Pangenome analysis of the genus Herbiconiux and proposal of four new species associated with Chinese medicinal plants

Five Gram-stain-positive, aerobic, non-motile actinobacterial strains designated as CPCC 205763T, CPCC 203386T, CPCC 205716T, CPCC 203406T, and CPCC 203407 were obtained from different ecosystems associated with four kinds of Chinese traditional medicinal plants. The 16S rRNA gene sequences of these five strains showed closely related to members of the genus Herbiconiux of the family Microbacteriaceae, with the highest similarities of 97.4–99.7% to the four validly named species of Herbiconiux. In the phylogenetic trees based on 16S rRNA gene sequences and the core genome, these isolates clustered into the clade of the genus Herbiconiux within the lineage of the family Microbacteriaceae. The overall genome relatedness indexes (values of ANI and dDDH) and the phenotypic properties (morphological, physiological and chemotaxonomic characteristics) of these isolates, readily supported to affiliate them to the genus Herbiconiux, representing four novel species, with the isolates CPCC 203406T and CPCC 203407 being classified in the same species. For which the names Herbiconiux aconitum sp. nov. (type strain CPCC 205763T = I19A-01430T = CGMCC 1.60067T), Herbiconiux daphne sp. nov. (type strain CPCC 203386T = I10A-01569T = DSM 24546T = KCTC 19839T), Herbiconiux gentiana sp. nov. (type strain CPCC 205716T = I21A-01427T = CGMCC 1.60064T), and Herbiconiux oxytropis sp. nov. (type strain CPCC 203406T = I10A-02268T = DSM 24549T = KCTC 19840T) were proposed, respectively. In the genomes of these five strains, the putative encoding genes for amidase, endoglucanase, phosphatase, and superoxidative dismutase were retrieved, which were classified as biosynthetic genes/gene-clusters regarding plant growth-promotion (PGP) functions. The positive results from IAA-producing, cellulose-degrading and anti-oxidation experiments further approved their potential PGP bio-functions. Pangenome analysis of the genus Herbiconiux supported the polyphasic taxonomy results and confirmed their bio-function potential.


