a selective medium for the isolation of fusarium spp ...

Phytophylactica 18,67459 (1986)

A SELECTIVE MEDIUM FOR THE ISOLATION OF FUSARIUM SPP. FROM SOIL DEBRIS P. S. VANWYK 1, D. J. SCHOLTZ I andO. LOS 2

ABSTRACf Key words: Fusarium spp., soil debris, selective medium A rose bengal-glycerine-urea medium was developed for the isolation of Fusarium spp. from soil debris. It was at least as selective for Fusarium as Komada and Nash-Snyder media. On one sample it was more selective. Advantages of the present medium are that it is easy to prepare, and the colonies are transparent. Transparency of the colonies allows direct observation to determine whether they consist of one or more species. A list IS given of Fusarium spp., isolated with this medium from soil debris, plant material and wheat seed. Vittreksel 'N SELEKTIEWE MEDIUM VIR DIE ISOLASIE VAN FUSARIUM SPP. VfT GRONDDEBRIS 'n Rose bengal-gliserien·ureum-medium is ontwikkel vir die isolasie van Fusarium spp. uit stukkies organiese materiaal in grond.Jiierdie medium was net so doeltreffend as die medium van Komada of Nash-Snyder vir die selektiewe isolasie van Fusarium-isolate. Vit een grondmonster egter was dit hoogs betekenisvol meer selektief. Hierdie medium het die addisionele voordele gehad dat dit vinnig en maklik is om te berei en dat kolonies wat hierop ontwikkel het deursigtig was en direkte ondersoek moontlik gemaak het om te bepaal of een ofmeer spesies betrokke is. 'n Lys van Fusarium spp. wat reeds met hierdie medium uit grond, plantmateriaal en koringsaad geilsoleer is, word gegee.

mg/dm3 streptomycin was added after autoclaving. The Komada and Nash-Snyder media (Nelson et al., 1983) were chosen as standard media for the evaluation of RbGU agar.

Reproduced by Sabinet Gateway under licence granted by the Publisher ( dated 2012)

INTRODUCfION

Much research effort has gone into the development and standardization of culture media for the isolation, enumeration and indentification of Fusarium spp. from soil (Nash & Snyder, 1962; Stover, 1962; Wensley & McKeen, 1962; Papavizas, 1967; Abawi & Lorbeer, 1971; Fisher et ai" 1982; Elad & Chet, 1983; Nelson et al., 1983). Fusarium oxysporum Schlecht and F. solani (Mart.) Appel & Wollenw. have received the most attention, though the medium developed by Nash & Snyder (1962) for F. solani is also generally used to isolate other Fusarium spp. (Nelson et al., 1983). In preliminary work on the iS91ation of Fusarium spp. from soil, we tested a number of media. They were more or less selective for Fusarium spp. but the colonies were dense and opaque, so that it was impossible to determine microscopically whethh one or more species was involved. This paper compares a Fusarium-selective medium developed by the authors with two commonly used Fusarium media.

Isolation from soil Soils were processed by the debris-isolation technique of Nelson et al., (1983). The following soils were sampled: A. Glen Agricultural Coliege, Glen, O.F.S.: wheat planted for 5 years. B. Farm near Bloemfontein, O.F.S.: wheat planted for 5 years. C. Farm near Theunissen, O.F.S.: non-cultivated grassland pasture. D. Tobacco and Cotton Research Institute, Rustenburg, Tv!.: wheat experimental plots. E. Farm near Okahandja, South West Africa/Namibia: non-cultivated tree/grassland pasture. The pieces of debris were porous and it was difficult to surface disinfest them. Debns samples were therefore air dried overnight on sterile paper towels and finally dried in a desiccator for 12 h. This helped to keep the bacterial populations down. One hundred pieces of debris were plated (10 pieces/ plate) on each of the three media. Plates were incubated at 25°C for 6-- 10 d. Colonies developing from the debris pieces on the isolation plates were examined under a light microscope at lOOx magnification. Colonies consisting of a single species were transferred to potato dextrose agar (PDA) containing 50 mg/dm3 streptomycin. In cases where colonies consisted of more than one species, each species was single-spored (Nelson et al., 1983) directly from the isolation plate. All isolates identified as Fusarium spp. on the PDA plates were transferred to carnation leaf agar (CLA) (Fisher et al., 1982), singlespored, and identified according to Nelson et al. (1983).

