Isolation of endophytic bacteria
A total of 348 endophytic bacteria were isolated from different tissues of Arctium lappa L. using ten different isolation media, of which 108 strains were from NB1 (roots, stems and leaves), 116 strains were from NB2 (roots, stems and leaves), 86 strains were from roots of NB3, and 38 strains were from roots of NB4. There were 230 strains in roots, 68 strains in leaves, and 52 strains in stems, respectively. Comparing the isolation efficiency of different media, we found that M8-M10 media included the most isolated bacteria, with 112, 84, and 65 strains, respectively (Fig. 1A), whereas 87 endophytic bacterial strains were obtained from the other seven different media.
Identification and diversity of culturable endophytic bacteria
By observing the morphology and color of colonies, mycelial and spore characteristics on ISP 2 and LB agar media, we compared the phenotypes of obtained 348 strains. The morphology and culture characteristics of some representative strains are shown in Figure S1. Based on the phenotypic dereplication, 140 strains (108 from roots, 16 from stems, and 16 from leaves) were carried out detailed identification via 16S rRNA gene sequencing. The similarity between the sequenced strains and their most recent type strains was 99-100%. One strain, WY0036, showed only 97.83% similarity (1,460 bp) with the type strain Sphingobacterium composti T5-12T, indicating its status of a potential new species (Table S1). These strains belonged to the four phyla: Actinobacteria (67.24%), Proteobacteria (22.86%), Firmicutes (9.29%) and Bacteroidetes (0.71%) (Fig. 2A), and Actinobacteria was the most predominant among the endophytic bacteria. All the sequenced strains distributed into 30 genera and 73 different species (Table S1), including 18 genera in Actinobacteria, such as Amycolatopsis, Arthrobacter, Brachybacterium, Cellulosimicrobium, Corynebacterium, Gordonia, Leucobacter, Microbacterium, Micrococcus, Nocardia, Nonomuraea, Plantibacter, Promicromonospora, Pseudoclavibacter, Pseudonocardia, Rhodococcus, Streptomyces and Tsukamurella. There are nine genera in Proteobacteria, including Brevundimonas, Methylobacterium, Rhizobium, Sphingomonas, Achromobacter, Cedecea, Enterobacter, Pantoea and Pseudomonas. There are two genera of Bacillus and Staphylococcus in Firmicutes and only one genus in the phylum Bacteroidetes (Fig. 3B). At the genus level, Streptomyces (22 isolates), Microbacterium (20 isolates), Promicromonospora (16 isolates), Bacillus (12 isolates), Pseudomonas (8 isolates) and Arthrobacter (8 isolates) were the most dominant genera (Fig. 2B).
The composition of endophytic bacteria in A. lappa L. was also affected by plant cultivars and geographical origin (Fig. 2C and D). At the phylum level, there is little difference in bacterial composition, and the most abundant is Actinobacteria, followed by Proteobacteria and Firmicutes (Fig. 2C). At the genus level, 18, 14, 11 and 18 different bacterial genera were obtained from the four samples, respectively (Fig. 1B and Table S2). However, plant cultivars and geographical factors affect the distribution of culturable endophytic bacteria. The genera Bacillus, Microbacterium, Pseudomonas and Streptomyces were common in the four samples. Among the three A. lappa L. cultivars sampled from Xuzhou, all the most dominant genera were Microbacterium and Streptomyces. However, abundant Promicromonospora was isolated from NB3 cultivar and rarely obtained from other samples. The genera Arthrobacter, Microbacterium and Pseudoclavibacter were found abundant in the Harbin sample NB4, which is different from the mainly composition of three Xuzhou samples (Fig. 2D).
Diversity of unculturable endophytic bacteria in roots of A. lappa L.
A total of 145,525 quality-filtered reads were obtained from four root samples (27,380-52,573 per sample). Root of NB3 harbors the most abundant OTUs (494) at the species level. The number of OTUs in other samples were 332, 449, and 221 for NB1_R, NB2_R and NB4_R, respectively. Diversity coverage estimations indicated more than 99.0% of bacteria was sequenced. Moreover, rarefaction analyses indicated that the curves nearly reached asymptotes, suggesting that measured diversity was nearly saturated (Fig. 3A). Alpha diversity indices were calculated and indicated that community diversity differentiate across the for roots samples, with NB2 and NB3 roots harbored more abundant bacterial endophytes (Table 1). A total of 100 OTUs were common present across four samples. In contrast, OTUs that were exclusive to each root tissue ranged from 43 to 141. NB3_R harbored the highest numbers of unique OTUs, followed by NB2_R and NB4_R (Fig. 3B).
