Candidate chemotactic AEB for RRD from P. notoginseng
We previously reported that 104 endophytic strains of P. notoginseng screened from leaf, petiole, stem, root, and seed samples grown in Wenshan region of Yunnan Province, China exhibited antagonistic properties against at least one RRD pathogen (Fusarium oxysporum, Ralstonia sp., and Meloidogyne hapla) (Ma et al. 2013), but previous studies have indicated that the abundance, diversity, and species assemblage of endophytes can be strongly influenced by the geographic regions they occur in (Deng et al. 2011; Yaish et al. 2015). These observations prompted us to further study the antagonistic endophytes associated with P. notoginseng growing in a different geographic area (Luxi County, Yunnan Province, China) to explore novel and more extensive antagonistic endophytes associated with RRD of P. notoginseng. Accordingly, 600 endophytic bacteria were isolated from different tissues (leaf, petiole, stem, and root) of P. notoginseng grown in Luxi County, different ecological region from Wenshan County in Yunnan Province, China, and were further evaluated in vitro for their antagonistic activity towards the pathogens F. oxysporum, Ralstonia sp., and M. hapla. Of the 600 isolates, 118 strains showed antagonistic activity against at least one of the tested pathogens (Table 1). PCR amplification using the P4P5.for and P4P5.rev primer pair revealed that 56 of the 222 isolates (104 previously isolated and 118 obtained from the present study) produced a characteristic cheA band (approximately 500 bp), which could be taken as chemotactic candidates (Table 1). The chemotactic ability of all 56 candidates that produced the cheA band was verified by the drop assay with aspartic acid, which was used as the attractant in a previously described chemotaxis assay (Singh et al. 2010). Results indicated that the 56 candidates could be taken as chemotactic strains because they all exhibited positive chemotaxis toward the tested aspartic acid (Fig. S1 and Table S1).
Phylogeny of P. notoginseng-associated chemotactic AEB
The 16S rRNA gene phylogenetic analysis was used to assign all 56 chemotactic AEB (CAEB) into three bacterial groups: Actinobacteria, Firmicutes, and Proteobacteria (Fig. 1 and Table 1). The most abundant class of CAEB was the Actinobacteria group, which contained 35 strains (62.5% of the total). In this group, Bacillus spp. represented the most dominant genus with 28 isolates (80.0% of the Actinobacteria group), and the majority of the Bacillus isolates were associated with the species of B. safensis (8 strains) and B. amyloliquefaciens subsp. plantarum (7), with similarities of 98.76–100% and 99.45–99.93%, respectively, whereas all other 13 Bacillus spp. isolates belonged to eight species with similarities of 98.37–100%: B. aerophilus (3 isolates), B. aryabhattai (2), B. methylotrophicus (2), B. tequilensis (2), B. cereus (1), B. mycoides (1), B. simplex (1), and B. xiamenensis (1). The seven remaining members of Actinobacteria were affiliated with five species from four genera with similarities of 97.85–100%: Staphylococcus hominis subsp. hominis (1), Staphylococcus arlettae (1), Lysinibacillus sphaericus (2), Paenibacillus taiwanensis (1), and Brevibacillus borstelensis (2). The 19 strains related to Proteobacteria made up the second-largest fraction (33.9% of the total) of the CAEB communities, and included alpha, beta, and gamma subdivisions with similarities of 97.99–99.93%. Of the 19 strains affiliated with Proteobacteria, the majority (16) exhibited high similarity to Gammaproteobacteria. However, only three strains were grouped into alpha and beta subdivisions of Proteobacteria, with 99.18–99.35% sequence similarity to Sphingomonas dokdonensis and Alcaligenes faecalis subsp. faecalis. Additionally, the 16 strains in the class Gammaproteobacteria consisted of six genera: Pseudomonas, Acinetobacter, Enterobacter, Enhydrobacter, Pantoea, Stenotrophomonas. Of the Gammaproteobacteria strains, most were related to four species in the genus Pseudomonas: P. chlororaphis subsp. aurantiaca (4), P. helmanticensis (2), P. moraviensis (1), P. plecoglossicida (1). Finally, two isolates were grouped into Actinobacteria, and they accounted for 3.6% of the total. Among them, one was found to belong to the family Micrococcaceae, showing 99.78% similarity with Micrococcus yunnanensis, and the other was most closely related to the lineage of Dermacoccacea in Actinobacteria, with 99.85% sequence similarity to Kytococcus sedentarius.
