Actinomycetes University of Udine, Mycology Department ISSN: 0732-0574 Vol. 4, Num. 1, 1993

B.PETROLINI, S.QUARONI, M.SARACCHI and P.SARDI

Institute of Plant Pathology, University of Milan, via Celoria 2, I-20133 Milan, Italy

Code Number: AC93002
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ABSTRACT. A sporangiate actinomycete (IPV-2867 = NCB 1173), isolated from decomposed leaf litter of Prunus persica using an Andersen sampler connected with a sedimentation chamber, has been studied. The characteristic features of spore-bearing structures differentiate it from all previously described genera. On the grounds Or the wall chemotype III and whole-cell sugar pattern B, this organism is regarded as new genus of the maduromycetes: Planopolyspora gen. nov., type species P. crispa sp nov.

The sure identification at the genus level of actinomycetes can rarely be made on the basis of morphology alone, nevertheless the morphological features of an unknown isolate can provide useful and rapid clues to its identity. The configuration of sporulating structures and the spore arrangement especially permit the recognition of previously described organisms. Therefore direct observations of these structures on isolation plates is a first step in the detection of rare or novel actinomycetes.

During isolations from decomposed leaf litter of different plants, carried out to detect rare genera of actinomycetes (Petrolini et al., 1992), a strain, characterized by unusual spore-bearing structures containing motile spores, was isolated.

The present paper reports a detailed description of this isolate. Morphological characteristics precluding its placement in any of the previously described genera, it is proposed to include it in a new genus, Planopolyspora, the type species being P. crispa.

MATERIALS AND METHODS

Bacterial strain. Strain IPV-2867 (= NCB 1173) was isolated from Prunus persica leaf litter, using an isolation procedure previously described (Petrolini et al., 1992). It was detected on a plate of WA25 (2.5% agar in tap water) containing 50 ppm of tunicamycin, after 14 dd of incubation at 26 C.

Cultural and morphological characterization. Cultural characteristics were determined after 7,14 and 21 dd of incubation at 26 C. The following media were used: tap water agar (WA25), ISP media 2, 3, 4 and 7 (Shirling and Gottlieb, 1966), nutrient agar Difco (NA), thin potato-carrot agar (TPC) (Higgins et al.,1967), modified Bennett agar (BA) (Jones, 1949), Czapek solution agar Difco (CA), Czapek yeast extract agar (CAY) (CA+2 g/l yeast extract). Colours of substrate and aerial mycelia were determined using the colour chart of Prauser (1964).

Morphological characteristics were examined by light microscopy and scanning electron microscopy (SEM). For SEM observations, small blocks of medium bearing good growth were removed from plates and prepared by a technique previously described (Locci and Petrolini Baldan, 1971). Suspensions of sporangia in ultrapure water (Milli-Q Plus Water Purification System, Millipore S.p.A.) were observed by light microscopy to study sporangial dehiscence and spore motility. Flagellation was examined by light microscope, staining the suspensions of motile spores as suggested by Heimbrook et a. (1989). For SEM specimen preparation, spore suspensions were washed by centrifugation in ultrapure water to remove the mucilaginous substances abundantly produced by the strain. Suspensions of sporangia were also vacuum dried immediately after wetting to study dehiscence. Observations were made using a Stereoscan 250 (Cambridge Sci. Instr. Ltd., UK).

Physiological characterization. The isolate was first incubated on ISP medium 3 at a variety of temperatures (4, 10, 26, 37 and 45 C) to determine the most favourable growing temperature. The other physiological tests employed were: carbohydrate utilisation on ISP medium 9 and melanin production on ISP media 6 and 7 (Shirling and Gottlieb, 1966), H2S production, nitrate reduction, tyrosine degradation, hydrolysis of gelatine, casein and starch (Williams et al., 1989), NaCl tolerance (3, 4, 5%). All the tests were carried out at 26 C.

Chemotaxonomic characterization. Chemotaxonomic analyses were carried out as reported by Petrolini et a. (1992).

RESULTS

Substrate mycelium of IPV-2867 does not bear spores; tufts of aerial hyphae producing sporangia with many motile spores develop on the colony surface.

The cultural characteristics on tested media are shown in Table 1. No growth, or just traces, is present after 21dd incubation on ISP medium 2. Substrate mycelium is colourless to pinkish or orange. Aerial growth does not occur on NA and ISP medium 7; only traces are present on ISP medium 2 and BA after 21dd of incubation. On the other media it is sparse and scanty, white to pale pink. Only ISP medium 3 gave rise to a good production of aerial mycelium. In 14dd old cultures on BA, NA and ISP medium 3, the surface of colonies becomes moist and glossy. A purplish diffusible pigment is produced in ISP medium 7 only.

