First Report of Colletotrichum aenigma Causing Anthracnose on Dragon Fruit in Korea.

Dragon fruit (Selenicereus undatus) is a valuable fruit crop in tropical and subtropical regions. It is renowned for its nutritional benefits, such as high sodium, potassium, and vitamin levels, and as a source of prebiotics and antioxidants (Balendres et al. 2019). In July 2023, anthracnose symptoms on stems were detected on dragon fruit plants in Jeju, South Korea. The typical anthracnose symptoms, such as sunken necrotic lesions (5-20 mm in diameter), were seen on the mature stems. The disease incidence ranged from 10% to 12% among the three surveyed greenhouses. To isolate the causative organism, infected stem samples were surface sterilized, cut into small pieces, and placed on potato dextrose agar (PDA). After two days of incubation at 24ºC, white hyphae appeared on the PDA around the plant tissues. Isolates CNU H23009 and CNU H23010 were purified from a single hypha under a stereoscope (e-Xtra Figure 1). Conidial morphology was examined from two-day-old fungal cultures grown on V8 juice agar. The conidia were transparent, aseptate, cylindrical to clavate, with a rounded apex and base, and measured 11.9 - 16.85 × 5.17 - 6.91 µm (mean = 15.28 × 5.93 µm, n = 30). No appressoria was observed. Morphological characteristics indicated the isolates were Colletotrichum sp. matching the description of the C. gloeosporioides species complex (Weir et al. 2012). To further identify the isolates, genomic DNA was extracted and the ribosomal internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT) were amplified using ITS1/ITS4, GDF/GDR, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). Based on phylogenetic analysis, the isolates clustered with C. aenigma (strains ICMP18608, ICMP18686, CSH2, and QSG1), with 71% bootstrap support, as determined using the maximum parsimony method in PAUP 4.0 (e-Xtra Figure 2). Based on morphological and molecular characteristics, isolates were identified as C. aenigma. Sequences of CNU H23009 and CNU H23010 were deposited in GenBank with accession numbers OR535144 and OR535145 for ITS, OR540725 and OR540726 for GAPDH, and OR540723 and OR540724 for ACT. The pathogenicity was tested on healthy dragon fruit stems using wound inoculation with mycelial plugs of the CNU H23009 isolate. Controls were inoculated with PDA plugs. The plants were covered with plastic bags to maintain humidity and incubated in a greenhouse at 25ºC. After two days, necrotic spots had developed on the inoculated tissues; after four days, black, irregular, and sunken necrotic lesions similar to those seen in the field were observed. No symptoms occurred in the controls. C. aenigma was re-isolated from the artificially inoculated plants and re-identified based on conidial morphology. The pathogenicity test was repeated three times with three replications for each treatment. Previous studies have reported that C. aenigma, C. gloeosoporioides, C. siamense, C. truncatum, and C. karsti cause anthracnose in dragon fruit. However, C. aenigma has been reported only in Thailand (Balendres et al. 2019; Meetum et al. 2015). To our knowledge, this is the first report of C. aenigma causing anthracnose in dragon fruit in Korea.

First report of tomato pith necrosis caused by Pseudomonas mediterranea in South Korea.

