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STUDIES ON THE STREPTOMYCETE POPULATION INHABITING PLANT ROOTS B. PETROLINI, S. QUARONI^1, M. SARACCHI and P. SARDI^1
Istituto di Patologia Vegetale dell'Universit… di Milano
and
Abstract. Streptomycetes were constantly recovered from surface- sterilised roots of 156 plant species. Following a grouping of the isolate population (1422 strains), a more detailed characterisation was carried out on 82 representative isolates. Identification by probabilistic methods showed the predominance of three Streptomyces clusters, namely S.anulatus, S.halstedii and S.albidoflavus, and allowed a physiological characterisation of the streptomycete population inhabiting plant roots.
Relationships between Streptomyces spp. and plants are well documented for their phytopathological implications, with particular reference to potato scab inducing agents (Locci, 1994). Occurrence of streptomycetes inhabiting the cortical tissues of root system (Quaroni et al., 1989; Sardi et al., 1992) as well as their involvement in the enhancement of plant growth (Ferri et al., 1996; Quaroni and Saracchi, 1994; Saracchi et al., 1992) have been previously reported.
The following report represents an attempt to identify the characteristics of root inhabiting streptomycetes so far isolated.
Materials and methods
Sampling, isolation and culture maintenance procedures. Strains were isolated, according to procedures previously described (Sardi et al., 1992), by sampling roots of 156 plant species, during a 7 year period (1987-1994). Altogether 205 plants, 50 of which were grapevines (Vitis vinifera), were collected from different habitats and locations. For each sample, the selection of isolates was performed on the grounds of phenetic differences. The high number of grapevine specimens were collected during the uprooting of a vineyard in northern Italy (Retorbido, province of Pavia). Plants were chosen at random and only roots growing below 40 cm of depth were sampled.
Cultures were preserved by lyophilization and/or by freezing spore suspensions in 10% (w/v) glycerol at -20 C. CAY (Czapek solution agar plus 0.2% yeast extract) was chosen as a suitable medium favouring sporulation.
Preliminary grouping of isolates. All selected isolates, attributed to Streptomyces spp. (1422 strains) on the basis of morphological features, were examined by recording morpho-cultural characteristics and some antimicrobial activities, as described by Sardi et al. (1992). This characterisation provided a suitable file of data that could be analysed by a dBase IV computer program (Ashton Tate) to group similar isolates. Inside each so formed group, strains originating from the same sample were considered as identical and only one of them was retained.
Selection of representative isolates. For a more detailed characterisation 82 isolates, whose morpho-cultural features recorded on oatmeal agar (ISP 3, Difco) are reported in Table 2, were chosen. All the dominant patterns (major groups), numerous minor groups and some single or two-membered groups were represented by at least one strain. Strains from roots of Vitis vinifera and of a restricted number of other plants were chosen preferentially because of the higher number of isolates in order to evaluate the existence of possible specificity of the streptomycete microflora.
Identification of isolates. Thirty-five out of the 82 representative isolates (group A, marked with an asterisk in Table 2) were subjected to probabilistic identification using the 50 characters (Williams et al., 1989) derived from numerical classification and identification studies of Streptomyces species (Williams et al., 1983a; 1983b). Cluster identification was assessed using the MATIDEN program and was regarded as acceptable when the similarity level was higher than 80%, with low scores for taxonomic distance and for its standard error (lower than 2.5). In addition distribution of characters within the 35 isolates, expressed as percentage of strains with positive character states, was analysed.
Analysis of the characteristics of the streptomycete root population. The distribution of 31 out of the 50 characters used in streptomycete identification (see Table 4) was studied with reference to all 82 representative isolates. Isolates of group B (47 strains) were investigated with regard to these characters. Eighteen of the characters (bold-faced in Table 4) were shown by previous numerical analysis to be typical for strains of group A, being expressed as positive character states by over 80 or under 20 per cent of all 35 strains.
Results
Isolation of actinomycetes from surface-sterilised roots resulted in a collection of 1755 strains. The most frequent isolates belong to the genus Streptomyces (1422 strains, 81%) and were detected inside the roots of all the sampled plants. Several strains (215) were identified as Micromonospora spp. (Williams et al., 1993), while other non-streptomycete actinomycetes rarely occurred. Twelve isolates, showing varied morphological features, belonged to the genus Actinoplanes (Petrolini et al., 1995) and 12 to Streptosporangium. The genera Streptoverticillium (Brambilla et al., 1995) and Saccharomonospora were represented by 2 and 1 strains respectively. Nocardioform actinomycetes (21 strains) and isolates never bearing reproductive structures (70 strains) were also isolated.
