First report of Puccinia xanthii causing rust disease on Xanthium orientale in Korea.

Puccinia xanthii Schw. is a microcyclic rust fungus, first found on Xanthium strumarium Lour in North Carolina, the United States. This rust fungus is native to the continental United States, Hawaii, Mexico, and the West Indies (Arthur 1934). It has become notoriously invasive and is now distributed in the Europe (Bulgaria, France, Hungary, Italy, Romania, Spain, and the former Yugoslavia), India, Indonesia, Australia, and South Africa (Parmelee 1969; Alcorn 1976; Wahyuno 2012). In East Asia, the fungus has been reported in Japan (Hiratsuka et al. 1992) and China (Zhao et al. 2014) but not in Korea. It has been reported mainly on the invasive weeds Xanthium and Ambrosia species. In addition, it rarely occurs on sunflowers (Helianthus spp.) in Australia (Alcorn 1976), South Africa (Pretorius et al. 2000), and North America (Gulya and Charlet 2002). In Korea, rust disease symptoms caused by a Puccinia fungus were first found on X. orientale L. at the roadside of Okcheon-gun, Chungcheongbuk-do (36 27'95.428"N 127 66'26.378"E) in October 2021 and were repeatedly observed in the same site in 2022. The similar symptom was additionally found on X. orientale in Yesan-gun, Oct. 2022. The symptoms were brown spots on round chlorotic haloes on the adaxial leaf surface and dark brown pustules on the abaxial leaf surface. Telia were brown to dark brown, round, mostly grouped, 0.28-0.61 mm in diameter, and mainly formed on the abaxial leaf surface but sometimes on the adaxial leaf surface. Teliospores were two-celled, pedicellate, and measured 37.6-110 × 12.4-21.5 µm in size; the wall was yellowish or almost colorless, smooth, 1.2-2.6 µm thick at the sides, and up to 7.4 µm thick at the apex. The morphological characteristics of the teliospores were identical to those of P. xanthii described by Arthur (1934) and Parmelee (1969). Based on phylogenetic analyses (e-Xtra 2) of the internal transcribed spacer (ITS) and partial large subunit (LSU) rDNA extracted from the teliospores, they were identified as P. xanthii. BLAST analysis showed that the sequences had high homologies (over 99.82%) with the reference strains of P. xanthii (EF635903 and KX999896). The representative specimens were preserved at the Animal and Plant Quarantine Agency Herbarium (PQK211005 for Okcheon-gun isolate and PQK220913 for Yesan-gun) and the sequences were deposited in GenBank (OR958716 and OR958692). A pathogenicity test was performed by dropping a suspension of germinating teliospores and basidiospores onto the adaxial leaf surfaces of apparently healthy X. orientale plants in Oct. 2022, using the isolate PQK220913 (OR958692). The three inoculated plants were placed together with three controls treated with only distilled water, in the dark at saturated humidity for 24 hours in an isolated greenhouse. After two weeks, typical rust symptoms were observed in the three infected plants, whereas no symptoms appeared in the control plants (e-Xtra 1). The causal fungus was identified as P. xanthii based on host relationships, successful experimental inoculation, morphological characteristics, and sequence similarity of partial DNA fragments. To our knowledge, this is the first report of P. xanthii on X. orientale in Korea. P. xanthii was additionally confirmed on X. orientale in Gumi-si, Boeun-gun, Seongju-gun, Naju-si, and Gunsan-si in 2023, indicating its wide distribution in Korea. It is expected that P. xanthii could be a candidate as a biological agent for controlling the invasive weed, X. orientale.

First report of a rust disease of Lycium chinense caused by Puccinia tumidipes in Korea.

