First Report of Bacterial Leaf Streak of Rice Caused by Pantoea ananatis in Guangdong Province, China.

Rice (Oryza sativa L.) is a crucial staple crop worldwide, and bacterial diseases are among the primary factors affecting rice yield. In late October 2022, bacterial leaf streak disease was observed on the leaves of the rice variety Meixiangzhan 2 across multiple fields (approximately 130 hm2) in Leizhou City, Guangdong Province, China. The incidence rate was up to 30% in each field. Infected rice leaves exhibited distinctive symptoms at the boundary between diseased and healthy tissue, featuring dark green to yellow-brown streaks, while most of the leaf margin exhibited symptoms of either leaf edge or sheath rot. Disease progression from the leaf tip inwards revealed gray-white or dehydrated lesions with a bluish-gray color. Some leaves exhibited wrinkling at the edges, and severe symptoms at the leaf tip resembled those of bacterial leaf blight in rice. Ten leaves were collected from 10 infected rice plants in three distinct fields, and leaf pieces at the border of diseased and healthy areas were surface disinfected with 75% anhydrous ethanol for 60 seconds, rinsed three times with sterile water, and then soaked in sterile water for 8 hours. The obtained bacterial suspension was diluted at a ratio of 1: 106, and 100 µL of the diluted samples were plated on Potato Dextrose Agar (PDA) plates. After incubation at 28°C for 48 hours, the yellow bacterial colonies that appeared, were purified on PDA plates. To confirm the bacterial species, the amplification of genes gyrB, leuS, rpoB, and 16S rDNA was performed on six randomly selected isolates from the three different fields using the primers 27F/1492R, gyrB-F/R, leuS-F/R and rpoB-F/R, as reported by Yu et al (2022), respectively. PCR products were sequenced. All six isolates had identical sequences for all genes sequenced.The gene sequences of 16S rDNA (960 bp), gyrB (953 bp), leuS (733 bp), and rpoB (877 bp) for LZ1, were deposited in the NCBI database under accession numbers PP048830 , PP068625 , PP068626, and PP068627, respectively. These sequences were subsequently compared using BLASTn tool against the NCBI nr/nt database. The 16S rDNA, gyrB, leuS, and rpoB of LZ1 showed similarities of 99.90%, 99.16%, 99.73%, and 99.89%, with the corresponding sequences of P. ananatis TZ39 (GenBank accession numbers MZ800600.1 for 16S rDNA, and CP081342.1 for gyrB, leuS and rpoB ). MLSA analysis using concatenated sequences of gyrB, leuS, and rpoB genes indicated that the isolated strain LZ1 belongs to P. ananatis. In the tillering stage of rice varieties Meixiangzhan 2 and Huahangyuzhan, P. ananatis LZ1 was inoculated at a concentration of 108 CFU/mL using the leaf-cutting method, with sterile water used as a control (Toh et al., 2019). After 14 days of bacterial inoculation, the inoculated leaves gradually became necrotic, changing from light green to brown showing identical symptoms as those in the field, while the control plants remained symptom-free. Subsequent 16S rDNA, gyrB, leuS and rpoB gene sequencing results further confirmed the identity of the pathogen as P. ananatis, thereby fulfilling Koch's postulates. Previous reports have already identified P. ananatis as the pathogen causing rice bacterial leaf streak (Kini et al., 2017; Arayaskul et al., 2019; Yu et al., 2022; Lu et al., 2022; Luna et al., 2023; Yuan et al., 2023). This is the first report of rice bacterial leaf streak caused by P. ananatis in Guangdong Province, China, laying the foundation for future research to establish strategies for the prevention and control of this disease.

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-ß-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.

Pantailocins: phage-derived bacteriocins from Pantoea ananatis and Pantoea stewartii subsp. indologenes.

IMPORTANCE: Phage-derived bacteriocins (tailocins) are ribosomally synthesized structures produced by bacteria in order to provide advantages against competing strains under natural conditions. Tailocins are highly specific in their target range and have proven to be effective for the prevention and/or treatment of bacterial diseases under clinical and agricultural settings. We describe the discovery and characterization of a new tailocin locus encoded within genomes of Pantoea ananatis and Pantoea stewartii subsp. indologenes, which may enable the development of tailocins as preventative treatments against phytopathogenic infection by these species.

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, 3,521 genes were annotated, of which 3,418 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.