Tomato (Solanum lycopersicum) is one of the most economically important vegetables worldwide, and its production is directly affected by several bacterial diseases (Singh et al., 2017). During a disease survey in 2020, pith necrosis-like symptoms, commonly caused by Pseudomonas spp., were observed in two commercial greenhouses in PyeongChang and Gyeongju, South Korea. Disease incidence ranged from 8 to 10%, and infected plants showed wilt symptoms, brown stem discoloration, leaf blight, and corrugated pith tissues (eXtra Fig. 1). Symptomatic stem tissues were surface disinfected, cut into small pieces, and macerated in sterile water. The resulting suspension was spread on nutrient agar, and incubated at 28°C. The dominant bacterial colony types were round, mucoid, and frequently produced yellow to brown pigments. Four bacterial colonies (CPB20664 - CPB20667), each from a different diseased plant, were selected for further study. All isolates were Gram-negative and did not produce fluorescent pigments on King's B medium. Biochemical profiles of the isolates were determined by the API20NE (Biomerieux, Durhan, NC, USA) and LOPAT test (eXtra Table 1). The bacterial isolates were further identified by PCR amplification of partial 16S rRNA, gyrB, and rpoD genes using primers 27F/1492R, UP-1E/AprU, and 70F/70R, respectively (Lane 1991, Yamamoto et al., 2000). The resulting sequences were deposited in GenBank under accession numbers MW602997 to MW603000 for 16S rRNA, MW602987 to MW602990 for gyrB, and MW602991 to MW602994 for rpoD. These sequences exhibited 99-100% nucleotide similarities with multiple Pseudomonas mediterranea sequences in Genbank. Additionally, the isolates were subjected to PCR assays using the P. mediterranea specific primers PC5/1-PC5/2 and the P. corrugata specific primers PC1/1-PC1/2 (Catara et al., 2002). All isolates produced a specific 600-bp band with the P. mediterranea primers, but did not produce any bands with the P. corrugata specific primers. The PCR amplicons were sequenced and BLAST queried against GenBank database. All isolates shared 100% identity with the type strain P. mediterranea DSM 16733 (acc No. LT629790.1). These results indicated that the bacteria isolated from the tomato plants with pith necrosis were P. mediterranea. Pathogenicity tests were conducted on 2-week-old tomato seedlings (cv. Yekwang) by wound inoculations. Single colonies were picked up using sterile toothpicks, and the stems of tomato seedlings were stabbed below the second leaves. As a negative control, a sterile toothpick was dipped in sterile water and used in the same manner. After inoculation, the plants were kept in a humidity box for 48 h, then moved to a plant growth room. After 15 days, light brown lesions had developed at the stab sites, and pith necrosis and slight wilting of plants were observed at 30 days (eXtra Fig. 1). Control plants remained asymptomatic. P. mediterranea was re-isolated from infected plants, fulfilling Koch's postulates. Five species of Pseudomonas are known to cause tomato pith necrosis (Alippi and Lopez, 2010, Cañizares and García-Pedrajas., 2015, Ruan et al., 2018) including P. corrugata previously reported from Korea (Choi and Han, 2004). This is believed to be the first report of P. mediterranea as the cause of tomato pith necrosis in Korea. Tomato pith necrosis disease reduces the quality and yield of tomato production and appropriate management strategies should be investigated to control this disease.

First report of bacterial leaf spot disease on sunflower (Helianthus annuus) caused by Pseudomonas viridiflava in South Korea.

In September 2019, bacterial leaf spot symptoms were observed on sunflowers in an experimental field in Eumseong, South Korea. The leaves of infected plants initially showed irregular brown spots surrounded by haloes; as the disease progressed, the spots became enlarged and darkened (eXtra Fig. 1a). At the flowering stage, leaves became dry and showed signs of blight including defoliation; dark brown spots were also observed on sunflower stems and petioles but not on floral discs (eXtra Fig. 1b). Disease incidence ranged from 5% to 30% in three surveyed plots of the field. Symptomatic leaf tissue was surface-sterilized, macerated with sterile distilled water, and cultured on nutrient agar plates at 28°C for 48 h. After incubation, nine bacterial isolates, representing individually collected samples from each field, were selected for further study. All nine isolates were Gram-negative and fluorescent pigments produced under UV on King's medium B. With the LOPAT test, the isolates were levan negative, oxidase negative, positive for pectinolytic activity, arginine dihydrolase negative, and positive for tobacco hypersensitivity. Based on 16s rRNA sequences, all isolates shared 100% identity with Pseudomonas viridiflava strain KNOX209.1 (GenBank accession no. AY604847). The 16s rRNA sequences of nine isolates were deposited in GeneBank (accession nos. MT393747, MW446479 to MW446486). Based on the phylogenetic analysis of the 16s rRNA, all isolates were grouped with P. viridiflava strains isolated from globe artichoke, Chinese cabbage, and rape (Myung et al. 2010, Sanver et al. 2019, and Liu et al. 2019). The isolates CPB 19362, CPB 19366 and CPB 19372, which represent each plot were selected for further phylogenetic analysis and pathogenicity assays. The identity of these isolates was confirmed by sequences of housekeeping genes of the gyrase B subunit (gyrB) and RNA polymerase σ70 factor (rpoD) (Yamamoto et al. 2000) (GenBank accession nos. MT409400, MW446487 and MW446494 for gyrB and MT409401, MW446495 and MW446502 for rpoD). Based on the phylogenetic analyses of gyrB and rpoD, the three isolates belong to the same clade as the P. viridiflava pathotypes and were distinguished from P. syringae complex (eXtra Fig. 2). These results indicated that the bacteria isolated from the spots on the sunflower plants were P. viridiflava strains. To confirm the pathogenicity, bacterial suspensions (approximately 108 CFU/mL) of three representative isolates sprayed onto 4-week-old sunflower (cv. Common) seedlings separately until runoff occurred. Sterile distilled water was used as a control and inoculated in the same manner. After inoculation, plants were covered with transparent plastic bags at room temperature for 24 h. Plastic bags were then removed and plants were grown on a plant growth shelf at 25°C in 50% relative humidity. The leaves of plants inoculated with the bacterial suspensions developed small brown spots after 24 h. After 3 days, brown spots surrounded by chlorotic or necrotic areas were observed on infected leaves (eXtra Fig. 1c). These spots gradually increased in size and formed brown lesions with haloes similar to those of infected field-grown plants (eXtra Fig. 1d), but not on the controls treated with sterile water. The pathogenicity test was repeated three times. Isolates recovered from infected leaves showed the same morphological, biochemical, and molecular characteristics as the original isolates from field samples. To our knowledge, this is the first report of bacterial leaf spot on sunflower caused by P. viridiflava in South Korea.

