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

Strains isolated from soft rot banana and clivia plants were classified as D. zeae based on phylogenetic analysis

D. zeae was reported to be the causal agent of banana soft rot disease in Guangzhou, China [10, 12]. In this study, strains MS2 and MS3 that caused severe soft rot disease were respectively isolated from the basal pseudostems of banana plants in Nansha and Panyu Districts in Guangzhou city in 2012; and strains JZL1, JZL2 and JZL7 were isolated from the decayed centre leaves of clivia plants collected at Fangcun flower market in Guangzhou in 2017. The pure cultures of these isolates were inoculated to the corresponding banana and clivia plants, and typical soft rot symptoms were noticed (data not shown), validating their roles as banana and clivia pathogens, respectively. To identify the taxonomic status of these pathogens, MLSA analysis was performed based on the partial sequences of 16S rRNA gene, and the other four housekeeping genes including atpD, gyrB, infB and rpoB. Results showed that the three JZL strains contain identical housekeeping gene sequences, thus were designated as strain JZL. The alignment results showed that strains JZL, MS2 and MS3 clustered in the same branch with known D. zeae strains both in the 16S rDNA tree (Fig. 1) and the joint phylogenetic tree built on the concatenated nucleotide sequences of atpD, gyrB, infB and rpoB from 30 Dickeya species and strains (Fig. 2).

The 16S rDNA sequences of strains MS2 and MS3 are fully identical to those of D. zeae MS1 (from banana) [12], 99% identical to those of D. zeae rice isolates EC1 [11, 43], ZJU1202 [19] and DZ2Q [20]. Strains MS2 and MS3 had the identical atpD and gyrB sequences, showing 99% identity to their counterparts of strain MS1. In contrast, while strains MS3 and MS1 contain the same infB and rpoB gene sequences, strain MS2 shared 99% identity to those of strain MS1. Cumulatively, these data indicate that strains MS2 and MS3 are members of D. zeae, closely related to the previously identified D. zeae strain MS1 [12].

The 16S rDNA sequence of strain JZL is 99% identical to those of D. zeae strains EC1, Ech586 (from Philodendron) and MK19 (from river water), and 98% identical to that of strain MS2. The assembled sequence of atpD, gyrB, infB and rpoB (3429 bp) of MS2 is 99% identical to those of strains MS1 and MS3, 98% identical to that of strain EC1, and 97% identical to that of strain Ech586; and the assembled sequence of strain JZL is 97% identical to those of strains EC1 and Ech586, and 98% identical to those of strains MS1, MS2, MS3 and MK19. In summary, these data establish that strain JZL also belongs to the species D. zeae. To our knowledge, this is the first report indicating clivia as another natural host plant of D. zeae. Thereafter, the natural host range of D. zeae is expanded.

D. zeae strains in Asian countries were usually isolated from monocots

D. zeae was reported to infect both monocots and dicots [7, 11]. In most cases, it was isolated from monocot natural hosts, such as maize, rice, banana, pineapple, Brachiaria and hyacinth [3, 10, 13, 14, 16, 18, 21, 23–26]. By analysing the distribution of D. zeae, we found that it was most geographically originated from southeast Asian countries, especially the southeast of China, on rice, maize and banana (Additional files 1 and 2). Apart from China, D. zeae was also isolated from Japan, South Korea, North Korea, Philippines, India, Indonesia and Bangladesh on rice plants, and from South Korea, Japan, Thailand and India on maize host. It was currently only found in China on banana host, and in Malaysia on pineapple (Additional file 2). Given that pineapple heart rot disease was also found in Philippines and Hawaii, the pathogen was probably a novel species different from D. zeae [21, 27]. These geographical distribution and natural host range findings suggest that D. zeae may have a high level of host specificity and geographic related evolution history.

