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Characterization and identification of rhizobacteria associated with Liberica and Robusta coffee rhizosphere

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  • Coffee is a viable agricultural commodity that makes a positive impact to the Philippine economy. However, with an increasing trend in domestic consumption, the local coffee production has declined. Chemical fertilization has been considered by many farmers to improve coffee production and yield but it causes a serious threat to public health and the environment. Biofertilizer using rhizobacteria has beneficial effects to improve the growth and yield of many crops, which is cost effective and safer than synthetic fertilizers. This study characterized the indigenous and beneficial rhizobacteria obtained from the Liberica and Robusta coffee rhizosphere, in terms of phosphate solubilization, biocontrol activities, and tolerance to abiotic stresses. Six rhizobacterial isolates were molecularly identified and belonged to genera Bacillus, Burkholderia, and Pantoea. These rhizobacteria solubilized inorganic phosphate with solubilization index ranging from 2.5 to 3.5 mm. For biocontrol activities, Bacillus sanguinis showed activity in terms of HCN and multiple hydrolytic enzymes production. Also, Burkholderia sp. demonstrated amylase, protease, and pectinase activities. Moreover, all isolates were found to be relatively tolerant to a wide range of pH and concentrations of salt and heavy metals. The performance of these rhizobacterial isolates in terms of phosphate solubilization, biocontrol activities, and tolerance to stresses is promising and shown to have potential in coffee cultivation in the Philippines.
  • Perilla frutescens (L.) Britton, belonging to Lamiaceae, is a variety of versatile economic crop, also commonly used in herbal medicine. It has been widely cultivated in China, Japan, South Korea and many other Asian countries in recent years[1]. The leaves of Perilla are used for the preparation of cold granules in Traditional Chinese Medicine (TCM), and as vegetables and spices in Asian countries[1,2]. The leaves of Perilla possess various chemicals, including terpenes, flavonoids, phenolic acids, etc.[3]. The medicinal value of Perilla has been officialized in the Chinese Pharmacopoeia and the catalog of affinal drugs and diet[1,4]. The essential oils of Perilla leaves are the major medicinal flavor components. They are also widely applied in the skin care and aromatization industry[5,6].

    The essential oils of Perilla leaves include an abundant diversity of chemical types, which are classified into monoterpene (MT)-type oils and phenylpropene (PP)-type oils[7,8]. Interestingly, there are multiple kinds of monoterpenes in the leaves of Perilla genus, which can be further classified into the following six chemotypes according to their principal constituents: perillaldehyde (PA), perillaketone (PK), perillene (PL), piperitenone (PT), citral (C) and elsholtziaketone (EK) types[9]. Among these monoterpenes, PA are the major aromatic medical ingredient for prescriptions in China and Japan, while PK were thought to be a potent lung toxin[10]. Besides these main monoterpenes, geraniol (GL) is an acyclic monoterpene also commonly found in a wide range of Perilla plants[11], and linalool (LL) can be found in all Perilla plants and maybe a dead-end compound in general monoterpene biosynthetic pathways[12]. Wherein, PA, PL, GL, and LL are commercially important for perfumery, food and medicine[13,14]. As multiple chemical types of monoterpenes are enriched in Perilla, Perilla hence has been considered to be a model system for the study of monoterpene metabolism.

    The biosynthesis of monoterpene is specially localized to the glandular trichomes and initiated from the mevalonate (MAV)/methylerythritol 4-phosphate (MEP) pathway in plants[1517]. Then the terpene synthases (TPS) catalyze prenyl pyrophosphates, the products of MVA and MEP pathways, to the formation of terpene compounds, and cytochrome P450s (CYP450s) further modify the backbones of these terpenes[17]. Recently, some TPSs and CYP450s involved in Perilla monoterpene biosynthesis have been reported, including limonene synthase, GL synthase, LL synthase, and mono-oxygenase[1820]. Enzymes participated in PA biosynthesis, such as CYP71D18 and CYP17A7146, have been characterized[1820] . Eight double-bond reductases (PfDBRs) that catalyze the conversion of isopyrone and soyone to PK were identified by enzymatic methods in vitro[21]. The recent high-quality and chromosome-scale Perilla genome data establishes a solid foundation for the characterization of Perilla monoterpene biosynthesis[22]. In the present study, transcriptome analysis was carried out on four different Perilla chemotypes some TPSs involved in monoterpene biosynthesis were identified, as well as multiple regulatory factors responsible for this biosynthesis pathway. Furthermore, the function of selected TPSs were characterized by a heterologous expression system and in vitro enzyme assay. These results collectively will help to understand the molecular mechanism of Perilla monoterpene biosynthesis and analyze the biosynthetic pathways of terpenes in Perilla.

    The Perilla cultivars of four chemotypes, including PA-, PK-, PL- and PT-types, were selected and planted in the greenhouse of Guangzhou University of Chinese Medicine (Guangzhou, Guangdong, China, 113°41' E; 23°07' N). The PA-type cultivar, with purple wrinkled leaves, belongs to P. frutescens var. crispa. The PK-, PL- and PT-type cultivars, with green non-wrinkled leaves, belong to P. frutescens var. frutescens (Fig. 1a). The leaves at seedling stages were collected for gas chromatography-mass spectrometry (GC-MS) analysis and RNA extraction. All samples are stored in liquid nitrogen immediately after sampling.

    Figure 1.  The analysis of volatile components in four Perilla cultivars. (a) Phenotype of four chemical types of Perilla; (b) GC-MS analysis of volatile essential components from four Perilla leaves; (c) Heatmap of metabolite contents in four Perilla leaves.

    Perilla leaves of four chemotypes were crushed. Then, 0.2 g leaves powder was extracted by petroleum ether and filtered for GC-MS analysis. Analysis conditions include: RXT-5 MS quartz capillary column (30 m × 0.25 μm × 0.25 μm); The front column pressure is 63.9 kPa; The initial temperature of the column was 80 °C and was retained for 1 min. After the heating rate of 15 °C/min, the column was increased to 300 °C and retained for 15 min. MS conditions: ionization mode EI, filament current 0.5 mA; Electron energy 70 eV; Multiplier voltage 0.86 kV; Ion source 230 °C, solvent delay 1 min; Plasma/nucleus ratio m/z: 40~500. The NIST spectrum library was retrieved by Agilent qualitative software, and the chemical structure analysis was combined to identify the species of components. The relative percentage of each chemical component of volatile oil was calculated by the peak area normalization method.

