Mycopathologia 99:9-13 (1987) 9 Martinus Nijhoff/Dr W.

Junk Publishers, Dordrecht - Printed in the Netherlands

Preliminary studies on the use of biochemical and physiological tests for the characterization of Fusarium isolates
E. H. Wasfy, 1 P. D. Bridge 2 & D. Brayford 2

1Faculty of Agriculture, Alexandria University, Egypt; 2CAB International Mycological Institute, Ferry Lane, Kew, UK
Received 26 August 1986; accepted in revised form 14 January 1987

Key words: Fusarium, enzymes, biochemistry, taxonomy

Abstract

Twenty-two isolates of Fusarium were examined for their ability to grow on a range of carbon sources, resistance to inhibitory chemicals, and their API ZYM enzyme profiles. Growth on common carbon sources showed little discrimination between isolates, but they could be differentiated on the basis of their enzymic activity and tolerance to inhibitors, particularly copper sulphate and malachite green.

Introduction

The identification of isolates in the genus Fusarium to species level is currently based mainly upon the characteristics of the spores produced and the conidiogenous cells from which they arise (2, 8, 9). Some species have races with restricted host ranges in that isolates may be assigned to special forms or pathotypes on the basis of host responses following infection (1). Given that members of this genus may have considerable economic importance in plant pathology, mycotoxicology and biotechnology and that there is often distinct variation between isolates of the same species, it is desirable to be able to accurately characterize specific strains. However, morphological features do not allow such precise differentiation to be made between isolates of the same species. Physiological and biochemical activities are used routinely in the characterization of bacteria and yeasts, and tests based upon these types of properties are now starting to be adopted for use with filamentous microfungi. Physiological tests have

been shown to be useful in the differentiation of isolates and species within a number of genera including PenicUlium (6) and Fusarium (11). The API ZYM system has been employed for some years in bacteriology and has recently produced encouraging results in Penicillium, Monascus, and some thermophilic fungi (4, 5, 10). This contribution presents preliminary results on the use of some physiological and biochemical characters within the genus Fusarium, carried out in order to determine whether similar approaches were likely to lead to improvements in the characterization and identification of such isolates.

Methods

Isolates studied
Twenty-two isolates belonging to 15 species were studied (Table 1), namely: E acuminatum Ell. & Everhart, E avenaceum (Fr.) Sacc., E chlamydosporum Wollenw. (syn. E fusarioides

10
Table 1. D i f f e r e n t i a l test results.
Isolate Host IMI a Raffinose Agar Liquid Final p H in citric acid Crystal violet Malachite green t W + + + + + + + W W + W W t + + + + + + t + Copper sulphate
t

G r o w t h in the p r e s e n c e o f

F. acuminatum F. acuminatum F. avenaceum F. chlamydosporum t7. culmorum F. decemcellulare F. equiseti F. equiseti F. equiseti F. graminearum F. graminearum F. lateritium F. oxysporum F. oxysporum F. pallidoroseum F. pallidoroseum F. pallidoroseum F. poae F. sambucinum F. solani F. sporotrichioides F. tricinctum

Oryza Arachis Dianthus

Unknown

136675 302356 299088 158405 164746 295901 102097 301088 302149 193939 299154 296351 299219 301173 157845b 302036 302039 105506 139165 299498 144270 175449

+ + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + +

<7 > 7 > 7 < 7 >7 <7 > 7 < 7 > 7 > 7 > 7 >7 >7 < 7 > 7 > 7 < 7 > 7 < 7 < 7 > 7 >7

W W W + + + + + + + W + + + + + t W + + t

Zea Theobrorna Arachis

Unknown

Cajanus Zea Zea Acer Hibiscus

Air

Citrus Acacia Acacia Triticum Populus A llium

Soil Unknown

+ = positive; - = negative; W = w e a k g r o w t h ; t = t r a c e g r o w t h . a A c c e s s i o n n u m b e r s in the culture collection o f the C A B I n t e r n a t i o n a l M y c o l o g i c a l Institute, K e w .