Lycium chinense Mill is a deciduous broad-leaved shrub belonging to the Solanaceae family and, is widely distributed throughout Korea. This plant is native to, or cultivated for various oriental medicinal purposes in, multiple regions of Asia, including Korea, China, and Japan (Lee 1982; Kim et al. 1994). Eleven Puccinia species have been reported to infect Lycium species (Otálora et al. 2018). In May and October 2022, symptoms of rust disease caused by Puccinia sp. were observed on almost all the leaves of about 60 sprawling stems of L. chinense plants on the seashore of Jeju island, Korea (33°14'15.0835″N, 126°30'53.40E). Brownish red (uredinium) or blackish brown (telium) pustules were observed on upper and lower surfaces of infected leaves. These symptoms were observed on about 40 L. chinense plants, barely growing between rocks on the seashore of Ulsan Metropolitan City, and on the about 20 L. chinense plants on a small home garden of Jindo-gun, Korea, in June and October 2023, respectively. Uredinia were amphigenous, individually scattered, but sometimes formed groups of two or three on leaves and sepals, ferruginous, pulverulent, and surrounded by a ruptured epidermis, often developing into blackish telia. Urediniospores were either ellipsoid or ovoid, approximately 29.3-34.9 × 17-24.3 µm, with yellowish walls, 1-2 µm thick. The germ pores were bizonate, and each band contained four pores covered by low papillae. Blackish-brown telia were observed on both leaf surfaces. Teliospores were broadly ellipsoidal, and rounded at the apex and towards the base. They were measured approximately 37.1-53.4 × 25-34.5 µm. The walls were light chestnut-brown and 2-3.7 µm thick, apically up to 5-9 µm thick. The swollen pedicel was persistent, basal, hyaline, smooth, and similar in length to the spores (Fig. 1). These morphological characteristics were similar to those of P. tumidipes, as described by Otálora et al. (2018). The representative specimens were preserved at the Animal and Plant Quarantine Agency Herbarium (PQK220531, -230605, and -231026). The fungal internal transcribed spacer (ITS2) and cytochrome oxidase subunit 3(CO3) regions were amplified from the total DNA of the isolates, using the primer pairs ITS5, ITS4, CO3F1, and CO3R1 for phylogenetic analysis (White et al. 1990; Vialle et al. 2009). PCR products were sequenced (Celemics, Seoul, Korea), and deposited in GenBank (Accession numbers are shown in Fig. 2.). The combined ITS2 and CO3 sequences were grouped with those of other isolates of P. tumidipes in the phylogenetic tree (Fig. 2). In November 2022, three pathogenicity tests were conducted using a urediniospore suspension made with the PQK220531 isolate in sterile distilled water. The suspension was smeared onto the upper surface of healthy L. chinense leaves. The three inoculated plants were kept in the dark at saturated moisture levels for 24 hours and placed in an isolated glasshouse together with the three control plants. After two weeks, uredinia of P. tumidipes were observed on the leaves of the inoculated plants, but not on the control plants. Although no spermogonial or aecial stage has been observed in Korea, our study has proven that P. tumidipes is the causal fungus of rust disease in L. chinense. This result is also the first discovery of the New-World P. tumidipes in Asia, showing this fungus is not limitedly distributed in America and suggesting further surveys be done on its exact geographical distribution.

Malva sylvestris, a new host of Puccinia modiolae in Korea.