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              Substrate mycelium     Aerial mycelium     
Medium                         
        Amount       Colour          Amount     colour
ISP2     traces                      traces          
ISP3     +++   Oc4a (pinkish)         ++       Oc6a (pale pink)    

ISP4     +++   Oc3a (orange-pinkish)  (+)      Oc6a (pale pink)    

ISP7     +     colourless              -             -     
WA25     +     colourless             (+)      white     
CA       +     Oc3a (orange-pinkish)  (+)      Oc6a (pale pink)    

CAY      ++    Oc3a (orange-pinkish)   +       Oc6a (pale pink)    

NA       ++    Oc4a (pinkish)          -             -     
TPC      +     Oc4a (pinkish)         (+)      Oc6a (pale pink)    

BA       ++    Oc2a (orange          traces    white     
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Table 1. Cultural characteristics and occurrence of sporing structures of strain IPV-2867

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           Soluble
Medium     pigment   Sporangia
     

ISP2        -          +
ISP3        -          +
ISP4        -          +
ISP7       purplish    -
WA25        -          +
CA          -          +
CAY         -          +
NA          -          -
TPC         -          +
BA          -          +
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--

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Non-fragmenting, branched vegetative mycelium grows profusely within the agar medium and forms on its surface a more or less compact layer, often coated by slime. The isolate does not generally produce a true aerial mycelium, but develops dense tufts of sporebearing structures. Hyphal aggregates can also be formed.

Tips of the aerial hyphae branch immediately and several times in all directionsFig. 1) and are never enclosed in a common sac-like structure. The initial sporangia have a finger-like configuration (Fig.1), afterwards they lengthen, become tubular, curly and twisted(Figs. 2, 4 and 5) and contain a large number of spores. Sometimes branched sporangia are also formed (Figs. 2 and 5), such as reported for Dactylosporangium (Thiemann, 1970). On CAY the sporing structures develop generally straight or slightly flexuous.

Sporangia do not reveal their contents when examined by light microscopy. Under SEM they show a smooth surface and in young sporangia the spores are not visible (Fig. 2). The formation of spores takes place through frequent septations of the sporogenous hypha. The wall of the sporangia remains distinct from the spores during their development, so that in the oldest ones the outline of individual spores is revealed by ondulation of the envelope (Figs. 4 and 5). The spores completely fill the sporangium (Figs. 4 and 5), as in Planomonospora and Planobispora (Thiemann, 1970), making it difficult to see the sporangial wall. The tightly adhering sporangial envelope holds the spores in a single row until maturity.

The abundant branching of the sporangiophores results in the formation of large tufts showing a typical claw-like rather loose structure, due to the shape and the remarkable length of the sporangia (Figs. 2, 3, and 7, 8 and 9).

On WA25, BA, TPC and CA, only tufts of sporogenous hyphae differentiate from the substrate mycelium (Fig. 3). On the other media hyphal aggregates with different degrees of organization are formed. These structures range from simple cushion-like masses to well cemented bundles. On CAY and ISP media 3, 4 and 7 sterile synnemata-like structures, generally sub-conical, protrude from the vegetative mycelium (Fig. 6). On the last medium these are the only structures formed. On ISP media 2, 4, 9 and CAY the hyphae sometimes group into strands often fused together to form larger ones (Figs. 7 and 8). Close strands form bridges and the agar surface appear covered by a loose network. Synnemata-like structures, each bearing a sporangial tuft, are formed on ISP medium 4 (Fig. 8). A true aerial mycelium is abundantly formed only on ISP medium 3 (Fig. 9).

The size of the tufts of sporangia varies depending on the medium (Figs. 3 and 7 and 9), being particularly large on WA25 (about 30 to 50 um) and smaller on the other media. In addition the tufts growing directly from substrate mycelium have larger size in respect of those originating from strands and synnemata that measure 10 to 15 um (Figs. 7 and 8).

The release of sporangiospores occurs immediately after immersion in water and the sporangial sheath quickly dissolves. Consequently it is difficult to follow sporangial dehiscence and only rarely can incontestable residues of sporangial envelopes be detected (Fig. 13). When sporangia are wetted and immediately vacuum dried, all the sporangia forming a tuft appear to be coated by a common slime which does not permit the recognition of individual sporangia. Soon afterwards, the surface of the whole mass appears brindled by a sort of viscous network and some spores are visible below through the mesh (Fig. 10).

