Ananatis Strains Research Articles

Role of hypothetical protein PA1-LRP in antibacterial activity of endolysin from a new Pantoea phage PA1.

Pantoea ananatis has emerged as a significant plant pathogen affecting various crops worldwide, causing substantial economic losses. Bacteriophages and their endolysins offer promising alternatives for controlling bacterial infections, addressing the growing concerns of antibiotic resistance. This study isolated and characterized the Pantoea phage PA1 and investigated the role of PA1-LRP in directly damaging bacteria and assisting endolysin PA1-Lys in cell lysis, comparing its effect to exogenous transmembrane domains following the identification and analysis of the PA1-Lys and the PA1-LRP based on whole genome analysis of phage PA1. Additionally, this study also explored how hydrophobic region of PA1-LRP (HPP) contributes to bacterial killing when combined with PA1-Lys and examined the stability and lytic spectrum of PA1-Lys under various conditions. Phage PA1 belonging to the Chaseviridae family exhibited a broad host range against P. ananatis strains, with a latent period of 40 minutes and a burst size of 17.17 phages per infected cell. PA1-Lys remained stable at pH 6-10 and temperatures of 20-50°C and showed lytic activity against various Gram-negative bacteria, while PA1-Lys alone could not directly lyse bacteria, its lytic activity was enhanced in the presence of EDTA. Surprisingly, PA1-LRP inhibited bacterial growth when expressed alone. After 24 h of incubation, the OD600 value of pET28a-LRP decreased by 0.164 compared to pET28a. Furthermore, the lytic effect of co-expressed PA1-LRP and PA1-Lys was significantly stronger than each separately. After 24 h of incubation, compared to pET28a-LRP, the OD600 value of pET28a-Lys-LRP decreased by 0.444, while the OD420 value increased by 3.121. Live/dead cell staining, and flow cytometry experiments showed that the fusion expression of PA1-LRP and PA1-Lys resulted in 41.29% cell death, with bacterial morphology changing from rod-shaped to filamentous. Notably, PA1-LRP provided stronger support for endolysin-mediated cell lysis than exogenous transmembrane domains. Additionally, our results demonstrated that the HPP fused with PA1-Lys, led to 40.60% cell death, with bacteria changing from rod-shaped to spherical and exhibiting vacuolation. Taken together, this study provides insights into the lysis mechanisms of Pantoea phages and identifies a novel lysis-related protein, PA1-LRP, which could have potential applications in phage therapy and bacterial disease control.

Characterization of Twelve Erwinia amylovora Bacteriophages

Erwinia amylovora bacteriophages are of interest as fire blight control agents. This paper presents data on the biology and molecular genetic properties of 12 E. amylovora tailed bacteriophages. Genome sequences of seven of them were determined and the phages were identified as the representatives of Caudoviricetes; Vequintavirinae, Ounavirinae and Autographiviridae families. The bacteriophages studied were active against E. amylovora, Pantoea agglomerans and Pantoea ananatis strains. The myovirus Hena1 had the narrowest host range lysing 12% of the bacterial cultures tested, the remaining myoviruses—had broader host ranges (56%) for this collection. The phages showed different reactions to the presence of a chelating agent in the cultivation medium. A significant proportion of phage-resistant E. amylovora cells were found both in infected liquid cultures and in the experiments on the plates with the phage agar (more than 20% in case of exposure to podoviruses), which correlates with the data of limited number of previous investigations of the phenomenon for E. amylovora interactions with phages. High prevalence of resistant cells in the host cultures suggest that they are formed not due to spontaneous mutations but result from another phenotypic or genetic dissociation mechanism(s) that remains to be identified.

First Report of Pantoea ananatis causing leaf streak disease on wheat (Triticum aestivum) in the United States of America.