    Total RNA from 12 Perilla leaves were extracted using the RNApre Pure plant RNA extraction kit (DP432) (Tiangen, Beijing, China). The mRNA sequencing library was constructed using the NEBNext® Ultra RNA Library Prep Kit (New England Biolabs Inc., Ipswich, MA, USA). Then, the sequencing library was analyzed using the Agilent 2100 Bioanalyzer and was sequenced by an Illumina HiSeq™ 2000 sequencing system (Illumina Inc., San Diego, CA, USA). The original transcriptome data has been uploaded to the NGDC database (National Genomics Data Center) (Number: PRJCA021059).

    The Perilla genome data came from the National Center for Biotechnology Information (NCBI, Accession No.: GCA_019511825.2)[22]. Trimmomatic software is used for quality control of transcriptome data[23]. STAR(v2.7.10a) software was used to build an index and clean data was compared to the Perilla genome[24]. The Python module HTseq is used for P. frutescens transcriptome data quantification[25]. Gene expression levels of fragments per kilobase of transcript per million mapped reads (FPKM) were calculated. Then differentially expressed genes (DEGs) were identified using DESeq2[26]. Genes with |log2 (Fold change) |≥ 1| and p < 0.01 were considered DEGs. The online tool eggnog (http://eggnog-mapper.embl.de) was used to annotate the whole genome protein of Perilla[27]. R package ClusterProfiler was used for GO (Gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis of differential genes[28].

    The two hidden Markov models (HMM) of TPS (Terpene_synthase, PF01397 and Terpene_synthase_C, PF03936) were downloaded from Pfam (http://pfam.xfam.org/) and the Perilla genome was searched[29]. The identified PfTPS proteins were further determined by online HMMER (www.ebi.ac.uk/Tools/hmmer) and a phylogenetic tree was constructed using the Neighbor-Joining method in MEGA X (Bootstrap 1000)[30]. Heatmaps of candidate genes were drawn using TBtools (v1.112) (https://github.com/CJ-Chen/TBtools)[31]. Intergenomic collinearity analysis using MCScanX[32]. Chromosomal localization and collinearity results were visualized using TBtools. According to the Annotation information of Metabolic pathway synthase in KEGG and eggnog, the encoded gens were identified in the MVA/MEP pathways in Perilla. The Python script is used to calculate the correlation coefficient between genes expression, using the Pearson correlation coefficient[33].

    The full-length transcripts of PfTPSs genes were derived by 5' RACE-PCR and/or 3' RACE-PCR. Then the PCR products were ligated to the PLB vector (Tiangen) and sequenced by Sangon Biotech. Primer3Plus (www.primer3plus.com/index.html) was used to design primers for PfTPS genes. The primers used for genes cloning were listed in Supplemental Table S1. The fluorescence quantitative reaction system was referred to Wu et al., three replicates were used in each group, and PfActin was used as the key gene for analysis[34]. For data analysis, refer to the 2−ΔΔCᴛ method[35].

    All the cloned PfTPSs genes were introduced into the heterologous expression vector pETDuet-1. Then the expression plasmids were transformed into C41 Escherichia coli (E. coli) strain. The positive colony were firstly cultivated in TB medium at 37 °C to an initial OD600 of 0.4-0.6, and then the cultures were induced by 1 mM IPTG for another incubation of 72 h at 16 °C. After the cultivation, the cultures were extracted for three times by equal volume of n-hexane, and then the extracts were concentrated by rotary evaporation instrument for gas chromatography-mass spectrometry (GC-MS) detection.

    The selected PfTPSs genes were introduced into pET28a vector and transformed into E. coli strain BL21 (DE3). The positive colony were incubated in LB medium at 37 °C to the initial OD600 of 0.4−0.6, and then the cultures were induced by 0.5 mM IPTG for another 12 h of cultivation. The cultured cells were harvested and resuspended in lysis buffer (50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, 20 mM β-mercaptoethanol, pH 8.0) for 30 min at 4 °C. Then the cells were disrupted by ultrasonication and the lysate was centrifuged at 13,000 g and 4 °C for 30 min. The crude proteins were inside the supernatant. For the soluble PfTPSs proteins, the crude proteins were purified by HIS nickel column.

    For in vitro enzymatic reaction, crude or purified PfTPSs proteins were added to the reaction buffer containing 200 mM Tris-HCl (pH 7.5), 40 mM MgCl2, 10% glycerol and 1 mM geranyl pyrophosphate (GPP) as the precursor. After incubation at 30 °C for 30 min, the reaction system was extracted using an equal volume of n-hexane and then detected by GC-MS.

    The volatile essential oils components of leaves from four Perilla cultivars were analyzed using GC-MS analysis (Fig. 1a, b). A total of 35 terpenes, including 22 monoterpenes and 13 sesquiterpenes were detected in these cultivars. The main monoterpenes are consistent with their chemotypes classification. 68.01% of PA was identified as the main compounds in PA-type cultivars, while 71.65% of PL, 88.76% of PK and, 61.20% of PT are the main compounds in PL-type, PK-type, PT-type varieties, respectively (Fig. 1b). The GL (0.07%−0.68%) and LL (0.03%−1.77%) were the ubiquitous and trace metabolites that existed in these Perilla cultivars. Besides these main chemicals, other monoterpenes and sesquiterpenes were also identified, including limonene, borneol, thujone, verbenol, citral, carvone, trans-shisool, terpine, thymol 2-pinen-4-on, caryophyllene, germacrene, farnesene, trans-nerlidol etc. (Fig. 1c; Supplemental Table S2).

    To explore the molecular mechanism involved in different monoterpenes biosynthesis, the transcriptome analysis was performed for the leaves of four chemotype cultivars. After sequence and data filtration, a total of 579 million clean reads, comprising 86.90 Gb nucleotide bases with an average 46.26% GC were obtained (Supplemental Table S3). The average 98% clean reads were assembled to the Perilla genome (GCA_019511825.2) (Supplemental Table S4). Then, the gene annotation and differential expression analysis were carried out among the four cultivars. The PA type are the main medicinal component of Perilla according to Chinese Pharmacopoeia. Hence, more attention is focused on the PA-type. In total, 236 specific up-regulated genes were compared with other three cultivars (Fig. 2a). In the specific up-regulated genes of PA-type, phylpropanoid, and monoterpene biosynthesis were enriched using the KEGG enrichment analysis (Fig. 2b; Supplemental Table S5). Meanwhile, the comparison analysis among the other three different chemotype cultivars, the 79, 92 and 155 specific up-regulated genes were identified in PK-type, PL-type, and PT-type, respectively (Fig. 2c, e & g). Genes involved in terpenes, including monoterpene, sesquiterpene, diterpene and triterpene were enriched in the corresponding chemical type (Fig. 2df; Supplemental Tables S6S8).

    Figure 2.  Different expression genes and KEGG enrichment analysis of four Perilla cultivars. (a), (c), (e), (g) The intersection of PA, PL, PK, PT4 chemotypes with the other three chemotypes are indicated in the Venn diagrams. (b), (d), (f), (h) KEGG enrichment analysis of special up-expressed genes in PA, PL, PK, PT-type, respectively.