Chemotaxis toward organic acids by P. notoginseng-associated CAEB
The chemotactic response toward the five organic acids (OAs; citric acid (CA), fumaric acid (FA), malic acid (MA), oxalic acid (OX), succinic acid (SA)) for CAEB from P. notoginseng was determined in drop medium plates containing 0.1 g of crystallized OA as a chemosubstrate at the center of each plate. The presence of one of more concentric rings radiating away from the tested compound was considered positive for chemotaxis. The strength of the bacterial chemotactic response was quantified by measuring the diameter of the chemotactic ring. The substrates tested and chemotactic responses of the representative 32 strains observed are listed in Fig. S1–6 and Table S1 to illustrate the scoring of the chemotactic response. Chemotactic ring diameters were used to measure response: >51 mm = strongest response, 31–50 mm = stronger response, 21–30 mm = strong response, and 1–20 mm = weaker response. A stronger response indicated that more cells accumulated near the attractants, and that they formed a dense ring. Overall, FA—followed by CA and MA—invoked the strongest positive chemotaxis in tested strains according the chemotactic response scoring (diameter of the chemotactic ring) (Fig. 2). As shown in Fig. 2, Fig. S3, and Table S1, all 32 tested strains showed chemotactic rings with diameters > 31 mm when FA was used as the chemoattractant. Additionally, the majority of the strains (23, 71.9% of the tested strains) displayed the strongest chemotactic response to CA (51–77 mm diameter) and three strains exhibited a stronger response (33–46 mm). Fifteen strains displayed the strongest chemotactic response to MA (54–70 mm) and eight strains exhibited a strong response (35.8–50.7 mm). In contrast, SA and OA elicited relatively lower positive chemotaxis in about half of the tested strains. For example, 12 strains exhibited the weaker positive chemotactic response to OA (diameter < 15 mm), and nine strains displayed the weaker chemotactic response to SA (diameter < 18 mm). Interestingly, among all the tested strains, four (KL1, YP1, KR4, and YR3) exhibited a stronger chemotactic response towards all tested OAs, as evident by the diameter of the chemotactic ring being > 40 mm (Fig. S2–6 and Table S1), which indicated that these strains could be candidates for further study.
Diverse chemotactic response profiles across P. notoginseng-associated CAEB
At the genus level, chemotactic response profiles of the CAEB strains varied greatly across all of the tested OAs, and there was no general trend in strong or weak chemotaxis for any of the phylogenetic relationships. Using hierarchical cluster analysis and a heatmap, CAEB strains belonging to different genera (12 genera) exhibited different chemotactic responses to the same tested OAs, even when the strains were responding to a positive control AS (Fig. 3). For example, strain L22 (affiliated with Acinetobacter calcoaceticus) exhibited the strongest chemotactic response to CA, as evident by the diameter of the chemotactic ring reaching 70 mm (Table S1 and Fig. S2). However, very weak chemotaxis of strain YS3 (Kytococcus sedentarius) was measured in the diameter of the chemotactic ring responding to CA (5 mm; Table S1, Fig. S2). Similarly, strain KS1—classified as Pantoea vagans—showed the strongest chemotactic response to MA, with a diameter of 70 mm (Table S1, Fig. S4). In contrast, strain NP3 (affiliated with Sphingomonas dokdonensis) exhibited very weak chemotaxis to MA (5 mm diameter; Table S1 and Fig. S4). There was no chemotaxis toward SA in the case of strain NP3 (affiliated with S. dokdonensis), whereas NR2 (Pseudomonas helmanticensis) displayed the strongest chemotactic response to SA, with a chemotactic ring 74.2 mm in diameter (Table S1 and Fig. S6).
Additionally, within the same genus, different species have diverse chemotactic response profiles, as demonstrated by Bacillus (Fig. S7 and Table S2). We found considerable diversity in chemotactic response profiles among the 15 strains of Bacillus using hierarchical cluster analysis and a heatmap (Fig. 4). Two strains—L14 and NL2, classified as B. aryabhattai and B. aerophilus, respectively—showed the strongest chemotactic response to CA, while another strain—R7, classified as B. amyloliquefaciens subsp. plantarum—exhibited very weak chemotaxis to CA. In the case of MA, three strains (L9, YR5, and KR2, affiliated with B. amyloliquefaciens subsp. plantarum, B. tequilensis, and B. aerophilus, respectively) displayed a weaker response (chemotactic ring diameter of 2.0, 2.0, and 5.0 mm, respectively). In contrast, three other strains (YR7, YP1, and R18, classified as B. mycoides, B. amyloliquefaciens subsp. plantarum, and B. methylotrophicus, respectively) exhibited the strongest chemotactic response, with a chemotactic ring > 60 mm in diameter. In response to the SA, the 15 strains of Bacillus displayed a range of chemotaxis profiles, from 0 to 69.6 mm in diameter; e.g., strain L14 (affiliated with B. aryabhattai) was nonchemotactic to SA, but strain YR5 (classified as B. tequilensis) showed the strongest chemotactic response towards SA. Interestingly, in our study, the chemotactic response towards specific chemoattractants was found to be diverse not only among species but also among strains of a single species. For example, we found a striking diversity in the responses to all tested substrates across all five strains of B. amyloliquefaciens subsp. plantarum, as all the strains displayed unique chemotaxis response profiles to tested substrates (Fig. 4 and Fig. S7 and Table S2).