Disruption of MiaA provides insights into the regulation of phenazine biosynthesis under suboptimal growth conditions in Pseudomonas chlororaphis 30-84.

Many products of secondary metabolism are activated by quorum sensing (QS), yet even at cell densities sufficient for QS, their production may be repressed under suboptimal growth conditions via mechanisms that still require elucidation. For many beneficial plant-associated bacteria, secondary metabolites such as phenazines are important for their competitive survival and plant-protective activities. Previous work established that phenazine biosynthesis in Pseudomonas chlororaphis 30-84 is regulated by the PhzR/PhzI QS system, which in turn is regulated by transcriptional regulator Pip, two-component system RpeA/RpeB and stationary phase/stress sigma factor RpoS. Disruption of MiaA, a tRNA modification enzyme, altered primary metabolism and growth leading to widespread effects on secondary metabolism, including reduced phenazine production and oxidative stress tolerance. Thus, the miaA mutant provided the opportunity to examine the regulation of phenazine production in response to altered metabolism and growth or stress tolerance. Despite the importance of MiaA for translation efficiency, the most significant effect of miaA disruption on phenazine production was the reduction in the transcription of phzR, phzI and pip, whereas neither the transcription nor translation of RpeB, a transcriptional regulator of pip, was affected. Constitutive expression of rpeB or pip in the miaA mutant completely restored phenazine production, but it resulted in further growth impairment. Constitutive expression of RpoS alleviated sensitivity to oxidative stress resulting from RpoS translation inefficiency in the miaA mutant, but it did not restore phenazine production. Our results support the model that cells curtail phenazine biosynthesis under suboptimal growth conditions via RpeB/Pip-mediated regulation of QS.

Pseudomonas chlororaphis Produces Two Distinct R-Tailocins That Contribute to Bacterial Competition in Biofilms and on Roots.

R-type tailocins are high-molecular-weight bacteriocins that resemble bacteriophage tails and are encoded within the genomes of many Pseudomonas species. In this study, analysis of the P. chlororaphis 30-84 R-tailocin gene cluster revealed that it contains the structural components to produce two R-tailocins of different ancestral origins. Two distinct R-tailocin populations differing in length were observed in UV-induced lysates of P. chlororaphis 30-84 via transmission electron microscopy. Mutants defective in the production of one or both R-tailocins demonstrated that the killing spectrum of each tailocin is limited to Pseudomonas species. The spectra of pseudomonads killed by the two R-tailocins differed, although a few Pseudomonas species were either killed by or insusceptible to both tailocins. Tailocin release was disrupted by deletion of the holin gene within the tailocin gene cluster, demonstrating that the lysis cassette is required for the release of both R-tailocins. The loss of functional tailocin production reduced the ability of P. chlororaphis 30-84 to compete with an R-tailocin-sensitive strain within biofilms and rhizosphere communities. Our study demonstrates that Pseudomonas species can produce more than one functional R-tailocin particle sharing the same lysis cassette but differing in their killing spectra. This study provides evidence for the role of R-tailocins as determinants of bacterial competition among plant-associated Pseudomonas in biofilms and the rhizosphere.IMPORTANCE Recent studies have identified R-tailocin gene clusters potentially encoding more than one R-tailocin within the genomes of plant-associated Pseudomonas but have not demonstrated that more than one particle is produced or the ecological significance of the production of multiple R-tailocins. This study demonstrates for the first time that Pseudomonas strains can produce two distinct R-tailocins with different killing spectra, both of which contribute to bacterial competition between rhizosphere-associated bacteria. These results provide new insight into the previously uncharacterized role of R-tailocin production by plant-associated Pseudomonas species in bacterial population dynamics within surface-attached biofilms and on roots.