    The MEP and MVA pathways are the basic terpene biosynthesis pathways. The 69 genes encoding 17 enzymes in MEP and MVA pathways were identified in the four cultivars (Fig. 3; Supplemental Table S9). The MEP pathway starts with pyruvate, which is catalyzed by DXS to form 1-deoxy-D-xylulose-5-phosphate. Subsequently, it is continuously catalyzed by DXR, MCT, CMK, MDS, HDS, and HDR to form MEcPP. There were two encoded genes of DXR, MCT, CMK, and MDS in Perilla which showed upregulated expression in PA-type. In the MVA pathway, Acetyl-CoA is catalyzed by AACT HMGS, HMGR, MVK, PMK, MPDC IPP, and DMAPP to IPP. Finally, the equilibrium between IPP and DMAPP are controlled by isopentenyl diphosphate isomerase (IPPI)-encoded genes and the further reaction synthesize by geranyl pyrophosphate synthase (GPPS)-encoded genes to produce the GPP in plastids (Fig. 3; Supplemental Table S10).

    Figure 3.  Synthesis pathway and single thread synthesis pathway of P. frutescens isoprene. The MEP pathway: 1-deoxy-D-xylulose 5-phosphate synthase (DXS); 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR); 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (MCT); 4 diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK); 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS/MECPS); (E)-4-hydroxy-3 methylbut-2-enyl-diphosphate synthase (HDS); 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase (HDR); The MVA pathway: acetyl-CoA C-acetyltransferase (AACT); hydroxymethylglutaryl-CoA synthase (HMGS); hydroxymethylglutaryl-CoA reductase (HMGR); mevalonate kinase (MVK); phosphomevalonate kinase (PMK); diphosphomevalonate decarboxylase (MPDC); isopentenyl-diphosphate Delta-isomerase (IPPI); geranyl diphosphate synthase (GPPS); Linalool synthase (LLS); Geraniol synthase(GS); limonene synthase (LMS); alcohol dehydrogenase (AD); Cytochrome P450 proteins (CYP); isopiperitenol dehydrogenase (ID); pulegone reductase (PR).

    Moreover, the biosynthesis pathway of PA and PT has been reported in Perilla and Mentha, respectively[1820,36]. Limonene is the common substrate for the synthesis of PA and PT. Two genes encoded limonene synthase (LMS) were identified in Perilla. For the biosynthesis of PA, limonene is catalyzed by CYP71D18 and CYP17A7146. Two genes encoded CYP71D18 were found and up-expressed in PA-type, while four paralogs encoded CYP17A7146 were identified and one of them showed upregulated expression in PA-type (Fig. 3; Supplemental Table S11). The biosynthesis of PT by isopiperitenol dehydrogenase (ID) and pulegone reductase (PR) were identified in Perilla, which possesses three and four encoded genes and shows different expression in four cultivars, respectively. (Fig. 3; Supplemental Table S11. Moreover, geraniol is catalyzed by GL synthase (GLS). Two genes encoded GLS and showed upregulated expression in PL. GL were further catalyzed by alcohol dehydrogenase (AD) to produce citral, which is the precursor of PL and PK. Besides them, LL synthase (LLS) are also the common monoterpene compounds in Perilla. Two encoded genes encoded LLS and up-expressed in PA-type were identified in Perilla (Fig. 3).

    The TPS use prenyl pyrophosphates as the substrate to synthesize terpenes, which are important for various chemotype formation in Perilla. In Perilla, a total of 109 TPS family members were identified using HMM search. The putative PfTPS proteins ranged from 230 to 817 amino acids in length (Supplemental Table S12), with the exon number from 3 to 15 (Fig. 4b). All members contained N-terminal (PF01397) and C-terminal (PF03936) conserved domains of TPS genes (Fig. 4c), and the RRX8W domain existed in the N-terminal, while the typical DDXXD conserved domain, as well as the typical functional domain RXR, existed in the C-terminal (Fig. 4d). The PfTPS genes displayed different expression trends in the four chemotypes (Fig. 4e).

    Figure 4.  The PfTPS Gene Family Characteristics in P. frutescens. (a)−(e) Phylogenetic evolutionary tree, conservative domain, gene structure, protein motifs and expression heatmap of PfTPS genes. (f) Subfamily classification of the PfTPS Family (LLS: linalool synthase; GS: geraniol synthase; LMS: limonene synthase). (g) Chromosomal localization and collinearity analysis of Perilla. (h) Collinearity analysis of TPS in Perilla and S. baicalensis.

    PfTPS family members were divided into five major subfamilies, including TPS-a (57 members), TPS-b (24 members), TPS-c (12 members), TPS-e/f (eight members), and TPS-g (eight members) (Fig. 4f). The number of TPS genes in Perilla (109) showed significant expansion compared to that of Arabidopsis thaliana[37], Solanum lycopersicum[38], and other lamiaceae species, including Mentha longifolia[39], Salvia miltiorrhiza[39], Ocimum tenuiflorum[39], and Lavandula angustifolialabiaceae[40]. Among the TPS in Perilla, the TPS-a and TPS-b accounts for the 57.29% and 22.02% proportion as main expanded sub-families in Perilla. (Supplemental Table S13). To explore the evolutionary relationship of TPS, chromosome mapping, and collinear block analysis were carried out. The PfTPS genes were unevenly distributed on the 18 chromosomes. As the tetraploid genome of Perilla, the distribution of allele genes in pairs is a normal phenomenon. There were nine PfTPS genes found on chromosome Chr10/11/12/13, 5/8 PfTPS in Chr5/12, and 4/6 PfTPS in Chr4/6, which showed obvious collinearity in the Perilla genome (Fig. 4g). The collinearity relationship between Perilla and S. baicalensis was further analyzed. Further analysis of the collinear relation between Perilla and S. baicalensis was performed. The collinear block in 11 Perilla chromosomes correspond with seven chromosomes in S. baicalensis. Universally, the TPS in Perilla showed tandem duplication, containing 45 indicating that there is an obvious evolutionary relationship between TPS of Perilla and S. baicalensis. However, it is especially that SbChr09 has obvious chromosome blocks corresponding with multiple chromosomes of Perilla (Fig. 4h).