Introduction
Anti-inflammatory herbs have been widely used in Chinese typical ethnic drugs. Even nowadays, these ethnic drugs have still contributed significantly to the health of the people living in the marginal impoverished regions. We supposed that microorganisms associated with the anti-inflammatory herbs may gain some potential abilities to produce anti-inflammatory substances resulted from their co-evolution with these plants. During screening of the microorganisms with anti-inflammatory activities, five Herbiconiux spp. were collected. We carried out polyphasic taxonomic study on these strains. The detailed phenotypic and genotypic properties of these strains supported us to propose four novel species of the genus Herbiconiux.
Previously, Herbiconiux spp. were classified as members of the genus Leifsonia within the family Microbacteriaceae (Park et al., 1994). Later, combined the phylogenetic analysis based on 16S rRNA gene sequences and the chemotaxonomic characterization, Behrendt et al. (2011) reclassified Leifsonia ginsengi as Herbiconiux ginsengi. In 2012, two more species, Herbiconiux moechotypicola and Herbiconiux flava were identified, and accordingly, the description of the genus was emended (Kim et al., 2012). Currently, four species with valid scientific names have been accommodated in the genus Herbiconiux, 1 sharing the taxonomic characteristics as follows. The peptidoglycan is of the B2γ type with D-and L-2,4-diaminobutyric acid as the diagnostic diamino acids, and glycine, alanine and threo-3-hydroxyglutamic acid may present. The predominant menaquinone is MK-11; major amounts of MK-10 may also be present. Cellular fatty acids mainly comprise iso-and anteiso-branched fatty acids, with anteiso-C 15 : 0 as a major component. Cyclohexyl-C 17 : 0 may be present. The DNA G + C content is 69-71% (Hamada et al., 2012).
It is so interesting that except the type strain of H. moechotypicola, all of the type strains of other three species of the genus Herbiconiux were isolated from the ecosystems associated with plants. In this study, five more isolates representing four novel Herbiconiux species were obtained from the ecosystems involved in anti-inflammatory herbs.
The primary goal of the present research was to study the properties of Herbiconiux members inhabiting Chinese traditional medicinal plants relating niches, to explore the genotypic properties regarding plant growth-promotion functions. As a result, in the genomes of strains CPCC 205763 T representing Herbiconiux aconitum sp. nov., CPCC 203386 T representing Herbiconiux daphne sp. nov., CPCC 205716 T representing Herbiconiux gentiana sp. nov., and CPCC 203406 T and CPCC 203407 representing Herbiconiux oxytropis sp. nov., putative encoding genes for endoglucanase, phosphatase, superoxidative dismutase and amidase were retrieved, and the corresponding phenotypic experiments further validated the putative functions. On the one hand, the microbes harbor the abilities to synthesize plant growth hormone, indole-3-acetic acid (IAA), remove excessive harmful oxygen negative ions, transform the substrates that are hard to be assimilated directly by plants, to promote plant growth; on the other hand, these anti-inflammatory plants may horizontally transfer the abilities to produce anti-inflammatory substances to the microorganisms. Our study preliminarily exemplified microbeplant interaction.
Frontiers in Microbiology 03 frontiersin.org within 1 week after collection. Isolation of microorganisms from these soils was carried out following the procedure described by Jiang et al. (2021). The medicinal plant samples IMB12384P and IMB11009P were sealed with sterile wax at the incisions before they were sent to the laboratory. The follow-up work was performed as described by Deng et al. (2022) to acquire the endophytic bacterial strains. Distinct colonies picked from the isolation plates were streaked into newly prepared PYG agar plates (g l −1 ; peptone 3, yeast extract 5, glycerol 10, betaine hydrochloride 1.25, sodium pyruvate 1.25, agar 15, pH 7.2), respectively. The purified isolates were maintained on PYG slants at 4°C and also as glycerol suspensions (20%, v/v) at -80°C. The Herbiconiux spp. were primarily identified according to the 16S rRNA gene sequence comparison following next steps. Genomic DNA was extracted according to the manual of a commercial genomic DNA extraction kit (TianGen, China). PCR amplification of the strains' 16S rRNA genes was carried out using the bacterial universal primers 27F and 1492R (Li et al., 2007). The obtained sequences were compared with available 16S rRNA gene sequences from GenBank using the BLAST program and the EzTaxon-e server 2 to determine an approximate taxonomic affiliation (Yoon et al., 2017a). Multiple sequence alignment and analysis of the data were performed by using the molecular evolutionary genetics analysis (MEGA) software package version X (Kumar et al., 2018). The phylogenetic trees were reconstructed by the software package MEGA version X using the neighbor-joining (Kimura, 1979), and confirmed by maximumlikelihood (Felsenstein, 1981) and maximum-parsimony (Kluge and Farris, 1969) tree-making methods. Bootstrap analysis with 1,000 replicates was performed to obtain the confidence level of the branches (Felsenstein, 1985).
The reference strains of Herbiconiux ginsengi KCTC 19440 T and Herbiconiux moechotypicola KCTC 19653 T were obtained from KCTC (Korean Collection for Type Cultures), and Herbiconiux flava NBRC 16403 T and Herbiconiux solani NBRC 106740 T were acquired from NBRC (NITE Biological Resource Center), respectively, and they were included in some assays in parallel.