First report of rice bacterial leaf blight disease caused by Pantoea ananatis in the United States.

In August 2021, bacterial leaf blight-like symptoms were observed on 14 out of 570 rice genotypes (Oryza sativa) in research field plots of global rice germplasm grown in Arkansas (eXtra Figure S1. A & B). The disease was characterized by spreading lesions on leaves, panicle sterility and reduced yield in highly susceptible, mature rice germplasm. No spread of disease to nearby plants was observed. Isolations were performed at Colorado State University, where soakates from symptomatic leaves were spread onto nutrient agar. After 72 h at 28°C, uniform, distinct, yellow-colored bacterial colonies were observed. To screen for the presence of common rice bacterial pathogens, PCR amplification directly from colonies or from DNA isolated from symptomatic field-collected leaves was performed. Primers specific for Xanthomonas oryzae pvs. oryzae and oryzicola (Lang et al., 2010), Burkholderia glumae (Echeverri-Rico et al., 2021), and Pseudomonas fuscovaginae (Ash et al., 2014) did not amplify indicating these organisms were not present. Sequencing of 16S rRNA gene (Weisburg et al., 1991) amplicons suggested the bacteria belonged to the genera Pantoea and Sphingomonas (NCBI accession no. OP683332 and OP683333, respectively). Amplicons resulting from primers specific to the gyrB gene region of P. ananatis (Kini et al., 2021) were sequenced and the fragment was compared to the P. ananatis PA13 reference genome using a BLAST analysis. One candidate (AR358) showed 100% identity with the P. ananatis gyrB region. Primers specific for Sphingomonas sp. (Bangratz et al., 2020) confirmed the second candidate (AR359) as a Sphingomonas sp. The identity of P. ananatis was confirmed by the Plant Pathogen Confirmatory Diagnostics Laboratory (Beltsville, MD, USA). To determine pathogenicity, leaves from 7-day-old seedlings of rice (Oryza sativa) cultivar Kitaake were scissor-clip inoculated (Kauffman et al., 1973) with four different treatments and compared to control leaves inoculated with sterile water. Treatments for the experiment consisted of bacterial suspensions (108 CFU/ml) of the two candidate organisms, P. ananatis (strain AR358) or Sphingomonas sp. (strain AR359), individually or in a 1:1 ratio of P. ananatis:Sphingomonas sp., or soakate from infected field tissue. Lesions similar to those observed in the field were only detected on leaves inoculated with P. ananatis or infected field tissue soakate at 7-days post-inoculation (eXtra Figure S1. C). Bacteria were recovered from the leaves of the artificially inoculated seedlings from three treatments (P. ananatis, P. ananatis:Sphingomonas sp. and soakate from the infected field tissue) and were determined to be P. ananatis based on colony morphology, amplification of 16s rRNA, and gyrB sequence data. Our results confirm the pathogenicity of P. ananatis to rice and fulfill Koch's postulates. P. ananatis was also recovered from several similarly diseased rice breeding lines at the University of Arkansas System Division of Agriculture Rice Research and Extension Center. We conclude that P. ananatis is the causal pathogen for leaf blight-like symptoms observed in the global rice cultivars grown in Arkansas. P. ananatis was previously reported as a pathogen on rice in several rice growing regions, including China (Yu et al., 2021), India (Reshma et al., 2022), and Africa (Kini et al., 2017), however, this is the first report of P. ananatis as a pathogen of rice in the United States.

A new l-serine binding orphan SerBP affects indole synthesis in Pantoea ananatis.

Indole is traditionally known as a metabolite of l-tryptophan and now as an important signaling molecule in bacteria, however, the understanding of its upstream synthesis regulation is very limited. Pantoea ananatis YJ76, a predominant diazotrophic endophyte isolated from rice (Oryza sativa), can produce indole to regulate various physiological and biochemical behaviors. We constructed a mutant library of YJ76 using the mTn5 transposon insertion mutation method, from which an indole-deficient mutant was screened out. Via high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR), the transposon was determined to be inserted in a gene (RefSeq: WP014605468.1) of unknown function that is highly conserved at the intraspecific level. Bioinformatics analysis implied that the protein (Protein ID: WP089517194.1) encoded by the mutant gene is most likely to be a new orphan substrate-binding protein (SBP) for amino acid ABC transporters. Amino acid supplement cultivation experiments and surface plasmon resonance revealed that the protein could bind to l-serine (KD = 6.149 × 10-5 M). Therefore, the SBP was named as SerBP. This is the first case that a SBP responds to l-serine ABC transports. As a precursor of indole synthesis, the transmembrane transported l-serine was directly correlated with indole signal production and the mutation of serBP gene weakened the resistance of YJ76 to antibiotics, alkali, heavy metals, and starvation. This study provided a new paradigm for exploring the upstream regulatory pathway for indole synthesis of bacteria.