    To further mine the functional TPS genes in various chemotypes, the gene co-expression analysis was performed. Interestingly, PfTPS18, PfTPS46, PfTPS47, and PfTPS49 as significant core genes were identified. To present the significant relationship between those TPSs, the co-expression network was present in the core TPS and the terpene biosynthesis genes and TFs, respectively. Firstly, PfTPS18 as core genes were significant co-expression with 201 the terpene biosynthesis genes and TFs, including GPPS, HMGS, HDR, AACT, and ERF, MYB, NAC etc. (Fig. 5a). Similarly, PfTPS46 and PfTPS47 were also co-expressed with IPPI, HDR, GPPS, FPPS, HMGS, and MPDC genes, which are important rate-limiting genes in the MVA/MEP biosynthesis pathway (Fig. 5b, c). PfTPS49 was co-expressed with other five PfTPSs, including PfTPS15, PfTPS24, PfTPS38, PfTPS39, and PfTPS63, and associated with The GPPS, HMGS, and other TFs (Fig. 5d).

    Figure 5.  The co-expression analysis and verification of PfTPSs. (a)−(d) Co-expression analysis with core PfTPSs, including (a) PfTPS18, (b) PfTPS46, (c) PfTPS47 , (d) PfTPS49 and five other PfTPSs. (e) qRT-PCR verification of PfTPS genes and MVA pathway genes (qRT-PCR results (left, line) and transcription results (right, bar)).

    Among them, 12 PfTPS genes and two MVA pathway genes were selected for expression verification using qRT-PCR. The significant coincident gene levels were identified in transcriptome sequencing and qRT-PCR (r > 0.9). Those PfTPSs present general transcription in four cultivars but showed high expression in one chemotype. Such as PfTPS18, PfTPS21, and PfTPS76 showed up-regulated expression in PA types, especially PfTPS49 presents a specific high expression level. PfTPS46, PfTPS47, and PfTPS62 showed up-regulated expression in PL types. The expression levels of PfTPS87, PfTPS93, and PfTPS108 were similar in the four chemical types (Fig. 5e).

    As PfTPS18, PfTPS46, PfTPS47, and PfTPS49 were the significant core genes according to the co-expression analysis, we selected these PfTPSs genes for further functional characterization. Due to the expression levels of the above PfTPSs in different chemotypes (Fig. 5e), genes with predominant expression levels in specific chemotypes were the only successful clones, such as the highest-expressed PfTPS18 and specific-expressed PfTPS49 in PA-type. Thus, the cloned PfTPSs genes were named with their chemotypes as follows: PfTPS46-PL, PfTPS46-PK, PfTPS18-PA, PfTPS47-PA, and PfTPS49-PA, respectively. To predict the possible catalytic functions of these PfTPSs, phylogenic analysis was performed.

    To identify the catalytic functions of the cloned PfTPSs, the CDSs of different TPS to the expression vectors were ligated and transformed into E. coli to characterize the functions of PfTPSs. After being cultivated for 3 d, the cultures were extracted by n-hexane and then the compounds were detected by GC-MS analysis. Strain with PfTPS46-PL produced one peak in GC-MS profile compared to the control group (strain with control vector) (Fig. 6a). The product was determined to be linalool by the comparison of the retention time in total ion chromatograms and the mass spectrum with authentic standard linalool (Fig. 6a; Supplemental Fig. S1a, S1b). As the signal peptide (SP) region in the N terminal of PfTPS46-PL might affect the catalytic activity of the enzyme inside E. coli cells, this region was removed in the CDS of PfTPS46-PL and its function explored using the truncated variant. The strains harboring the truncated PfTPS46-PL brought the same linalool product in GC-MS analysis (Fig. 6a; Supplemental Fig. S1c). Next, to further confirm the catalytic function of PfTPS46-PL, the purification of the PfTPS46-PL protein was attempted and its function characterized using an in vitro enzymatic reaction. The proteins of PfTPS46-PL were not obtained due to its insolubility. Thus, the crude proteins of PfTPS46-PL were used with geranyl pyrophosphate (GPP) as precursor. Consistent with the result in the heterologous expression system, the crude PfTPS46-PL protein also produced the sole product linalool (Fig. 6b). According, PfTPS46-PL is a linalool synthase.

    Figure 6.  Functional characterization of four PfTPSs. (a) Heterologous functional characterization of PfTPS46-PL and (b) the in vitro enzymatic reaction of PfTPS46-PL. (c) Heterologous functional characterization and (d) the in vitro enzymatic reaction of PfTPS46-PK, PfTPS18-PA, and PfTPS49-PA. (e) Heterologous functional characterization and (f) the in vitro enzymatic reaction of PfTPS47-PA. (g) Catalytic model of four PfTPSs.

    Next, the functions of other candidate monoterpene synthases were characterized using the same strategy. The products of PfTPS46-PK, PfTPS18-PA, and PfTPS49-PA were all found to be linalool in the heterologous expression system, as well as the truncated enzymes (Fig. 6c; Supplemental Figs S2, S3). The crude proteins of PfTPS18-PA were selected as the representative for the in vitro enzymatic analysis. The crude proteins also produced the sole product linalool (Fig. 6d; Supplemental Fig. S4). The results indicated that these PfTPSs are linalool synthases.

    For the function characterization of PfTPS47-PA, two products were detected in PfTPS47-PA and truncated PfTPS47-PA-harboring strains, with the major product citronellol and the minor product geraniol (Fig. 6e; Supplemental Figs S5, S6). However, the purified PfTPS47-PA protein catalyzed GPP to the sole product geraniol (Fig. 6f). Here, we speculated that some certain enzyme inside E. coli cells catalyzed geraniol, the product of PfTPS47-PA, to citronellol. The results showed that PfTPS47-PA was highly similar to geraniol synthase while other PfTPSs were assigned to the linalool synthase category. Collectively, we identified four linalool synthases and one geraniol synthase in different Perilla chemotypes (Fig. 6g).

    The essential oils of Perilla are well-recognized aromatic compounds and possess multiple pharmacological effects. They are also the valuable genetic materials of monoterpene biosynthesis and regulation for multiple kinds of chemical types. In the present study, four monoterpene chemotype cultivars were selected. The important monoterpene biosynthesis pathway and important candidate TPSs were analyzed and verified using transcriptome sequence and heterologous expression verification.

    Terpene biosynthesis initiates from the MVA and MEP pathway in plants[16]. Compared with Arabidopsis, the number of encoded genes in the MVA and MEP pathways increased significantly in Perilla. The gene amplification could induce gene differentiation and affect the biosynthesis of terpenes[38]. The genes encoded HMGS, PMK, and MDPC in the MVA pathway, DXR, MCT, CMK, and MDS in the MEP pathway, and IPPI, and FPPS in the cross-flow pathway were found to have obvious expansion in Perilla. Interestingly, most MEP pathway genes were up-regulated in the PA type, which implied the high-efficiency biosynthesis in the PA type of Perilla.