Wheat (Triticum aestivum) loses 21.5% yield to pests and diseases annually (Savary et al. 2019). Among the wheat diseases, bacterial leaf streak (BLS) is a growing problem, costing $78.5 million in losses (https://cropprotectionnetwork.org/). In July 2022, we sampled winter wheat leaf samples at Volga (44.30, -96.92), South Dakota, USA with an estimated disease incidence of 40% (n=100). The typical symptoms were water-soaking with large necrotic and chlorotic streaks extending the length of the leaves and were strikingly similar to BLS. To isolate the pathogen, leaves were cut lengthwise into 1 cm pieces and surface-sterilized using a 10% NaOCl solution for 3 min, followed by 70% ethanol for 3 min, and then rinsed with sterile distilled water and placed in 500 ul of sterile distilled water for 5 min and using a sterile loop the water was streaked over a plate of Nutrient Agar (NA). Following Duveiller et al. (1997), the streaked plate was incubated in the dark at 28℃ for 48 h. Observed single colonies were sub-cultured thrice onto fresh NA plates to obtain a pure culture. We named the culture SD101. Bacteria were found to be gram-negative with a colony morphology initially raised, smooth, and white that later turned yellow. DNA was extracted using the Wizard HMW DNA Extraction Kit (Promega, Madison, WI) following the manufacturer's protocol, and sequenced using Nanopore MinION R9.4 (Oxford Nanopore Technology). We used the Rapid Annotation Using Subsystems Technology approach (Aziz eal. 2008) to extract the 16S rDNA, DNA gyrase subunit B (gyrB), and translation initiation factor IF-2 (infB) gene sequences that were deposited in GenBank under accession numbers PP329908.1 for 16S rDNA, PP496481 for infB, and PP328920.1 for gyrB. Homology analysis using CLC Genomics Workbench 22.0.2 (QIAGEN) and BLASTn against the GenBank nucleotide database resulted in a 99.74% match (1543/ 1547 bp) of the 16S sequence, 99.59% match (2674/ 2685 bp) of the infB sequence, and 99.42% match (2396/ 2410 bp) of the gyrB sequence with Pantoea ananatis strain AJ13355 (AP012032). To test pathogenicity, seeds of spring wheat breeding line SD4892 were planted in 30 cm × 30 cm pots in a greenhouse under a 16 h light photoperiod. The inoculum was prepared from 48-h-old NA plates of SD101 rinsed with 1X Phosphate Buffer Saline (PBS buffer), adjusted to an OD600 = 1.0, and amended with two drops of Tween 20 (polyoxyethylene sorbitol ester, Millipore Sigma). PBS with Tween 20 was used as a negative control. The inoculum was sprayed on 15 replicates of 15-day-old seedlings, kept at 95% relative humidity for 48 h, then moved to the greenhouse at 23 to 25°C. The symptoms appeared as water soaking that later turned to necrotic streaks with surrounding chlorosis on all 15 inoculated plants while control plants remained healthy. The pathogen was re-isolated from the leaves as described above. The 16S rDNA, infB, and gyrB sequences amplified and sequenced were identical to the gene sequences from the whole genome sequencing. The experiment was repeated with the same results, completing Koch's postulates. Although P. ananatis is pathogenic on corn, rice, and other plant species in the United States (Coutinho et al. 2009), and was reported pathogenic on wheat in Poland (Krawczyk et al. 2020), this is the first report of its pathogenicity on wheat in the United States. The prevalence, and incidence of BLS disease caused by P. ananatis in wheat is needed to estimate its threat to wheat production and to develop management strategies.

Complete Genome Sequence of Pantoea ananatis strain LCFJ-001, Isolated from Bacterial Wilt Mulberry.

A Pantoea ananatis strain, named LCFJ-001 (GDMCC: 1.6101), was isolated for the first time from bacterial wilt-diseased roots of mulberry (Morus atropurpurea) in the western part of the Guangxi Zhuang Autonomous Region, China. Moreover, through Koch's postulates, it was proven that LCFJ-001 can cause mulberry wilt, which is one of the pathogens of mulberry bacterial wilt. Here, we report a complete, annotated genome sequence of P. ananatis LCFJ-001. The entire genome sequence of P. ananatis strain LCFJ-001 was a 4,499,350 bp circular chromosome with 53.50% GC content. In total, 3521 genes were annotated, of which 3418 were assigned protein-coding genes. In addition, 22 ribosomal RNAs and 81 transfer RNAs were identified. The presented resource will help explore the pathogenetic mechanisms of mulberry wilt disease caused by the genus Pantoea.

The Genetic Requirements for HiVir-Mediated Onion Necrosis by Pantoea ananatis, a Necrotrophic Plant Pathogen.

Pantoea ananatis is an unusual bacterial pathogen that lacks typical virulence determinants yet causes extensive necrosis in onion foliage and bulb tissues. The onion necrosis phenotype is dependent on the expression of the phosphonate toxin, pantaphos, which is synthesized by putative enzymes encoded by the HiVir (high virulence) gene cluster. The genetic contributions of individual hvr genes in HiVir-mediated onion necrosis remain largely unknown, except for the first gene, hvrA (phosphoenolpyruvate mutase, pepM), whose deletion resulted in the loss of onion pathogenicity. In this study, using gene-deletion mutation and complementation, we report that, of the ten remaining genes, hvrB to hvrF are also strictly required for the HiVir-mediated onion necrosis and in-planta bacterial growth, whereas hvrG to hvrJ partially contributed to these phenotypes. As the HiVir gene cluster is a common genetic feature shared among the onion-pathogenic P. ananatis strains that could serve as a useful diagnostic marker of onion pathogenicity, we sought to understand the genetic basis of HiVir-positive yet phenotypically deviant (non-pathogenic) strains. We identified and genetically characterized inactivating single nucleotide polymorphisms in the essential hvr genes of six phenotypically deviant P. ananatis strains. Finally, inoculation of cell-free spent medium of the isopropylthio-β-galactoside (IPTG)-inducible promoter (Ptac)-driven HiVir strain caused P.ananatis-characteristic red onion scale necrosis as well as cell death symptoms in tobacco. Co-inoculation of the spent medium with essential hvr mutant strains restored in-planta populations of the strains to the wild-type level, suggesting that necrotic tissues are important for the proliferation of P. ananatis in onion. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

First Report of leaf spot on Cucumber Caused by Pantoea ananatis in Hainan of China.