Assessment in the chemotaxis of the representative P. notoginseng-associated CAEB strain B. amyloliquefaciens subsp. plantarum YP1 in the presence of OAs
Taking the antagonism, chemotactic response profiles, strength of chemotaxis, and affiliation to the genus Bacillus into consideration, B. amyloliquefaciens subsp. plantarum YP1 was selected as the representative strain to evaluate the role of OAs—identified previously in root exudates of P. notoginseng (Li et al. 2015), in the biocontrol traits of the P. notoginseng-associated CAEB assemblage—concerning chemotaxis, growth, and antagonistic activity. A modified capillary assay was performed to assess the effects of OAs on the chemotaxis of YP1. The results showed that all tested OAs at concentrations of 10 to 100 µM significantly induced chemotactic activity in YP1 (Fig. 5). The concentrations of OAs (10 to 100 µm) tested in quantitative chemotaxis studies were chosen, as several studies have shown that OAs detected in REs at this concentration range lead to positive and apparent chemotaxis responses in Bacillus, and thus this range is commonly employed in capillary assays (Ling et al. 2011; Tan et al. 2013). The effects of OAs on the chemotactic response of YP1 differed depending on the OAs used. In general, CA and FA stimulated chemotaxis in YP1 markedly more than other OA treatments. FA had particularly marked effects on YP1 chemotaxis at various tested concentrations, and the RCR values obtained at concentrations from 10 to 100 µM were 1.1- to 1.3-fold higher in the FA treatment (Fig. 5A) than the CA one (Fig. 5B). Meanwhile, additional comparative analyses showed that FA and CA induced the chemotaxis of YP1 in a concentration-dependent manner within a dose range of 10 to 50 µM, and a 50 µM concentration yielded the highest chemotaxis in both cases (RCR = 6.73 for FA and RCR = 5.20 for CA, Fig. 5A and B, respectively). The quantitative reverse transcription-PCR (qRT-PCR) regarding cheA relative gene expression levels in YP1 demonstrated that FA at 50 µM induced the highest increase in cheA gene transcript expression (Fig. 6).
Effects of FA on the growth of B. amyloliquefaciens subsp. plantarum YP1 in vitro
The results of the capillary assay suggested that the most potent chemoattractant among tested OAs for strain YP1 was FA, as evident by the observation that FA invoked the highest chemotaxis in YP1, especially at a concentration of 50 µM (RCR = 6.73) (Fig. 6A). Therefore, we putatively selected FA as the target to evaluate the effects of OAs on the growth and biocontrol activity of P. notoginseng-associated CAEB. Compared to the control (0 µM), exogenous applications of different concentrations of FA had different but stimulating effects on YP1 growth at concentrations of 25 to 100 µM (Fig. 7). Early in incubation (12 h), YP1 had a similar growth tendency in the presence of FA and CK. Subsequently, the results showed that FA significantly stimulated the growth of YP1 at 25 and 50 µM after 12 h incubation, and the OD600 of cell biomass reached its highest level at 28 h under a 50 µM FA concentration. Moreover, the stimulation effect at 50 µM was significantly (p < 0.05) higher than at other concentrations.
The effect of FA on the biocontrol activity of B. amyloliquefaciens subsp. plantarum YP1
YP1 growth in vitro was highest with 50 µM FA, and this was thus chosen as the appropriate concentration for further analysis. Results of in vitro assays showed that treatments with 50 µM of FA significantly enhanced the antagonism of YP1 towards RRD of P. notoginseng compared to the control treatment without the FA (personal communication). Thus, to perform an in-depth analysis on the effects of FA on the antagonistic activity of YP1, the relative expression levels of the genes involved in biocontrol activity were analysed in YP1 grown in the presence of FA. As illustrated in Fig. 8, the QRT-PCR results suggested that FA at 50 µM could increase the transcription levels of all tested biocontrol-related genes, and significantly enhance the transcription levels of srfAA and sft genes in YP1, with increases of 2.4- and 3.6-fold up-regulation compared to the control (without FA), respectively.