    Various volatile oil components as chemical type of Perilla have been researched. In the early stage, the genetic basis for the monoterpene chemical type in Perilla was verified using artificial hybrids method. The chemical composition is controlled by a series of multiple alleles (G1, G2, g) and an independent pair of alleles (H, h)[41]. In the present research, 109 TPS members in Perilla genomes were identified. They also showed obvious gene expansion, especially, the expansion of TPS-a and TPS-b, reached 57.29% and 22.02% proportion, more than other majority of Lamiaceae plants. The expression and function of TPSs were also significantly differentiated. In the past few years, more geraniol synthases, linalool synthases, and limonene synthases have been acquired in Perilla[1820]. The biosynthetic pathway of piperitenon in Mentha longifolia was also reported. Based on genome-wide identified, more GS, LLS, LMS, and the PA and PT biosynthesis encoded genes were also explored. Their expression trends are in accordance with volatile oil components. LMS and GS for example had high expression in PA-types and LLS had high expression in PL-types. Based on co-expression analysis, the four TPSs act as core genes in various chemical types. The high expression in PA types and PL types were selected for function verification.

    The heterologous functional characterization and in vitro enzymatic reaction are two important methods for the functional characterization of TPSs. PfTPS18, PfTPS46, and PfTPS49 were characterized as linalool synthases and PfTPS47 was characterized as geraniol synthases, respectively. Linalool and geraniol are general compounds. The core genes in co-expression analysis were characterized as linalool and geraniol synthases. Further research will be carried out to identify more chemotype-related TPSs. Moreover, the genotype in different cultivars of a certain species is unique and widely used in the recognition of different cultivars in many plants (Supplemental Figs S7 & S8)[42,43]. For example, the polymorphic variant of one sesquiterpene synthase, VvTPS24, in grape conferred the cultivar a different product in the chemotype, which was distinguishable from other grape cultivars (Supplemental Fig. S9)[44]. However, the identified isozymes in different Perilla chemotypes, such as the linalool synthases, including PfTPS46-PL, and PfTPS46-PK, showed no obvious variations in their amino acid sequences. As the constitutions of compounds in different cultivars are determined by the enzymes and their expressions, we guess that the diverse content of linalool in different cultivars is caused by the regulatory elements.

    The authors confirm contribution to the paper as follows: conceptualization, methodology: Yang SM, An TY, Shen Q; formal analysis: Yang SM, Wang YX, Lin GB; investigation: Yang SM, Wang YX, Lin GB, Chu HY; software: Yang SM, Chu HY, Wang YX; data curation, validation: Wang YX; supervision, writing – review & editing: An TY, Shen Q; resources, writing – original draft: Yang SM, Chu HY; funding acquisition: Shen Q. All authors reviewed the results and approved the final version of the manuscript.

    The datasets presented in this study are publicly available. RNA-seq data are available via NGDC with accession No. PRJCA021059.

    This work was funded by the National Natural Science Foundation of China Grant (U22A20446) and the National Natural Science Foundation for Regional Fund (31860391).

  • The authors declare that they have no conflict of interest.

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  • Cite this article

    Navarro GVD, Quirong DD, Maghanoy GA, Cortes AD. 2023. Characterization and identification of rhizobacteria associated with Liberica and Robusta coffee rhizosphere. Technology in Horticulture 3:24 doi: 10.48130/TIH-2023-0024
    Navarro GVD, Quirong DD, Maghanoy GA, Cortes AD. 2023. Characterization and identification of rhizobacteria associated with Liberica and Robusta coffee rhizosphere. Technology in Horticulture 3:24 doi: 10.48130/TIH-2023-0024

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Characterization and identification of rhizobacteria associated with Liberica and Robusta coffee rhizosphere

Technology in Horticulture  3 Article number: 24  (2023)  |  Cite this article

Abstract: Coffee is a viable agricultural commodity that makes a positive impact to the Philippine economy. However, with an increasing trend in domestic consumption, the local coffee production has declined. Chemical fertilization has been considered by many farmers to improve coffee production and yield but it causes a serious threat to public health and the environment. Biofertilizer using rhizobacteria has beneficial effects to improve the growth and yield of many crops, which is cost effective and safer than synthetic fertilizers. This study characterized the indigenous and beneficial rhizobacteria obtained from the Liberica and Robusta coffee rhizosphere, in terms of phosphate solubilization, biocontrol activities, and tolerance to abiotic stresses. Six rhizobacterial isolates were molecularly identified and belonged to genera Bacillus, Burkholderia, and Pantoea. These rhizobacteria solubilized inorganic phosphate with solubilization index ranging from 2.5 to 3.5 mm. For biocontrol activities, Bacillus sanguinis showed activity in terms of HCN and multiple hydrolytic enzymes production. Also, Burkholderia sp. demonstrated amylase, protease, and pectinase activities. Moreover, all isolates were found to be relatively tolerant to a wide range of pH and concentrations of salt and heavy metals. The performance of these rhizobacterial isolates in terms of phosphate solubilization, biocontrol activities, and tolerance to stresses is promising and shown to have potential in coffee cultivation in the Philippines.

    • Coffee is among the most widely consumed pharmacologically active beverage world wide and its consumption has become part of everyday life[1]. Many coffee-producing countries in the coffee belt are benefiting from this agricultural commodity because their locations support the ideal growth and production of coffee, serving as a major source of income[2]. In particular, the Philippines is an ideal place to grow quality coffee of different types. However, despite the 2.1% increase in consumption, the local coffee production has been decreasing by 3.5% per year for the last 10 years[3].

      Coffee cultivation is usually associated with agri-chemical inputs, such as fertilizers and pesticides, to increase soil fertility and crop growth. However, overuse of chemical fertilizers causes soil nutrient imbalance and environmental contamination[4]. Soil acidification due to unabsorbed chemical fertilizers may lead to plant toxicity, resulting in growth deterioration and low yield of crops. In addition, chemical fertilizers are expensive and may be a burden for smallholder coffee farmers. Sustainable strategies in agricultural farming are being introduced to combat such threats, such as the exploitation of beneficial microbes as a biofertilizer[5]. Rhizospheric microorganisms are being characterized to explore their potential role in food safety and sustainable crop production.

      Rhizobacteria with plant growth promoting attributes and biocontrol activities are ubiquitous and highly abundant in the plant rhizosphere, which primarily colonize the roots and promote plant growth. They can act as biocontrol agent and their effects can occur via local antagonism to soil-borne pathogens or by induction of plant systemic resistance against pathogens[6]. Plant growth promotion can be attributed to their ability to produce phytohormones (e.g., indole acetic acid, cytokinins, and gibberellins), fix atmospheric nitrogen, solubilize phosphate, and mineralize organic substances[79].