Cucumber (Cucumis sativus L.) is one of the most important vegetables cultivated in the world. It is widely cultivated and mostly grown under greenhouse conditions (Sallam et al. 2021). Cucumber has a long growth cycle and is particularly susceptible to bacterial diseases. In May 2021, bacterial leaf spot was found on cucumbers of the variety Lyuyou NO.3 in Hainan Province, China. In the early stage of the disease, the leaves showed small yellow-brown spots in the shape of water stains. When exposed to light, a yellow halo around the disease spots could be seen. In later stages, the lesions gradually become larger and more yellow. The leaf veins around the disease site also gradually turned yellow (Figure 2a). In serious cases, the whole leaf turned yellow, resulting in leaf death. We collected plants with the same symptoms from 25 different farms in Hainan Province. Five plants were selected from each farm by the classic five-point sampling method and three leaves were selected from each plant, for a total of 15 leaves collected from each farm. Then three leaves were randomly selected from the 15 leaves on each farm for isolation of the pathogen, and a total of 75 leaves were isolated. We found that the incidence of the disease was from 20% to 30% based on a diagnostic test, which conducted on 75 cucumber leaves samples suspected of same symptom of cucumber, collected from Hainan Province. Using microscopy, bacterial streaming was observed to tentatively identify the causal agent as a bacteria. Tissue isolation was used to isolate the responsible pathogens. A 5 mm × 5 mm sample of tissue at the junction of diseased and healthy sections was collected. First, the surface of the tissue was disinfected in a 75% ethanol solution for 30 sec; then it was soaked in 2% NaOCl for 5-7 min, and finally, it was washed thrice in sterile distilled water. The tissues were inoculated onto lysogen broth culture media (LB) and cultured in a 28℃ incubator for 2 days. Bacterial colonies that emerged from the tissues were cultured in LB. Four isolated colonies were selected for verification. The colonies of isolated from the diseased leaves of cucumber are round, egg yellow and slightly sticky (Figure 2c). The isolate named PA-1 was identified by PCR amplification and sequencing of the partial 16S rRNA gene with the primer 27F/1492R (Lane 1991) and gyrB gene (Li et al. 2019). Sequences were stored in GenBank with the accession numbers OK576932.1 (16S rRNA, PA-1) and OL978577 (gyrB); BLASTn was used to compare these with other GenBank sequences. Sequencing of the 16S rRNA gene showed that PA-1 had a sequence length of 1403bp, with 99.78% genetic similarity to Pantoea ananatis strain MZ007857.1. Sequencing of the gyrB gene showed that the sequence length of PA-1 was 1136bp, with 99.29% genetic similarity to P. ananatis strain MW981331.1. Then, a pathogenicity text was conducted to verify Koch's postulates, which was done by first inoculating P. ananatis into LB liquid medium (shake culture at 28°C, 180 r/min). The log phase cell was collected by centrifugation (5,000 r/min for 2 min at 4°C), and inoculated strains were resuspended in sterile water at OD600 = 0.5. The bacterial suspension was inoculated on healthy cucumber leaves with a syringe. The control was sterile water, which was injected onto healthy cucumber leaves using the same methodology. The plants were placed in a greenhouse with a diurnal temperature difference of 21- 27°C and were observed daily. After two weeks, all bacterial inoculated plants developed symptoms of shriveling and necrosis (Figure 2b), while the control group showed no symptoms. From the symptomatic plants, the pathogen was isolated again and identified by morphological and molecular characterization. The sequences of the isolates recovered from the inoculated experiment matched 100% the sequences of the isolate PA-1. Koch's postulates were completed by following the previously described method. To our knowledge, this is the first report of P. ananatis causing leaf spot of cucumber.

Genome-wide association and dissociation studies in Pantoea ananatis reveal potential virulence factors affecting Allium porrum and Allium fistulosum × Allium cepa hybrid.