Morphology, Molecular Identity, and Pathogenicity of Verticillium dahliae and V. longisporum Associated with Internally Discolored Horseradish Roots.

During 2008 to 2009, 255 isolates of Verticillium were obtained from internally discolored horseradish roots collected from California, Illinois, and Ontario. Twenty representative isolates were selected according to morphological features and geographic origin for further characterization. Based on the conidial size, the isolates were divided into two groups: Verticillium dahliae (4.4 ± 1.23 µm) and V. longisporum (7.8 ± 1.76 µm). Genetic diversity of the isolates was determined by sequence analysis of the internal transcribed spacer (ITS) and two mitochondrial genes (cytochrome oxidase subunit III [cox3] and NADH dehydrogenase subunit I [nad1]). Based on ITS analysis, Verticillium isolates were divided into two clades: V. dahliae and V. longisporum. However five isolates of V. longisporum (identified based on conidial size) were clustered with a V. dahliae clade, whereas the other five isolates formed a distinct V. longisporum clade. Combined analysis of the mitochondrial genes cox3 and nad1 showed that the two genetic clades of V. longisporum in ITS region analysis corresponded to the previously reported V. longisporum lineage A1/D3 and A1/D2. Pathogenicity tests revealed that all tested Verticillium isolates caused internal discoloration of horseradish roots, and there were no significant differences in either incidence or severity of root discoloration among the genetic groups.

Adaptation genomics of a small-colony variant in a Pseudomonas chlororaphis 30-84 biofilm.

The rhizosphere-colonizing bacterium Pseudomonas chlororaphis 30-84 is an effective biological control agent against take-all disease of wheat. In this study, we characterize a small-colony variant (SCV) isolated from a P. chlororaphis 30-84 biofilm. The SCV exhibited pleiotropic phenotypes, including small cell size, slow growth and motility, low levels of phenazine production, and increased biofilm formation and resistance to antimicrobials. To better understand the genetic alterations underlying these phenotypes, RNA and whole-genome sequencing analyses were conducted comparing an SCV to the wild-type strain. Of the genome's 5,971 genes, transcriptomic profiling indicated that 1,098 (18.4%) have undergone substantial reprograming of gene expression in the SCV. Whole-genome sequence analysis revealed multiple alterations in the SCV, including mutations in yfiR (cyclic-di-GMP production), fusA (elongation factor), and cyoE (heme synthesis) and a 70-kb deletion. Genetic analysis revealed that the yfiR locus plays a major role in controlling SCV phenotypes, including colony size, growth, motility, and biofilm formation. Moreover, a point mutation in the fusA gene contributed to kanamycin resistance. Interestingly, the SCV can partially switch back to wild-type morphologies under specific conditions. Our data also support the idea that phenotypic switching in P. chlororaphis is not due to simple genetic reversions but may involve multiple secondary mutations. The emergence of these highly adherent and antibiotic-resistant SCVs within the biofilm might play key roles in P. chlororaphis natural persistence.

Microbacterium azadirachtae CNUC13 Enhances Salt Tolerance in Maize by Modulating Osmotic and Oxidative Stress.

Soil salinization is one of the leading threats to global ecosystems, food security, and crop production. Plant growth-promoting rhizobacteria (PGPRs) are potential bioinoculants that offer an alternative eco-friendly agricultural approach to enhance crop productivity from salt-deteriorating lands. The current work presents bacterial strain CNUC13 from maize rhizosphere soil that exerted several PGPR traits and abiotic stress tolerance. The strain tolerated up to 1000 mM NaCl and 30% polyethylene glycol (PEG) 6000 and showed plant growth-promoting (PGP) traits, including the production of indole-3-acetic acid (IAA) and siderophore as well as phosphate solubilization. Phylogenetic analysis revealed that strain CNUC13 was Microbacterium azadirachtae. Maize plants exposed to high salinity exhibited osmotic and oxidative stresses, inhibition of seed germination, plant growth, and reduction in photosynthetic pigments. However, maize seedlings inoculated with strain CNUC13 resulted in significantly improved germination rates and seedling growth under the salt-stressed condition. Specifically, compared with the untreated control group, CNUC13-treated seedlings exhibited increased biomass, including fresh weight and root system proliferation. CNUC13 treatment also enhanced photosynthetic pigments (chlorophyll and carotenoids), reduced the accumulation of osmotic (proline) and oxidative (hydrogen peroxide and malondialdehyde) stress indicators, and positively influenced the activities of antioxidant enzymes (catalase, superoxide dismutase, and peroxidase). As a result, CNUC13 treatment alleviated oxidative stress and promoted salt tolerance in maize. Overall, this study demonstrates that M. azadirachtae CNUC13 significantly enhances the growth of salt-stressed maize seedlings by improving photosynthetic efficiency, osmotic regulators, oxidative stress resilience, and antioxidant enzyme activity. These findings emphasize the potential of utilizing M. azadirachtae CNUC13 as a bioinoculant to enhance salt stress tolerance in maize, providing an environmentally friendly approach to mitigate the negative effects of salinity and promote sustainable agriculture.