(Frag. & Cif.) C. Booth), F. culmorum (W.G. Sm.) Sacc., E decemcellulare Brick, F. equiseti (Corda) Sacc., F.. graminearurn Schwabe, E lateritium Nees, F. oxysporum Schlecht. emend. Snyder & Hansen, E pallidoroseum (Cooke) Sacc. (syn. E semitectum Berk. &Rav.), E poae (Peck) Wollenw., E sambucinurn Fuckel, F. solani (Mart.) Sacc. emend. Snyder & Hansen, F. sporotrichioides Sherb., and F. tricinctum (Corda) Sacc. Isolates were grown initially on Czapek-Dox (Cz) agar at room temperature before subculturing onto tap-water agar. Inoculation was by agar plugs (4 mm diam) from cultures grown on tap-water agar at room temperature ( - 2 0 ~ for 7 days.

Carbon sources
The ability to grow on particular carbon sources was tested using both solid and liquid culture media. The basal medium used in each case was Czapek-Dox medium with the inclusion of 10 g1-1 o f one of the required carbon sources. The carbon sources used for solid media were glucose, maltose, raffinose and sucrose. The carbon sources used for the liquid media were lactose, maltose, raffinose and sucrose. The ability to grow and cause a pH rise on citric acid was tested using liquid medium LB containing 0.05% (w/v) bromocresol purple as described by Bridge (3). All carbon assimilation tests were compared to negative controls consisting of the basal medium with no added carbon source. Incubation was at room temperature for 14 d in the case of liquid media and 21 d for solid media.

11
Enzymic activities

Catalase activity was tested by placing 3 drops of 3~ (v/v) hydrogen peroxide onto the edge of a 7-day-old culture on Cz agar. A positive reaction was seen as effervescence. The ability to degrade RNA was tested using the method of Bridge (3). The ability to degrade starch was tested on Cz agar containing 1% (w/v) soluble starch as the sole carbon source. The utilization of the starch was seen as growth and a clear zone around the colony after flooding with Gram's iodine after 14 days incubation. The API ZYM system (API Ltd) was used to test for the presence of extracellular enzymes in culture fluid. Cultures to be tested were grown in liquid Cz medium with sucrose as the sole carbon source at room temperature for 4 weeks. The mycelium was removed from the culture fluid by centrifugation and the culture fluid was used to inoculate the API ZYM strip directly. Incubation of the strips was for 4 h at 37 ~ and they were then read according to the manufacturers instructions.

ble 1). All isolates with the exception of E avenaceum (IMI 299088) were able to grow on raffinose in both liquid and solid media. This result was confirmed by repetition. The results for the API ZYM tests are given in Table 2. Differential responses were detected for phosphoamidase, cegalactosidase, /3-glucosidase and N-acetyl-8-glucosaminidase. Where several strains of the same species were tested, marked differences in response were detected. This might be expected since the strains used were deliberately chosen to encompass something of the range of morphological variation within the species. Isolate IMI 157845b of E pallidoroseum did not produce reliable results on the API ZYM strips, results being very weak and irreproducible. Another isolate of E pallidoroseum (IMI 302039) produced copious amounts of a dark red pigment which prevented the API ZYM colour changes being read. The results of these two isolates are therefore not included in Table 2.

Discussion
[nhibitors

Filter-sterilized compounds were included into Cz agar plates. These plates were inoculated and incubated for 14 days at room temperature. The inhibitors used, and their final concentrations, were copper sulphate (0.5 g 1-1), crystal violet (0.01 & 0.05 g 1-1), malachite green (0.003 g 1-I), sodium azide (0.1 g 1-1) and zinc sulphate (3 g 1 1).

