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

Preliminary Investigations on the Streptomyces Flora of Grapevine Berries

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

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

Code Number: AC90002
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Abstract.

Streptomycetes were constantly recovered during an investigation on yeasts and acetic bacteria inhabiting the grapevine carposphere. Preliminary data on their frequency and probabilistic identity are illustrated.

Research work supported by CNR, Italy. Special grant I.P.R.A. - Sub-project 1

Apart from one study (Kurtboke et al., 1986), to our knowledge the actinomycete fraction of the carposphere microflora of grapevine (Vitis vinifera L.) has never been studied systematically.

Actinomycete colonies were constantly present on media intended for the isolation of other organisms (yeasts and acetic bacteria). Preliminary results on their frequency and their probabilistic identity are reported.

Materials and Methods

Grape samples were collected in a Riesling Italico vineyard in Northern Italy (Montebello della Battaglia, province of Pavia) at the following phenological stages: blossoming initiation, setting, beginning of berry touch, start of veraison, full veraison, harvest. Each sample consisted of 200 bunches: 100 of which were shaken manually in sterile dist.water for 10 minutes. The rest, after being suspended in sterile water, were ultrasound treated at 50 khz for 45 seconds. The two suspensions were diluted to 10^-10. Three media were used for isolation: NAP (Nutrient Agar Difco + 20 ml/l of a 0.025% pimaricin solution, pH 6.8-7.0), TSB (a bacteria selective medium, pH 4.5) and TSL (a yeast selective medium, pH 3.0-3.5). TSB and TSL have the same basic composition (g/l): Bacto Autolyzed Yeast (Difco), 5; Bacto Casamino acids (Difco), 5; glucose, 10; agar, 20. In addition 10 ml/l of tomato juice were added to TSB. After autoclaving 15 ml/l of a 0.75% alcoholic solution of diphenyl were added to TBS; 10 ml/l of a 0.25% solution of penicillin (250,000 IU) and 20 ml/l of a 0.25% pimaricin solution to TSL.

Plates were observed for 15 days and representatives of each morphocultural type isolated on PDA (potato dextrose agar).

Isolates were identified using the probability matrix of Williams et al. (1983). The MATIDEN program (Sneath, 1979) provided the identification scores for each strain.

Results

Isolation frequency. The results are listed in Table 1. The number of Streptomyces colonies refers to one gram of fresh weight.

Streptomycetes were recovered at all phenological stages sampled. The maximum number of colonies was found on TSB during the setting stage, a decrease was observed as the season progressed.

No remarkable differences were observed between the two methods used for propagule detachment from the berries.

Probabilistic identification of the isolates. The 58 streptomycetes isolated were recovered in seven of the phena proposed by Williams et al. (1983) as shown in Table 2.

Table 1. Streptomyces colonies (no./g fresh weight) recovered on different media by two detachment method.

method       Medium  BBL  SET  BBT  BVE  FVE  HAR
-------------------------------------------------
Ultrasounds  TSL       6   33    3    4    0    0
             NAP       0   63    7   39    2   40
             TSB       0  177    3    9    1    2
Manual       TSL       0   49    4    1    0    0
 shaking     NAP       0    0   49   24    1    0
             TSB       0    0   30   53    5    0

Grapevine berry phenological stages. BBL: beginning of
blossoming, SET: setting, BBT: beginning of berry touch, BVE:
beginning of veraison, FVE: full veraison, HAR:
harvest.

Table 2. Distribution of Streptomyces isolates according to Williams et al. (1983) phena.

                           Number 
                             of       percentage 
Phenon                     Isolates
-------------------------------------------------
Streptomyces albidus       95           44.0
     "       griseoruber   14           24.1
     "       diastaticus    8           13.7
     "       chromofuscus   5            8.6
     "       rochei         3            5.1
     "       albus          1            1.8
     "       atroolivaceus  1            1.8

Six of the 26 strains grouped in the phenon S. albidoflavus show a low Willcox probability and differ from the others in some cultural characteristics. Inside S. griseoruber the identification scores of 5 out of 14 isolates are not completely satisfactory. Strains recovered in the S. diastaticus duster can be separated into two groupings, one of which is characterized by pigment production. One of the five pigmented strains does not show good agreement with identification scores. Identification of the other three strains is less satisfactory.

One strain, out of the five attributed to the phenon S. chromofuscus, does not show a correct Willcox probability.

S.atroolivaceus is represented by a single strain, however considering the low identification scores its attribution to the phenon is merely tentative.

Discussion

The present survey on the actinoflora of grapes is the result of isolations carried out during a study of particular fungal and bacterial populations of the berries, as is apparent from the media employed. Although these were not chosen with the primary view of isolating actinomycetes, it was deemed useful to determine their characteristics and report their preliminary identification, with the view of planning ad hoc investigations using media selective for actinomycetes.

However, in spite of the limitations, it appears possible to gain an idea of their frequency and specificity, though definitive conclusions will have to await better isolation methodologies. In addition the adoption of surface reference parameters, as an alternative to ponderal ones, requires further investigation.

Anyway the use of probabilistic identification methods has permitted a better systematic definition of the isolates with reference to previous gross groupings based on dichotomic keys (Kurtboke et al., 1986). The purpose of the study and the limitations mentioned above did not justify at this stage more critical attributions of some isolates.

The study shows that, although low in number, actinomycetes are constantly present on the berry surface. In addition their fluctuation during the growing season suggests that they are not only present in a dormant state but may play a role in the particular habitat. The organisms were isolated during a search for saprophytic yeasts and bacteria capable of inducing the "sour rot" alteration of grapevine berries (Vercesi et al., 1986). Their role as potential antagonists deserves particular attention.

Results obtained stress the need for a more direct approach to the problem of the presence of actinomycetes on grapes, both with reference to the use of adequate methodologies and in loco observation using suitable techniques (Vercesi and Locci, 1984). Work in this direction is in progress.

References

Kurtboke, I., B.Petrolini, S.Quaroni, R. Cardillo, G.Nasini & R. Locci (1986). Actinomycetes of the carposphere of Vitis vinifera. In: G.Szabo, S.Biro & M.Goodfellow (eds.) Biological, Biochemical and Biomedical Aspects of Actinomycetes. Akademiai Kiado, Budapest, Part B, pp. 743- 745

Vercesi, A. & R.Locci (1984). Impiego della microscopia elettronica a scansione nello studio della microflora del grappolo della vite. Vignevini, 11 (6): 31-33

Vercesi, A., F.Zerbetto, M.Bisiach & R. Locci (1986). On the grouping of yeasts associated with grapevine sour rot. Ann. Microbiol., 36:23-34

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

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