Malva sylvestris (Malvaceae), known as common mallow, is native to Europe, western Asia, and northern Africa. It was intentionally introduced to Korea as an ornamental plant in the early 20th century, and has become partly naturalized in several areas including the woods (Jung et al. 2017). Among nine microcylic Puccinia species attacking the Malvaceae plants, three species of P. heterospora, P. malvacearum, and P. modiolae have been reported on M. sylvestris (Classen et al. 2000, Colenso 1885, McKenzie 1998 and Melo et al. 2012). Only P. modiolae has been found on Alcea rosea and M. verticillata, and not M. sylvestris in Korea (Lee et al. 2022; Ryu et al. 2022). In August 2022, rust disease symptoms of a Puccinia fungus were observed on some overgrown seedlings of M. sylvestris, which were neglected in containers after sales at a wholesale nursery (36°50'19.8″N, 128°55'28.7″E) in Bonghwa, Korea. Typical rust spots were observed around 60% (on 111 seedlings of the 186 seedlings of M. sylvestris). The brown spots were produced on round chlorotic haloes on the adaxial leaf surface, and brown to dark brown pustules on the abaxial. Subepidermal spermogonia on the adaxial, were obovoid, and 112.1-160.0 × 88.7-149.3 µm in size. Telia were golden-brown to dark brown, round, mostly grouped, and 0.30-0.72 mm in diameter, and mainly hypophyllus. Fusoid teliospores were two-celled, rarely one- or three-celled, 36.2-92.3 × 10.6-19.3 µm in size, with many anomalies appearing notched at apex; wall was yellowish or almost colorless, smooth, 1.0-2.6 µm thick at the sides, and up to 6.8 µm thick at the apex; pedicel was hyaline, thick wall, persistent, and (39.3-)60.4-154.6(-189.9) µm long. Based on these morphological features together with the results of the phylogenetic analyses (e-Xtra 2) using internal transcribed spacer (ITS) and partial large subunit (LSU) sequences according to the method described by Ryu et al. (2022), the fungus was identified as an autoecious P. modiolae, recently reported on M. verticillate and A. rosea in Korea (Lee et al. 2022; Ryu et al. 2022). A representative sample was deposited in the Animal and Plant Quarantine Agency Herbarium (PQK220818). Pathogenicity tests were done using three host plants: M. sylvestris, M. verticillate and A. rosea. Three to four leaf discs with basidiospore-bearing telia were placed on the upper surfaces of healthy young leaves of the seedlings. Three replicates of each host plant set including an untreated control were tested. The plants were kept in an isolated glass house. At ten to twelve days after inoculation, typical telial spots of P. modiolae were recovered, but not in the control plants, showing all three tested species were highly susceptible (e-Xtra 1). The ITS and LSU sequences obtained from the genomic DNAs of each newly recovered rust spot were consistent with that of the inoculum (accession no. OQ542745). The previous A. rosea isolate (OP369290 by Ryu et al. 2022) also showed the pathogenesis on M. sylvestris and M. verticillata by the same tests, mentioned above (e-Xtra 1). To date, only one collection of P. modiolae on M. sylvestris has been reported in Louisiana, the United States (Aime and Abbasi 2018). The results of this study show that P. modiolae is firstly confirmed as the causal rust fungus of M. sylvestris and the same causal agent of M. verticillate and A. rosea rust disease, recently reported in Korea.

Identification and characterization of gut-associated lactic acid bacteria isolated from the bean bug, Riptortus pedestris (Hemiptera: Alydidae).

Lactic acid bacteria (LAB) are beneficial bacteria for humans and animals. However, the characteristics and functions of LAB in insects remain unclear. Here, we isolated LAB from the gut of Riptortus pedestris, a pest that is a significant problem in soybean cultivation in Korea, and identified two Lactococcus lactis and one Enterococcus faecalis using matrix-associated laser desorption/ionization-time of flight and 16S rRNA analyses. All three LAB strains survived at pH 8, and L. lactis B103 and E. faecalis B105 survived at pH 9 for 24 h. In addition, these strains survived well in simulated gastric juice of humans containing pepsin and exhibited high resistance to bile salts. Two strains of L. lactis and one of E. faecalis maintained constant density (> 104 colony-forming units [CFU]/mL) at pH 2.5, but viability at pH 2.2 was strain-dependent. The three LAB were reinoculated into second-instar nymphs of R. pedestris and colonized well, reaching a constant density (> 105 CFU/gut) in the adult insect gut. Interestingly, feeding of these LAB increased the survival rate of insects compared to the negative control, with the largest increase seen for L. lactis B103. However, the LAB did not increase the weight or length of adult insects. These results indicate that insect-derived LAB possess the traits required for survival under gastrointestinal conditions and have beneficial effects on insect hosts. The LAB infection frequency of the wild bean bug populations was 89% (n = 18) in Gyeongsangnam-do, South Korea. These LAB can be utilized as a novel probiotic in the cultivation of beneficial insects. This study provides fundamental information about the symbiosis between insects and LAB, and a novel concept for pest control.

Toxoflavin contamination in rice samples from rice processing complexes in South Korea.