Polyphasic taxonomic study Morphological and physiological characterization
Growth conditions of the strains were tested using ISP 2 (Shirling and Gottlieb, 1966), Tryptic soy agar (TSA, Difco), Reasoner's 2A agar (R2A, Difco), nutrition agar (NA) and PYG agar. The growth of the strains was monitored at 4, 10,15,20,28,30,32,35,37 and 40°C using TSB medium for cultivation for 2 weeks. The pH range (5.0-11.0, at intervals of 1 pH unit) for growth was observed in TSB using the buffer system described by Xu et al. (2005). Tolerance to NaCl was examined using TSB as the basal medium with different total NaCl concentrations [0-10% (w/v; at 1% intervals)]. Colony characteristics were recorded after 3-day incubation on TSA at 28°C. The Gram reaction was tested by the Gram-staining method as described by Magee et al. (1975), and the capsule-staining was performed according to the protocols described previously (Breakwell et al., 2009). The cells were observed using light microscopy (BH-2, Olympus). Motility of cells was examined on TSA semi-solid agar medium (0.3%, w/v) and then double checked using hanging drop method (Bernardet et al., 2002). The morphological characteristics of the exponentially-growing cells were observed using transmission electron microscopy (JEOL JEM-1010).
The assimilation of carbon compounds by the isolates was tested at 28°C using Biolog GEN III Microplates and observed in an Omnilog device (BIOLOG Inc., Hayward, CA, United States). Other metabolic properties were examined by API 50CH, API 20NE and API ZYM test kits (bioMerieux) according to the manufacturer's instructions. Results were evaluated after incubation at 28°C for 72-144 h. Activities of catalase, oxidase and urease, hydrolysis of Tweens and starch were investigated according to the procedures as previously described (Zhou et al., 1998;Yuan et al., 2008). The cellulose degradation activity was examined using CMC-Na screening medium (Reinhold-Hurek et al., 1993). The ability of the strains to produce IAA (indole-3-acetic acid) was tested using colorimetric methods (Bric et al., 1991) and recorded as described by Jiang et al. (2022). The scavenging effect of these strains on DPPH (2,2-diphenyl-1-picrylhydrazyl) radical was studied following the method described earlier (Singh and Rajini, 2004) and slightly modified according to the manufacture's instruction of DPPH scavenging kit (Yuanye Bio-Technology Co., Ltd., Shanghai, China) as follows. Cells were harvested and washed with 0.1 M PBS solution for twice, and then suspended in 0.1 M PBS solution to the final concentration of 10 5 cells ml −1 . The cells were sonicated in ice bath at 200 W for 3 s, followed by 10 s break. Repeated 30 rounds. The broken cells were discarded by centrifugation at 10000 r.p.m. for 10 min to collect the supernatant. Then added 50 μl of the supernatant into 450 μl of DPPH solution (0.1 mM in 95% ethanol) and incubated the mixture in dark at room temperature for 30 min. The absorbance of the resulting solution was read at 517 nm against a blank. The scavenging activity of vitamin C standard solution (2, 4, 6, 8, 10 mg L −1 ) was taken as the abscissa and the absorbance value of the corresponding concentration at 517 nm as the ordinate to draw a scatter diagram, and a linear trend line was added to obtain the scavenging activity of vitamin C standard curve. The radical scavenging activity was measured as a decrease in the absorbance of DPPH and was calculated using the following equation: A b , A c and A s is the absorbance of the blank (PBS), control (ethanol) and the sample, respectively.

Chemotaxonomic properties
Biomass for chemotaxonomic studies of the strains was obtained by cultivation in flasks on a rotary shaker (180 r.p.m.) using GYM broth at 28°C for 4 days except that cellular fatty acids extraction and analysis were conducted using the cultures harvested from Tryptic soy broth (TSB). The diagnostic isomers of diaminopimelic acid in the whole cell hydrolysates (4 N HCl, 100°C, 15 h) of these strains were subjected to thin-layer chromatography on cellulose plates using the solvent system of Schleifer and Kandler (1972). The sugar analysis of the whole cell hydrolysates followed procedures described Frontiers in Microbiology 04 frontiersin.org by Staneck and Roberts (1974). Polar lipids were extracted and examined by two-dimensional TLC and identified using previously described procedures (Minnikin et al., 1984). Menaquinones were isolated using the method of Collins et al. (1977) and were analyzed by HPLC (Groth et al., 1997). Analysis of the cellular fatty acid pattern followed the described methods using the MIDI system (Microbial ID, Inc., Newark, Del;Kroppenstedt, 1985;Meier et al., 1993). MIDI Sherlock Version 6.0 and ACTIN1 database were employed for the analysis.

Genomic traits
Genome sequencing and assembly The whole-genome sequencing was implemented using an Illumina HiSeq 4,000 system (Illumina, SanDiego, CA, United States) at the Beijing Genomics Institute (Beijing, China). Genomic DNA was sheared randomly to construct three read libraries with length of 300 bp by a Bioruptor ultrasonicator (Diagenode, Denville, NJ, United States) and physico-chemical methods. The paired-end fragment libraries were sequenced according to the Illumina HiSeq 4,000 system's protocol. Raw reads of low quality from paired-end sequencing (those with consecutive bases covered by fewer than five reads) were discarded. The sequenced reads were assembled using SOAPdenovo v1.05 software. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between these strains and other related strains were calculated using the Genome-to-Genome Distance Calculator (GGDC, version 3.0; http://ggdc.dsmz. de/ggdc.php;Auch et al., 2010) and with the ezbiocloud platform (Yoon et al., 2017b), respectively. VKM Ac-2,851) were downloaded from NCBI database and were also corrected as functionally annotated by the KEGG database. The Interpro database 4 and the UniProt database 5 were used for validation of putative functional genes. The tRNAscan-SE (Lowe and Eddy, 1997), RNAmmer, and the Rfam databases were employed for sorting of tRNA, rRNA and sRNAs, respectively.