Pantoea ananatis is a member of a Pantoea species complex that causes center rot of bulb onions (A. cepa) and also infects other Allium crops like leeks (Allium porrum), chives (Allium schoenoprasum), bunching onion or Welsh onion (Allium fistulosum), and garlic (Allium sativum). This pathogen relies on a chromosomal phosphonate biosynthetic gene cluster (HiVir) and a plasmid-borne thiosulfinate tolerance cluster (alt) for onion pathogenicity and virulence, respectively. However, pathogenicity and virulence factors associated with other Allium species remain unknown. We used phenotype-dependent genome-wide association (GWAS) and phenotype-independent gene-pair coincidence (GPC) analyses on a panel of diverse 92 P. ananatis strains, which were inoculated on A. porrum and A. fistulosum × A. cepa under greenhouse conditions. Phenotypic assays showed that, in general, these strains were more aggressive on A. fistulosum × A. cepa as opposed to A. porrum. Of the 92 strains, only six showed highly aggressive foliar lesions on A. porrum compared to A. fistulosum × A. cepa. Conversely, nine strains showed highly aggressive foliar lesions on A. fistulosum × A. cepa compared to A. porrum. These results indicate that there are underlying genetic components in P. ananatis that may drive pathogenicity in these two Allium spp. Based on GWAS for foliar pathogenicity, 835 genes were associated with P. ananatis' pathogenicity on A. fistulosum × A. cepa whereas 243 genes were associated with bacterial pathogenicity on A. porrum. The Hivir as well as the alt gene clusters were identified among these genes. Besides the 'HiVir' and the alt gene clusters that are known to contribute to pathogenicity and virulence from previous studies, genes annotated with functions related to stress responses, a potential toxin-antitoxin system, flagellar-motility, quorum sensing, and a previously described phosphonoglycan biosynthesis (pgb) cluster were identified. The GPC analysis resulted in the identification of 165 individual genes sorted into 39 significant gene-pair association components and 255 genes sorted into 50 significant gene-pair dissociation components. Within the coincident gene clusters, several genes that occurred on the GWAS outputs were associated with each other but dissociated with genes that did not appear in their respective GWAS output. To focus on candidate genes that could explain the difference in virulence between hosts, a comparative genomics analysis was performed on five P. ananatis strains that were differentially pathogenic on A. porrum or A. fistulosum × A. cepa. Here, we found a putative type III secretion system, and several other genes that occurred on both GWAS outputs of both Allium hosts. Further, we also demonstrated utilizing mutational analysis that the pepM gene in the HiVir cluster is important than the pepM gene in the pgb cluster for P. ananatis pathogenicity in A. fistulosum × A. cepa and A. porrum. Overall, our results support that P. ananatis may utilize a common set of genes or gene clusters to induce symptoms on A. fistulosum × A. cepa foliar tissue as well as A. cepa but implicates additional genes for infection on A. porrum.

Genetic Engineering of Escherichia coli BL21 (DE3) with a codon-optimized insecticidal toxin complex gene tccZ.

A toxin complex consists of a high-molecular-weight group of toxins that exhibits insecticidal activity against insect pests. These toxins are a promising alternative to Bacillus thuringiensis (Bt) toxins that have been extensively utilized in insect pest control. Herein, a codon-optimized insecticidal gene (tccZ) (381 bp) identified in Pantoea ananatis strain MHSD5 (a bacterial endophyte previously isolated from Pellaea calomelanos) was ligated into the pET SUMO expression vector and expressed in Escherichia coli BL21 (DE3). We report the success of cloning the tccZ gene into the pET SUMO vector and ultimately the transformation into E. coli BL21 (DE3) competent cells. However, despite conducting a time course of expression as well as isopropyl β-d-1-thiogalactopyranoside (IPTG) dosage optimization to determine optimal conditions for expression, TccZ protein expression could not be detected on Stain-Free and Coomassie-stained SDS-PAGE gels.

First Report of Onion (Allium cepa) Bulb Rot Caused by Pantoea agglomerans in China.