Plate. 1. Morphology of Planopolyspora crispa: development of branching hyphal tips, finger-like initial sporangia (Fig. 1) and young claw-like sporangial tuft (Fig. 2) on CA at 7 dd; sporangial tufts on WA25 at 14 dd (Fig. 3); arrangement in single row of sporangiospores in 14 dd old cultures on ISP 3 (Fig. 4) and WA25 (Fig. 5). In Fig. 5 branched sporangia are visible. Sterile synnema-like structures on ISP 4 at 14 dd (Fig. 6). Bar markers equal 20 um in Figs. 3 and 6, 4 um in the others.

Plate 2. Morphology of Planopolyspora crispa: strands and synnema-like structures bearing sporangial tufts on ISP 4 at 14 dd (Figs. 7 and 8); aerial mycelium and tufts of sporangia on ISP 3 at 21 dd ()Fig. 9). Sporangial dehiscence: melting of sheath in sporangia vacuum dried after 1 minute wetting (Fig. 10); flagella (Fig. 11) and shape Or the spores ()Figs. 11 to 13). The sporangial wall of 3 months old sporangia is visible in Fig. 13. Bar markers equal 20 um in Figs. 7 and 9, 4 um in the others.

Sporangiospores are rod-shaped and frequently curved (Figs. 11-13). Their volume increases in the presence of water: spores inside the sporangia measure 0.43/0.76 x 0.96/2.28 um (average 0.63 x 1.43um), while free spores measure 0.62/1.03 x 1.66/2.72 um (average 0.79 x 2.21 um). The swelling of the sporangiospores is another important factor in the dehiscence and, added to the solvent effect of the water, speeds up the process .

The spores of young sporangia become highly motile after a few minutes and swim vigorously in ultrapure water, sometimes remaining attached together and moving one behind the other. The observation of flagella by SEM was not successful. Stained preparations of motile spores revealed the presence of 2 to 6 flagella (Fig. 11). No clear indications could be obtained concerning the type of flagellation. However some observations suggest that the flagella are more probably subpolarly inserted rather than peritrichous.

The rapidity of sporangial dehiscence and the beginning of spore motility, as well as the number of motile spores, progressively decrease with the age of the cultures. The release of a large number of motile spores is immediate in 3-6 dd old cultures; 1 month old sporangia release their spores after a few minutes but motility occurs after more than 1 hr; 3 month old sporangia release the spores slowly, few of them become motile after about 2 hrs. Because of the slowness of dehiscence of old sporangia, it was possible to obtain clear evidence of the sporangial envelope (Fig. 13).

Growth is good at 26 C, moderate at 10 C, poor at 37 C, and absent at 4 and 45 C .

D-glucose, D-mannose, D-mannitol, L-rhamnose, sucrose, D-fructose, mesoinositol, D-xylose, D-lactose, L-arabinose, D-galactose, maltose and salicin are utilized for growth; there is no growth with raffinose, adonitol, xylitol, D-melibiose, dextran and glycerol. H2S is produced; melanin is not produced on ISP media 6 and 7; tyrosine crystals, gelatine, casein and starch are hydrolyzed. Nitrate is reduced to nitrite. The isolate tolerates 3% NaCl, but no growth occurs on media containing 4 and 5% NaCl.

The walls of strain IPV-2867 contain meso-diaminopimelic acid and madurose has been detected in whole-cell hydrolysates. Traces of galactose are also present. The chemical composition of cell walls therefore conforms with chemotype III and sugar pattern B according to the classification scheme proposed by Lechevalier and Lechevalier (1970).

CONCLUSIONS

The ability of hyphal filaments to coalesce forming more or less complicated aggregates is well documented in actinomycetes. Particularly the formation of sporulating synnemata is an important feature of the genus Actinosynnema (Hasegawa et a., 1978; 1989), characterised by true synnemata and motile arthrospore. Members of this genus do not form sporangia and lack madurose in whole-cell hydrolysates.

On the grounds of its wall chemotype III, whole-cell sugar pattern B and formation of sporangia containing motile spores, strain IPV-2867 is related to the sporangiate maduromycetes with motile elements, which include the genera Planomonospora, Planobispora and Spirillospora (Goodfellow, 1989; Goodfellow and Cross, 1984).

Generic classification of sporangia-forming actinomycetes is currently based on morphology of the sporangia, number of spores and their arrangement within the sporangium. On the basis of these characteristics the placement of the new isolate IPV-2867 in any of the above mentioned genera is not possible.

