Actinomycetes University of Udine, Mycology Department ISSN: 0732-0574 Vol. 3, Num. 1, 1992

ON THE PRESENCE OF STREPTOMYCES SPP. IN THE GRAPEVINE CARPOSPHERE

A.VERCESI, E.VOLPI and R. LOCCI*

Institute of Plant Pathology, University of Milan and *Chair of Mycology, University of Udine, Italy '

Code Number: AC92002
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ABSTRACTS. Studies carried out using a selective medium confirmed the regular presence of streptomycetes on grapevine berry surfaces during the entire cycle of development of the fruit. Identification by probabilistic methods shows the predominance of the S. albidoflavus phenon at all developmental stages. Implications of the presence of actinomycetes on the grapevine carposphere are discussed.

Research supported by National Research Council of Italy, Special Project RAISA, Sub-project N.2, Paper N. 286

During previous investigations on grapevine microflora, aimed at isolating yeasts, the constant presence of strepto- mycetes was ascertained (Vercesi et al., 1990). Following this observation, an analysis of grapevine carposphere actinoflora was carried out employing more selective media.

MATERIALS and METHODS

Collection and treatment of the samples of Vitis vinifera, cv. Riesling Italico, were carried out according to a previously illustrated methodology Vercesi et al., 1990). Isolations were carried out on the selective medium proposed by Kuster and Williams (1964). Actidione (50 ppm) and nystatin (50 ppm) were added to the sterilized medium. Plates were dried after medium solidification for 30 min in a laminar flow cabinet and for 1 hr at 50 C. After inoculation plates were dried again for 30 min in a laminar flow cabinet.

Plates were incubated for up to 1 month at 25 C and colonies counted weekly. One or more representatives of each morphological type were isolated on PDA (potato dextrose agar) or CYA (Czapek Yeast Extract Agar). Strains were identified using the probability matrix of Williams and co-workers (1984); identification scores were calculated according to the MATIDEN program (Sneath, 1979).

RESULTS

Streptomyces spp. are present on the bunches during the entire vegetative season (Table 1).

A conspicuous number of streptomycetes were recovered from the grapevine carposphere at the beginning of blossoming. Subsequently they showed a progressive decrease until the beginning of veraison, when the minimum number of colonies was found. A slight increase was observed in corre- spondence to full veraison. At harvest streptomycetes reached the highest level.

The efficiency of the two methods used for propagule detachment showed notable differences at the phenological stages of beginning (BVE) and full veraison (FVE) and of harvest (HAR). In particular the number of colonies detached by manual shaking at BVE and at HAR was 25 and 5 times as great respectively as that recovered from samples treated with ultrasounds.

Only one out of the 160 strains isolated did not belong to the genus Streptomyces. On the basis of its morphology as determined by scanning electron microscopy the organism appears to belong to the family Actinoplanaceae.

Figure 1. Streptomyces colonies (n/g fresh weight) recovered during the different phenological stages by two detachment methods. Grapevine berry phenological stages: BBL, beginning of blossoming; SET, setting; BBT, beginning of berry touch; BVE, beginning of veraison; FVE, full veraison; HAR, harvest.

Correctly identified streptomycetes belong to 9 phena. Number of isolates and relative percentages are detailed in Table 2.

-----------------------------------------------------
Phenon               No. of            Percentage 
                     Isolates 
-----------------------------------------------------
S.albidoflavus        98                 61.63 
S. chromofuscus       8                  5.03 
S. exfoliatus         8                  5.03 
S. rochei             8                  5.03 
S.diastaticus         7                  4.40 
S.cyaneus             5                  3.14 
S.griseoruber         5                  3.14 
S.atroolivaceus       3                  1.90 
S.phaeochromogenes    2                  1.27
-----------------------------------------------------

Distribution of the nine phena in each phenological stage (Table 3) shows that the larger proportion of the population is constantly represented by Streptomyces albidoflavus, while S.chromofuscus is only absent at the SET stage. S. diastaticus and S.rochei show good frequency, while S.cyaneus and S.phaeochromogenes are characteristically present late in the season. The remaining three phena are detected only sporadically.

CONCLUSIONS

The selective medium used for the isolation of streptomycetes permits a better characterization of this component of the grapevine microflora.

The streptomycetes are relatively abundant at blossoming and setting. However their number decreases progressively during the two following phenological stages. From full veraison on, the actinoflora increases and the maximum number of colonies is isolated at harvest. This pattern seems to be due to the saprophytic habit of streptomycetes: the flower residues present on the bunch during blossoming and setting and the senescent structures at the end of the season could be more easily colonized by the organisms. On the contrary the young tissues of the berry in the central phenological stages are not a suitable substrate for the actinoflora.