Onion (Allium cepa L.) is one of the most cultivated vegetables throughout the world. With an average annual production quantity of 18 million kg in recent 21 years, China is the world's biggest onion producer (Hanci F., 2018). Among them, onion is mainly cultivated in the provinces of Gansu, Shandong, Yunnan, Jiangsu, Zhejiang and Henan. A survey in Gansu province in last several years showed that the incidence of onion bulb rot was 30%-80%. In April 2020, bulbs displayed water-soaked, and then rot symptoms observed during storage in Lanzhou City, Gansu Province, China. The initial symptoms of bulb rot disease were yellowish brown, and produced an abundant exudate in the inner bulb scales when cut. Gradually, symptomatic bulbs became soft, watery and decayed. In severe infections, the onions showed total rot of the bulb. Therefore, we sampled some diseased onions and isolated pathogenic bacteria from the junction of lesion along with healthy parts on Luria-Bertani (LB) medium. Three representative single colonies were obtained on LB medium, and the culture characteristics were raised elevation, mucoid texture, round, and smooth with entire margin, the brown at the beginning and turned yellow later, and scanning electron microscopy (SEM) observations showed that these isolates were short rod-shaped (Fig. 1A). The physiological and biochemical determination revealed that the isolates were positive for yellow pigment, v-p test, growth at 37 ℃, nitrate reduced, catalase, glucose, sucrose, D (-)-salicin, starch hydrolysis, motility, pellicle. On the contrary, they were negative for indole production, methyl red, lactose, gelatin liquefaction, glycerol, gram staining (Gavini et al., 1989; Nabrdalik et al., 2018). Based on these morphological, physiological and biochemical characteristics, three isolates were initially identified as Pantoea agglomerans (Guo et al., 2020). A representative isolate L1 was selected to extract DNA, and the conserved sequences of the pathogen gene were sequenced according to 23S ribosomal RNA (23S rRNA), DNA gyrase subunit B (gyrB), elongation factor G (fusA) (She et al., 2021) housekeeping gene. The sequence alignment of the 23S rRNA gene (P. agglomerans, MZ314289, 930bp) showed that the homology between the strain L1 and P. agglomerans (CP016889) with similarity of 99.54%, and based on the sequence alignment of gyrB (P. agglomerans, MZ337547, 1189bp) and fusA (P. agglomerans, MZ350961, 1037bp) genes, the similarity with P. agglomerans (FJ617386 and MG845872) was 100%. A phylogenetic analysis based on the 23S, gyrB, fusA housekeeping gene sequences was performed by using the neighbor-joining methods in MEGA 7.0 under the p-distance (Kumar et al, 2016), which included P. agglomerans strains AR1a, TH81, L15, ASB05, P. eucalypti strain LMG 24197, P. dispersa strains BJQ0007 and DSM 32899, P. ananatis strains LMG 20103 and AJ13355, P. vagans strains C9-1, LMG24199 and PV989. The phylogenetic distribution generated five primary phylogroups, and strain L1 formed a clade with the other four P. agglomerans strains (Fig. 2). Thus, the strain L1 was identified as P. agglomerans. To satisfy Koch's postulates, ten onions were divided into two groups, five in each group, and needle punctured wound on the surface of each onion. In the experimental group, 400 μL bacterial suspensions were injected with sterile syringe, and the other five onions were injected with the same amount of sterile distilled water as the negative conrol. Inoculated onions were incubated in the greenhouse incubator (28 ℃, humidity > 80%). After 4 days of incubation, all onions inoculated with strain L1 appeared water-soaked, rot symptoms, and no symptoms were observed in the negative control (Fig. 1B). Subsequently, pathogens were re-isolated from inoculated bulbs and identified as P. agglomerans according to molecular identification described above. To the best of our knowledge, this is the first report that the bulb rot disease of stored onion caused by P. agglomerans in China.

Complete Genomic Data of Pantoea ananatis Strain TZ39 Associated with New Bacterial Blight of Rice in China

Pantoea ananatis is a phytopathogen infecting many economically important crops, including rice worldwide. Here, we report the complete genome of P. ananatis strain TZ39 identified as causative agent of a new bacterial blight of rice that emerged in China in 2020. The assembled genome consists of one circular chromosome of 4,483,976 bp and two plasmids of 135,135 and 276,579 bp. This complete genome of the first Chinese pathogenic P. ananatis strain will provide new insights into the traits of pathogenicity on genomic level from China and worldwide.

Utilization of Various Organic Wastes as Liquid Biofertilizer Carrier Agents towards Viability of Bacteria and Green Bean Growth

The problem in the production of biofertilizers is that raw materials are cheap, easy to get and apply. Another problem is determining the viability of the consortium microbes in a biofertilizer formulation. This study aims to determine the bacterial viability of various liquid media originating from organic waste as a liquid biofertilizer carrier. Three indigenous bacterial strains under consortium for phosphate soluble (Pantoea ananatis strain 53 (BC32)), non-symbiostic Nitrogen fixation (Bacillus licheniformis strain S45) and stabilizing soil aggregate (Pseudomonas plecoglossidastrain PR19) were added to liquid biofertilizer. The study evaluated 10 treatments using a randomized design with three replicates. The treatments are as follows: Peptone,molasses, Compost wash from seaweed waste, Vermiwash, molasses + glycerol, compost wash from seaweed waste + glycerol, vermiwash + glycerol, molasses + PEG (PolyEthylene Glycols)1%, compost wash seaweed waste + PEG 1%, and Vermiwash + PEG 1%. This biofertilizer formulation (liquid) was kept for 16 weeks at optimumpH 5.5. VP3 (Vermiwash made from vermicompost + PEG 1%) treatment showed thebest viability of bacterial strains during the 16-week storage period. The pathogenicitytest using green bean seeds Vima-1 showed that all liquid formulations of biological fertilizers with the three consortium bacterial isolates did not show signs of diseases and demonstrated better growth than the control treatment. Compared to other treatments, the best growth of bacterial strains was detected with MP2 (Molasses + glycerol) treatment. Formulations using vermiwash and PEG appear to maintain bacterial viability in the formulation effectively. However, the formulation of molasses and glycerol exerts a stimulating effect on sprouts growth.