Establishment and application of the Recombinase-Aided Amplification-Lateral Flow Dipstick detection method for Pantoea ananatis on rice.

Pantoea ananatis is a major pathogen that causes the new bacterial blight in rice, and its symptoms very similar to rice bacterial blight. Therefore, there is a dire need for an accurate and rapid method for detecting P. ananatis. In this study, an early and rapid visual detection method for P. ananatis was established. Using GyrB gene as the target sequence, an innovative recombinase-aided amplification detection system integrated with a lateral flow dipstick (RAA-LFD) was constructed. The optimized RAA-LFD detection method can be initiated at body temperature and does not rely on precise instruments. It does not require DNA extraction and can be used directly with plant tissue fluids. The results can be visualized after 10 minutes of amplification. The specificity and sensitivity tests showed that the RAA-LFD method could detect P. ananatis, whereas other common plant pathogens were not detected, and its detection sensitivity for P. ananatis DNA reached 100 copies/µL. The detection of diseased tissues indicated that this method could accurately detect P. ananatis in artificially inoculated rice tissues in the early stages of infection before symptoms. The RAA-LFD detection system established in this study is simple and fast, with visual results, excellent specificity, and high sensitivity. It is semi-quantitative and should be used for the early detection and rapid field diagnosis of new leaf blight, which provides technical support for the early warning and real-time detection of field samples.

Negatively Regulated Aerobactin and Desferrioxamine E by Fur in Pantoea ananatis Are Required for Full Siderophore Production and Antibacterial Activity, but Not for Virulence.

Pantoea ananatis is an emerging plant pathogen that causes disease in economically important crops such as rice, corn, onion, melon, and pineapple, and it also infects humans and insects. In this study, we identified biosynthetic gene clusters of aerobactin and desferrioxamine E (DFO-E) siderophores by using the complete genome of P. ananatis PA13 isolated from rice sheath rot. P. ananatis PA13 exhibited the strongest antibacterial activity against Erwinia amylovora and Yersinia enterocolitica (Enterobacterales). Mutants of aerobactin or DFO-E maintained antibacterial activity against E. amylovora and Y. enterocolitica, as well as in a siderophore activity assay. However, double aerobactin and DFO-E gene deletion mutants completely lost siderophore and antibacterial activity. These results reveal that both siderophore biosynthetic gene clusters are essential for siderophore production and antibacterial activity in P. ananatis PA13. A ferric uptake regulator protein (Fur) mutant exhibited a significant increase in siderophore production, and a Fur-overexpressing strain completely lost antibacterial activity. Expression of the iucA, dfoJ, and foxA genes was significantly increased in the Δfur mutant background, and expression of these genes returned to wild-type levels after fur compensation. These results indicate that Fur negatively regulates aerobactin and DFO-E siderophores. However, siderophore production was not required for P. ananatis virulence in plants, but it appears to be involved in the microbial ecology surrounding the plant environment. This study is the first to report the regulation and functional characteristics of siderophore biosynthetic genes in P. ananatis. IMPORTANCE Pantoea ananatis is a bacterium that causes diseases in several economically important crops, as well as in insects and humans. This bacterium has been studied extensively as a potentially dangerous pathogen due to its saprophytic ability. Recently, the types, biosynthetic gene clusters, and origin of the siderophores in the Pantoea genus were determined by using genome comparative analyses. However, few genetic studies have investigated the characteristics and functions of siderophores in P. ananatis. The results of this study revealed that the production of aerobactin and desferrioxamine E in the rice pathogen P. ananatis PA13 is negatively regulated by Fur and that these siderophores are essential for antibacterial activity against Erwinia amylovora and Yersinia enterocolitica (Enterobacterales). However, siderophore production was not required for P. ananatis virulence in plants, but it appears to be involved in the microbial ecology surrounding the plant environment.

First Report of Pantoea ananatis Causing Crown Necrobiosis on Strawberry in China.