Preliminary grouping of isolates. After discarding identical isolates, the initial number of streptomycetes (1422) was reduced to 1080, of which 103 were isolated from roots of Vitis vinifera.
On the basis of morpho-cultural features and antimicrobial activity (Sardi et al., 1992), the population of root endophytic streptomycetes was divided into 177 groups of similar isolates and 224 single-membered groups, for a total of 401 profiles. Inside each group the number of strains corresponds to that of the plants from which the same profile was recovered, since just a single strain was selected from each plant.
Most groups so defined consist of few strains and the two or three-membered ones (69 and 33 respectively) are particularly numerous. Twenty-four have 9 or more members, the largest 37. This indicates that some patterns occur more frequently within the streptomycetes inhabiting plant roots, but no profile appears to be plant taxon specific.
Major groups are similar to those encountered during a previous investigation carried out on 482 strains isolated from 28 plant species (Sardi et al., 1992). Groups in that preliminary research were more numerous since identical strains were not removed, owing to the low number of isolates.
A picture of the whole population is given in Table 1 which shows the distribution of recorded characters among strains allocated to colour series according to Tresner and Backus (1963). A large number of isolates (43%) form a grey spore mass. Grey and red-coloured streptomycetes constitute about 70 per cent of the population. The most frequent antimicrobial activity is against Micrococcus luteus. Yellow strains are most active (49% against M. luteus and 30% against Fusarium oxysporum f. sp. cyclaminis). Red isolates show the highest number of strains active against Escherichia coli (more than 12%).
Grapevine isolates are distributed in several different profiles, though always together with isolates from other sources. So, even if the streptomycete population obtained from this plant species is better represented inside some profiles than others, it was not possible to single out any one profile consisting only of V.vinifera isolates. This confirms previous data regarding the distribution of streptomycetes in tested plants.
--------------------------------------------------------------
Table 1. Distribution of morpho-cultural characters of the
streptomycete root population according to colour series.
Values are expressed as number of strains with positive
character states (^+ number of strains; * spore chain
morphology recorded on 2.5% water agar; a.m.: aerial
mycelium).
--------------------------------------------------------------
Spore mass colour on ISP 3
------------------------------------------------
Character White Blue Grey Yellow Red no a.m. Total
81^+ 92 466 136 276 29 1080
--------------------------------------------------------------
Different spore mass colour on
CAY and ISP 3 31 61 117 14 82 8 313
Distinctive mycelial
pigment 3 8 78 5 37 2 133
Diffusible pigment:
brown 23 30 115 24 68 10 270
red 4 0 36 3 16 0 59
yellow 7 9 62 30 19 1 128
other 0 9 49 4 5 1 68
Spore chain:
Pseudoverticillate
2 30 27 1 10 2* 72
Rectiflexibiles 46 4 226 100 150 6* 532
Spirales 20 79 151 31 44 9* 334
Retinaculiaperti 15 7 88 5 82 8* 205
Activity against:
Micrococcus luteus 28 26 169 66 87 9 385
Escherichia coli 5 4 31 15 34 2 91
Fusarium oxysporum
f.sp. cyclaminis 8 8 72 41 55 5 189
--------------------------------------------------------------
Table 2. Grouping of the 82 representative strains
according to morpho-cultural characteristics.
--------------------------------------------------------------
Represent- Spore Spore Mycelial Diffusible No. of No. of
atives colour chains pigment pigment isolates sample
strains (a) (b) (c) (d)
--------------------------------------------------------------
white
AM12*, MR01*, RF 31 22
VT092
VT023 S brown 7 6
VT113 RA brown 8 5
--------------------------------------------------------------
blue
EP11*, VT101*, S 37 26
VT414, VT461
VT422 S yellow 7 5
VT064, VT254, S brown 26 24
VT273
VT423 RA yellow 1 1
VT222 RA brown 3 3
grey
AZ144*, CN09*, RF 94 52
FT05*, MR19,
RMX04, RMX24,
RMX44, VT144
LMP72*, VT214, RF blue, violet, green 10 8
ZEA07*, ZEA15*
AST48*, MR06 RF yellow 36 28
AZ112*, RMX64 RF brown 46 35
AST25* RF + yellow 2 2
CU13, RMX27 S 51 34
VT105* S yellow 15 13
VT013, VT363, S brown 43 31
VT493
MR05, VT482 RA 46 34
LMP64*, VT504 RA yellow 9 9
LMP74*, VT492 RA brown 17 13
PO03* RA red 9 8
--------------------------------------------------------------
yellow
MR02, MR07, RF 56 36
RMX01, RMX13,
SG10*, VT098*,
VT111*
CX11* S 17 8
VT173 S yellow 6 6
VT373 RA yellow 1 1
--------------------------------------------------------------
red
AST32*, CU03, RF 79 51
QR26*, RMX02,
RMX42, VT183
VT104 *, VT494 RF yellow 14 14
CMJ60*, EP15*, RF brown 41 35
VT110, VT142,
VT161, VT361,
VT365
AZ117* RF + yellow 1 1
CMJ58* RF + brown 1 1
ZEA13* S 25 17
ZEA17* S brown 11 10
CMJ59* S + brown 3 3
ER08, RMX16, RA 48 24
RMX29
CX14*, VT491 RA brown 14 13
FS01* RA + 13 9
EP17* RA + red 6 6
--------------------------------------------------------------
no a.m.