Toxoflavin contamination was investigated in broken rice produced as a by-product of domestic rice processing complexes (RPCs) in 2011 in South Korea. Of the 68 RPCs investigated, toxoflavin contamination was confirmed in 12 from three provinces: Gangwon, Gyeonggi, and Gyeongsang. Isolation of toxoflavin-producing bacteria independent of toxoflavin contamination was also performed in this study. We obtained 25 toxoflavin-producing bacterial isolates from rice samples; these samples were collected from the same 12 RPCs mentioned above. All 25 toxoflavin-producing bacteria were identified as Burkholderia glumae by 16S rRNA gene sequencing. Toxoflavin-producing ability differed slightly among the 25 isolates, but they all inhibited rice seed germination and induced seed rot. This is the first report of toxoflavin contamination and the toxin-producing bacterium B. glumae in broken rice produced during the rice milling process. Because toxoflavin has stable physical properties even above a boiling temperature of 100°C, it can pose a problem even if rice is cooked or processed. These results will serve as baseline data aiding comprehensive management of toxoflavin contamination during the post-harvest storage and processing of rice.

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.

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.

A novel toxoflavin-quenching regulation in bacteria and its application to resistance cultivars.

The toxoflavin (Txn), broad host range phytotoxin produced by a variety of bacteria, including Burkholderia glumae, is a key pathogenicity factor of B. glumae in rice and field crops. Two bacteria exhibiting Txn-degrading activity were isolated from healthy rice seeds and identified as Sphingomonas adhaesiva and Agrobacterium sp. respectively. The genes stdR and stdA, encoding proteins responsible for Txn degradation of both bacterial isolates, were identical, indicating that horizontal gene transfer occurred between microbial communities in the same ecosystem. We identified a novel Txn-quenching regulation of bacteria, demonstrating that the LysR-type transcriptional regulator (LTTR) StdR induces the expression of the stdA, which encodes a Txn-degrading enzyme, in the presence of Txn as a coinducer. Here we show that the bacterial StdRTxn -quenching regulatory system mimics the ToxRTxn -mediated biosynthetic regulation of B. glumae. Substrate specificity investigations revealed that Txn is the only coinducer of StdR and that StdA has a high degree of specificity for Txn. Rice plants expressing StdA showed Txn resistance. Collectively, bacteria mimic the mechanism of Txn biosynthesis regulation, employ it in the development of a Txn-quenching regulatory system and share it with neighbouring bacteria for survival in rice environments full of Txn.

Genetic Diversity and Distribution of Korean Isolates of Burkholderia glumae.

Burkholderia glumae causes panicle blight of rice (grain rot in Japan and Korea), and the severity of damage is increasing worldwide. During 2017 and 2018, 137 isolates of B. glumae were isolated from symptomatic grain rot of rice cultivated in paddy fields throughout South Korea. Genetic diversity of the isolates was determined using transposase-based PCR (Tnp-PCR) genomic fingerprinting. All 138 isolates, including the B. glumae BGR1 strain, produced toxoflavin in various amounts, and 17 isolates produced an unidentified purple or orange pigment on Luria-Bertani medium and casamino acid-peptone-glucose medium, respectively, at 28°C. Transposase-based PCR genomic fingerprinting was performed using a novel primer designed based on transposase (tnp) gene sequences located at the ends of the toxoflavin efflux transporter operon; this method provided reliable and reproducible results. Through Tnp-PCR genomic fingerprinting, the genetic groups of Korean B. glumae isolates were divided into 11 clusters and three divisions. The Korean B. glumae isolates were mainly grouped in division I (73%). Interestingly, most of the pigment-producing isolates were grouped in divisions II and III; of these, 10 were grouped in cluster VIII, which comprised 67% of this cluster. Results of a phylogenetic analysis based on tofI and hrpB gene sequences were consistent with classification by Tnp-PCR genomic fingerprinting. The BGR1 strain did not belong to any of the clusters, indicating that this strain does not exhibit the typical genetic representation of B. glumae. B. glumae isolates showed diversity in the use of carbon and nitrogen sources, but no correlation with genetic classification by PCR fingerprinting was found. This is the first study to analyze the geographical distribution and genetic diversity of Korean B. glumae isolates.

Pantoea ananatis carotenoid production confers toxoflavin tolerance and is regulated by Hfq-controlled quorum sensing.

