Insect pathogenicity in plant-beneficial pseudomonads: phylogenetic distribution and comparative genomics

Abstract

Bacteria of the genus Pseudomonas occupy diverse environments. The Pseudomonas fluorescens group is particularly well-known for its plant-beneficial properties including pathogen suppression. Recent observations that some strains of this group also cause lethal infections in insect larvae, however, point to a more versatile ecology of these bacteria. We show that 26 P. fluorescens group strains, isolated from three continents and covering three phylogenetically distinct sub-clades, exhibited different activities toward lepidopteran larvae, ranging from lethal to avirulent. All strains of sub-clade 1, which includes Pseudomonas chlororaphis and Pseudomonas protegens, were highly insecticidal regardless of their origin (animals, plants). Comparative genomics revealed that strains in this sub-clade possess specific traits allowing a switch between plant- and insect-associated lifestyles. We identified 90 genes unique to all highly insecticidal strains (sub-clade 1) and 117 genes common to all strains of sub-clade 1 and present in some moderately insecticidal strains of sub-clade 3. Mutational analysis of selected genes revealed the importance of chitinase C and phospholipase C in insect pathogenicity. The study provides insight into the genetic basis and phylogenetic distribution of traits defining insecticidal activity in plant-beneficial pseudomonads. Strains with potent dual activity against plant pathogens and herbivorous insects have great potential for use in integrated pest management for crops.

Figure 1

Phylogeny of the P. fluorescens group based on the core genome. Genomes sequenced in this study and high-quality genomes that are publicly available by February 2015 were used to generate a core genome tree in EDGAR. Strains investigated in this study are depicted in bold. Sub-clades were defined after Loper et al. (2012). Sub-clade 1 corresponds to the P. chlororaphis subgroup, sub-clade 3 to the P. fluorescens subgroup and sub-clade 2 comprises strains belonging to three different subgroups within the P. fluorescens group according to Mulet et al. (2012), see also Supplementary Figure 1.

Figure 2

Overview on insecticidal activity, pathogen suppression and presence of associated gene clusters in 26 strains of the P. fluorescens group. Colored boxes represent activity against insects and plant pathogens as assessed within this study: high activity, medium activity, no activity. Insecticidal activity was assessed in injection assays against G. mellonella larvae and feeding assays against P. xylostella and S. littoralis larvae, and depicted activities are based on the results presented in Figure 3,Table 2, Supplementary Figure S2 and Supplementary Table S4. Disease suppression was assessed in a cucumber-Pythium ultimum assay and activities are based on the data depicted in Supplementary Table S5. Strains indicated by an asterisk were reported to have biocontrol activity against plant diseases in earlier studies (Table 1). In vitro inhibition of mycelial growth was assessed on two media against P. ultimum and Fusarium oxysporum f. sp. radicis-lycopersici and activities are based on the results shown in Supplementary Figure S3. Gray boxes represent presence of selected genes/gene clusters that were found to be associated with insecticidal strains (this study) or that are required for the production of the indicated antifungal metabolites. present, partially present, absent. Exact loci, which were checked for presence/absence, are indicated in Supplementary Table S1. There, additional genes as well as all additional strains are presented. ^aSelected genes that were identified by comparative genomics to be specific for strains that show insecticidal activity. A complete list is presented in Supplementary Table S6. P. fluorescens insecticidal toxin-cluster (fit), chitinase C (chiC), phospholipase C (plcN), metallopeptidase AprX (aprX), rebB-cluster (rebB), psl-cluster (psl). ^bGenes that were shown to contribute to insecticidal activity in this study (chiC and plcN) or elsewhere (fit) (Péchy-Tarr et al., 2008; Ruffner et al., 2013). ^cPresence/absence of gene clusters required for the production of the indicated antifungal metabolites. DAPG, 2,4-diacetylphloroglucinol; Phz, phenazine; HCN, hydrogen cyanide; Prn, pyrrolnitrin; Plt, pyoluteorin; HPR, 2-hexyl-5-propyl-alkylresorcinol.

Figure 3

Oral and systemic insecticidal activity is restricted to strains of specific phylogenetic subgroups within the P. fluorescens group. (a) Systemic activity against G. mellonella. Larvae were injected with 4 × 10⁴ bacterial cells. Bars show average mortality of three replicates with 10 larvae after 48 h. The experiment was repeated twice and highly similar results were obtained. (b) Oral activity against P. xylostella. Larvae were exposed to artificial diet covered with 8 × 10⁷ bacterial cells. Bars show average mortality of four replicates with eight larvae after 3 days. The experiment was repeated and similar results were obtained (Supplementary Table S4). Error bars show s.e.m. Asterisks indicate strains that were significantly different from control larvae treated with 0.9% NaCl based on multiple comparisons by Kruskal–Wallis adjusted by Bonferroni–Holm (P⩽0.05). (c) Typical melanization symptoms observed after 32 h in infections with P. protegens CHA0^T, P. chlororaphis subsp. piscium PCL1391, Pseudomonas sp. CMR5c, but not with P. fluorescens DSM 50090^T. (d) Although larvae injected with Pseudomonas sp. MIACH do not die, they become slightly melanized compared with control larvae. P. chl., Pseudomonas chlororaphis.

Figure 4

A derivative of P. protegens CHA0 deficient for a specific chitinase is reduced in oral, but not in injectable activity against insect larvae. (a) Systemic activity against G. mellonella. Thirty larvae per treatment were injected with 2 × 10³ bacterial cells and survival was recorded hourly. (b) Oral activity against P. xylostella. Larvae were exposed to artificial diet inoculated with 4 × 10⁶ bacterial cells. Significant differences according to a Log-Rank test (Survival Package in R) between treatments with the wild-type CHA0 and the chitinase C-negative mutant (ΔchiC) are indicated with ***P<0.0001. Each mutant was tested at least three times with similar results. A repetition of the feeding assay is depicted in Supplementary Figure S5. (c) Chitinase activity of wild-type CHA0 and its chiC mutant was assessed using a chitinase assay kit (Sigma, St Louis, MO, USA). Three different substrates were used to test for exo- (β-N-acetylglucosaminidase and chitobiosidase) and endochitinase activity. Treatments indicated by an asterisk are significantly different based on a t-test (P⩽0.05). CHA0, wild type; ΔchiC, chitinase C-negative mutant; ΔgacA, GacA-negative mutant; 0.9% NaCl served as negative control in the virulence assay; a positive control for chitinase activity was provided by the chitinase assay kit.