Actinomycetes University of Udine, Mycology Department ISSN: 0732-0574 Vol. 1, Num. 1, 1990

Problems In The Selective Isolation Of Streptoverticillium Species From Soil

S.CARDINALI & G.FIRRAO

Chair of Mycology, University of Udine, Italy

Code Number: AC90004
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Abstract.

Selective isolation of streptoverticillia from natural habitats still poses a problem. Recently claimed solutions to this difficulty do not seem reproducible.

Physiological and biological homogeneity between streptoverticillia and streptomycetes (Collins et al., 1977; Lechevalier et al. 1977; Minnikin and O'Donnell, 1984; Pridham and Tresner, 1974; Kroppenstedt et al., 1981) is recognized.

At present about one hundred actinomycete strains are placed in the genus Streptoverticillium and some fifty specific epithets have been proposed inside the genus (Baldacci and Locci, 1974).

The situation is obviously biased in proportional terms. However it should be mentioned that most species of the genus Streptoverticillium have been proposed by industrial firms and secondly that isolation of streptoverticillia is problematic.

In ecological terms the situation is disturbing. The majority of described species consist of antibiotic producing microorganisms, fortuitously collected during screening for biologically active metabolite producers.

Hanka et al. (1985) suggested a method for the selective isolation of streptoverticillia using oxytetracycline (10 ug/ml) to inhibit streptomycetes. According to them this method augments the number of recoverable streptoverticillia from 6 to 20%. An increase in the oxytetracycline level to 25 ug/ml according to Hanka and Schaadt (1988), improves streptoverticillia recovery by a further 35%. In addition the authors claim that by using lysozyme (activity not specified) isolation rates can be raised as high as 60%.

According to Attwell et al. (1985) most streptoverticillia are resistant to lysozyme, in contrast to the majority of streptomycetes.

In this connection experiments were run in order to check isolation methods for the organisms.

Materials And Methods

Techniques proposed by Hanka and collaborators (Hanka et al., 1985; Hanka and Schaadt, 1988), together with some possible variations, were tested.

Cycloheximide and nystatin at a concentration of 50 ug/ml were used to limit fungal growth. Specific inhibitors were sterilized by filtration and added to the molten medium.

To reduce bacterial growth soil samples were heat-treated and soil suspensions spread over Millipore membranes (pore size 0.45 um) placed on the agar.

Heat treatments (dry and moist) were carried out at 55 and 70 C (the latter for moist samples only) for 5, 10, 20 and 40 minutes. Five repetitions for each treatment were made. Millipore membranes were removed after seven days and the plates incubated for a further week (Hirsch and Christensen, 1983).

Following incubation actinomycete and bacterial colonies were counted and results statistically analyzed.

The ability of oxytetracycline to discriminate between streptomycetes and streptoverticillia was investigated by testing several culture collection strains and by screening 30 soil-samples from different environments in Friuli (Northern Italy). The use of lysozyme, suggested by Attwell et al. (1985), was also taken into account and tests were carried out using the enzyme alone or in combination with oxytetracycline.

Table 1. Collection strains used in sensitivity tests (ATCC=American Type Culture Collection, Maryland, USA; CBS=Centraalbureau for Schimmelcultures, The Netherlands; DPDU=Culture Collection of the Department of Plant Defence, University of Udine, Italy; HACC=Hindustan Antibiotics Co., Pimpri, India; SMP=Department of Biological Sciences, Manchester Polytechnic, UK).

 Streptomyces alboflavus, DPDU 0721 (CBS123)
 Stm. albus, DPDU 0777 (ATCC25427). Type strain
 Stm. cespitosus, DPDU 0152 (ATCC 27422)
 Streptomyces sp., DPDU 0850, DPDU 0853 and DPDU 0868
 Stm. viridochromogenes, DPDU 0058 (ATCC14923)
 Streptoverticillium baldaccii, DPDU 0819 (ATCC 23654). Type
  strain
 Sty. cinnamoneum var.cinnamoneurn, DPDU 0093 (ATCC 11874)
 Stv. cinnarnoneum subsp. lanosum, DPDU 0281 (ATCC 25187)
 Stv. hiroshimense, DPDU 0100 (ATCC 19772)
 Stv. mashuense, DPDU 0258 (ATCC 23934)
 Stv. roseoverticillatum var.albosporum, DPDU 0138 (HACC227)
 Streptoverticillium sp., DPDU 0820 (SMP 1), DPDU 0863 and
  DPDU 0826 (SMP 3)

Sixteen strains (Tab. 1) were tested for their ability to grow in the presence of 5, 10, 15 and 20 p.g/ml of oxytetracy- cline and of 480, 750, 1500, 2250 and 3000 units/ml of lysozyme. Four repetitions were made for each and the results were statistically analyzed.

