Dickeya zeae strains isolated from rice, banana and clivia rot plants show great virulence differentials

Abstract

Background: Dickeya zeae is the causal agent of maize and rice foot rot diseases, but recently it was also found to infect banana and cause severe losses in China. Strains from different sources showed significant diversity in nature, implying complicated evolution history and pathogenic mechanisms.

Results: D. zeae strains were isolated from soft rot banana plants and ornamental monocotyledonous Clivia miniata. Compared with D. zeae strain EC1 isolated from rice, clivia isolates did not show any antimicrobial activity, produced less extracellular enzymes, had a much narrow host ranges, but released higher amount of extracellular polysaccharides (EPS). In contrast, the banana isolates in general produced more extracellular enzymes and EPS than strain EC1. Furthermore, we provided evidence that the banana D. zeae isolate MS2 produces a new antibiotic/phytotoxin(s), which differs from the zeamine toxins produced by rice pathogen D. zeae strain EC1 genetically and in its antimicrobial potency.

Conclusions: The findings from this study expanded the natural host range of D. zeae and highlighted the genetic and phenotypic divergence of D. zeae strains. Conclusions can be drawn from a series of tests that at least two types of D. zeae strains could cause the soft rot disease of banana, with one producing antimicrobial compound while the other producing none, and the D. zeae clivia strains could only infect monocot hosts. D. zeae strains isolated from different sources have diverse virulence characteristics.

Keywords: Dickeya zeae; Host range; Pathogenicity; Virulence factor.

Fig. 1

Phylogenetic tree based on the 16S rRNA sequences of Dickeya species. Consensus sequences were aligned with ClustalW and trimmed in size of 654 bp. Bootstrap value after 1000 replicates is expressed as percentages. Pectobacterium atrosepticum SCRI1043 is included as an outgroup. Bar, 0.1% substitution rate per site

Fig. 2

Joint phylogenetic tree based on the concatenated nucleotide sequences of atpD, gyrB, infB and rpoB of Dickeya strains. Consensus sequences were aligned with ClustalW and trimmed in the following sizes: atpD, 642 bp; gyrB, 745 bp; infB, 1042 bp; rpoB, 1000 bp. All the sequences of a same strain were assembled for constructing the joint Neighbor-joining tree. Bootstrap values after 1000 replicates are expressed as percentages. P. atrosepticum SCRI1043 was included as an outgroup. Bar, 2% substitution rate per site

Fig. 3

The number of bacterial cells of all the tested D. zeae strains invading into potato (a), cabbage (b), banana (c) and clivia (d), respectively. Healthy plant materials were surface-sterilized and inoculated with 2 μL of bacterial overnight cultures (OD₆₀₀ = 2.0) in LB medium, and incubated at 28 °C. Tissues were taken out after 12 h and 24 h, respectively. And the diseased and surrounding healthy tissues in equal weight were cut and ground, and then added with 10 mL of sterilized 0.85% NaCl solution, stirred evenly, and 1 mL of which was diluted in series gradients, and 100 μL in each gradient was spread evenly onto LB agar plates in triplicates and kept at 28 °C for 24 h. Colonies between 30 to 300 CFU were counted. LB medium was used as a negative control. Each assay was repeated three times with duplicates

Fig. 4

Major virulence factors produced by D. zeae strains. a Growth curves of D. zeae strains in LB and LS5 media. b Extracellular cell wall degrading enzymes produced by D. zeae strains. Samples of 40 μL bacterial cells (OD₆₀₀ = 1.8) were added to the assay plate wells (4 mm in diameter) and incubated at 28 °C. Pel and Peh assay plates were treated with 4 N HCl after 11 h and 14 h respectively. Cel assay plate was stained with 0.1% (w/v) Congo Red for 15 min after 14 h, and decolored with 1 M NaCl twice. Prt assay plate was taken photos after 24 h without any further treatment. c Production of extracellular polysaccharides of D. zeae strains. Samples of 3 mL bacterial cultures (OD₆₀₀ = 1.8) were applied into 300 mL LB medium and grown with shaking at 200 r/m for 12 h, which were centrifuged at 8000 rpm for 40 m, and then at 4000 rpm for 20 m to obtain 250 mL supernatants. Double volumes of absolute ethanol were added to the supernatants, mixed thoroughly, stored at 4 °C overnight for precipitation, and centrifuged at 8000 rpm for 40 m. Finally, supernatants were discarded and pellets were weighed after drying at 55 °C overnight. d Phytotoxins produced by D. zeae strains. The bioassay plate was prepared as previously described [44]. Samples of 20 μL of bacterial cultures (OD₆₀₀ = 1.5 in LS5 medium) were added into the toxin bioassay plate wells (4 mm in diameter), and incubated overnight at 37 °C

Fig. 5

Phytotoxins produced by Dickeya strains. a, Bioassay of toxin production. b, Inhibitory activity of toxins from Dickeya strains against rice seed germination. Bacteria were grown in LB medium till OD₆₀₀ = 1.5, and 20 seeds of rice variety CO39 were added to 5 ml of every bacterial culture and incubated at room temperature for 5 h, which were then cleaned and transferred onto a Petri dish with filter paper on it, and then incubated at 28 °C under 16 h light and 8 h dark conditions. Rice seeds incubated with same amount of D. dadantii 3937 were used as a control. c, The toxin produced by MS2 is an extracellular nonprotein metabolite. Strain MS2 was grown in LS5 medium till OD₆₀₀ = 1.5 (c), and supernatant of the culture (s) was collected, which was treated by boiling at 100 °C for 10 min (b) or digestion with protease K at 37 °C for 30 min (k). Finally, the inhibition activity against the growth of E. coli DH5α was measured. d, PCR detection of zeamines biosynthesis genes from zmsO to zmsN. Based on the coding sequences of zms gene cluster in strain EC1 [43], 18 pairs of primers corresponding to zmsO to zmsN were designed to detect the zeamine biosynthsis gene cluster in D. zeae strains, which are presented in Additional file 4. The DNA marker is DL2000

Fig. 6

Worm-killing assay of D. zeae strains towards C. elegans. a Slow-killing; b Fast-killing. Strains EC1 and MS2 were grown in LB medium at 28 °C and the C. elegans food-source E. coli OP50 (negative control) at 37 °C overnight, and 50 μL of the liquid culture was spotted onto the center of NGM (slow-killing) (a) or PGS (fast-killing) (b) agar plates and allowed to dry thoroughly. In the slow-killing assay, 50 μM of floxuridine (FudR, Sigma) was added into NG agar to inhibit hatching of nematode eggs [58]. The plates containing bacteria were incubated at 28 °C and 37 °C respectively overnight and cooled for at least 2 h at room temperature before adding 30 L4 stage or adult hermaphrodite worms. The plates were kept at 20 °C, and live worms were scored

Fig. 7

Cell motility of D. zeae strains. One microlitre of bacterial culture (OD₆₀₀ = 1.5 in LB medium) was spotted onto the centre of a plate containing about 20 mL of semisolid swimming or swarming medium, which was then incubated at 28 °C for 20 h before measurement of the diameters of bacterial motility zone