Analysis of Pan-genome, functional genes and biosynthetic gene clusters
The protein sequences from each genome were followed by functional genes retrieval and pan-genome analysis. For pathway analysis, the predicted proteins sequences were uploaded to KEGG Automatic Annotation Server. Bacterial Pan-genome Analysis (BPGA) pipeline was applied for analysis of the genomic diversity of the Herbiconiux population. Pan-genome analysis was performed by BPGA 1.3 using default settings (Chaudhari et al., 2016). Predictions of gene clusters for natural products were performed using antiSMASH (antibiotic & Secondary Metabolite Analysis Shell, http://antismash.secondarymetabolites.org; Blin et al., 2021).

Results and analysis Phenotypic characteristics
Strains designated as CPCC 205763 T and CPCC 205716 T were recovered from herb rhizospheric-niche samples IMB15132S and IMB20178S, respectively. Plant endophytic strains CPCC 203386 T , CPCC 203406 T , and CPCC 203407 were isolated from the samples IMB12384P (the stem of a medicinal plant Daphne aurantiaca) and IMB11009P (the leaves of Oxytropis falcata), respectively.
Good growth of these five isolates was observed on tested media ISP 2, TSA, R2A, and PYG, at 28-32°C and pH 7.0. NaCl was not required for growth. Light to bright yellow colonies formed on TSA with diameter of 0.8-1.2 mm in diameter. Capsulated cells were Gramstaining-positive, non-motile and rod-shaped. Detailed physiological and biochemical characteristics results from Biolog and API assay kits were listed in Table 1 and species description.
The clear transparent circle was observed around the strains on plate with CMC-Na as the sole carbon source, which demonstrated these strains were cellulose-degrading microbes. IAA was detected in the fermentation broth of strains CPCC 205763 T , CPCC 203386 T , CPCC 205716 T , CPCC 203406 T , and CPCC 203407. As shown in Supplementary Figure S1, the linear regression equation y = 0.0255x + 0.0411, r 2 = 0.9992, gave a good fit. Accordingly, the IAA content produced by the test strains was calculated. The IAA content yielded from the fermentation broth of CPCC 205763 T , CPCC 203386 T , CPCC 205716 T , CPCC 203406 T , and CPCC 203407 was 5.89 ± 0.91 mg L −1 , 0.94 ± 0.00 mg L −1 , 4.61 ± 0.02 mg L −1 , 2.17 ± 0.02 mg L −1 and 2.10 ± 0.05 mg L −1 , respectively. Total DPPH scavenging potential of the test strains was measured and depicted in Supplementary Figure S2. The linear regression equation of the radical scavenging activity of vitamin C standard curve y = 11.65x + 17.724, r 2 = 0.9999, had a good fit. Strains CPCC 205763 T , CPCC 203386 T , CPCC 205716 T , CPCC 203406 T and CPCC 203407 were capable of neutralizing the DPPH free radicals via hydrogen donating activity by 38.3 ± 0.9%, 78.6 ± 5.8%, 51.8 ± 3.3%, 65.4 ± 10.9%, and 33.8 ± 4.1% at the concentration of 10 5 cells mL −1 , respectively. Accordingly, the Herbiconiux strains harbored a higher antioxidant activity than that of 4 mg L −1 vitamin C.

Phylogenetic analysis
The almost full-length 16S rRNA gene sequences (1, (Figure 1), which showed almost the same case in the maximum-parsimony tree, maximum-likelihood tree and phylogenetic trees based on the concatenated core genes ( Figure 2) and pan-matrix by pan-genome analysis (Supplementary Figure S4).
The genomic G + C content of these five strains ranged in 68.6-71.0%. ANI values calculated between strains CPCC 205763 T , CPCC 203386 T , CPCC 205716 T , CPCC 203406 T , and other type strains of Herbiconiux species were all less than 89.2%, and the corresponding dDDH values were below 68.1% (Supplementary Table S3). These values were lower than the thresholds used to delineate bacterial species (i.e., ANI < 95% and dDDH <70%; Kim et al., 2012). While strains CPCC 203407 and CPCC 203406 T shared a high ANI value of 100% and a high dDDH value of 99.9% (Supplementary Table S3), which were consistent with the high level of 16S rRNA gene sequence similarity (100%) between the two strains, consistently indicating the assignment of these two strains to a same species of the genus Herbiconiux (Kim et al., 2012).
Filled circles indicate that the corresponding nodes were also recovered in the trees generated with the maximum-likelihood and maximum-parsimony methods. Bootstrap values (those above 50%) are shown as percentages of 1,000 replicates. Cellulomonas flavigena DSM 20109 T (GenBank accession no. X83799) was used as an outgroup. Bar, 0.01 nt substitution per nt.