Strawberry (Fragaria × ananassa) is an economically important crop in Zhejiang, China. In the autumn of 2021, crown necrobiosis and angular leaf spot was observed in commercial strawberry fields (cultivar 'fenyu') in Cixi, Ningbo, Zhejiang, China (N30°9'55″, E121°21'13″). The disease incidence ranged from 5 to 8 % in the field, but could reach 50 to 60 % in some heavily affected plastic tunnels. In the affected field, this disease could reduce strawberry production by 50%. Early symptoms were water-soaked lesions around the vein of the abaxial leaves; subsequently, reddish-brown irregular spots and coalesced lesions developed. In humid conditions, a sticky bacterial ooze exuding from lesions was observed. Finally, the crown of the diseased plant was necrotized, and several pockets were observed inside the crown after dissection. To isolate the causal agent, the infected leaves and crown tissues from six different plants were surface-sterilized with 75% ethanol for 1 min, rinsed twice with sterile distilled water, cut into small pieces, and soaked in 5 ml of sterile distilled water for 20 min. The supernatant from the cut-up pieces was serially diluted and spread on nutrient agar medium. After 2 to 3 days at 28℃, several yellow colonies were grown on the medium. The colonies from five infected plants were gram-negative, anaerobic rods, yellow, viscous, and gloss, which are typical characteristics of Erwinia anana (Wells et al. 1986). To confirm the identity of the causal bacteria, PCR was conducted for six randomly selected colonies to amplify 16S rRNA (Monciardini et al. 2002), fusA, and gyrB (Stice et al. 2002). The amplicons were sequenced and blasted, and the results showed that the six colonies were identical. The 16S rRNA, fusA, gyrB sequences of the isolate CM3 were deposited in GenBank with accession number ON754076.1, OP587277, and OP587278; BLAST search showed 99.93% (1445 bp out of 1446 bp), 100% (746 bp out of 746 bp), 99.64% (1371 bp out of 1376 bp) similarity with strains of Pantoea ananatis (KT741001.1, MH015093.1 and CP066803.1 accessions, respectively). The resulting concatenated data set of 16S rRNA-fusA-gyrB was used to build a multilocus phylogenetic analysis (MLSA) by maximum likelihood criteria. The MLSA tree indicated that the isolate CM3 belonged to Pantoea ananatis. The isolate's identity was further confirmed by P. ananatis-specific primers pagyrB-F/R (Xiao et al. 2022). Thus, this isolate was designated as P. ananatis CM3. To fulfill Koch's postulates, two old leaves were broken off each of the ten 2-month-old strawberry (cultivar 'fenyu') plants to create wounds, each plants was sprayed with a cell suspension of P. ananatis (107CFU/ml, 0.5 ml) on the stem base. Ten plants were sprayed with water to serve as a control. All plants were kept at 28/25°C (day/night) under a 12-h/12-h photoperiod. All plants were covered with transparent plastic bags to maintain humidity. After 48 h, the bags were removed. After 2 weeks, water-soaked lesions on some leaves were observed similar to those in the field . Three to five weeks after inoculation, the crown of the inoculated plants was necrotized, which was similar to the symptoms in the field. No symptoms were observed in the control plants. The experiment was repeated three times. The bacteria were successfully reisolated from the inoculated crown tissues and leaves and confirmed as CM3 according to the same methodologies used for the initial identification. Bacterial leaf blight in strawberry caused by Pantoea ananatis has been reported in Nova Scotia, Canada, and Egypt (Bajpai et al. 2019; Abdel-Gaied et al. 2022). To our knowledge, this is the first report of Pantoea ananatis causing crown necrobiosis on strawberry in China. This report provides a basis for further research on this disease and its management and control.

First Report of Maize White Spot Disease Caused by Pantoea ananatis in China.