Results
All isolates tested were able to grow on citric acid, glucose, lactose, maltose, starch and sucrose as sole carbon sources. Catalase activity was detected in all of the strains and all were able to degrade RNA. Similarly all isolates were inhibited by sodium azide but were able to grow in the presence of crystal violet (0.01 g 1-1) and zinc sulphate. However, the results for final pH on citric acid and growth with crystal violet (0.05 g 1-1), malachite green and copper sulphate were differential between strains (Ta-

In Penicillium and Monascus, characteristics such as substrate utilization, inhibition and API ZYM tests have been used successfully in taxonomic schemes (4, 5). The results of this preliminary study indicate that similar methods may prove to be of value in the characterization of Fusarium isolates. Further Fusarium isolates now merit examination to determine if the discriminatory tests hold for isolates of different origins. The results of the carbon assimilation tests showed that these have a very low discriminatory value. The carbon utilization results are in agreement with others given in the literature for species of Fusarium (7). An interesting result from this category is the ability of the isolate of E avenaceum studied to grow significantly more than the negative control on raffinose when on a solid, but not in a liquid medium. A number of species of Fusarium have been reported to grow poorly on raffinose (7) and it appears that this is better seen in liquid media, where trace levels of available nutrients are not provided by agar.

12
Table 2. Isolate
API ZYM

results.
IMI API ZYM

test number a 4
-

1 F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. F. acuminatum acuminatum avenaceum chlamydosporum culmorum decemcellulare equiseti equiseti equiseti graminearum graminearum lateritium oxysporum oxysporum pallidoroseum poae sambucinum solani sporotrichioides tricinctum
136675 302356 299088 158405 164746 295901 102097 301088 302149 193939 299154 296351 299219 301173 302036 105506 139165 299498 144276 175449 W + W W W W W + + + W + W W + + W W W W

2
W W + W + W + + + + W + + + + + + + W W

3
W W W W W W + W + + W + W W + + W W W W

5
+ W W W W W W W + W + + W W W + W W + W

6
W W W + W W W W + W W W W W + W W W + W

9
-

10
+ + +

11
W W W + W + W W

12

13

14

15

16
W

17

18

19

W
W

. . . .

+ + + + W

+ + + + W + W + W W

W + + +

W + + W

+ W + + + + + + W

+ W W W

W W W

+ = positive reaction between 3 & 5 on the manufacturers scale; - = no discernible colour change. a 'Enzymes tested for: 1 = alkaline phosphatase; 2 = e s t e r a s e ( C 4 ) ; 6 = valine arylamidase; 7 = Cystine arylamilase; 8 = T r y p s i n ;
12 = a - g a l a c t o s i d a s e ; 13 = / 3 - g a l a c t o s i d a s e ;

W =

positive reaction between

1 & 3

on the manufacturer's scale;

3 = esterase/lipase 9 = Chymotrypsin;

(C4); 4 = Lipase (C14); 5 = 10=

acid phosphatase;

14 = / 3 - g l u c u r o n i d a s e ;

15 = c ~ - g l u c o s i d a s e ;

leucine arylamidase; phosphoamidase; 16 = / 3 - g l u c o s i d a s e ; 17 = N-acetyl-~11 =

glucosaminidase; 18=c~-mannosidase; 19=c~-fucosidase.

A rise in pH associated with growth on various specific carbon and nitrogen sources has been detected in a number of previous studies (39). The mechanisms involved have not been fully explained, but the pH rise has proved to be a reproducible characteristic which may be taxonomically useful (3, 6). In this study, the pH rise was only seen after approximately 10 d incubation when the cultures were approaching stationary phase, and so may be associated with ammonium excretion or autolysis

(6).
The results for growth with inhibitory compounds appear useful for discrimination between isolates and may be of value in the in vitro separation of special forms. Also, since malachite green and copper sulphate are used commercially as fungicides, albeit in widely different contexts, an abil-

ity to tolerate these compounds may have considerable practical implications. The API ZYM results indicate, at this preliminary stage, that enzyme profiles may have more potential in isolate characterization than in species delimitation. This is seen by the variation shown between the isolates of E acuminatum and also of F. equiseti. The enzyme profiles shown in this study are derived from culture fluid and so will consist entirely of extracellular enzymes associated with the isolate's growth on the basal medium. This probably accounts for the lowered activity and restricted range of results seen here in comparison to those given by conidial suspensions in Monascus and Penicillium (4, 5). The results do however show higher activities than those seen with thermophilic fungi and suggest that further evaluation of this

