0034-7744 0034-7744 S0034-77442000000200024 Italy Italy 00 06 2000 00 06 2000 48 2-3 511 517

The influence of air pollution on the phyllosphere microflora
composition of Tillandsia leaves (Bromeliaceae)

Luigi Brighigna1  Alessandro Gori2  Silvia Gonnelli2 and Franco Favilli.2

Received 8-XII-1998. Corrected 30-XI-1999. Accepted 1-XII-1999.

Abstract

The effect of air pollution on total phyllospheric microflora from two species of the epiphytic neotropical genus Tillandsia (Bromeliaceae) was studied by comparing unpolluted plants living in a forest (Escazú, San José) with polluted ones from an urban site of Costa Rica (San José city). Dilutions of homogenized leaf samples were plated on media suitable for each microbial group. For each microorganism group, total counts were performed and purified strains of randomly chosen colonies were identified. There was a global reduction in the number of living microorganisms due to pollution effects, especially yeasts and bacteria, while nitrogen-fixing microorganisms and fungi were less affected. Our results showed that the phyllosphere microflora of Tillandsia plants living in a tropical urban environment changes in terms of number and species composition of yeasts and bacteria with respect to plants living in unpolluted environment.
 

Key words

Tillandsia; phyllosphere; air pollution; microflora
  ]]>  

Air pollutants influence biological systems in different ways at a global level. At regional or local levels these effects are more significantly detectable, especially in urban and industrial areas or when the pollution is associated with roadways. Studies concerning air pollution impacts are difficult to undertake because of the different nature of the pollutants, the meteorology and the orography of the considered area. Even the complexity of the affected biological systems can be a cause of difficulty. The microbial ecosystems established on plant surfaces are strongly influenced by pollutants (Manning 1976, Dowding 1986, Fenn et al. 1989) and the capacity of these microbial populations to withstand environmental changes can influence their activity and consequently the whole host plant condition. The presence of such microbial populations on plant leaves and their role in global plant welfare are not well understood. It is known that some of these microorganisms fix atmospheric nitrogen (Ruinen 1974, Sucoff 1979, Murty 1983, Favilli and Messini 1990), produce plant growth regulators (Buckley and Pugh 1971), and can control plant parasites either by stimulating plants to synthesize phytoalexins (Last and Warren 1972) or by producing antibacterial (Mc Cormack et al. 1994) and/or antifungal compounds (Starmer et al. 1987). In some cases such a community can also produce air contaminants (Smith 1976).

Tillandsia (Bromeliaceae), a vascular neotropical epiphytic genus, represents a suitable model for phyllospheric studies because the microbial community that lives on these plant leaves, especially the nitrogen fixing species, greatly contributes to the plant nutrition (Brighigna et al. 1992). Tillandsia leaves are covered by characteristic trichomes, which play a role in the absorption of water and nutrients for the plants. In addition, the trichomes conform an ecological niche that shelters a plentiful microbial population (Brighigna 1992) whose metabolic activity, together with the organic and inorganic nutrients coming from different sources (air, host leaching), play a fundamental role in the whole plant nutrition. This nutritional activity is especially important in Tillandsia plants that live in extreme conditions such as high-tension wires. In general the species of Tillandsia either lack roots or the roots are unable to absorb nutrients and serve only as an adhesion system. To date, investigations on phyllosphere organisms have mainly considered the effects of pollution on yeasts and fungi of different plant species (Manning 1976, Dowding 1986, Fenn et al. 1989). Since the phyllospheric microflora plays a fundamental role in Tillandsia nutrition, we have investigated the influences of air pollution on the total phyllospheric microflora of Tillandsia leaves, with the main purpose to evaluate how it affects the phyllosphere microbial composition, and trying to establish whether a relationship exists between the nutrition of the plants and the associated phyllospheric microflora. This first approach to the problem of the relationships between the phyllosphere composition in Tillandsia and the air pollution was the consequence of a direct observation, not foreseen in advance.
 

Materials and Methods

Material.- Visiting San Josè, the capital of Costa Rica, the authors observed that all the Tillandsia plants living into the urban area were, as a consequence of intense roadway traffic fumes, markedly covered by a dark coat made by dust and ash particles retained by trichomes. In contrast Tillandsia plants living in open air sites were not coated. Many Tillandsia species were present on the host tree, Thabebuia spp. (Bignoniaceae), but we prefer do not collect tank species, because of their capacity to retain water, prefering bulbous species as Tillandsia caput-medusae and the very common intermediate species Tillandsia schiedeana. Thus plants of Tillandsia caput medusae Morr, and Tillandsia schiedeana Morr, were collected at the heigh of 3.0-3.5 m in two different areas: one, an urban area, with high level of air pollution (the Avenida Segunda of San José, close to the border line between the municipalities of San Josè and San Pedro) and the other one without pollution, an open air site 10 Km away from any urban area, factories and road trafic but with the same epiphyte-host combination (inside a wood; along the road to Escazu’, 84° 05’ W; 9° 56’ N).