D. zeae strain JZL showed a significantly narrower host range than other D. zeae strains

D. zeae clivia strains showed weak aggressiveness on potato and cabbage, but were comparable with rice and banana strains on banana and clivia

To understand whether the decreased virulence of D. zeae clivia strains on hosts was due to their weak aggressiveness, we performed pathogenicity tests on two dicotyledonous and two monocotyledonous plants in equal weight, and calculated the numbers of bacterial cells invading into the plant tissues. Results showed that the JZL strains could not propagate in potato or cabbage tissues after inoculation for 12 h and 24 h, while EC1, MS2 and MS3 strains grew rapidly in potato in 12 h, and slowly in cabbage (Fig. 3a and b). In banana tissues, the cell number of all the strains were similar no matter at 12 h or 24 h post inoculation (Fig. 3c). In clivia, JZL strains initially grew slower than strains EC1, MS2 and MS3, but caught up at 24 h post inoculation (Fig. 3d).

D. zeae strains from clivia and banana differ in production of extracellular enzymes and polysaccharides

From the above results of pathogenicity tests, we inferred that the narrower host range of JZL strains may be due to its less virulence than the other strains, and strains EC1, MS2 and MS3 probably possess some additional virulence factor(s) than the three JZL strains. To compare the major virulence factors produced by different D. zeae strains, we first measured their growth dynamic process. Results showed that all the strains shared a similar growth pattern reaching the peak from 12 h to 16 h both in LB and LS5 media, though the cell density of JZL strains could not reach OD₆₀₀ = 1.3 in LS5 medium (Fig. 4a). We then measured the production of cell wall degrading enzymes and extracellular polysaccharides (EPS), which are two categories of virulence factors produced by the rice pathogen D. zeae strain EC1 [44, 59]. Results showed that JZL strains produced lower amount of pectate lyases (Pel), polygalacturonases (Peh), proteases (Prt) and cellulases (Cel) than the others, especially hardly any protein degrading zone was visible. In contrast, the two banana strains, MS2 and MS3, produced higher amounts of the above-mentioned extracellular enzymes than those produced by the rice strain EC1 except polygalacturonases (Fig. 4b). In a somewhat different pattern to the production of cell wall degradation enzymes, the rice pathogen strain EC1 produced significantly less EPS than the MS strains from banana and the JZL strains from clivia plants (Fig. 4c).

D. zeae banana strain MS2 produces novel antibiotic-like toxin(s) different from zeamines with slightly weaker ability to inhibit rice seed germination and kill nematodes

Our previous study found that D. zeae rice strain EC1 produces zeamines as major virulence factors in both rice plants and potato tubers [11, 60]. The phytotoxins are encoded by a zms gene cluster only found in D. zeae rice strains including ZJU1202 [19] and DZ2Q [20], some D. solani and Serratia plymuthica strains [43, 61], but were absent in other D. zeae strains isolated from other hosts including strain MS1 from banana [12, 43]. The results from this study showed that JZL strains could not produce zeamines, neither could strain MS3 (Fig. 4d), consistent with our previous findings [43].

Surprisingly, strain MS2 produced an antibiotic-like toxin(s) to inhibit the growth of E. coli DH5α, resulting in a halo with a vague edge, quite different from the halo with a clear edge caused by strain EC1 (Fig. 5a). Given that zeamines are able to inhibit rice seed germination, we tested the inhibitory activity of strain MS2 against rice seeds. The results indicated that strain MS2 had a moderate inhibitory effect on seed germination, with an inhibitory rate being about 50%, less than that of 87% by strain EC1 (Fig. 5b). Consistent with its negative antimicrobial activity phenotype (Fig. 3d, Fig. 5a), strain MS3 had no effect on seed germination (Fig. 5b). It is inferred that the inhibitory activity against seed germination is probably due to the toxin(s) produced by strain MS2, which is supposed to be a kind of extracellular nonprotein metabolite since it did not loss the activity after boiling at 100 °C for 10 min or treating with Protease K at 37 °C for 30 min (Fig. 5c).

To define whether the phytotoxin(s) produced by strain MS2 are zeamines or derivatives, we detected the zeamine biosynthesis genes from zmsO to zmsN based on their DNA sequences in strain EC1 [43], and results showed that no corresponding bands could be amplified if using the genomic DNA of strain MS2 or MS3 as a template (Fig. 5d). In combination with the sequencing results of MS2 genome (unpublished results), we confirmed that the phytotoxin(s) produced by D. zeae MS2 is probably novel, encoded by biosynthesis genes different from the zms gene cluster [43].