Maize (Zea mays L.) is one of the most important crops in China. Since 2020, a new leaf spot disease has occurred in southwest China in areas such as Yunnan, Sichuan and Hubei provinces. Typical symptoms appeared after tasseling. The spots are scattered on the leaf surface, round to oval in shape with diameter range 3-20 mm. Spots are initially water-soaked, gradually turning yellow or white. In 2021, diseased leaf samples with typical white spot were collected for pathogen isolation and identification in Qujing, Yunnan province. Four small pieces of leaf tissue (about 0.25 cm2 in area) were excised from the edge of the necrotic lesion of each plant, surface sterilized with 75% ethanol for 1 min, rinsed three times with sterile distilled water, and soaked in sterile distilled water for 5 min. The solution was plated on Luria Broth medium (LB) plate (Shin et al. 2022) . After incubation at 28°C for 24 h, round, smooth-edged yellow colonies appeared in the LB plate. The bacterium isolated was gram-negative, negative for oxidase, positive for peroxidase, indole, citrate (Wells et al. 1987). Three strains (PA21QJ01, PA21QJ02 and PA21QJ03) showed identical colony morphology. PA21QJ01 was used for further molecular analyses. DNA was extracted from fresh colonies cultured in LB(Shin et al. 2022), and the fragments at the 16S rDNA, gyrB and rpoB loci were amplified using primers 27F/1492R (Galkiewicz and Kellogg 2008), UP-1/UP-2r (Yamamoto and Harayama 1995) and rpoBCM81-F/rpoBCM32b-R (Brady et al. 2008), respectively. The sequences of fragments of 16S rDNA, gyrB and rpoB from isolate PA21QJ01 were was deposited in GenBank (accession number: OM184301.1, OM302500, OM302499). A search for homologous sequences using BLAST resulted in 99.9, 99.6 and 99.8% identity of 16S rDNA, gyrB and rpoB, respectively, with sequences from the NN08200 of Pantoea ananatis (GenBank accession numbers: MK415050.1 for 16S rDNA, CP035034.1 for gyrB and CP035034.1.1 for rpoB). Above molecular results indicated that PA21QJ01 isolated from maize white spot is P. ananatis. Pathogenicity tests were performed on tasseled plants of the suscptible maize variety Wugu1790. After culture in LB medium plate at 30°C for 12 h, the bacterial solution was used for inoculation at a concentration of 1 × 108 CFU ml-1. After 7 days of inoculation, the leaves of the plants appeared water-soaked. After 10 days, white spot developed with brown margin. In contrast, the control plants remained healthy and symptomless. The same P. ananatis was reisolated in the inoculated maize plants, fulfilled Koch's law. In the last decade, P. ananatis has been reported to cause maize white spot in South Africa, Mexico, Poland, Argentina, Brazil (Sauer et al. 2015), and Ecuador (Toaza et al.2021). It has caused crop diseases with other crops, such as onion, rice, pineapple, melon, and sorghum, and others (Sauer et al. 2015). It caused leaf blight and leaf steak in rice in China (Yu et al. 2021). This is the first report of maize white spot caused by P. ananatis in China. However, to our knowledge, this is the first report of maize white spot disease in China. Attentions should be paid to screening for disease-resistant resources and breeding disease-resistant hybrids. Reference: Wells, J. M. et al. 1987. Int. J. Syst. Bacteriol. 37:136-143. Shin, G. Y. et al. 2022. Plant Dis. Doi: 10.1094/PDIS-08-21-1810-SC. Brady, C., et al. 2008. Syst. Appl. Microbiol. 31:447. Galkiewicz, J. P., and Kellogg, C. A. 2008. APPL ENVIRON MICROB, 74.24: 7828-7831. Toaza, A. et al. 2021. Plant Dis. Doi:10.1094/PDIS-02-21-0298-PDN Yamamoto, S., and Harayama, S. 1995. APPL ENVIRON MICROB, 61:1104.L. Sauer, A. V. et al. 2015. Agronomy Science and Biotechnology. Doi:10.33158/ASB.2015v1i1p21 Yu et al. 2021. Plant Dis. Doi:10.1094/PDIS-05-21-0988-PDN.

First report of a new bacterial stem rot disease of strawberry (Fragaria×ananassa) caused by Pantoea ananatis in Jiangsu, China.