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                                    PHENOLOGICAL STAGE 
PHENON        -----------------------------------------------
                   BBL    SET    BBT    BVE    FVE    HAR 
-------------------------------------------------------------
S.albidoflavus  65.9   84.0   70.5   45.9   65.6  56.1 
S.atroolivaceus 2.3                                3.7 
S.chromofuscus  31.8   12.0          22.5   14.4   0.5 
S.cyaneus                            2.0           3.1 
S.diastaticus          1.3           3.1    2.2    0.3 
S.exfoliatus           2.7                         19.1 
S.griseoruber                 22.7                 3.3 
S.phaeochromogenes                          1.1    0.3 
S.rochei                      2.3           4.5    2.8 
Streptomyces spp.             4.5    26.5   26.5   10.8
-------------------------------------------------------------

At the same time possible interferences from fungicides, employed during the growing season, against downy and powdery mildews, cannot be disregarded. In fact no antifungal spraying was carried out in the particular vineyard during the last two phenological stages.

Manual shaking was generally more effective than ultrasounds in detaching streptomycete propagules from berries, with the exception of the isolation carried out at the setting stage, thus confirming results obtained previously (Vercesi et al., 1990). This could possibly be due to the biological status of actinomycete propagules at the moment of sampling. Possibly if the prevailing propagules in the carposphere were represented by spores, these could be de- tached more effectively by ultrasounds. This was confirmed indirectly by coating berries with spore suspensions of S. albidoflavus strain R85. Ultrasound treatment (45 to 90 sec) equalled in recovery the effects of manual shaking (10 min). Recovery doubled with longer ultrasound treatment (180 sec) and started to decline after 360 sec.

The use of a selective medium allowed the isolation of a larger range of phena than with non-specific substrata used previously (Vercesi et al., 1990).

In particular S.cyaneus, S.exfoliatus and S.phaeochromogenes were isolated only on the medium of Kuster and Williams (1964). Similarly phena such as S. chromofuscus, S.diastaticus and S.rochei, isolated only sporadically, were frequently recovered using the se- lective medium. On the other hand a notable decrease in S.griseoruber was observed. S. albidoflavus is the major component of the streptomycete community during the entire growing season and represents the only phenon constantly present on the berries. S. chromofuscus, S. diastaticus and S.rochei are also regular inhabitants of the carposphere, while S.cyaneus and S.phaeochromogenes tend to appear towards the end of the season. S.atroolivaceus, S.exfoliatus and S.griseoruber are present more sporadically. However the presence of sphere has been confirmed, due to the good identification scores of the isolated strains.

The strain assigned to Actinoplanaceae was isolated at harvest. The isolation is occasional, however it could support the hypothesis that these organisms are able to colonize plant tissues before coming into contact with soil (Willoughby, 1969).

The present results demonstrate the ability of streptomycetes to settle in the aerial portions of plants, as already found in some members of the Solanaceae (Sinha, 1971).

Because of the very limited data available on the actinomycete-grapevine carposphere association, it is presump- tuous at this stage to put forward any hypothesis as to the significance of the presence of streptomycetes. However, considering their metabolic potential, it is not improbable that, in addition to occupying carposphere niches during the growing season, they could also affect the final product. Some reports of Italian researchers in the past (Peglion, 1900; Ciferri, 1922; Baldacci, 1941) point out the role of some actinomycetes as occasional producers of unpleasant tastes in wines. To our knowledge no further investigations have been carried out on the subject, which, in the light of similar findings in other habitats (Lacey, 1988), could be worthwhile investigating.

REFERENCES

Baldacci, E. (1941). Gli attinomiceti nell'enologia. Progr. Vinic. Oleario, 17:3056-3057

Ciferri, R. (1922). Studi sul muffito dei vini e le muffe delle cantine. Riv.Ampelogr., 3:3-23

Kuster, E. & S.T.Williams (1964). Selection of media for isolation of streptomycetes. Nature, 202:928-929

Lacey, J. (1988). Actinomycetes as biodeteriogens and pollutants of the environment. In: M.Goodfellow, S.T.Williams & M.Mordarski (eds.) Actinomycetes in Biotechnology. Academic Press, London, pp.359-432

Peglion, V. (1900). Sulla possibile origine del sapore terroso dei vini. Atti Staz.Agr. Sper. Ital., 33:525-531

Sinha, S. (1971). The microflora of leaves of Capsicum annuum (L.) Watt E.D. Solanum melongena L., Solanum tuberosum L. and Lycopersicum esculentum Mill. In: T.F.Preece & C.H.Dickinson (eds.) Ecology of Leaf Surface Microorganisms. Academic Press, London, pp. 175-189

Sneath, P.H.A. (1979). Basic program for character separation indices from an identification matrix of percent positive characters. Comp. & Geosc., 5:349-357

Vercesi, A., E.Volpi & R.Locci (1990). Preliminary investigations on the Streptomyces flora of grapevine berries. Aclinomycetes, 1:7-9

Williams, S.T., M.Goodfellow, E.M.H. Wellington, J.C.Vickers, G.Alderson, P.H.A. Sneath & M J.Sackin (1983). A probability matrix for identification of some streptomycetes. J.gen.Microbiol., 129:1815-1830

Willoughby, L.G. (1969). A study on aquatic actinomycetes, their allochthonous leaf component. Nova Hedwigia, 18:45-113

Copyright 1992 CETA

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