To investigate whether this phytotoxin(s) is toxic to nematodes, like zeamines produced by S. plymuthica A153 [62], we tested the nematode-killing dynamics of the two strains producing toxins. The results showed that both strains EC1 and MS2 had nematocidal activities, with similar slow-killing activity on NGM medium (Fig. 6a), whereas, strain EC1 had faster speed in killing C. elegans on PGS medium than strain MS2 (Fig. 6b). We inferred that the toxins produced by strains MS2 and EC1 may have similar killing effect on nematodes in the NGM medium. In the fast-killing assay, the worms treated with strain EC1 quickly became immobilized, and were completely dead in 12 h after treatment, while worms on strain MS2 lawn seemed more energetic and remained alive for a longer time compared with those on strain EC1 lawn, with only about 51% of worms died at 12 h, and 100% death occurred at 72 h after treatment (Fig. 6b), suggesting that the toxin(s) produced by strain MS2 was less toxic to C. elegans than zeamines produced by strain EC1 in the PGS medium.

D. zeae JZL strains have stronger cell motility than other strains

Bacterial motility is of important pathological significance during the early stage of infection. In this study, the mobile capability of D. zeae strains was measured including swimming and swarming motility. Results showed that all the strains were capable of swimming and swarming, but in general, the strains isolated from clivia swarmed and swam faster than the other strains, except that strain JZL7 swarmed similar to strain MS2 (Fig. 7). Given that JZL strains were weaker than the other three strains in production of various virulence factors but showed comparable virulence against Clivia miniata (Fig. 4, Table 1, Additional file 3), we reasoned that the cell motility of JZL strains might contribute substantially in their pathogenicity against the clivia plants, which awaits further investigations.

D. zeae strains collection, isolation and identification

Bacterial strains used in this study were listed in Additional file 4. D. zeae strain EC1 was isolated from diseased rice stem foot [11]. Strains MS2 and MS3 were isolated from the basal pseudostems of banana plants with soft rot disease respectively collected from Nansha and Panyu Districts in Guangzhou, Guangdong Province in 2012. D. zeae JZL strains were isolated from Clivia miniata (belonging to Amaryllidaceae) plants with soft rot symptoms at Fangcun flower market in Guangzhou city, Guangdong Province, China in 2017. D. dadantii 3937 isolated from African violets was also used for testing the virulence factors as a control.

MS2, MS3 and JZL strains that fulfilled Koch’s postulates were identified by sequencing of 16S rRNA gene using 27F/1492R primers [46], and atpD, gyrB, infB and rpoB genes using primers and PCR conditions listed by Brady et al. [47]. DNA fragments were amplified using EasyTaq DNA polymerase (Transgen) and cloned into pUC19-T vector for sequencing. The sizes of the resultant amplicons were as follows: 16S rRNA, 1507 bp; atpD, 884 bp; gyrB, 974 bp; infB, 1124 bp; rpoB, 1090 bp.

Multilocus sequences analysis (MLSA) and phylogenetic analysis

Sequence similarities of all genes were determined by using BLASTn program, and all the sequences of related strains were obtained from GenBank database. Consensus sequences were aligned with ClustalW and trimmed in the following sizes: 16S rRNA, 654 bp; atpD, 642 bp; gyrB, 745 bp; infB, 1042 bp; rpoB, 1000 bp. All the sequences of a same strain except 16S rRNA were assembled for constructing a joint phylogenetic tree. Neighbor-joining trees were constructed by bootstrap analysis with 1000 replicates using MEGA5 package [48]. The GenBank accession numbers of 16S rRNA, atpD, gyrB, infB and rpoB gene sequences of strain MS2 are MF973080, MG018807, MG018810, MG018813 and MG018816, respectively, those of strain MS3 are MF973081, MG018808, MG018811, MG018814 and MG018817, respectively, and those of strain JZL are MF973082, MG018809, MG018812, MG018815 and MG018818, respectively.