      This study focused on the isolation, characterization, and identification of rhizobacteria with phosphate solubilization ability from the coffee rhizosphere and we evaluated their biocontrol activities and tolerance to different abiotic stresses. Their potential use as a biofertilizer will provide new insights in coffee cultivation and production in the Philippines.

    • Soil samples were randomly collected at 35-cm soil depth along the roots of Coffea liberica (Liberica) and Coffea canephora (Robusta) trees at the Coffee Genebank of National Coffee Research, Development and Extension Center (NCRDEC) in Cavite State University. A total of three 100 g of rhizospheric soil sub-samples from each coffee tree were pooled to make one composite sample per tree. A portion of the collected samples were immediately processed for microbiological analysis and the rest were air-dried at room temperature for soil physicochemical analysis.

    • Dried soil samples were sent to the Agricultural System Institute, University of the Philippines Los Baños, for the analysis of pH, moisture content, and organic matter content. The pH of the soil was found to be at 5.2 and 5.7, moisture content was 41.6% and 42.3%, and organic matter content was about 2.88% and 3.28% in the Liberica and Robusta coffee rhizosphere, respectively.

    • We selectively isolated rhizobacteria with phosphate solubilization activity on tricalcium phosphate medium. Briefly, a total of 10 g of fresh composite soil samples were mixed in 90 mL sterile distilled water and was serially diluted to the 106 dilution. One mL from each dilution was spread-plated on Pikovskaya's agar medium, containing 0.5 g·L−1 yeast extract, 10.0 g·L−1 dextrose, 5.0 g·L−1 tricalcium phosphate, 0.5 g·L−1 ammonium sulphate, 0.2 g·L−1 potassium chloride, 0.1 g·L−1 magnesium sulphate, 0.0001 g·L−1 manganese sulphate, 0.0001 g·L−1 ferrous sulphate, and 15.0 g·L−1 agar. Agar plates were incubated at 30 °C for 5 d and the clear zone around colonies were observed as indication of phosphate solubilization. The colonies with a clear zone were purified on Nutrient Agar (NA) plates. The colony characteristics of the isolates, such as elevation, margin, shape, and color were recorded. In addition, the purity of the cultures was verified using Gram stain reaction, where bacterial shapes, arrangement, and Gram reaction were also recorded. Pure cultures were then stored at 4 °C for further analysis.

      The phosphate solubilization index of rhizobacteria was measured, following the spot-plating method[10]. Initial numbers of cells were adjusted to 0.5 McFarland standard that is approximately 1.5 × 108 CFU·mL−1, this was used in all experimental assays of the study. A total of 4 μL 24-h bacterial suspension grown in Nutrient Broth (NB) was spot inoculated on Pikovskaya's agar plates containing tricalcium phosphate and incubated at 30 °C for 5 d. The colony and clear zone diameters (mm) were measured, then the solubilization index was calculated by dividing the sum of colony diameter and clear zone over the colony diameter.

    • All isolates were grown in a 5-mL glycine-supplemented NB medium. Initially, a Whatman filter paper was saturated with picric acid solution, containing 2% sodium carbonate and 0.5% picric acid, and was immediately placed at the inner top of the screw cap tubes. Tubes were sealed with parafilm and incubated with shaking for 4 d at room temperature. A color change from yellow to reddish brown in the filter paper indicate a positive HCN production.

    • The bacterial inoculum was spot inoculated, in triplicates, on starch agar medium (containing 0.5 g·L−1 peptone, 3 g·L−1 beef extract, 0.5 g·L−1 NaCl, 1% starch powder, and 12 g·L−1 agar) and incubated for 48 h at 30 °C. The plates were then flooded with iodine solution for 1 min and then drained off. The appearance of a clear zone around colonies indicate a positive for amylase activity.

    • The protease activity of the isolates was screened using milk agar medium (containing 5 g·L−1 peptone, 3 g·L−1 yeast extract, 100 mL·L−1 UHT non-fat milk and 12 g·L−1 agar). A total of 4 μL fresh inoculum was spot inoculated on the medium, in triplicate, and incubated for 48 h at 30 °C. A clear zone around the colonies indicates a positive protease activity.

    • Pectinase activity of isolates was screened using the pectin agar medium (containing 0.5 g·L−1 peptone, 0.3 g·L−1 beef extract, 0.5 g·L−1 NaCl, 4 g·L−1 citrus pectin, and 12 g·L−1 agar). Spot inoculation was similarly used but incubation was extended to 96 h. A 50 mM potassium iodide-iodine solution was then flooded on the surface of the agar plates. A positive result was indicated by the appearance of a clear zone around colonies.

    • The ability of the isolates to tolerate abiotic stresses, such as acidity, salinity, and heavy metal contents (i.e., lead, copper, and manganese) was screened. Briefly, the isolates were subjected to varying pH level, namely 4, 5, 7, 9 and 11. Uniform initial number of cells were grown in NB for 24 h with shaking and bacterial suspension and spot inoculated on NA plates. Also, NA medium was supplemented with varying sodium chloride concentrations (i.e., 1%, 3%, 5%, 7%, 9%, and 11%) and was used to screen for salt tolerance of the isolates. Lastly, the ability of the isolates to tolerate heavy metals such as lead, copper, and manganese was screened using NA medium supplemented with varying concentrations (i.e., 100, 150, 200, 400, 800, and 1,600 ppm) of lead acetate (Pb(C2H3O2)2), copper sulphate (CuSO4), and manganese sulphate (MnSO4), respectively. Growth was observed after 48 h at room temperature, indicating resistance/tolerance to varying levels of pH, salt, and heavy metals.

    • A total of six isolates were subjected to DNA extraction using Vivantis GF-1 Bacterial DNA Extraction Kit, following the manufacturer's protocol. The quality of the isolated genomic DNA was verified in 0.8% agarose gel (dissolved in 0.5X TAE buffer), following gel electrophoresis (Mupid One) and gel documentation (Vilber Lourmat). The purity and concentration of DNA were quantified using a NanoDrop 2000c UV-Vis Spectrophotometer (Thermo Scientific™).

      Genomic DNA samples were subjected to polymerase chain reaction (PCR) amplification using universal primers to target the 16S rRNA gene, which are 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). PCR was performed in a 50-μL reaction containing 1X Taq Master Mix (Vivantis), 2 mM MgCl2, 0.2 μM each of 27F and 1492R primers, and 100 ng of DNA template. A final volume of 50 μL was adjusted with molecular grade water. PCR reactions were performed using MiniAmp™ Plus Thermal Cycler (Applied Biosystems™) with the following conditions: initial denaturation step at 95 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, annealing at 55 °C for 30 s, and 72 °C for 1 min, with a final extension step of 72 °C for 10 min. The PCR products were ran using 1.2% agarose gel (stained with GelRed® nucleic acid dye) under 100 V for 35 min and verified under UV transilluminator.