Pangenome analysis of the genus Herbiconiux
A total of 51,271 protein-coding genes (Table 2) were sorted from the genomes of these 14 strains of the genus Herbiconiux, which were divided into 15,209 homologous families by cluster analysis. According to the presence or absence of a certain homologous gene in these 14 genomes, we defined the homologous gene families conservation values (HGFCV, be abbreviated to CV). Each homologous family was given a conserved value (CV) based on its frequency of occurrence in each of the 14 genomes. Histograms were constructed according to different CVs ( Figure 4A). In the pan-genome profile of the genus Herbiconiux, there were a total of 753 core genes commonly shared by these 14 strains (CV = 14), accounting for about 5.0% of the total number of homologous gene families. The accessory genes (6,160 genes; CV = 2 ~ 13) accounted for about 40.5% of the homologous gene families in the genus Herbiconiux. The proportion of the unique genes (8,296 genes; CV = 1) was about 54.5%. The relationship between the pan-genome size and the number of genomes of the genus, and the relationship between the number of core genes and the number of genomes were deduced ( Figure 4B) by using all the protein sequences extracted from these 14 strains of the genus Herbiconiux. The functional relationship between pan-genome size (f pan ) and the number of genomes (n) was obtained by fitting, as follows: It could be observed from the pan-genome fitting curve in the Figure 4B that, with the increasing number of sequenced genomes, the pan-genome size become larger and larger, instead of tending to a plateau. Accordingly, it could be inferred that the pan-genome of the genus Herbiconiux is the open type, which suggests that the genus Herbiconiux has a strong ability to accept horizontal gene transfer events.

Secondary metabolite biosynthesis gene clusters analysis
The results from antiSMASH database showed that in these 14 strains of the genus Herbiconiux, three to eight secondary metabolite gene clusters with moderate similarities to previously described secondary metabolite biosynthetic gene clusters were Heatmap of putative functional genes predicted from the 14 genomes of the genus Herbiconiux according to the copy number of the genes from the KEGG annotation.  Table S5).

Discussion
Obviously, the 16S rRNA gene sequences alignment and the phylogenetic analysis indicated these five isolates affiliated to the genus Herbiconiux, which was supported by the chemotaxonomic traits. The genome relatedness indexes, i.e., values of ANI and dDDH between strains CPCC 205763 T , CPCC 203386 T , CPCC 205716 T , CPCC 203406 T , and all the type strains of the validly named species in the genus Herbiconiux well differentiated them from each other. The ANI and dDDH values between strain CPCC 203406 T and CPCC 203407 suggested to classify these two strains into a same species. Integrated the morphological properties, physiologic characteristics, chemotaxonomic profiles and phylogenetic analysis of these five strains, it is reasonable to propose four novel species of the genus Herbiconiux, i.e., Herbiconiux aconitum sp. nov. with CPCC 205763 T as the type strain, Herbiconiux daphne sp. nov. with CPCC 203386 T as the type strain, Herbiconiux gentiana sp. nov. with CPCC 205716 T as the type strain, and Herbiconiux oxytropis sp. nov. with CPCC 203406 T as the type strain.
Rhizospheric and endophytic microorganisms live together with the plants for a time, and co-evolve with the host plant, where microbes and plants shared the common special mico-ecosystem, so that they form a mutually beneficial symbiotic relationship during the evolution process. On the one hand, plants provide photosynthetic products and minerals for the growth of these microbes. In turn, once a kind of beneficial bacteria colonizes in a niche associated with a plant, it can influence the physiologic traits of the plant through various mechanisms. In this study, strains CPCC 205763 T , CPCC 203386 T , CPCC 205716 T , CPCC 203406 T , and CPCC 203407 were isolated from different ecosystems associated with four kinds of Chinese traditional medicinal plants, Aconitum carmichaelii, Daphne aurantiaca, Gentiana rigescens and Oxytropis falcate, respectively. As well, the strains Herbiconiux flava DSM 26474 T , Herbiconiux ginsengi CGMCC 4.3491 T , Herbiconiux solani NBRC 106740 T , Herbiconiux sp. SALV-R1, Herbiconiux sp. VKM Ac-1786 and Herbiconiux sp. VKM Ac-2,851 were reported to inhabit the niches associated with plants. Based on the genomic information, we summarized genetic characteristics of these Herbiconiux spp. The detailed phenotypic properties illustrated the abilities of these strains to make contribution to their associated medicinal plants. For instance, indole-3-acetic acid (IAA), a kind of