Method.- The sampling was limited to few plants only and the researchs carried out on a representative sample obtained homogenizing three plants collected in each site.

The collected plants were put in sealed polyethylene bags, stored at 0°C, in a portable refrigerator, and sent to the lab in Firenze (Italy) where they arrived 96 hours after the collection. Samples of each unpolluted and polluted Tillandsia species collected were prepared immediatly after the arrival of the plants in the lab, selecting leaves from the same position with the assumption that their age was comparable.

Chemical analisys of the leaves samples, carried out as reported elsewhere (Brighigna et al. 1997), collected in the urban area showed high concentration of heavy metals (169.0; 2.9, 69.0 µg g -1 respectively for Pb, Cd, and Cu) indicating pollution by motor vehicles and industries while in the leaves collected in the open air site the concentration of the same heavy metals was negligible.

Each sample was obtained by homogenizing, in a mortar with sterile quartz sand and 9 ml of sterile saline (9 %.), one gram of fresh weight of leaves taken from each Tillandsia specimens collected. Samples were replicated three times. ]]> The dilutions were plated on different media suitable for each microbial group. Developed colonies were counted and then randomly chosen and purified. The media used were: (1) tryptone yeast extract for bacteria (5 g tryptone, 3 g yeast extract, 15 g agar, with volume made up to 1 l with distilled water, pH 6.8); (2) caseinate-asparagine agar for actinomycetes (2 g sodium caseinate, 0.1 g asparagine, 4 g sodium propionate, 0.5 g dipotassium phosphate, 0.1 g magnesium sulphate, 0.001 g ferrous sulphate, 15 g agar, with volume made up to 1 l with distilled water, pH 8.0); (3) potato dextrose agar supplemented with streptomycin for fungi and yeast (potato infusion from 200 g potatoes, 20 g dextrose, 15 g agar with volume made up to 1 l with distilled water, pH 5.6); (4) semisolid N- free Rennie medium modified [14] replacing sucrose by glucose for nitrogen fixing bacteria (5 g glucose, 5 g mannitol, 0.5 g sodium lactate, 0.8 g dipotassium phosphate, 0.2 g potassium diphosphate, 0.2 g magnesium sulphate, 0.06 g calcium cloride, 0.1 g sodium cloride, 0.1 g yeast extract, 0.025 g sodium molibdate, 0.028 g sodium-ferrous EDTA, 5 µg biotine, 10 µg p-aminobenzoic acid, 1.75 g agar with volume made up to 1 l with distilled water, pH 7.0). Nitrogen-fixing bacteria were counted with the MPN method combined with the acetylene reduction assay (Burris 1974). The bacterial pellicle developed was diluted and plated on modified N-free solid Rennie medium (Brighigna et al. 1992). After 36 h of incubation several colonies were isolated. These bacteria were considered nitrogen fixing bacteria. All the bacterial strains isolated were identified using the criteria suggested by Krieg and Holt (1984) in Bergey's Manual of Systematic Bacteriology as well as by using either the API (Appareil et Proceides d'Identification API system S.A.A., France) tests 20 E, 20 NE (Rennie 1980) and 50 CHB (Anonimous 1979, Mc Carley and Rennie 1980, Logan and Berkeley 1981, 1984) or the BIOLOG method (Garland and Mills 1991). The yeasts were identified according to Kreger Van Rij (1984) and by using the API systems ID 32C, while the isolated fungi were identified using the criteria recommended by Domsh et al. (1980).
 

Results

The main effect of the pollution by heavy metals, retaines by the leaves on the phyllosphere microflora was the strong inibition of each group of microorganisms (Table 1). The degree of inibition was ranging from the 100% for yeasts and 97% for the bacteria in both Tillandsias to nearly 60% and 50% either for the fungal population or for the nitrogen fixing bacteria in Tillandsia caput-medusae and Tillandsia schiedeana respectively..
 


Table 1.

Total counts of microorganisms on Tillandsia leaves (CFU/g fresh weight)*.

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Tillandsia caput-medusae
Tillandsia schiedeana
 
 
 
 
 ]]>  
 
Not polluted
Polluted
Not polluted
Polluted
 
 
 
 
 ]]>  
Bacteria
103 x 104
2.7 x 104
25 x 106
50 x 105
 
 
 
 
 
N2 Fixing ]]>  
 
 
 
Bacteria
2.5 x 104
1.5 x 104
4.5 x 104
2.5 x 104
 
 
 ]]>  
 
 
Fungi
18 x 104
5.5 x 104
5 x 104
2.4 x 104
 
 
 
 ]]>  
 
Yeasts
66 x 104
0
1.6 x 104
0