MATERIALS AND METHODS

Isolation media A rose bengal-glycerine-urea (RbGU) medium was formulated with: (I) Rose bengal and streptomycin as bacterial suppressors; (2) Pentachloronitrobenzene (PCNB) to restrict the growth of Rhizopus and other Mucorales; (3) Urea and glycerine to restrict the growth of aerial mycelium, as with Verticillium dahliae (Van Wyk, 1969); (4) L-alanine to stimulate the production of conidia, as with V. dahliae (Van Wyk, 1969). The composition of the medium was: glycerine 10 g, urea I g, L-alanine 0,5 g, PCNB I g, rose bengal 0,5 g, agar IS g and water I 000 cm'. The ingredients were mixed and autoclaved at 121°C for 20 min and 50

Isolations from wheat seed and plant material Wheat seed was surface disinfested for I min in 3,5 % sodium hypochlorite (NaOCI), rinsed twice with sterile water and plated (IO/plate) on RbGU agar. Other material was surface disinfested for I min in 3,5 % NaOCI solution, rinsed twice with sterile water, and plated on RbGU agar.

Department of Plant Pathology, University of the Orange Free State, P.O. Box 339, Bloemfontein 9300 2 Glen Agricultural College, Private Bag XOI, Glen 9360 Received 18 June 1985; accepted for publication 28 October 1985 I

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TABLE I Total number of fungi and number of Fusarium spp. isolated from five soil samples' on three selective mediab ~

A

Total number of fungal isolates Total number of Fusarium isolates Selectivity fof Fusarium isolates (%) Selectivity Range RbGU Mean Komada Mean N-S Mean

8

RbGU

Medium Kom

N-S

103 84 8l,55A

153 1I8 77,12A

89 72 80,90A

RbGU 89 77 86,528

en

Soil Sample C

Medium Kom

N-S

138 91 67,418

99 83 83,848

tTl

E

0

RbGU

Medium Kom

N-S

RbGU

Medium Kom

N-S

RbGU

Medium Kom

N-S

110 110 lOOC

173 171 98,84C

165 148 89,69C

38 30 78,950

75 47 62,670

86 57 66,620

95 87 91,58E

76 57 75,OF

109 78 71,56F


~

e::::0 2l

::c

>-i

78,95-100 87,75 62,67-98,84 76,21 66,2-89,69 78,43

fii

C;;

0

r

~ 0 Z 0

...,

'T1

c::

~c::

:::en

TABLE 2 Number of Fusarium spp. isolated from five soil samples' with three selective mediab 00

< tTl

• See text for source b RbGU-Rose bengal-glycerine-urea Kom-Komada N-S-Nash-Snyder , Percentages followed by the same letter in each soil sample do not differ significantly as analysed by the normal z test for proportions

'"

qr

~ ~

Soil Sample

02903710

0

:::: en

A 8 C

0

P

D

0

tTl c:l

E

Fusarium spp. isolated RbGU

Medium Kom

I I 26

I 60

41 12 I 2

F. acuminatum F. chlamydosporum F. equiseti F. graminearum F. moniliforme F.oxysporum F. scirpi F. scirpi var. compactum F. solani Species I' Species II Species ill Species IV Total Fusarium isolates • See text for source of sample b RbGU = Rose bengal-glycerine-urea; Kom , Unidentified Fusarium spp.

84

RbGU

Medium Kom

2 32 2

I 25

8 40

34

52

30

I 36

37

31

3 2

5

6

9

1I8

N-S

72

= Komada; N-S = Nash-Snyder

N-S

RbGU

Medium Kom

N-S

16 48

34 62

30 66

Medium Kom

N-S

II

I 26

I 22

6 7

5 12

6 15

3 3

I 2

I 10

RbGU

RbGU

2

8 5 I

77

7

91

83

26 2 9 2 7

110

7 33 13 7 8 7

171

18 I 13 I 10 9

184

47

57

N-S

Total isolates

6 3

13 16

3 8

44 3 I 19 2

22

40

4 2

4 12

9

1I

3 1I3 479 2 25 444 19 86 59 38 16 2 20

57

78

1310

2 30

Medium Kom

87

~ en

P. S. VANWYK,D. J. SCHOLTZ &0. LOS

medium for the isolation of Fusarium spp. from a variety of soil samples. During this experiment colonies of two Fusarium spp. developed on the Nash-Snyder medium but not on the RbGU medium (Table 2). However, these species occurred infrequently and were isolated on the RbGU medium from other sources (Table 3). It seems therefore that it was not that the RbGU medium suppressed these species but rather that these species were not present in the debris plated on this medium. Advantages of a medium on which transparent colonies develop are that colonies which consist of more than one species can be recognised and isolated in pure culture. The isolates can be checked, grouped together and only representative colonies isolated. This is an important advantage in dealing with large numbers of isolates.