Carotenoids are widely used in functional foods, cosmetics, and health supplements, and their importance and scope of use are continuously expanding. Here, we characterized carotenoid biosynthetic genes of the plant-pathogenic bacterium Pantoea ananatis, which carries a carotenoid biosynthetic gene cluster (including crtE, X, Y, I, B, and Z) on a plasmid. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the crtEXYIB gene cluster is transcribed as a single transcript and crtZ is independently transcribed in the opposite direction. Using splicing by overlap extension with polymerase chain reaction (SOE by PCR) based on asymmetric amplification, we reassembled crtE-B, crtE-B-I, and crtE-B-I-Y. High-performance liquid chromatography confirmed that Escherichia coli expressing the reassembled crtE-B, crtE-B-I, and crtE-B-I-Y operons produced phytoene, lycopene, and ß-carotene, respectively. We found that the carotenoids conferred tolerance to UV radiation and toxoflavin. Pantoea ananatis shares rice environments with the toxoflavin producer Burkholderia glumae and is considered to be the first reported example of producing and using carotenoids to withstand toxoflavin. We confirmed that carotenoid production by P. ananatis depends on RpoS, which is positively regulated by Hfq/ArcZ and negatively regulated by ClpP, similar to an important regulatory network of E. coli (HfqArcZ →RpoS Í° ClpXP). We also demonstrated that Hfq-controlled quorum signaling de-represses EanR to activate RpoS, thereby initiating carotenoid production. Survival genes such as those responsible for the production of carotenoids of the plant-pathogenic P. ananatis must be expressed promptly to overcome stressful environments and compete with other microorganisms. This mechanism is likely maintained by a brake with excellent performance, such as EanR.

Dual-Purpose Inoculants and Their Effects on Corn Silage.

This study was conducted to screen dual-purpose lactic acid bacteria (LAB) from uncontrolled farm-scale silage, and then we confirmed their effects on corn silage. The LAB were isolated from eight farm-scale corn silages, and then we screened the antifungal activity against Fusarium graminearum and the carboxylesterase activity using spectrophotometer with p-nitrophenyl octanoate as substrate and McIlvane solution as buffer. From a total of 25 isolates, 5M2 and 6M1 isolates were selected as silage inoculants because presented both activities of antifungal and carboxylesterase. According 16S rRNA gene sequencing method, 5M2 isolate had 100.0% similarity with Lactobacillus brevis, and 6M1 isolate had 99.7% similarity with L. buchneri. Corn forage was ensiled in bale silo (500 kg) for 72 d without inoculant (CON) or with mixture of selected isolates at 1:1 ratio (INO). The INO silage had higher nutrient digestibility in the rumen than CON silage. Acetate was higher and yeasts were lower in INO silage than in CON silage on the day of silo opening. In all days of aerobic exposure, yeasts were lower in INO silage than CON silage. The present study concluded that Lactobacillus brevis 5M2 and L. buchneri 6M1 confirmed antifungal and carboxylesterase activities on farm-scale corn silage.

Grey mould control by oxalate degradation using non-antifungal Pseudomonas abietaniphila strain ODB36.

Grey mould is an important necrotrophic fungal pathogen that causes huge economic losses in agriculture. Many types of bacteria are used for biological control of grey mould via competition for space or nutrients and/or the production of antifungal metabolites. Oxalate is a key component of virulent necrotic fungal pathogens. In this study, we isolated non-antifungal oxalate-degrading bacteria (ODB) from the surfaces of oxalate-rich spinach and strawberries to investigate their ability to control necrotic fungal pathogens such as grey mould. A total of 36 bacteria grown on oxalate minimal (OM) agar plates were tested for oxalate-degrading activity. Five isolates exhibiting the highest oxalate degradation activity were subjected to molecular identification using 16S rRNA gene sequencing. Two isolates exhibiting non-antifungal activity were subjected to disease suppression assays using Arabidopsis-Botrytis systems. The isolate Pseudomonas abietaniphila ODB36, which exhibited significant plant protective ability, was finally selected for further investigation. Based on whole-genome information, the pseudomonad oxalate degrading (podA) gene, which encodes formyl-CoA transferase, was analysed. The podA- mutant did not inhibit Botrytis infection and oxalate toxicity; the defects were recovered by podA complementation. Purified PodA-His converted oxalate to formate and eliminated oxalate toxicity. These results indicate that P. abietaniphila ODB36 and PodA enzyme are associated with various aspects of grey mould disease inhibitory effects.