Spirillospora is the only genus forming multispored sporangia. These are usually spherical, rarely elongated or club-shaped and vermiform (Couch, 1963; Vobis and Kothe, 1989). In addition to spores formed in sporangial envelopes, the species described by Couch (1963) may produce regular or irregular coils of arthrospores among the aerial hyphae. When flooded with water, these coils break up into rod-shaped to curved elements which also show motility. At the beginning of sporangium formation in Spirillospora the tip of an aerial hypha coils terminally into a spiral and soon after branches. The first twistings seem to be free, but later on they are enclosed in a common envelope (Locci and Petrolini Baldan, 1970; Vobis, 1986; Vobis and Kothe, 1985), as do all groups of sporangiate actinomycetes with spherical, cylindrical to irregular, multispored sporangia (Bland and Couch, 1981). Finally the sporogenous hyphae, arranged in orderly coils, are converted into spores which shortly before dehiscence are probably irregularly arranged inside the sporangium (Locci and Petrolini Baldan, 1971).

In IPV-2867 the development of the sporangium begins with the formation of an aerial hypha which immediately branches several times, and forms some sporogenous hyphae, never enclosed into a common envelope. Each of them, growing inside its sheath, gives origin to one tubular curly sporangium of remarkable length, containing one long single row of numerous sporangiospores. This pattern of development is typical of finger-like or cylindrical sporangia (Bland and Couch, 1981) and allows spores to remain arranged in chain until the dehiscence of sporangia, similarly to what happens in Planomonospora and Planobispora (Thiemann, 1970; Vobis, 1989a; 1989b). These genera are characterized by formation on aerial mycelium of short and slender cylindrical sporangia containing respectively a single and a longitudinal pair of motile spores. In addition to the different number of spores sporangia of strain IPV 2867 are very tighter and longer than those of the members of the two above mentioned genera.

In conclusion strain IPV-2867 appears well differentiated from previously described genera and the culture represents a new morphological type of sporangiate actinomycetes included in maduromycetes group, justifying the creation of the new genus Planopolyspora, of which the type species is P. crispa sp. nov.

Description of Planopolyspora gen. nov.

Pla.no.po'ly.spo.ra. Gr. adj. planos wanderer; Gr. adj. polys many; Gr. n. spora a seed; M. L. fem. n. Planopolyspora a motile, multispored organism.

Non-fragmenting substrate mycelium well developed growing within the agar medium and forming a more or less compact layer at its surface. Aerial mycelium moderately developed or absent. Tubular elongated sporangia, containing one single row of numerous sporangiospores motile by means of 2 to 6 apparently subpolar flagella, born on aeral mycelium only. Sporangia arising as tufts directly from substrate mycelium and/or from hyphal strands or synnema-like structures. Cell wall countering meso-diaminopimelic acid and whole-cell hydrolysates containing madurose. Type species: Planopolyspora crispa.

Planopolyspora crispa sp. nov.

cri'spa. L. adj. crispus curly.

No growth, or just traces, on ISP medium 2; on other media substrate mycelium, often coated by slime, colourless to pinkish or orange. Aerial growth absent on NA and ISP medium 7; occasionally in traces on BA and ISP medium 2; sparse and scanty, white to pale pink on the other media. True aerial mycelium formed only on ISP medium 3; on the other media powdery, spore-bearing aerial mycelium arising as dense tufts from the substrate mycelium and/or from hyphal aggregate. Sporangia very elongated, tubular in shape, curled, grouped in large sized tufts showing a typical clew-like rather loose structure. Branched sporangia sometimes formed. Sporangiospores rod-shaped, frequently curved (average 0.63 x 1.43 um within the sporangium), increasing in volume in presence of water (average 0.79 x 2.21 um); motile by means of 2 to 6 apparently subpolar flagella. Soluble purplish pigment produced only on ISP medium 7. Peculiar process of sporangial dehiscence due both to the spore swelling in presence of water and to the dissolution by water of the sporangial sheath. Large hyphal strands and sterile sub-conical synnema-like structures formed, depending on cultural media. On ISP medium 4 sporulating-synnemata also produced from mycelial strands. Temperature range 10 to 37 C, with an optimum of growth around 26 C. D-glucose, D-mannose, D-mannitol, L-rhamnose, sucrose, D-fructose, meso-inositol, D-xylose, D-lactose, L-arabinose, D-galactose, maltose and salicin utilized; no growth with raffinose, adonitol, xylitol, D-melibiose, dextran and glycerol. H2S produced; melanin not produced on ISP media 6 and 7; tyrosine crystals, gelatine, casein and starch hydrolyzed. Reduction of nitrate positive. Growth with maximum 3% NaCl.

The species description single isolate and strain NCB 1173) represents the type strain P.crispa.

The type culture has been deposited in the National Culture Bank, as NCB 1173.

REFERENCES

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Copyright 1993 C.E.T.A.

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