FIGURE 4
Overview of the pan-genomic results generated by BPGA using the 14 genomes of the genus Herbiconiux. (A) The gene family frequency spectrum.  (Unwin et al., 2004), aromatic-L-amino-acid/L-tryptophan decarboxylase (ddc) and monoamine oxidase (MAO, aofH). What's more, in the core genome of the genus Herbiconiux, the amidase coding gene (Unwin et al., 2004) was retrieved. In addition, according to the distribution of IAA-producing genes in the five strains, the pathway of IAA production by the annotation of the KEGG database could be predicted ( Figure 6). The metabolites produced by rhizospheric microbes or endophytes, such as IAA, can stimulate the growth and development of the plants that shared the common ecosystems with them, accordingly, improve the resistance of the plants to biotic or abiotic stresses. Therefore, some metabolites from these microorganisms are of significance for plant growth and development. In order to explore the useful metabolite-producing candidates, microbiologists are increasingly employing genome sequencing of a wide variety of such microbes. Here, we identified biosynthetic gene clusters (BGCs) by antiSMASH. From the antiSMASH results of these strains, we found that 100% (14/14) of the Herbiconiux genomes contain the carotenoid gene clusters (Table 3), with the 50 -66% similarities. The carotenoids belong to the isoprenoids and contain eight isoprene units, namely, tetraterpenoids. For one thing, carotenoids have the ability to absorb and transfer electrons, and play an important role in scavenging superoxide anion free radicals produced during photosynthesis (Bartley and Scolnik, 1995). In addition, carotenoids, such as astaxanthin (Chang and Xiong, 2020), fucoxanthin and zeaxanthin (Firdous et al., 2015) were reported to have anti-inflammatory functions. In this research, the four kinds of Chinese traditional medicinal plants, Aconitum carmichaelii (Zhao et al., 2018), Daphne aurantiaca (Nie et al., 2021), Gentiana rigescens (Zhao et al., 2015) and Oxytropis falcate The assigned metabolic pathways associated with the core, accessory and unique genes among the genus Herbiconiux from the KEGG database (14 strains).
Frontiers in Microbiology 12 frontiersin.org (Chen et al., 2011), were reported to have anti-inflammatory activities. Could we suppose that these microbes isolated from these ecological niches might be endowed with the anti-inflammatory traits by their corresponding medicinal plants?
From the antiSMASH results of the strain CPCC 203406 T and CPCC 203407, the herboxidiene gene clusters were retrieved. Herboxidiene (GEX1A) is a potent phytotoxic polyketone compound. On the one hand, as a potent splicing inhibitor in plants, GEX1A could trigger abiotic stress responses and ABA (abscisic acid) signaling in plants. Splicing stress signaling generated by GEX1A treatment is differentially regulated to ensure plant adaptation to stress conditions (AlShareef et al., 2017). On the other hand, as a novel polyketide which selectively and effectively controls several annual weed species, GEX1A could improve the biological competitiveness of the medicinal plant. In the antiSMASH results of the strain Herbiconiux sp. L3-i23 and Herbiconiux sp. SYSU D00978, the BGCs of alkylresorcinol were identified. Alkylresorcinols are phenolic lipids widely distributed in plants and bacteria. Different degrees of substitution yield various compounds that have been reported to possess antioxidant (Gliwa et al., 2011), anti-inflammatory (Wei et al., 2022), antimicrobial and antitumor activities. Given a variety of alkylresorcinols compounds have antimicrobial activity, it is possible to conclude that these compounds act as defensive agents in plants. Moreover, phenolic compounds (such as 5-n-alkylresorcinols, phlorizin, resveratrol, ferulic acid, et al.) are part of the plant's protection system against various pests and are therefore considered natural alternatives to protective agents (Patzke and Schieber, 2018). In the annotation results of the KEGG, the spermidine/putrescine transport related Putative overview of indole-3-acetic acid-producing pathway in the tryptophan metabolism pathways of the strains CPCC 205763 T , CPCC 203386 T , CPCC 205716 T , CPCC 203406 T , and CPCC 203407. The squares colored in green, blue, red, pink and light blue represents the strain CPCC 205763 T , CPCC 203386 T , CPCC 205716 T , CPCC 203406 T , and CPCC 203407, respectively.
Frontiers in Microbiology 13 frontiersin.org genes (potA, potB, potC, and potD) were retrieved in the genomes of the new species. In plants, it has been shown that increasing polyamine levels minimizes harmful effects caused by biotic and abiotic stresses (Yoda et al., 2003). Reactive oxygen species (ROS), generated by the plant, might be neutralized by the production of enzymes such as superoxide dismutases, catalases and alkyl hydroperoxide reductases in microorganisms. In the genomes of the strains CPCC 205763 T , CPCC 203386 T , CPCC 205716 T , CPCC 203406 T , CPCC 203407, and other members of the genus Herbiconiux included in our study, various antioxidant encoding genes were retrieved, such as genes coding for superoxide dismutase (sodA), putative manganese catalase (ydbD), catalase coding gene (katA), alkyl hydroperoxide reductase E (ahpE) and redox-sensitive transcriptional activator SoxR (soxR). The carotenoids and phytohormones mentioned above also play an important role in antioxidant activities. Therefore, the strains of the genus Herbiconiux may play an important role in their associated plants responding to oxidative stress.
Microorganisms promote the growth of their associated plants by obtaining nutrients from the environments. Such as the acquisition of organic phosphorus and inorganic phosphorus. At the genomic level, phosphate starvation-inducible protein coding gene (phoH), alkaline phosphatase coding genes (phoA, phoB), phosphate transport-related genes (phnB, phoP, phoR, phoU, pstA, pstB, pstC, and pstS) and polyphosphate kinase coding gene (ppk) were retrieved from all these 14 genomes. We also found some genes (entS) related to the production and export of the siderophore enterobactin and ironsiderophore transport system permease protein related genes (fecE, fecF, fepB, fepD, fepF, and fepG.) in the genomes of the genus Herbiconiux (Figure 3). Accordingly, we speculate that the new species of genus Herbiconiux may have beneficial effects on the growth of their associated medicinal plants, in addition to production of IAA, partially through the production of phytohormones and siderophore, solubilization of phosphorus, or by production of SOD to partly mitigate the oxidative pressure.