All isolates from plant material or seeds were transferred to PDA. Species identified as Fusarium were transferred to CLA (Fisher et at., 1982) single-spored, and identified according to Nelson et at. (1983). TABLE 3 Fusarium spp. isolated from necrotic plant parts, soil debris and wheat seed on RbGU agiu"

Fusarium spp.

Isolated from:

F. acuminatum F. chlamydosporum F. crookwellense F. culmorum F. equiseti F. graminearum

F. moniliforme F. oxysporum F.poae F. proliferatum F. sambucinum F. scirpi F. scirpi.var. compactum F. semitectum F. solani


F. subglutinans

wheat seed soil debris wheat seed soil debris wheat seed wheat plant. wheat seed soil debris wheat seed wheat plant wheat seed soil debris soil debris wheat seed soil debris soil debris plant materials (varietY of) wheat seed asparagus

Origin of sample Bethlehem Meiringspoort Bethlehem Several localities Bethlehem Bethlehem Bethlehem Several localities Bloemfontein Bloemfontein Bethlehem Bethlehem Bloemfontein Bloemfontein Several localities Several localities Several localities

papaya soil debris soil debris

Bethlehem Phutaditjhaba, QuaQua Somerset West Several localities Several localities

soil debris plant material soil debris wheat seed soil debris orchid leafspot

Several localities Several localities Several localities Bethlehem Kroonstad Saldanha Bay

ACKNOWLEGEMENTS

We thank Sentraalwes (Cooperative) and the Department of Agriculture and Water Supply for financial support, and Dr W. F. O. Marasas, National Research Institute for Nutritional Diseases, Medical Research Council, Tygerberg, for identifying some of the Fusarium isolates. REFERENCES ABAWI, G. S. & LoRBEER, J. W., 1971. Populations of Fusarium oxysporum f. sp. cepae in organic soils in New York. Phytophathology 61: 883-1039. ELAD, Y. & CHET, I., 1983. Improved selective media for isolation of Trichoderma spp. or Fusarium spp. Phytoparasitica 11: 55-58. FISHER, NANCY L., BURGESS, L. W., TOUSSOUN, T. A. & NELSON, P. E., 1982. Carnation leaves used as a substrate and for preservation of cultures of Fusarium species. Phytopathology 72: 151-153. NASH, SHIRLEY M. & SNYDER, W. C., 1962. Quantitiveestimation by plate counts of propagules of the bean rot Fusarium in field soil. Phytopathology 52: 567-572. NELSON, P. E., TOUSSOUN, T. A. & MARASAS, W. F. 0., 1983. Fusarium species: an illustrated manual for identification. Pennsylvania State Univ. Press. University Park. PAPAVIZAS, G. C., 1967. Evaluation of various media and microbial agents for the isolation of Fusarium 'from soil. Phytopathology 57: 848-852. STOVER, R. H., 1962. Fusarial wilt (Panama disease) of bananas and other Musa spp. Commonwealth Mycol. Inst., Phytopathalogical PaperNo. 4. VAN WYK, P. S., 1969. Die inhibisie van Verticillium dahliae. M.Sc Thesis, University of the Orange Free State. WENSLEY, R. N. & MCKEEN, C. D., 1962. A soil suspension-plating method for estimating populations of Fusarium oxysporum. f. melonis in muskmelon wilt soils. Canadian Journal of Microbiology 8: 57-{i4.

• Rose-bengal-glycerine-urea agar RESULTS AND DISCUSSION

The three media were equally selective for Fusarium isolates (Table 1) from four different soil samples (A-D), but RbGU agar was more selective for Fusarium from the remaining soil sample (E) than either of the other media. It seems therefore that the RbGU medium is selective for Fusarium spp. and can be used as a general

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