Pathogenicity assay against monocotyledonous and dicotyledonous plants

The tested strains were grown in LB medium till OD₆₀₀ = 1.5, and different plant organs of monocotyledons and dicotyledons listed in Table 1 were selected using different inoculation methods. For rice (Oryza sativa), banana (Musa sapientum ABB), Gladiolus gandavensis and Alocasia macrorrhiza, every 200 μL of bacterial cultures was injected into the basal stems of the seedlings, and into the bases of clivia leaves. For radish (Raphanus sativus), carrot (Daucus carota), potato (Solanum tuberosm), Zingiber officinale, and Colocasia esculenta, tubers were washed with tap water and dried with a paper towel, subsequently, surface-sterilized with 70% ethanol and then sliced evenly about 5 mm in thickness. Each slice was placed in a tray with moistened filter paper. Other plant materials to be inoculated were surface-sterilized. Bacterial cells of 2 μL were applied to the inoculated parts after piercing them with pipette tips except eggplant (Solanum melongena) and tomoto (Lycopersicon esculentum) inoculated with 100 μL of bacterial cultures. All trays were kept at 28 °C till symptoms appeared. Same volume of LB medium was inoculated as a negative control. Each assay was repeated three times with triplicates. The area of lesions were measured using Image J software.

Tests on aggressiveness of D. zeae strains

The tested strains were grown in LB medium till OD₆₀₀ = 2.0. Healthy potato tubers, cabbage petiole, banana pseudostems and clivia leaves were selected, washed with tap water, surface-sterilized with 70% ethanol, and placed onto the moistened filter paper in plates in biosafety cabinet under UV sterilization for 15 min. The plant tissues were pierced and inoculated with 2 μL of bacterial cultures. Plates were incubated at 28 °C. Same volume of LB medium was inoculated as a negative control. Tissues were taken out after 12 h and 24 h of incubation, respectively, where the diseased and surrounding healthy tissues in equal weight were cut and ground, then added with 10 mL of sterilized 0.85% NaCl solution, stirred evenly, and 1 mL of which was taken and diluted into 10³, 10⁴, and 10⁵ folds, and then 100 μL in each dilution gradient was spread evenly onto LB agar plates in triplicates. Plates were kept at 28 °C for 24 h, and colonies between 30 to 300 CFU (Colony-Forming Unit) were counted. Each assay was repeated three times with duplicates.

Measurement of bacterial growth curves

Bacterial strains to be tested were grown in LB medium overnight at 28 °C. All the bacterial cultures were adjusted to OD₆₀₀ = 2.0 and diluted into fresh LB medium in 1:100 ratio. Dilutions were mixed thoroughly and aliquots of 500 μL were transferred into 2.0 mL tubes. Bacteria were grown with shaking at 200 r/min under 28 °C and cell density was measured at 0, 4, 8, 12, 16, 20, 24, 28 and 40 h respectively. The experiment was repeated three times in triplicate.

Measurement of cell wall degrading enzymatic (CWDE) activities

The activities of cell wall degrading enzymes were measured using the medium recipe described previously [49–51]. Briefly, assay medium was prepared, and 30 mL of each medium was poured into a 12 × 12 cm square plate. Subsequently, wells (4 mm in diameter) were punched after solidification. Samples of 40 μL bacterial cells were added into the wells after they had grown to OD₆₀₀ = 1.8. All plates were incubated at 28 °C. Pectate lyase (Pel) and polygalacturonase (Peh) assay plates were covered with 4 N HCl after 11 h and 14 h respectively. Cellulase (Cel) assay plate was stained with 0.1% (w/v) Congo Red for 15 min after 14 h, and treated with 1 M NaCl twice. Protease (Prt) activity was indicated by the transparent halos surrounding the wells after 24 h incubation. The experiment was repeated three times with duplicates.

Assay of extracellular polysaccharide (EPS) production

Extracellular polysaccharides (EPS) are one of the important virulence factors for bacterial phytopathogens and the main toxic factors leading to water-logging and wilting on plants after infection [52–54]. For measuring the production of EPS, single colonies of the tested strains were picked and transferred into 10 mL LB medium for culture overnight at 28 °C until OD₆₀₀ = 1.8, afterwards, 3 mL of which was applied into 300 mL LB medium, and grown with shaking at 200 r/m for 12 h. Cultures 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 subjected to centrifugation at 8000 rpm for 40 m. Finally, supernatants were discarded and pellets were weighed after drying at 55 °C overnight. The experiment was repeated three times in triplicate.