Results And Conclusions

Preliminary heat-treatment to eliminate vegetative growth of bacterial forms did not seem to be effective. On the other hand the use of Millipore membranes can be recommended, but taking the precaution of drying them first for 15 minutes in a sterile-flow cabinet to avoid diffusion of bacteria.

With regard to the medium composition it was found that there were no real differences between that suggested by Hanka et al. (1985) for selective isolation of streptoverticillia and the one proposed by Vickers et al. (1984) for the isolation of streptomycetes; it must be pointed out that variations in carbon and/or nitrogen sources are ineffective for the selective isolation of strepto- verticillia.

In several instances oxytetracycline did not seem to discriminate between organisms belonging to the genus Streptomyces and the genus Streptoverticillium (Tab. 2). The growth of representatives of both genera is limited by the suggested concentrations of oxytetracycline.

Similarly the use of lysozyme does not seem to produce the expected results, at least at the suggested concentration; resistance is more widely distributed among streptomycetes than hitherto believed (Tab. 2).

By adopting the suggested procedure to screen a series of some 30 soil samples only occasionally Streptoverticillium representatives were isolated. It thus appears that the problem of selective isolation of Streptoverticillium is still far from solved.

Table 2. Oxytetracycline and lysozyme sensitivity in Streptoverticillium and Streptomyces strains, expressed as percentage of colonies growing on test media. Because of the results scattering, only variations of more than 15% are significant.

STRAIN              OXYTETRACYCLINE   LYSOZYME
                       (ug/ml)        (units/ml)
No(DPDU)             5   10  15  20    480  750 1500 2250 3000
--------------------------------------------------------------
Streptoverticillium 
0138                100  52  37   9    100  100  100  100  100
0820                 46  25   4   0    100  100   88   99   94
0826                 40  13   4   0    100  100  100  100  100
0258                 90  73  83  76    100   95  100  100  100
0093                100 100 100 100    100  100  100  100  100
0863                 97  76  46  33     76   68   71   76   78
Streptomyces 
0850                 50  75  51  30    100   38   10    6    3
0058                  0   0   0   0     98   25    0    0    0
0152                 55  31  10   0    100  100   44    1    0
0868                 75  50  58  53     83   45   16    9    6
0853                 90  80  70  65     94   42   17   10    8
0721                 76  73  51   0     98   51    0    0    0
0777                 88  84  69  70     74   92   53   31   22

References

Attwell, R.W., A. Surrey & T.Cross (1985). Lysozyme sensitivity in Streptoverticillium species. System.Appl. Microbiol., 6, 239-242

Baldacci, E. & R.Locci (1974). Genus II. Strep- toverticillium Baldacci, Farina and Locci, 1966, 168. In: (R.E.Buchanan & N.E.Gibbons, eds.) "Bergey's Manual of Determinative Bacteriology", 8th ed., The Williams & Wilkins Co., Baltimore, pp.829-842

Collins, M.D., T.Pirouz, M.Goodfellow & D.E. Minnikin (1977). Distribution of menaquinones in actinomycetes and corynebacteria. J. gen. Microbiol., 100, 221-230

Hanka, L.J., P.W.Rueckert & T.Cross (1985). Method for isolating strains of the genus Streptoverticillium from soil. FEMS Letters, 30, 365-

Hanka, L.J. & R.D.Schaadt (1988). Methods for isolation of streptoverticillia from soils. J. Antib., 41,576-578

Hirsch, C.F. & D.L.Christensen (1983). Novel method for selective isolation of actinomycetes. Appl. Env. Microbiol., 46, 925-929 Kroppenstedt, R.M.F.Korn-Wendisch, V.J.

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Lechevalier, M.P., C.DeBievre & H.A.Lechevalier (1977). Chemotaxonomy of aerobic actinomycetes: phospholipid composition. Biochem. System. Ecol., 5, 249-260

Minnikin, D.E. & A.G.O'Donnell (1984). Actinomycete envelope lipid and peptidoglican composition. In: (M.Goodfellow, M.Mordarski & S.T.Williams, eds.) "The Biology of the Actinomycetes". Academic Press, London, pp. 335-388

Pridham, T.G. & H.D.Tresner (1974). Streptomyces Waksman and Henrici 1943. In: (R.E.Buchanan and N.E. Gibbons, eds.) "Bergey's Manual of Determinative Bacteriology". The Williams & Wilkins Co., Baltimore, 8th ed., pp. 747-829

Vickers, J.C., S.T.Williams & T. Cross (1984). A taxonomic approach to selective isolation of streptomycetes from soil. In: (L.Ortiz-Ortiz, L.F.Bojalil & V.Yakoleff, eds.)"Biological, Biochemical, and Biomedical Aspects of Actinomycetes". Academic Press, London, pp.553-561.

Copyright 1990 C.E.T.A., The International Centre for Theoretical and Applied Ecology, Gorizia