Strawberry (Fragaria×ananassa Duch.) is an important economic fruit crop in the world. With the continuous expansion of strawberry planting area, strawberry disease is one of the most important limiting factors, which seriously affects the agronomic performance and leads to significant economic losses. In November 2020, an infected stem rot disease of strawberries was detected in the strawberry growing area of Donghai County, Jiangsu Province, China. The disease incidence ranged from 30 % to 45 %. Initially, infected plants included stunted growth of new leaves, leaflet asymmetry, and holes in the vertical section of the stem, resulting in leaf blight and death in severe cases. To isolate the pathogen, two symptomatic plants were randomly collected. And then infected plants were surface sterilized with 75 % ethanol for 1 min, followed by 2 % sodium hypochlorite for 6 minutes. After that, the infected plants were washed 4-5 times with double sterilized distilled water, cut into 3-5 mm small pieces, and soaked in 2 ml of sterile water for 15 min, after which 100 µl of liquid suspension were spread onto Luria-Bertani medium (LB) and incubated at 28 °C for 12-16 h. All isolates showed yellow, viscous, round, and smooth (Figure S1, C) and the isolates were designated as JX1 and JX2. To identify the pathogen, the genomic DNA were extracted from isolates using the Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech, China) and the fragments of gyrB, rpoB and leuS gene were amplified using the primer pairs UP-1S/UP-2Sr (Yamamoto and Harayama 1995), rpoB-F/rpoB-R and leuS-F/leuS-R (Yu et al. 2022), respectively. Sequence analyses showed that the nucleotide sequences of gyrB, rpoB, and leuS fragments of the isolates shared 99.72 %, 99.67 % and 98.37 % identity with the Pantoea ananatis type strain LMG 2665 (KF482590.1, EF988972.1 and KF482626.1, respectively ), which suggests that the isolate could be Pantoea ananatis. To further verify that P. ananatis was identity of these isolates, the whole genome was sequenced using PacBio sequel II technology. The Average Nucleotide Identity (ANI) calculation showed that the whole-genome sequence was 99.0% similar to that of the Pantoea ananatis type strain LMG 2665 (Jain et al. 2018). The isolates were therefore recognized as P. ananatis. To confirm pathogenicity, roots of strawberry plants were inoculated by wounding as described (Wang et al. 2017) with bacterial suspensions (108 CFU/ml) for 30 min, and transplanted into 10 cm ×8.5 cm pots filled with substrate (peat: perlite: vermiculite =3:1:1). The negative control plants were inoculated with sterile distilled water (20 individual plants per group). All infected plants were placed in a greenhouse under the following environmental conditions: 30 ℃/25 ℃ day/night, >70 % relative humidity, 16-h/8-h photoperiod. The experiment was carried out three times. After 3 to 4 weeks of inoculation, the new leaves of the plants were smaller and asymmetrical, while the negative plants remained healthy. After 8 weeks, a significant stem rot pocket developed on all inoculated plants, similar to the symptoms observed in the field. In contrast, no symptoms were observed in negative plants (Figure S2). To fulfill Koch's postulates bacteria were further isolated, purified and identified from the greenhouse inoculated plants. The results proved that the causative agent of strawberry stem rot was P. ananatis. In recent decades, P. ananatis has been found to cause bacterial leaf blight in strawberries (Bajpai et al. 2020). It has also caused other crop diseases, such as maize white spot, peach soft rot and others (Cui et al.2022; Liao et al. 2015). Although other crop diseases caused by P. ananatis, a bacterial pathogen, there has been no report of P. ananatis causing the symptoms of stem rot disease in strawberry. To our knowledge, this is the first report of P. ananatis causing stem rot in strawberry. This study provides solid evidence that strawberry stem rot disease in China can also be caused by the novel pathogen Pantoea ananatis. In conclusion, this report will provide a theoretical reference for the prevention and control measures of P. ananatis causing strawberry stem rot disease in the future.

The Role of Pyoluteorin from Pseudomonas protegens Pf-5 in Suppressing the Growth and Pathogenicity of Pantoea ananatis on Maize.

The rhizospheric bacterium Pseudomonas protegens Pf-5 can colonize the seed and root surfaces of plants, and can protect them from pathogen infection. Secondary metabolites, including lipopeptides and polyketides produced by Pf-5, are involved in its biocontrol activity. We isolated a crude extract from Pf-5. It exhibited significant surface activity and strong antibacterial activity against Pantoea ananatis DZ-12, which causes maize brown rot on leaves. HPLC analysis combined with activity tests showed that the polyketide pyoluteorin in the crude extract participated in the suppression of DZ-12 growth, and that the lipopeptide orfamide A was the major biosurfactant in the crude extract. Further studies indicated that the pyoluteorin in the crude extract significantly suppressed the biofilm formation of DZ-12, and it induced the accumulation of reactive oxygen species in DZ-12 cells. Scanning electron microscopy and transmission electron microscopy observation revealed that the crude extract severely damaged the pathogen cells and caused cytoplasmic extravasations and hollowing of the cells. The pathogenicity of DZ-12 on maize leaves was significantly reduced by the crude extract from Pf-5 in a dose-dependent manner. The polyketide pyoluteorin had strong antibacterial activity against DZ-12, and it has the potential for development as an antimicrobial agent.

