Culturable bacterial diversity from the chestnut (Castanea sativa Mill.) phyllosphere and antagonism against the fungi causing the chestnut blight and ink diseases

Abstract

The phyllosphere supports a large and complex bacterial community that varies both across plant species and geographical locations. Phyllosphere bacteria can have important effects on plant health. The sweet chestnut (Castanea sativa Mill.) is an economically important tree species affected worldwide by the fungal pathogens Cryphonectria parasitica and Phytophthora cinnamomi. We examined the culturable phyllosphere bacterial community of the sweet chestnut at two nearby locations in Central Spain in order to know its geographical variability and to explore its potential as source of biological control agents against these two pathogenic fungi. The bacterial diversity at strain level was high but it varied significantly between locations; however, phylotype richness and diversity were more comparable. The isolates were affiliated with the phyla Actinobacteria, Firmicutes and Proteobacteria. Most of them were members of recognized bacterial species, with a notable proportion of representative of the genera Dietzia and Lonsdalea, but a small fraction of the strains revealed the existence of several potential novel species or even genera. Antagonism tests showed the occurrence in the chestnut phyllosphere of bacterial strains potentially useful as biological control agents against the two pathogenic fungi, some of which belong to species never before described as fungal antagonists. Chestnut phyllosphere, therefore, contains a great diversity of culturable bacteria and may represent an untapped source of potential biocontrol agents against the fungi causing blight and ink diseases of this tree species.

Keywords: Castanea sativa; Cryphonectria parasitica; Phytophthora cinnamomi; bacterial diversity; bacterial-fungal antagonism; phyllosphere.

Figure 1.. TP-RAPD (a) and M13-RAPD (b) profiles separated on ethidium bromide stained agarose gel (inverse display) of representative bacterial strains isolated from the chestnut phyllosphere. TP-RAPD patterns are noted in roman numerals. Representative strains and their taxonomic designations are given in Table 2. The DNA molecular weight marker (lane M) is Standard VI (Boehringer-Roche, Indianapolis, IN, USA) with 2176, 1766, 1230, 1033, 653, 517, 453, 394, 298, 234, and 154 bp.

Figure 2.. Observed and estimated OTU richness of culturable bacterial populations from the chestnut phyllosphere at the C and CH locations versus sampling size. OTUs as defined by M13-RAPD (a) and TP-RAPD (b) fingerprinting. The Chao1 estimated OTU richness (C ▪; CH □) and rarefaction curves (C —— ; CH – – – –) averaged over 50 simulations are shown.

Figure 3.. Rank-order plot showing the relative frequency of each OTU as defined by M13-RAPD (a, b) and TP-RAPD (c, d) patterns in the phyllosphere of chestnut trees growing at locations C (a, c) and CH (b, d).

Figure 4.. Phylogenetic neighbor-joining tree based on partial 16S rRNA gene sequences showing the relationship between representative strains of each TP-RAPD pattern (in bold types) and closely related type strains. GenBank accession numbers are in parentheses. The significance of each branch is indicated by a bootstrap value calculated for 1000 subsets; only boostrap values greater than 50% are shown. Bar, 2 substitutions per 100 nt.

Figure 5.. Cumulative radial growth of fungal colonies of Cryphonectria parasitica (a) and Phytophthora cinnamomi (b) on PDA plates in presence of different bacterial strains from the phyllosphere of chestnut. Data are Mean ± SD (N = 3).