CH02* RF brown 5 2
--------------------------------------------------------------
(a.m.: aerial mycelium; RA, RF and S: Retinaculiaperti, Rectiflexibiles and Spirales spore chains; *strain subjected to probabilistic identification; ^(a) spore mass colour on ISP3; ^(b) distinctive mycelial pigment. The last two columns show the number of represented isolates^(c) and of different plant samples^(d) on which each profile was detected. Strains are labelled according to isolation sources, AM: Amaryllis belladonna; AST: Aster sp.; AZ: Azalea sp.; CH: Chelidonium majus; CMJ: Camellia japonica; CN: Phragmites communis; CU: Cyclamen persicum; CX: Carex sp.; EP: Euphorbia sp.; ER: Erica carnea; FS: Festuca rubra; FT: Triticum aestivum; LMP: Rubus idaeus; MR: Vaccinium myrtillus; PO: Allium porrum; QR: Quercus sp.; RMX: Rumex sp.; SG: Secale cereale; VT: Vitis vinifera; ZEA: Zea mays).
--------------------------------------------------------------
Table 3. Distribution of the 35 isolates of group A
according to Williams et al. (1989) clusters.
Correctly identified strains are *
--------------------------------------------------------------
Best-fit cluster Isolates %
--------------------------------------------------------------
S.albidoflavus AST32*, MR01*, VT104, VT105*, VT111, 17.1
ZEA13
S.anulatus AM12*, AZ112*, AZ117, AZ144*, CMJ58, 25.7
CX11*, CX14*, SG10*, VT098*
S.chromofuscus CMJ59 2.8
S.cyaneus EP11, VT101, ZEA17 8.6
S.diastaticus QR26*, ZEA15 5.7
S.exfoliatus AST48, FS01*, FT05* 8.6
S.halstedii AST25*, CN09*, EP15, LMP64*, LMP72*, 20.0
LMP74, ZEA07*
S.rochei EP17, CMJ60, PO03* 8.6
S.violaceus CH02* 2.8
--------------------------------------------------------------
Table 4. Distribution of characters within the 35
strains of group A subjected to numerical analysis (A), the 47
strains of group B (B) and all the 82 isolates together
(C).
--------------------------------------------------------------
Character A B C
--------------------------------------------------------------
Spore chain
Rectiflexibiles 69 47 56
Spirales* 11 28 21
Spore mass
red 31 28 29
grey 37 36 37
Red-orange m.p. * 11 0 5
Diffusible pigment 46 51 49
Yellow-brown d.p. 28 49 40
Melanin on PYA * 14 4 9
Melanin on tyrosine agar* 17 9 12
Antibiosis against:
Bacillus subtilis 31 34 33
Micrococcus luteus 51 45 48
Candida albicans 37 15 24
Saccharomyces cerevisiae 57 19 35
Streptomyces murinus 37 45 41
Aspergillus niger 26 17 21
Lecithinase activity 34
Lipolysis 71
Pectin hydrolysis 63 21 39
Nitrate reduction* 89 74 80
H2S production* 100 100 100
Hippurate hydrolysis 23
Elastin degradation 77
Xanthine degradation 74
Arbutin degradation* 97 91 94
Resistance to (mg/ml):
Neomycin (50)* 0 11 6
Rifampicin (50) 31 45 39
Oleandomycin (100)* 100 89 94
Penicillin G (10 I.U.)* 100 100 100
Growth at 45 C* 11 2 6
Growth in the presence of (w/v):
NaCl 7%* 91 60 74
Sodium azide 0.01% 77
Phenol 0.1% 74
K tellurite 0.001%* 100 96 98
Thallous acetate 0.001% 71
Utilisation of:
DL-a-Amino-n-butyric acid 40
L-Cystein* 91 96
94 L-Valine 77
L-Phenylalanine* 91 94 93
L-Histidine 54
L-Hydroxyproline 63
Sucrose 46
meso-Inositol 63
Mannitol* 86 79 82
L-Rhamnose* 94 79 86
Raffinose 43
D-Melezitose 63
Adonitol 29
D-Melibiose 46
Dextran 37
Xylitol 6 11 9
Values are expressed as percentage of strains with positive
character states. Characters common to more than 80% of
strains of group A are * (d.p.: diffusible pigment; m.p.:
mycelial pigment; PYA: peptone-yeast-iron agar; TA: tyrosine
agar).