Complete Genome Sequence Resources for the Onion Pathogen, Pantoea ananatis OC5a.

Here, we report on the genomic sequence and annotation for Pantoea ananatis OC5a, a strain that was isolated from an onion bulb grown in New York and that is pathogenic to onion, causing center rot of onion. OC5a is the first P. ananatis strain pathogenic to onion from New York to be completely assembled and sequenced. Having been assembled using long PacBio reads and high-fidelity Illumina reads, this genome is closed, complete, and of high quality.

Genomics-Informed Multiplex PCR Scheme for Rapid Identification of Rice-Associated Bacteria of the Genus Pantoea.

The genus Pantoea forms a complex of more than 25 species, among which several cause diseases of various crop plants, including rice. Notably, strains of Pantoea ananatis and P. stewartii have been repeatedly reported to cause bacterial leaf blight of rice, whereas other authors have observed that P. agglomerans can also cause bacterial leaf blight of rice. The contribution of these and perhaps other species of Pantoea to plant diseases and yield losses of crop plants is currently not well documented, partly due to the lack of efficient diagnostic tools. Using 32 whole-genome sequences of the three major plant-pathogenic Pantoea spp., a set of PCR primers that detect each of the three species P. agglomerans, P. ananatis, and P. stewartii was designed. A multiplex PCR scheme which can distinguish these three species and also detects members of other Pantoea spp. was further developed. Upon validation on a set of reference strains, 607 suspected Pantoea strains that were isolated from rice leaves or seed originating from 11 African countries were screened. In total, 41 P. agglomerans strains from 8 countries, 79 P. ananatis strains from 9 countries, 269 P. stewartii strains from 9 countries, and 218 unresolved Pantoea strains from 10 countries were identified. The PCR protocol allowed detection of Pantoea bacteria grown invitro, in planta, and in rice seed. The detection threshold was estimated as total genomic DNA at 0.5 ng/µl and heated cells at 1 × 104 CFU/ml. This new molecular diagnostic tool will help to accurately diagnose major plant-pathogenic species of Pantoea. Due to its robustness, specificity, sensitivity, and cost efficiency, it will be very useful for plant protection services and for the epidemiological surveillance of these important crop-threatening bacteria.

Pan-Genome-Wide Analysis of Pantoea ananatis Identified Genes Linked to Pathogenicity in Onion.

Pantoea ananatis, a gram negative and facultative anaerobic bacterium is a member of a Pantoea spp. complex that causes center rot of onion, which significantly affects onion yield and quality. This pathogen does not have typical virulence factors like type II or type III secretion systems but appears to require a biosynthetic gene-cluster, HiVir/PASVIL (located chromosomally comprised of 14 genes), for a phosphonate secondary metabolite, and the ‘alt’ gene cluster (located in plasmid and comprised of 11 genes) that aids in bacterial colonization in onion bulbs by imparting tolerance to thiosulfinates. We conducted a deep pan-genome-wide association study (pan-GWAS) to predict additional genes associated with pathogenicity in P. ananatis using a panel of diverse strains (n = 81). We utilized a red-onion scale necrosis assay as an indicator of pathogenicity. Based on this assay, we differentiated pathogenic (n = 51)- vs. non-pathogenic (n = 30)-strains phenotypically. Pan-genome analysis revealed a large core genome of 3,153 genes and a flexible accessory genome. Pan-GWAS using the presence and absence variants (PAVs) predicted 42 genes, including 14 from the previously identified HiVir/PASVIL cluster associated with pathogenicity, and 28 novel genes that were not previously associated with pathogenicity in onion. Of the 28 novel genes identified, eight have annotated functions of site-specific tyrosine kinase, N-acetylmuramoyl-L-alanine amidase, conjugal transfer, and HTH-type transcriptional regulator. The remaining 20 genes are currently hypothetical. Further, a core-genome SNPs-based phylogeny and horizontal gene transfer (HGT) studies were also conducted to assess the extent of lateral gene transfer among diverse P. ananatis strains. Phylogenetic analysis based on PAVs and whole genome multi locus sequence typing (wgMLST) rather than core-genome SNPs distinguished red-scale necrosis inducing (pathogenic) strains from non-scale necrosis inducing (non-pathogenic) strains of P. ananatis. A total of 1182 HGT events including the HiVir/PASVIL and alt cluster genes were identified. These events could be regarded as a major contributing factor to the diversification, niche-adaptation and potential acquisition of pathogenicity/virulence genes in P. ananatis.

Enterobacter asburiae and Pantoea ananatis Causing Rice Bacterial Blight in China.