Conclusion
Our research investigated the taxonomic characteristics of Herbiconiux, and discovered the genetic basis of Herbiconiux producing secondary metabolites, based on pan-genome analysis and experimental validation, specifically, the PGP function of the Herbiconiux spp. associated with plants. Results from this study indicated diversity of novel Herbiconiux members are abundant in the ecosystems associated with plants, and are a group of plants-friendly microbes. Based on these results, we expect to accumulation of sufficient Herbiconiux cultures from diverse ecosystems to compare the components of secondary metabolite gene clusters in different Herbiconiux species, and to reveal new gene elements associated with secondary metabolism and explore important secondary metabolites from Herbiconiux.
Description of Herbiconiux aconitum sp. nov.
Herbiconiux aconitum (a.co.ni'ti. N.L. gen. n. aconiti, of Aconitum, a plant genus name, referring to the site related to a plant of Aconitum carmichaelii, from which the type strain was isolated).

Description of Herbiconiux daphne sp. nov.
Herbiconiux daphne (daph'nis. N.L. gen. n. daphnis, of Daphne, a plant genus name, referring to the isolation of the type strain from a plant of Daphne aurantiaca).

Description of Herbiconiux gentiana sp. nov.
Herbiconiux gentiana (gen.ti.a'nae. N.L. gen. n. gentianae, of Gentiana, a plant genus name, referring to the site related to a plant of Gentiana rigescens, from which the type strain was isolated).

Description of Herbiconiux oxytropis sp. nov.
Herbiconiux oxytropis (o.xy.tro'pis. N.L. gen. n. oxytropis, of Oxytropis, a plant genus name, referring to the isolation of the type strain from a plant of Oxytropis falcata).

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material. Microbiology  16 frontiersin.org