      The PCR products were sent to Apical Scientific Sdn Bhd (Selangor, Malaysia) for Sanger sequencing. The electropherogram of the sequences was inspected and verified using Sequence Analyzer of MEGAX software. Homologous partial 16S rRNA gene sequences with > 97% similarity were mined in the GenBank database using BLAST algorithm. In MEGAX, ClustalW was used to perform multiple sequence alignment of the unknown and homologous nucleotide sequences. Phylogenetic trees were constructed using Neighbor joining method with 1,000 replicated bootstrap values. The identity of the rhizobacterial isolates was verified based on the clustering of target sequence with the closest annotated sequence (type strain).

    • A total of six rhizobacteria with phosphate solubilization activity (Fig. 1a) were recovered and were subjected to morphological characterization. Two isolates were Gram positive and four were Gram negative, by which all are rod-shaped cells. Colony shapes vary from irregular to circular; elevation is either flat, raised, convex, and crateriform; margin varies from being entire, serrate, undulate, and curled; and the color is yellow, cream, or white (Table 1). The solubilization index (SI) ranges 2.5 to 3.5 mm. Isolate PCL 2.1 and PCR 1.1 obtained the highest phosphate SI of 3.5 mm, whereas isolate PCL 1.3 obtained the lowest (2.5 mm) (Table 2).

      Figure 1. 

      Representatives of rhizobacteria (a) capable of phosphate solubilization as indicated by the clear zone around the colony (arrow) and (b) not capable of phosphate solubilization grown on Pikovskaya's agar medium for 5 d at 30 °C.

      Table 1.  Morphological characteristics of rhizobacteria from the coffee rhizosphere.

      Rhizosphere sourceIsolateColony shapeColony elevationColony marginColony colorGram reactionCell shape
      LibericaPCL 1.2IrregularRaisedEntireWhiteNegativeRods
      LibericaPCL 2.1CircularFlatSerrateWhitePositiveRods
      LibericaPCL 2.3IrregularConvexEntireWhiteNegativeRods
      RobustaPCR 1.1CircularRaisedEntireYellowNegativeRods
      RobustaPCR 1.3IrregularCrateriformUndulateCreamPositiveRods
      RobustaPCR 1.5CircularFlatCurledWhiteNegativeRods

      Table 2.  Six rhizobacterial isolates showing phosphate solubilization, biocontrol activities, and abiotic stress tolerance.

      IsolatePhosphate
      solubilization
      index (mm)
      Biocontrol activityAbiotic stress tolerance
      HCN productionAmylaseProtease PectinaseNaCl (%)pHMnSO4 (ppm)CuSO4 (ppm)Pb(C2H3O2)2 (ppm)
      PCL 1.23.1 ± 0.574 – 111,600400800
      PCL 2.13.5 ± 0.5+74 – 111,600400800
      PCL 2.33.1 ± 0.534 – 111,600400800
      PCR 1.13.5 ± 0.5+34 – 111,600800800
      PCR 1.32.5 ± 0.5+++54 – 11800400800
      PCR 1.52.8 ± 0.5+++34 – 111,600400800
      HCN, hydrogen cyanide; NaCl, sodium chloride; MnSO4, manganese sulphate; CuSO4, copper sulphate; Pb(C2H3O2)2, lead acetate.
    • Among the six rhizobacterial isolates, only PCR 1.3 produced HCN. For the hydrolytic enzyme production, PCL 2.1 produced amylase, PCR 1.1 produced protease, PCR 1.3 produced amylase and protease, and PCR 1.5 produced amylase, protease, and pectinase (Table 2).

    • For pH tolerance, all isolates were tolerant to a wide range of pH (4 to 11). For salt tolerance, PCL 1.2 and PCL 2.1 were tolerant to 7% NaCl, while the lowest tolerance was observed at 3% NaCl. For heavy metal tolerance, all rhizobacteria tolerated 1600 ppm of MnSO4, except PCR 1.3 that only tolerated 800 ppm. PCR 1.1 was able to tolerate up to 800 ppm of CuSO4, while the rest were only tolerant to 400 ppm. Meanwhile, all of them were tolerant to 800 ppm of Pb(C2H3O2)2 (Table 2).

    • Based on the 16S rRNA gene analysis of six promising rhizobacterial isolates, PCL 1.2, PCL 2.1, PCL 2.3, PCR 1.1, PCR 1.3, and PCR 1.5 were identified as Pantoea rwandensis (97.44%), Bacillus pseudomycoides (97.23%), Pantoea sp. (93.62%), Bukholderia cepacia (97.17%), Bacillus sanguinis (99.51%), and Bukholderia sp. (86.66%), respectively (Table 3). Based on the phylogenetic tree analysis, each of the unknown sequence clustered to its closest neighbor (type strain) with bootstrap values (Fig. 2).

      Table 3.  Molecular identity of rhizobacteria isolates from coffee rhizosphere.

      IsolateIdentityClosest neighbor (type strain)Similarity (%)Accession no.
      PCL 1.2Pantoea rwandensisPantoea rwandensis strain LMG 2627597.44NR_118121.1
      PCL 2.1Bacillus pseudomycoidesBacillus pseudomycoides97.23NR_114422.1
      PCL 2.3Pantoea sp.Pantoea agglomerans strain JCM123693.62NR_111998.1
      PCR 1.1Burkholderia cepaciaBurkholderia cepacia ATCC 25416 strain LMG 122297.17NR_114491.1
      PCR 1.3Bacillus sanguinisBacillus sanguinis strain BML-BC00499.51NR_175555.1
      PCR 1.5Burkholderia sp.Burkholderia pseudomallei strain ATCC 2334386.66NR_043553.1

      Figure 2. 

      Neighbor joining tree of rhizobacteria isolates and their closely related species generated using BLAST. Bootstrap values are based on 1000 replications analyzed using MEGAX.

    • Rhizosphere is a nutrient-rich region consisting of a wide variety of microorganisms living in the small area of soil that surrounds and is associated with plant roots. The organisms in the rhizosphere microbiome can have a significant influence on the development, nutrition, and health of plants[11]. Rod-shaped bacteria are commonly found in the rhizosphere and known to have plant growth-promoting attributes, including those species that belong to the genera Bacillus[6], Burkholderia[12], and Pantoea[13], which were isolated and characterized in our study.