--------------------------------------------------------------
Morpho-cultural profiles of the 82 more extensively characterised strains are reported in Table 2. Expression of morpho-cultural features alone allowed 111 profiles varying in size (from 1 to 94 members, obtained from 1 to 52 different plants) to be recognised, 48 of which are single or two- membered (29 and 19 respectively). Morpho-cultural profiles include more profiles with unlike antimicrobial activities, therefore inside major and minor groups more than one strain was chosen, in order to characterise isolates with different physiological properties. Selected strains, belonging to 37 morpho-cultural patterns, represent 839 isolates corresponding to about 78 per cent of the root population.
Identification of isolates. Results of identification by numerical analysis are summarised in Table 3, where the twenty-one strains (60%) with good identification scores are asterisked.
The 35 isolates of group A are distributed among 9 major clusters of Williams and his co-workers (1989) as best-fit taxon, 7 of them including correctly identified strains. Physiological heterogeneity of the preliminarily recognised groups was confirmed by the fact that the representatives of the same morpho-cultural pattern were identified as belonging to different clusters. This is true except for strains LMP72 and ZEA07, both belonging to the Streptomyces halstedii cluster. The majority of strains was assigned to 3 clusters (S.albidoflavus, S.anulatus and S.halstedii), in particular S.anulatus groups the largest number of identified organisms and it is also the most frequently occurring cluster as nearest alternative.
The distribution of 50 characters among strains of group A is given in column 1 of Table 4. All strains are resistant to oleandomycin and penicillin G and sensitive to neomycin, grow in the presence of potassium tellurite, produce hydrogen sulphide and, except for one, degrade arbutin. A few strains show positive melanin reaction, particularly on tyrosine agar. Growth rarely occurs at 45 C. Most isolates grow in the presence of sodium chloride, reduce nitrate and utilise L- cysteine, L-phenylalanine, mannitol, L-rhamnose but not xylitol.
Considering the rather large number of common properties (18, mainly physiological ones), it is justifiable to argue that they determine the convergence of most strains on, or around, only three clusters. Subsequent allocation of isolates into one of these taxa is due to other characters. In particular the most diagnostic properties that assist in separation are spore-mass grey, antimicrobial activity against M.luteus, Candida albicans, Saccharomyces cerevisiae, hippurate and pectin hydrolysis, nitrate reduction.
Analysis of special characteristics of the root population. Comprehensive results are given in Table 4. The importance of 16 of the 18 characters which are most representative of and consistent within the isolates of group A (column 1) has been confirmed for the whole root population (column 3), also considering the distribution among other strains (column 2). Morpho-cultural features were of low diagnostic potential. Red-orange mycelial pigment can be considered as the only diagnostic property, characterising 5% of all strains. Spore chain morphology does not seem to be important, although only a small number of isolates (21%) forms spores borne in spiral chains. Among physiological properties, only the ability to grow in the presence of 7% NaCl is below 80 per cent, since only 28 strains of group B gave positive answers to the test, a lower percentage (60%) in comparison with that of group A (91%). Out of the 5 characters expressed by all the strains of group A, only two (hydrogen sulphide production and resistance to penicillin G) were characteristic of the whole population, the others showing a very high frequency. Resistance to each tested antibiotic was particularly homogenous, except for rifampicin.
