Microbial phyllosphere populations are more complex than previously realized

Abstract

Phyllosphere microbial communities were evaluated on leaves of field-grown plant species by culture-dependent and -independent methods. Denaturing gradient gel electrophoresis (DGGE) with 16S rDNA primers generally indicated that microbial community structures were similar on different individuals of the same plant species, but unique on different plant species. Phyllosphere bacteria were identified from Citrus sinesis (cv. Valencia) by using DGGE analysis followed by cloning and sequencing of the dominant rDNA bands. Of the 17 unique sequences obtained, database queries showed only four strains that had been described previously as phyllosphere bacteria. Five of the 17 sequences had 16S similarities lower than 90% to database entries, suggesting that they represent previously undescribed species. In addition, three fungal species were also identified. Very different 16S rDNA DGGE banding profiles were obtained when replicate cv. Valencia leaf samples were cultured in BIOLOG EcoPlates for 4.5 days. All of these rDNA sequences had 97--100% similarity to those of known phyllosphere bacteria, but only two of them matched those identified by the culture independent DGGE analysis. Like other studied ecosystems, microbial phyllosphere communities therefore are more complex than previously thought, based on conventional culture-based methods.

Figure 1

PCR-DGGE 16S rDNA banding profiles of microorganisms from the phyllosphere of nine different plant crops. Lanes: 1–3, OroBlanco; 4–6, Valencia orange; 7–9, navel orange; 10–12, cotton; 13–15, corn; 16–18, sugar beet; 19–21, green bean.

Figure 2

Cluster analysis of 16S rDNA banding profiles for epiphytic bacteria from the phyllosphere of OroBlanco, Valencia orange, navel orange, cotton, corn, sugar beet, and green bean.

Figure 3

(A) PCR DGGE 16S rDNA banding profiles of epiphytic bacteria on citrus Valencia trees either directly extracted for DGGE or extracted after 4.5 days growth in different wells of a BIOLOG plate at 25°C. Lanes Val1, Val2, and Val3 are epiphytic bacteria directly extracted from Valencia orange trees 1, 2, and 3. Lanes: A6 and A10, BIOLOG with β-methyl-d-glucoside; B1, B5, and B9, BIOLOG with pyruvic acid methyl ester; D2, D6, and D10, BIOLOG with d-mannitol; E2, E6, and E10, BIOLOG with n-acetyl-d-glucosamine; G2, G6, and G10, BIOLOG with glucose-1-phosphate; H1, H5, and H9, BIOLOG with α-d-lactose. Lanes B1, D2, E2, G2, and H1 were samples from Valencia tree 1. Lanes A6, B5, D6, E6, G6, and H5 were samples from Valencia tree 2. Lanes A10, B9, D10, E10, G10, and H9 were from Valencia tree 3. (B) PCR DGGE 16S rDNA banding profiles of epiphytic bacteria from citrus Valencia leaves directly extracted for DGGE. Lanes Val1, Val2, and Val3 are epiphytic bacteria directly extracted from Valencia orange trees 1, 2, and 3. (C) PCR DGGE 16S rDNA banding profiles of phylloplane bacteria directly extracted from Valencia trees or extracted after 4.5 days growth in different wells of a BIOLOG plate at room temperature. Lanes: A11, BIOLOG with d-galactonic acid γ-lactone; B6 and B10, BIOLOG with d-xylose; B7 and B11, BIOLOG with d-galacturonic acid; B8 and B12, BIOLOG with l-asparagine; C5, BIOLOG with Tween 40; C10, BIOLOG with I-erythritol; D4 and D12, BIOLOG with l-serine; F6, BIOLOG with d-glucosaminic acid; F7, BIOLOG with itaconic acid; F12, BIOLOG with glycyl-l-glutamic acid; G1 and G9, BIOLOG with d-cellobiose; H8, BIOLOG with putrescine. Lanes D4 and G1 were samples from Valencia tree 1. Lanes B6, B7, B8, C5, F6, F7, and H8 were samples from Valencia tree 2. Lanes A11, B10, B11, B12, C10, D12, F12, and G9 were from Valencia tree 3. Lane Val3 is epiphytic bacteria directly extracted from Valencia orange trees 3.