      The microbes in the rhizosphere play key roles in nutrient acquisition and assimilation, improved soil texture, secreting, and modulating extracellular molecules such as hormones, secondary metabolites, antibiotics, and various signal compounds, all leading to enhancement of plant growth[14]. Our study initially screened phosphate solubilizing bacteria, because their activity is essential to address plant phosphate requirements of various plants. These bacteria may provide the plants with available phosphorus from sources that would otherwise be scarce due to a wide range of mechanisms. They can also contribute to the natural biogeochemical cycle of nutrients in the rhizosphere[10,15]. Besides, inoculation of phosphate-solubilizing bacteria species has reportedly improved phosphorus absorption and grain production of Triticum aestivum[16] and it significantly facilitated the growth of Vitis vinifera under greenhouse conditions[17]. Besides, the indigenous bacteria with phosphate solubilization activity obtained from coffee rhizosphere were able to stimulate the growth of the Arabica coffee seedlings under nursery conditions[18]. In addition, phosphate solubilizing microbes generally improved the growth and nutrient uptake of Robusta coffee grown in phosphorus deficient soil[19]. The ability of the rhizobacterial isolates to solubilize inorganic phosphate indicates their potential role to improve the growth of crops. It is recommended to evaluate the amount of phosphorus that these rhizobacteria will contribute to the coffee plants once inoculated.

    • In this study, Bacillus sanguinis demonstrated HCN production, suggesting its potential role as a biological agent against plant pathogens. HCN is mostly synthesized by Bacillus species, which is believed to disrupt several cellular processes such as the electron transport chain, correct functioning of enzymes and even impedes the action of cytochrome oxidase of the target pathogens[20]. HCN production of phosphate-solubilizing Bacillus isolates was also observed as a biochemical trait of potential biofertilizer agent for coffee production[21].

      On the other hand, Bacillus sanguinis and Burkholderia sp. had promising potential as a biocontrol agent due to its capability to produce multiple lytic enzymes, which is an essential strategy for fungal inhibition. Production of lytic enzymes of rhizobacteria is considered as a plant disease inhibitor since these enzymes are involved in cell wall degradation of plant pathogens present in the soil environment[22,23]. Lytic enzymes can be used as biocontrol agents to inhibit fungal pathogens that cause diseases in crops[24].

    • Abiotic stresses such as salinity, acidity or alkalinity, and heavy metal contamination may have a detrimental effect on agricultural production by affecting plant growth, nutritional imbalance, and physiological and metabolic changes. Rhizobacteria may help in plant growth promotion and alleviation of the stress-induced changes in the host plant[25]. Rhizobacteria that tolerate high concentrations of NaCl can help plants thrive in saline soil through their mechanisms such as osmolytes regulation, nitrogen fixation, solubilization of phosphate, as well as formation of auxin, siderophore and exopolysaccharides[26]. Meanwhile, plants are vulnerable to heavy metal stress exposure, which may came from industrial and other environmental pollutants. Rhizobacteria are essential for the reduction of toxic heavy metals in plants, they can contribute to the improvement of heavy metal tolerance and plant growth through improved phytoremediation and metal accumulation inside the plant[27]. The pH is also considered as a limiting condition for plants but stress tolerant rhizobacteria considerably enhance the seed germination of crops in acidic or alkaline soils[28]. The tolerance of isolates at low pH is expected since the pH of the soil used in the present study is acidic. The ability of the rhizobacteria to withstand a wide range of abiotic stresses indicates their potential to promote plant growth and protect plants from the detrimental effects of abiotic stresses in the soil.

    • The bacterial isolates obtained from the Liberica rhizosphere are Pantoea rwandensis, Bacillus pseudomycoides, and Pantoea sp. that exhibited tolerance to high salt concentration and wide range of pH and heavy metal concentrations, but only B. pseudomycoides exhibited amylase activity. On the other hand, the Robusta rhizobacterial isolates identified in our study were Burkholderia cepacia, Bacillus sanguinis and Burkholderia sp. that showed promising performance, specifically the B. sanguinis that has the ability to produce multiple lytic enzymes and HCN. The result of this study indicates that both Liberica and Robusta rhizosphere contained beneficial rhizobacteria that establish mutual symbiotic relationship with the host plant. Previously, phosphate solubilizing species under the genera Bacillus and Burkholderia and the species of endophytic Pantoea were reported to be abundant in the rhizosphere of forest-grown Coffea arabica[12,29]. These rhizobacterial isolates demonstrated a great potential to be utilized as a biofertilizer to improve the growth and yield of coffee.

      The bacterial genus Pantoea was found to be abundant in the rhizosphere microbiome and bean fermentation of coffee[30]. This genus comprises many versatile endophytic species that possess a variety of biosynthetic and biodegradative capabilities. They are found to be useful for biocontrol of plant pathogens, bioremediation of contaminated environments, biosensors, and a source of therapeutic drugs[31]. In addition, Bacillus species was discovered to be a phosphate-solubilizer and enhanced the growth of coffee seedlings and the availability of phosphorus in the soil[32]. Specifically, B. pseudomycoides was found to be a good choice in phytoremediation and an active agent in biofertilizers or biofungicide[33]. Meanwhile, B. sanguinis was similarly found to produce multiple lytic enzymes such as amylase, cellulase, protease, and xylanase[34], indicating its important role against plant pathogens. Lastly, Burkholderia species in general can establish symbiotic relationships with terrestrial plants, functioning as active rhizosphere components, endophytic plant colonizers, or microsymbionts in legume root nodules[35].

    • Our work successfully screened indigenous and beneficial rhizobacteria associated with Robusta and Liberica coffee rhizosphere. They belong to the genera Bacillus, Burkholderia, and Pantoea, which were reported to have plant growth-promoting attributes, biocontrol activities, and tolerance to abiotic stresses. These bacteria can be utilized as an alternative to chemical fertilization and be used as a potential biofertilizer for coffee cultivation in the Philippines. The performance of these isolates as a biofertilizer may vary, but their effectivity can only be verified upon application to crops.

    • All the authors confirm equal contributions for the following: study conception and design, data collection, analysis and interpretation of results, and manuscript preparation.

    • All data generated or analyzed during this study are included in this published article.

      • The study was funded by the Research Center of Cavite State University, Philippines through the Small Scale CvSU Research Grant. The authors are grateful to CvSU-Research Center for allowing them to conduct experiments in the Bacteriology and Genetics Laboratories of the Interdisciplinary Research Building.

      • The authors declare that they have no conflict of interest.

      • Copyright: © 2023 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (2)  Table (3) References (35)
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    Navarro GVD, Quirong DD, Maghanoy GA, Cortes AD. 2023. Characterization and identification of rhizobacteria associated with Liberica and Robusta coffee rhizosphere. Technology in Horticulture 3:24 doi: 10.48130/TIH-2023-0024
    Navarro GVD, Quirong DD, Maghanoy GA, Cortes AD. 2023. Characterization and identification of rhizobacteria associated with Liberica and Robusta coffee rhizosphere. Technology in Horticulture 3:24 doi: 10.48130/TIH-2023-0024

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