Emphasis was placed, during this study, on antagonistic tests owing to the importance of some antimicrobial activities in separating the three clusters on which most strains converge. Antimicrobial activity spectra appeared rather heterogeneous, so there is no antibiosis profile dominant or typical of the examined microflora. A summary of antimicrobial activity pattern distribution among all representative isolates is given in Table 5. Activity against at least one of the test organisms is shown by 67% of strains and against all by 4%. Antibacterial activity is typical of 25 strains, while 14 are active against all test bacteria. This later profile is the most frequent, characterising 17 per cent of the population (corresponding to about 26% of active strains). Antifungal activity is less frequent, being shown by only 7 isolates, 4 of which were active against yeasts and 3 against all three tested fungi. Activity against Aspergillus niger is the least frequent, being shown by 17 isolates, all characterised by rather large antimicrobial activity spectra (3 to 6 positive answers).
-------------------------------------------------------------
Table 5. Antimicrobial activity profiles of the 82
representative isolates.
--------------------------------------------------------------
Number of Activity against
strains ----------------------------------------------------
B. M. S. C. S. A.
subtilis luteus murinus albicans cerevisiae niger
--------------------------------------------------------------
27 - - - - - -
4 - + - - - -
3 - - - - + -
2 - - + - - -
2 + + - - - -
2 + - - - + -
1 - - - + + -
3 - + + - - -
14 + + + - - -
1 - + - + + -
3 - - - + + +
4 + + + - + -
1 + - + + + -
1 - + + + + -
1 - + + - + +
5 - + - + + +
4 - - + + + +
1 + + + + - +
3 + + + + + +
--------------------------------------------------------------
Conclusions
Application of the isolation technique proposed in a previous paper (Sardi et al., 1992) to the current large-scale investigation permitted detection of a high number of actinomycetes inhabiting plant roots, mostly belonging to genus Streptomyces (over 80% of the isolates). This study provided evidence that streptomycetes are constantly present in cortical tissues of roots and that, despite heterogeneity in individual features, can be regarded as a population that is reasonably consistent in character states, having some common, well defined physiological peculiarities. This suggests a possible significance of an ecological role of these organisms. At present, the most important feature of the close relationship between streptomycetes and plants appears to be connected with plant growth stimulation and biological control of root diseases (Ferri et al., 1996; Quaroni and Saracchi, 1994; Reggiori et al., 1992; Saracchi et al., 1992).
It is interesting to note that actinomycetes were detected on grapevine roots collected at a depth where the microbial population is scarce. It appears that growing inside the cortical layer, they are able to colonise even the deeper parts of the root system.
Results appear to confirm data obtained during previous investigations on a smaller number of plants (Sardi et al., 1992). The distribution of various patterns is ubiquitous and the composition of the streptomycete population does not vary considerably from plant to plant, notwithstanding their botanical diversity.
Most isolates have been found to belong to Streptomyces albidoflavus, S.anulatus and S.halstedii clusters, taxa which are closely related and regarded by Williams and co-workers (1983a) as three sub-clusters of S.albidoflavus, the largest streptomycete taxon, approximately equivalent to the "griseus" groups previously recognised by several authors. Subgeneric relationships among the above-mentioned taxa have been also evaluated in the course of a more extensive numerical characterisation of the genera Streptomyces and Streptoverticillium by means of physiological tests (K„mpfer et al., 1991) and using other taxonomic methods as, for example, those based upon DNA homology (Mordarski et al., 1986) and analysis of protein profiles (Manchester et al., 1990).
Morpho-cultural features, although useful in preliminary grouping of isolates, appeared to be of little importance as diagnostic characters for the streptomycete root population. On the other hand the same Williams clusters are heterogeneous with respect to these traditional characters that are generally regarded as too variable for use as taxonomic criteria and can be difficult to determine (Kutzner, 1981; Shirling and Gottlieb, 1977; Szab¢ and Marton, 1976; Williams and Wellington, 1980; Williams et al., 1989).
Physiological characters of all examined the strains are responsible for their congregation around a few taxa and diversity of antibiotic spectra appears to be in agreement with the distribution of isolates in the three above-mentioned taxa. According to Williams et al. (1989) the physiological peculiarities examined are the most common among the strains grouped in the three clusters, while the antimicrobial activities play a role as diagnostic characters discriminating among the same taxa.
Information from the database on streptomycetes has already proved useful for studies of an ecological nature, particularly for the development of selective procedures designed to isolate members of streptomycete communities different from those usually detected on conventional media (Vickers et al., 1984; Williams et al., 1984).
The identification matrix for Streptomyces species (Williams et al., 1989), as used in the present investigation, provided a useful tool for diagnostic selection in the presence of a large amount of data. Consequently information derived from numerical analysis appears very useful in providing a workable system for a simplified description of natural populations.
Acknowledgements. The authors wish to thank Mrs. Jacqueline Rogers for the revision of the English text.
References
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