Rice bacterial blight is a devastating bacterial disease threatening rice yield all over the world and Xanthomonas oryzae pv. oryzae is traditionally believed to be the pathogen. In recent years, we have received diseased rice samples with symptoms of blighted leaves from Sichuan and Guangdong provinces, China. Pathogen isolation and classification identified two different enterobacteria as the causal agents, namely Enterobacter asburiae and Pantoea ananatis. Among them, E. asburiae was isolated from samples of both provinces, and P. ananatis was only isolated from the Sichuan samples. Different from rice foot rot pathogen Dickeya zeae EC1 and rice bacterial blight pathogen X. oryzae pv. oryzae PXO99A, strains SC1, RG1, and SC7 produced rare cell wall degrading enzymes (CWDEs) but more extrapolysaccharides (EPS). E. asburiae strains SC1 and RG1 produced bacteriostatic substances while P. ananatis strain SC7 produced none. Pathogenicity tests indicated that all of them infected monocotyledonous rice and banana seedlings, but not dicotyledonous potato, radish, or cabbage. Moreover, strain RG1 was most virulent, while strains SC1 and SC7 were similarly virulent on rice leaves, even though strain SC1 propagated significantly faster in rice leaf tissues than strain SC7. This study firstly discovered E. asburiae as a new pathogen of rice bacterial blight, and in some cases, P. ananatis could be a companion pathogen. Analysis on production of virulence factors suggested that both pathogens probably employ a different mechanism to infect hosts other than using cell wall degrading enzymes to break through host cell walls.

The Distribution of Onion Virulence Gene Clusters Among Pantoea spp.

Pantoea ananatis is a gram-negative bacterium and the primary causal agent of center rot of onions in Georgia. Previous genomic studies identified two virulence gene clusters, HiVir and alt, associated with center rot. The HiVir gene cluster is required to induce necrosis on onion tissues via synthesis of pantaphos, (2-hydroxy[phosphono-methyl)maleate), a phosphonate phytotoxin. The alt gene cluster aids in tolerance to thiosulfinates generated during onion tissue damage. Whole genome sequencing of other Pantoea species suggests that these gene clusters are present outside of P. ananatis. To assess the distribution of these gene clusters, two PCR primer sets were designed to detect the presence of HiVir and alt. Two hundred fifty-two strains of Pantoea spp. were phenotyped using the red onion scale necrosis (RSN) assay and were genotyped using PCR for the presence of these virulence genes. A diverse panel of strains from three distinct culture collections comprised of 24 Pantoea species, 41 isolation sources, and 23 countries, collected from 1946–2019, was tested. There is a significant association between the alt PCR assay and Pantoea strains recovered from symptomatic onion (P < 0.001). There is also a significant association of a positive HiVir PCR and RSN assay among P. ananatis strains but not among Pantoea spp., congeners. This may indicate a divergent HiVir cluster or different pathogenicity and virulence mechanisms. Last, we describe natural alt positive [RSN+/HiVir+/alt+] P. ananatis strains, which cause extensive bulb necrosis in a neck-to-bulb infection assay compared to alt negative [RSN+/HiVir+/alt–] P. ananatis strains. A combination of assays that include PCR of virulence genes [HiVir and alt] and an RSN assay can potentially aid in identification of onion-bulb-rotting pathogenic P. ananatis strains.

Fermentative production of enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis

BackgroundLinalool, an acyclic monoterpene alcohol, is extensively used in the flavor and fragrance industries and exists as two enantiomers, (S)- and (R)-linalool, which have different odors and biological properties. Linalool extraction from natural plant tissues suffers from low product yield. Although linalool can also be chemically synthesized, its enantioselective production is difficult. Microbial production of terpenes has recently emerged as a novel, environmental-friendly alternative. Stereoselective production can also be achieved using this approach via enzymatic reactions. We previously succeeded in producing enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis, a member of the Enterobacteriaceae family of bacteria, via the heterologous mevalonate pathway with the highest linalool titer ever reported from engineered microbes.ResultsHere, we genetically modified a previously developed P. ananatis strain expressing the (S)-linalool synthase (AaLINS) from Actinidia arguta to further improve (S)-linalool production. AaLINS was mostly expressed as an insoluble form in P. ananatis; its soluble expression level was increased by N-terminal fusion of a halophilic β-lactamase from Chromohalobacter sp. 560 with hexahistidine. Furthermore, in combination with elevation of the precursor supply via the mevalonate pathway, the (S)-linalool titer was increased approximately 1.4-fold (4.7 ± 0.3 g/L) in comparison with the original strain (3.4 ± 0.2 g/L) in test-tube cultivation with an aqueous-organic biphasic fermentation system using isopropyl myristate as the organic solvent for in situ extraction of cytotoxic and semi-volatile (S)-linalool. The most productive strain, IP04S/pBLAAaLINS-ispA*, produced 10.9 g/L of (S)-linalool in “dual-phase” fed-batch fermentation, which was divided into a growth-phase and a subsequent production-phase. Thus far, this is the highest reported titer in the production of not only linalool but also all monoterpenes using microbes.ConclusionsThis study demonstrates the potential of our metabolically engineered P. ananatis strain as a platform for economically feasible (S)-linalool production and provides insights into the stereoselective production of terpenes with high efficiency. This system is an environmentally friendly and economically valuable (S)-linalool production alternative. Mass production of enantiopure (S)-linalool can also lead to accurate assessment of its biological properties by providing an enantiopure substrate for study.