The distribution of endophytic bacteria at the phylum level in different root samples is shown in Fig. 4C. A total of 17 phyla of bacteria were detected, among which Proteobacteria (47.98%-96.72%) was the most dominant, followed by Firmicutes, Actinobacteria, Bacteroidetes and Cyanobacteria. The phyla Firmicutes (23.03% and 32.02%) and Actinobacteria (6.843% and 12.01%) in NB2_R and NB3_R samples showed much higher relative abundance than other two samples (Fig. 3C). The composition of endophytic bacteria in different roots varied greatly at genus level. A total of 207 different known genera were detected by HTS technique. In addition to the unclassified genera, the genus Pantoea accounted for 53.19% in NB1, and Pseudomonas was the most abundant genus in NB2 and NB4 samples, accounting for 19.24% and 76.94% respectively, whereas Hyphomicrobium was more abundant in NB2, accounting for 7.35% (Fig. 3D). Except for the unclassified genera, the top ten core genera in each root tissue are shown in Table 2. We found that Pseudomonas, Bacillus, Pandora and Streptomyces were distributed in almost all samples, and they were the most common genera in A. lappa L. roots.
Phylogenetic affiliation of the 16S rRNA gene sequences revealed more abundant endophytic bacteria than those obtained by pure culture method. Specifically, only 4 of the 17 phyla detected by HTS technique were obtained by cultivation. At the genus level, 83-154 known genera were found in four kinds of roots, far more than the maximum of 18 genera obtained by pure culture technique (Fig. 1B). Many genera detected by HTS were also obtained by pure culture, including Amycolatopsis, Brachybacterium, Enterobacter, Methylobacterium, Microbacterium, Nonomuraea, Promicronospora, Rhizobium, etc. The core genera, including Pseudomonas, Pantoea, Arthrobacter, Bacillus, Streptomyces, Pseudomonas and Rhizobium, were also cultured by culture-dependent methods. However, some genera were not detected by high-throughput sequencing, such as Achromobacter, Cedecea, Cellulosimicrobium, Nocardia, Plantibacter, Pseudoclavibacter, Rhodococcus, Sphingobacterium and Staphylococcus, which may be related to the quality of DNA extraction and the bias of PCR amplification in high-throughput sequencing.
Plant growth promotion traits of culturable endophytic bacteria
After checking the PGP traits of the 140 strains, the positive for the ACC deaminase and ammonia production was up to 55% (77 strains) and 54.3% (76 strains), respectively, followed by siderophore production (48.6%, 68 strains), nitrogen fixation (46.4%, 65 strains), IAA production (41.4%, 58 strains), and phosphate solubilization (26.4%, 37 strains). Comparing the origin of these strains, we found that the endophytic bacteria from NB1 showed the most PGP characteristics, almost all more than 20 strains, followed those from NB2 and NB3, and the number of active strains from NB4 was the least (Fig. 4A). The positive results of representative strains are shown in Fig. 4C. For IAA quantitative detection, the yield of strain Pantoea SP. WY068 was the highest, reaching 37.8 mg/L. In addition, 47 isolates (33.6%) possessed amylase activity, 46 isolates (32.9%) possessed cellulase activity, and 45 isolates (32.1%) possessed protease activity. Whereas xylanase and chitinase producers were 28 isolates (20%) and 22 strains (15.7%), respectively. In comparison, NB3 possessed the largest number of enzymes producing bacteria, more than 10 strains for each hydrolytic enzyme (Fig. 4B). The enzyme activity screening of representative strains is shown in Fig. 4D.
Plant growth promoting activity of selected bacterial endophytes
Four representative endophytic bacteria, WY035 (Bacillus sp.), WY068 (Pantoea sp.), WY358 (Microbacterium sp.), and WY297 (Pseudomonas sp.), which showed multiple PGP characteristics (Table 3) of the most prevalent genera were selected for PGP evaluation. After seeds inoculation and 45 days growth in soil, seedlings growth was evaluated. There was no significant difference in seed germination rate after the treatment of the four strains. However, the results showed that all the strains stimulated the growth of A. lappa L. seedlings. Endophytes treated seedlings showed significant increase (P < 0.05) in seedlings length, root length and fresh weight (Fig. 5). The former three strains exhibited the highest PGP activities, and their results in pots were shown in Fig. 5D. Seedlings length was increased by 68.3% and 69.4%, respectively, after WY035 and WY068 inoculation (Fig. 5A). In addition, isolates WY068 and WY035 also greatly increased the root lengths by 133.9% and 94.3%, respectively (Fig. 5B). Furthermore, the fresh weight of the seedlings was increased at least by 4.5-folds in the presence of the endophytes (Fig. 5C).