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

BACKGROUND: Linalool, 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. RESULTS: Here, 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 ß-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. CONCLUSIONS: This 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.

Beneficial effects of endophytic Pantoea ananatis with ability to promote rice growth under saline stress.

AIMS: Soil salinization severely inhibits plant growth, leading to a low crop yield. The aim of the current study was to isolate endophytic bacteria with the ability to promote rice growth under saline conditions. METHODS AND RESULTS: We isolated eight salt-tolerant endophytic bacteria from rice roots. An isolated strain D1 was selected due to its ability to stimulate rice seed germination in the presence of NaCl, which was identified as Pantoea ananatis D1. It exhibited multiple plant growth-promoting traits including phosphate solubilization, production of indole-3-acetic acid, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and siderophore. Inoculation of P. ananatis D1 obviously enhanced the rice root and shoot growth under normal and saline conditions. It also significantly increased the contents of chlorophyll, total soluble protein, and proline in salt-stressed rice seedlings. Moreover P. ananatis D1 could ameliorate the oxidative stress in rice induced by NaCl and Na2 CO3 treatment. The malondialdehyde content and various antioxidant enzyme activities were decreased by P. ananatis D1 inoculation in salt-affected rice. In addition, P. ananatis D1 showed a positive potential for limiting the Na+ accumulation and enhancing the K+ uptake, leading to an increase of 1·2-1·7 fold in K+ /Na+ ratio under saline environment. CONCLUSIONS: Pantoea ananatis D1 has the ability to improve the salt tolerance of rice seedlings. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of plant growth-promoting bacteria (PGPB) is an eco-friendly strategy to improve plant tolerance towards abiotic stresses. We demonstrated that P. ananatis D1 could be used as an effective halotolerant PGPB to enhance rice growth in different salt-affected soils.

When Carotenoid Biosynthesis Genes Met Escherichia coli : The Early Days and These Days.

Nowadays, carotenoid biosynthetic pathways are sufficiently elucidated at gene levels in bacteria, fungi, and higher plants. Also, in pathway engineering for isoprenoid (terpene) production, carotenoids have been one of the most studied targets. However, in 1988 when the author started carotenoid research, almost no carotenoid biosynthesis genes were identified. It was because carotenogenic enzymes are easily inactivated when extracted from their organism sources, indicating that their purification and the subsequent cloning of the corresponding genes were infeasible or difficult. On the other hand, natural product chemistry of carotenoids had advanced a great deal. Thus, those days, carotenoid biosynthetic pathways had been proposed based mainly on the chemical structures of carotenoids without findings on relevant enzymes and genes. This chapter shows what happened on carotenoid research, when carotenoid biosynthesis genes met non-carotenogenic Escherichia coli around 1990, followed by subsequent developments.

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.

Bacterial Disease Complex Including Bleached Spot, Soft Rot, and Blight on Onion Seedlings Caused by Complex Infections.

In 2018, a bacterial disease complex composed of bleached spots and soft rot-blight on onion seedlings was observed in nursery beds in Changnyeong, a major onion-producing county in South Korea. Four bacteria isolated from the diseased lesions were identified: Pseudomonas viridiflava, Acidovorax avenae subsp. avenae, Pantoea ananatis, and Xanthomonas axonopodis, respectively. We referred to the four strains as a "bacterial disease complex" because they were isolated from the same sample with multiple symptoms. We examined the synergistic activity among the four strains to understand their relationships and roles. We monitored in vivo bacterial population density and disease progression after artificially inoculating the bacteria on onion seedlings at a temperature of 22 or 28°C. The disease pattern progressed sooner at 28 than at 22°C (by an average of 4 to 6 days). The rate of disease progression induced by inoculation of P. ananatis alone was consistent with that induced by coinoculation of P. ananatis with the other strains, regardless of the temperature (22 or 28°C). The in vivo growth of P. ananatis on onion seedlings was not different after inoculation alone versus together with the other strains. The rate of disease progression induced by P. viridiflava was similar when inoculated alone and when inoculated with other tree strains at 28°C, but disease progression induced by inoculation alone was slower at 22°C. The in vivo growth of P. viridiflava or X. axonopodis on onion seedlings decreased rapidly or gradually, respectively, when inoculated with the other strains. Coinfection with the other three strains had repression effects on the growth of P. viridiflava, a slight effect on X. axonopodis, and no effect on P. or A. avenae subsp. avenae in vivo. These results indicate that the strains coexist or interact antagonistically, rather than synergistically, depending on the conditions. These results were consistent with the results of the in vitro growth inhibition assay, in which P. viridiflava growth was inhibited by X. axonopodis or P. ananatis. These results also confirmed that X. axonopodis is present on bleached spots and P. viridiflava on soft rot-blight lesions, and that P. viridiflava and P. ananatis cause soft rot-blight but do not coexist. A. avenae subsp. avenae is a minor causative pathogen of bleached spots on onion seedlings, but it is not significantly affected by temperature and has no antagonistic or synergistic interactions with X. axonopodis.