Iron oxide nanoparticles ameliorated the cadmium and salinity stresses in wheat plants, facilitating photosynthetic pigments and restricting cadmium uptake

Cadmium and salinity are the major threats to environmental resources and agricultural practice worldwide. The present work aims green synthesis, characterization, and application of iron oxide nanoparticles for co-alleviation of Cd and salt stresses in wheat plants. The iron oxide NPs were synthesized from a native bacterial strain, Pantoea ananatis strain RNT4, yielding a spherical FeO-NPs with a size ranging from 19 to 40 nm evidenced by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images. Results showed that application of 100 mg kg−1 of the bioengineered FeO-NPs in an original saline soil stimulated wheat plant growth, gaining 36.7% of additional length as compared with the control scenarios, via alleviating the detrimental effects of abiotic stresses and thereby reprogramming the morpho-physiological state of wheat plants. In addition, the presence of FeO-NPs in soil significantly increased the nutrient concentrations of N, P and K+, while reducing the Na+ and Cl− components in the wheat grain. Interestingly, application of the FeO-NPs in Cd-polluted soils eventually reduced wheat plant uptake of Cd by 72.5%, probably due to the adsorption of Cd onto the large surface of NPs and thereby, constraining Cd bioavailability to the plants. It provides the first evidence that a FeO-NPs-based treatment could be a candidate agricultural strategy for mitigating the Cd and salt stresses in Cd-polluted saline soils for safe agriculture practice.

Pantoea ananatis, A New Bacterial Pathogen Affecting Wheat Plants (Triticum L.) in Poland.

Wheat (Triticum aestivum) is one of the most economically important crops in the world. During the routine monitoring of wheat pest, the cereal leaf beetle (CLB, Oulema melanopus, Coleoptera, Chrysomelidae), in the Greater Poland region, it was observed that some leaves wounded by CLB also displayed brownish lesions with clear margins and yellow halo, disease symptoms resembling a bacterial infection. The aim of this study was therefore to investigate those symptoms to establish a causal agent of the disease. The identification based on the results of the Biolog’s Gen III system, 16S rRNA, and gyrB genes sequencing, revealed the presence of eight strains of Pantoea ananatis bacteria. Four strains were derived from wheat leaves (Ta024, Ta027, Ta030, Ta046), and four from the CLB’s oral secretion (OUC1, OUD2, OUF2, and OUG1). They shared the nucleotide identity ranging from 99 to 100% to P. ananatis strains deposited in the GenBank database. Additionally, the multi-locus sequence analysis (MLSA) of concatenated sequences of partial atpD, fusA, gyrB, rplB, and rpoB genes was performed. All P. ananatis strains isolated in Poland, grouped into one cluster supported with high bootstrap value. Pathogenicity tests performed on four varieties of wheat plants have identified P. ananatis strains as a causal agent of wheat disease. To our knowledge, this is the first report of P. ananatis affecting wheat plants.

Transmission of Pantoea ananatis, the causal agent of leaf spot disease of maize (Zea mays), by western corn rootworm (Diabrotica virgiferavirgifera LeConte)

The aim of this study was to determine the role of Diabrotica virgifera virgifera (western corn rootworm, WCR) as a potential insect vector of the pathogenic bacterium Pantoea ananatis. The experiment included a greenhouse test to investigate if WCR was able to transfer the pathogenic bacteria from infected to healthy maize plants. Adult WCR specimens collected from maize fields near Rzeszów were used in the experiment. The plant materials were sweetcorn plants of the Waza variety. Pure cultures of previously verified P. ananatis strain M241 were the source of the inoculum. Insects caught under natural conditions were incubated in an isolator containing pathogen-negative plants. Randomly selected insects were then examined for the presence of bacterial pathogens of maize in their digestive tract. Pathogen-negative insects were used in the next stage of the experiment still carried out in isolators, in which the insects foraged on maize seedlings previously infected with P. ananatis. The control group consisted of healthy, uninfected insects and plants. After the incubation period, the presence of bacterial pathogens in the gastrointestinal tract of the WCR specimens was confirmed. Subsequent insects that acquired P. ananatis were bred on pathogen-negative maize plants. After incubation, the presence of pathogenic bacteria in the body of the examined WCR beetles was confirmed, and the presence of symptoms of the bacterial disease was noted on maize plants, fulfilling Koch's postulates and indicating that the WCR is a vector of P. ananatis on maize plants. This is the first report on the vectorization of P. ananatis by D. virgifera globally.