13 t e c h n i q u e s h o u l d be c a r r i e d o u t in F u s a r i u r n . T h i s p r e l i m i n a r y s t u d y s h o w s t h a t t h e relatively rapid used physiological in recent and biochemical with other techniques filamentous studies 4. ochemical methods as an aid to the characterization of subsection Fasciculata. J. G. Microbiol. 131:1887-1895, 1985. Bridge PD, Hawksworth DL: The API ZYM enzyme testing system as an aid to the rapid identification of Penicillium isolates. Microbiological Sciences 1:232-234, 1984. Bridge PD, Hawksworth DL: Biochemical tests as an aid to the identification of Monascus species. Letters in Applied Microbiology 1:25-29, 1985. Bridge PD, Hawksworth DL, Kozakiewicz Z, Onions AHS, Paterson RRM, Sackin M J: An integrated approach to Penicillium systematics. In: Samson RA, Pitt JI (eds) Advances in Penicilliurn and Aspergillus Systematics. Plenum, New York, 1986 ['1985'1, pp 281-309. Domsch KH, Gams W, Anderson T - H : Compendium of Soil Fungi, VoI. 1. Academic Press, London, 1980. Gerlach W, Nirenberg H: The genus Fusarium - a pictorial atlas. Mitteilungen aus der Biologischen Bundesanstalt f/it Land- und Forstwirtschaft, Berlin-Dahlem 209:1-406, 1982. Nelson PE, Toussoun TA, Marasas WFO: Fusarium Species. An Illustrated Manual for Identification. Pennsylvania State University Press, University Park & London, 1983. Satyanaryana T, Chavant L, Montant C: Applicability of API ZYM for screening enzyme activity of thermophilic moulds. Transactions of the British Mycological Society 85:727-730, 1985. Thrane U: The ability of common Fusariurn species to grow on tannin-sucrose agar. Letters in Applied Microbiology 2:33-35, 1986.

Penicillium

m i c r o f u n g i m a y be u s e f u l for t h e c h a r a c t e r i z a t i o n o f i s o l a t e s o f F u s a r i u r n . T h e s e t e c h n i q u e s m a y be o f use in i s o l a t i n g s p e c i f i c p r o p e r t i e s in s c r e e n i n g p r o c e d u r e s o r ' s p o t tests' in p r a c t i c a l s i t u a t i o n s a n d r e q u i r e little specialist e q u i p m e n t . 6. 5.

Acknowledgements
7. E.H.W. w o u l d like to t h a n k t h e B r i t i s h C o u n c i l for t h e s u p p o r t o f his visit to t h e C A B I n t e r n a t i o n a l M y c o l o g i c a l I n s t i t u t e at Kew in M a r c h - M a y 1986 9. w h e n t h e w o r k r e p o r t e d here was u n d e r t a k e n . T h e t e c h n i q u e s e m p l o y e d in this s t u d y were d e v e l o p e d at t h e I n s t i t u t e as p a r t o f t h e S E R C c o n t r a c t no. SO/17/84. 10. 8.

References
1. Armstrong GM, Armstrong JK: Formae speciales and races of Fusariurn oxysporurn causing wilt diseases. In: Nelson PE, Toussoun TA, Cook RJ (eds) Fusarium - Diseases, Biology and Taxonomy. Pennsylvania State University Press, University Park and London, 1981, pp 391-399. 2. Booth C: The Genus Fusariurn. Kew: Commonwealth Mycological Institute, 1971. 3. Bridge PD: An evaluation of some physiological and bi-

11.

Address f o r offprints:

Dr D. Brayford CAB International Mycological Institute Ferry Lane, Kew Surrey TW9 3AF, UK