and the fourth medium tested was Czapek's liquid medium which was not modified. Reagent grade chemicals were used to make up the sever-

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al media. All of the glassware in contact with the media was cleanned in hot, concentrated nitric acid- and thoroughly rinsed. Ten fresh isolates of A. nyctanthi and A. tropaeoli from the leaf spots of Nyctanthes arbortristis and Tropaeolum majus were made and single spore cultures of each were grown on Czapek's liquid medium in watch-glasses in the dark at 28 ° C for 7 days. These cultures were examined for growth and sporulation. Single spore cultures were grown on Czapek's medium under nonsporulating conditions of irradiation at 26--30 ° C for 4 days. Sodium nitrate (0.1%) with or without sucrose (3%), was added to these cultures which then were placed under sporulating conditions of 20 ° C in the dark immediately or 2 hr later for 24 hrs. I

RESULTS

In continuous darkness, A. nyctanthi and A. tr@aeoli sporulated on PDA, but abundantly on 2% vegetable juice agar. On PDA, tile fungus responded markedly to short exposures to lamp irradiation, b u t on vegetable juice agar it responded only slightly. The test species did not grow on White's medium and slight modifications or on the synthetic medium and its modification. The chemical composition of the simplest modidication was qualitatively similar to Czapek's medium. TABL~ II

Variation in growth and sporulation of A. nyctanthi and A. tropaeoli on different media. Isolate

Synthetic

White's

Czapek's

Vegetable juice

At 1

+

++

+++

+++

At 2 At 3

+ +

+ +

+++ +++

+++ ++

+ + +

+ ++ +

A. nyctanthi

A. tropaeoli At 4 At 5 At 6 +++ = Abundant sporulation.

sporulation;

++ +++ +++

+ + = Moderate

sporulation;

++ +++ + = Sparse

The test species of Alternaria reproduced to the same degree on Czapek's medium and also on the synthetic vegetable juice medium, indicating that minimum requirements of the fungus are very simple. The fungus reproduced to the same degree on Czapek's and on

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the synthetic vegetable juice medium, indicating that minimum requirements of the fungus are very simple. A. tropaeoli and A. nyctanthi on Czapek's solution revealed a wide range of responses. The other isolates grew well but sporulated poorly, whereas all other isolates grew exceptionally well and also sporulated on Czapek's solution. The addition of N a N Q , with or without sucrose, to cultures which had been inhibited from sporulating by irradiation at 26 ° C did not prevent subsequent sporulation. A. nyctanthi sporulated well at 24 ° C while A. tropaeoli sporulated well at 26 ° C in all solutions. DISCUSSION

In this study, vegetable juice agar supported abundant fungal sporulation, particularly in continuous darkness. A. nyctanthi and A. tropaeoli grew and sporulated well on Czapek's medium whereas both the species did not previously grow on White's medium and a synthetic medium which contain glycine and 4 vitamines and added minor elements. This indicated that Alternaria species varies in capacity to grow and sporulate well on Czapek's medium. Certain isolates, freshly obtained grew and sporulated well on Czapek's medium; others sporulated poorly or did not grow at alt. This virtually eliminated the possibility that specific sporulation metabolites for Atternaria spp. were present in vegetable juice. The chemical requirements for sporulation of these species were further tested by studeying the effect of nitrogen availability.

Summary Fresh, single-spore isolates of Alternaria nyctanthi spec. nov. and Alternaria tropaeoli DESHPANDE & N. R. RAJDERKAR from the infected leaves varied widely in response when cultured on Coon's, PDA, Richard's and Czapek's medium. Some isolates did not produce macroscopically visible colonies, although the spores germinated. A few isolates grew poorly without sporulating; most of the others grew well and sporulated abundantly on PDA and Czapek-Dox medium. Cultures that had been inhibited from sporulating by irradiation at 26 ° C sporulated when subsequently placed in a 22 ° C incubator, even though nitrogen availability was varied by adding solutions of N a N Q , with or without sucrose.

Acknowledgements The author is grateful to Prof. M. N. KAMAT, Head of the Mycology and Plant Pathology Lab., M. A. C. S., Poona for valuable suggestions and to the Principal, Govt. College of Science, Nagpur for help, and to the Marathwada University for laboratory facilities.

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References COCHRANE, V. W. 1958, Physiology of Fungi. J o h n Wiley & Sons. Inc. 524 p. LEwis, R. W. 1952. The v i t a m i n n u t r i t i o n of Allernaria solani. Phytopathology 4 2 : 657--659. LILLY, V. G. 88 BARNETT, H. L. 1951. Physiology of t h e Fungi. p. 464. l~icGrawHill, New York. NEWTON, W. 1946. The growth of Sclerotinig sclerotiorum and Alternaria solani. Canad. J. agri. Sci. 26: 803--304. RAJDERKAR, N. R. 1966. The influence of nitrogen nutrition o n growth and sporulation of AIternaria solani (ELL. & MART.) JONES & GROUT. Mycopathol. et Mycol. appl. 29, 1-2: 55--58. STEINBERG, R. A. 1935. The nutritional requirements of the fungus Aspergillus niger. Bull. Torrey Bot. Club. 62: 81--90.