Application of inorganic phosphate limitation to efficient isoprene production in Pantoea ananatis.

AIMS: Establishment of an efficient isoprene fermentation process by adopting inorganic phosphate limitation as the trigger to direct metabolic flux to the isoprene synthetic pathway. METHODS AND RESULTS: We constructed isoprene-producing strains of Pantoea ananatis (a member of the Enterobacteriaceae family) by integrating a heterologous mevalonate pathway and a metabolic switch that senses external inorganic phosphate (Pi) levels. This metabolic switch enabled dual-phase isoprene production, where the initial cell growth phase under Pi-saturating conditions was uncoupled from the subsequent isoprene production phase under Pi-limiting conditions. In fed-batch fermentation using our best strain (SWITCH-PphoC/pIspSM) in a 1-l bioreactor, isoprene concentration in the off-gas was maintained between 300 and 460 ppm during the production phase and at 20 ppm during the cell growth phase, respectively. The strain SWITCH-PphoC/pIspSM produced totally 2·5 g l-1 of isoprene from glucose with a 1·8% volumetric yield in 48 h. CONCLUSIONS: This proof-of-concept study demonstrated that our Pi-dependent dual-phase production system using a P. ananatis strain as a producer has potential for industrial-scale isoprene fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This Pi-dependent dual-phase fermentation process could be an attractive and economically viable option for the production of various commercially valuable isoprenoids.

Genome Resources of Three West African Strains of Pantoea ananatis Causing Bacterial Blight and Grain Discoloration of Rice.

Members of the genus Pantoea have been reported as pathogens for many economically important crops, including rice. Little is known about their host-pathogen interactions at the molecular level and the lack of comprehensive genome data impedes targeted breeding strategies toward resistant rice cultivars. Here, we describe the structural and functional annotation of the draft genome sequences of three rice-pathogenic Pantoea ananatis strains, ARC272, ARC310, and ARC311, which were isolated in Burkina Faso, Togo, and Benin, respectively. The genome sequences of these strains will help in developing molecular diagnostic tools and provide new insight into common traits that may enable P. ananatis to infect rice.

Identification and characterization of a novel bacterial carbohydrate esterase from the bacterium Pantoea ananatis Sd-1 with potential for degradation of lignocellulose and pesticides.

OBJECTIVE: Identification and characterization of a novel bacterial carbohydrate esterase (PaCes7) with application potential for lignocellulose and pesticide degradation. RESULTS: PaCes7 was identified from the lignocellulolytic bacterium, Pantoea ananatis Sd-1 as a new carbohydrate esterase. Recombinant PaCes7 heterologously expressed in Escherichia coli showed a clear preference for esters with short-chain fatty acids and exhibited maximum activity towards α-naphthol acetate at 37 °C and pH 7.5. Purified PaCes7 exhibited its catalytic activity under mesophilic conditions and retained more than 40% activity below 30 °C. It displayed a relatively wide pH stability from pH 6-11. Furthermore, the enzyme was strongly resistant to Mg2+, Pb2+, and Co2+ and activated by K+ and Ca2+. Both P. ananatis Sd-1 and PaCes7 could degrade the pesticide carbaryl. Additionally, PaCes7 was shown to work in combination with cellulase and/or xylanase in rice straw degradation. CONCLUSIONS: The data suggest that PaCes7 possesses promising biotechnological potential.