Inhibition of Salmonella enterica growth by competitive exclusion during early alfalfa sprout development using a seed-dwelling Erwinia persicina strain EUS78.

Salmonella enterica outbreaks in sprouts originate from contaminated seeds; conventional prevention technologies have been reported from many research institutes. In this study, we applied a biological control approach to inhibit S. enterica growth using the seed-dwelling non-antagonistic bacteria. We isolated non-antibacterial seed-dwelling bacteria from vegetable sprouts. A total of 206 bacteria exhibiting non-antibacterial activity against S. enterica were subjected to alfalfa sprout development tests. Eight isolates exhibiting no deleterious effect on the growth of alfalfa sprouts were tested for S. enterica growth inhibition on alfalfa seeds and sprouts, and an isolate EUS78 was finally selected for further investigation. Based on 16S rRNA, gyrB, and rpoB gene sequence analyses, strain EUS78 was identified as Erwinia persicina. In population competition, the S. enterica population increased by >3 log CFU/g after 6 days of alfalfa sprout growth, whereas S. enterica growth was significantly inhibited by treatment with EUS78 (P < .05). This effect of S. enterica growth inhibition by EUS78 was sustained until the end of the alfalfa sprout harvest. Overall, bacterial strain EUS78 significantly reduced S. enterica growth on alfalfa sprouts in a manner consistent with competitive exclusion. These findings led us to monitor EUS78 behavior on seeds during early sprout development using fluorescence and scanning electron microscopy. Strain EUS78 initially colonized alfalfa sprout seed coat edges, cotyledons, and finally root surfaces during early sprout germination. As alfalfa sprouts grew, EUS78 bacterial cells established colonies on newly emerged plant tissues such as root tips. The results of this study suggest that strain EUS78 has potential as a biological control agent to inhibit S. enterica contamination in the sprout food industry.

Simple and economical biosensors for distinguishing Agrobacterium-mediated plant galls from nematode-mediated root knots.

Agrobacterium-mediated plant galls are often misdiagnosed as nematode-mediated knots, even by experts, because the gall symptoms in both conditions are very similar. In the present study, we developed biosensor strains based on agrobacterial opine metabolism that easily and simply diagnoses Agrobacterium-induced root galls. Our biosensor consists of Agrobacterium mannitol (ABM) agar medium, X-gal, and a biosensor. The working principle of the biosensor is that exogenous nopaline produced by plant root galls binds to NocR, resulting in NocR/nopaline complexes that bind to the promoter of the nopaline oxidase gene (nox) operon and activate the transcription of noxB-lacZY, resulting in readily visualized blue pigmentation on ABM agar medium supplemented with X-gal (ABMX-gal). Similarly, exogenous octopine binds to OccR, resulting in OoxR/octopine complexes that bind to the promoter of the octopine oxidase gene (oox) operon and activate the transcription of ooxB-lacZY, resulting in blue pigmentation in the presence of X-gal. Our biosensor is successfully senses opines produced by Agrobacterium-infected plant galls, and can be applied to easily distinguish Agrobacterium crown gall disease from nematode disease.

Aversion center blackening of muskmelon fruit caused by Pseudomonas oryzihabitans, an opportunistic pathogen of humans and warm-blooded animals.

This is the first report of bacterial center blackening in muskmelon fruit caused by Pseudomonas oryzihabitans, which is known as an opportunistic pathogen of humans and warm-blooded animals. The aim of this study was to investigate the microbiological characteristics of this infection. Bacterial center blackening, which can cause aversion in consumers, was observed in muskmelon fruit in South Korea in the fall of 2017. Symptoms included severe black pigmentation in the pulp surrounding the seeds inside muskmelon fruit. Dark brown pigmentation and gram-negative, non-spore-forming, rod-shaped pseudomonads were consistently recovered from the black pigmented pulp tissue of muskmelons. The symptoms after artificial inoculation were the same as those of the natural infection, while the control fruit exhibited no symptoms of infection. Using pathogenicity tests, analytical profile index (API) tests, whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and 16S rRNA gene and gyrB region sequencing, the dominant species was identified as P. oryzihabitans. The recent outbreak indicates that P. oryzihabitans poses a potential threat to the global production and transportation of muskmelon as well as food safety.