Toxin bioassay

The tested strains were streaked on LB plates and single colonies were transferred into LS5 medium [55] to grow at 28 °C overnight. Cell density was adjusted to OD₆₀₀ = 1.5 prior to bioassay. The bioassay plate was prepared as previous described [44]. Wells of 4 mm in diameter were punched in the plate and 20 μL of the bacterial cultures were added into the wells. The plate was incubated overnight at 37 °C.

To preliminarily determine the chemical property of the toxin, supernatant of strain MS2 overnight culture was collected, and treated by boiling at 100 °C for 10 min and by digestion with protease K at 37 °C for 30 min, respectively. Finally, the inhibition activity against the growth of E. coli DH5α was measured. The experiment was repeated three times in triplicate.

Inhibition of phytotoxins 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 kept at room temperature for 5 h, then taken out to clean in sterilized water and transferred onto the moistened filter paper in a Petri dish, which was then kept 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. The experiment was repeated three times in triplicate.

Detection of zeamine biosynthesis gene cluster

Based on the coding sequences of zms gene cluster in strain EC1 [43], we designed 18 pairs of primers (Additional file 5) corresponding to zmsO to zmsN to detect the zeamine biosynthsis gene cluster in D. zeae strains, which are presented in Additional file 4. DNA fragments were amplified using EasyTaq DNA polymerase (Transgen) using conditions as following: 95 °C, 2 min; 30 cycles of 94 °C, 30 s, 55 °C, 30 s and 72 °C, 1 min; 72 °C, 5 min.

Antimicrobial activity assay

Pathogenic microorganisms used in this study were listed in Table 2, in which, bacterial pathogens were grown in LB medium overnight and fungi were grown on PDA medium at 28 °C until colonies covered the Petri dishes. For antibacterial activity, the same method of toxin bioassay against E. coli described in previous section was used against other bacterial pathogens, and the diameters of the antibacterial halos were measured. For antifungal activity, fungal dishes in diameter of 4 mm were punched and placed onto the center of PDA plate, and 2 μL of EC1, MS2, MS3 and 3937 strain overnight cultures were respectively spotted onto each of the four sides of the fungal dishes (3 cm away from the edge of the fungal dishes). Plates were incubated at 28 °C until fungal colonies on blank plates covered the petri dishes, and the distance between bacterial colonies and the hyphal edge of the tested fungi was measured. The experiment was repeated three times.

Nematode killing activity

Wild type Caenorhabditis elegans were maintained according to the methods as previously described [56], and the medium recipe for worm-killing assays was referred to in literature reported by Tan et al. [57]. D. zeae strains EC1 and MS2 were routinely grown in LB medium at 28 °C and the C. elegans food-source E. coli OP50 at 37 °C overnight, and 50 μL of the liquid culture was spotted onto the center of PGS (fast-killing) or NGM (slow-killing) 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]. E. coil OP50 culture was used as a negative control. 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. Each trial was repeated three times in triplicate.

Measurement of cell motility

To determine the cell motility, media for swimming (per litre contains 10 g bactotryptone, 5 g NaCl and 3 g agar) and swarming (per litre contains 5 g peptone, 3 g yeast extract and 4 g agar) assay were prepared. One microlitre of overnight bacterial culture (OD₆₀₀ = 1.5) was spotted onto the centre of a plate containing about 20 mL of .each medium. The plates were incubated at 28 °C for 20 h before measurement of the diameters of bacterial motility zone. Each experiment was repeated at least three times in triplicate [44].

Statistic analysis

All the experiments were repeated in three times with duplicates or triplicates. For statistic analysis, GraphPad Prism 5.0 software was used to performed Student’s t-test, and the data of D. zeae strains were normalized to those of strain EC1. * indicates P < 0.05, ** indicates P < 0.001, and *** indicates P < 0.0001.