Development of the Droplet Digital PCR Method for the Detection and Quantification of Erwinia pyrifoliae.

Black shoot blight disease caused by Erwinia pyrifoliae has serious impacts on quality and yield in pear production in Korea; therefore, rapid and accurate methods for its detection are needed. However, traditional detection methods require a great deal of time and fail to achieve absolute quantification. In the present study, we developed a droplet digital polymerase chain reaction (ddPCR) method for the detection and absolute quantification of E. pyrifoliae using a pair of species-specific primers. The detection range was 103 - 107 copies/ml (DNA templates) and cfu/ml (cell culture templates). This new method exhibited good linearity and repeatability and was validated by absolute quantification of E. pyrifoliae DNA copies from samples of artificially inoculated immature pear fruits. Here, we present the first study of ddPCR assay for the detection and quantification of E. pyrifoliae. This method has potential applications in epidemiology and for the early prediction of black shoot blight outbreaks.

Involvement of IDA-HAE Module in Natural Development of Tomato Flower Abscission.

The unwanted detachment of organs such as flowers, leaves, and fruits from the main body of a plant (abscission) has significant effects on agricultural practice. Both timely and precise regulation of organ abscission from a plant is crucial as it influences the agricultural yield. The tomato (Solanum lycopersicum) has become a model system for research on organ abscission. Here, we characterized four tomato natural abscission variants named jointless (j), functionally impaired jointless (fij), functionally impaired jointless like (fij like), and normal joint (NJ), based on their cellular features within the flower abscission zones (AZ). Using eight INFLORESCENCE DEFICIENT IN ABSCISSION (SlIDA) genes and eight HAESA genes (SlHAE) identified in the genome sequence of tomato, we analyzed the pattern of gene expression during flower abscission. The AZ-specific expression for three tomato abscission polygalacturonases (SlTAPGs) in the development of flower AZ, and the progression of abscission validated our natural abscission system. Compared to that of j, fij, and fij like variants, the AZ-specific expression for SlIDA, SlIDL2, SlIDL3, SlIDL4, and SlIDL5 in the NJ largely corelated and increased with the process of abscission. Of eight SlHAE genes examined, the expression for SlHSL6 and SlHSL7 were found to be AZ-specific and increased as abscission progressed in the NJ variant. Unlike the result of gene expression obtained from natural abscission system, an in silico analysis of transcriptional binding sites uncovered that SlIDA genes (SlIDA, SlIDL6, and SlIDL7) are predominantly under the control of environmental stress, while most of the SlHSL genes are affiliated with the broader context in developmental processes and stress responses. Our result presents the potential bimodal transcriptional regulation of the tomato IDA-HAE module associated with flower abscission in tomatoes.

Differential regulation of phenazine biosynthesis by RpeA and RpeB in Pseudomonas chlororaphis 30-84.

RpeA is a two-component sensor protein that negatively controls biosynthesis of phenazines, which are required for biological control activity by Pseudomonas chlororaphis 30-84. In this study, we identified the cognate response regulator RpeB and investigated how RpeA and RpeB interact with the PhzR/PhzI quorum sensing system and other known regulatory genes to control phenazine production. Quantitative real-time PCR revealed that, in contrast with an rpeA mutant, expression of the phenazine biosynthetic genes as well as the pip and phzR genes were significantly reduced in an rpeB mutant, suggesting positive control of phenazines by RpeB. Complementation assays showed that overexpression of pip in trans rescued phenazine production in an rpeB mutant, whereas multiple copies of rpeB genes were unable to restore phenazine production in a pip or phzR mutant. These results indicate that RpeA and RpeB differentially regulate phenazine production and act upstream of Pip and PhzR in the phenazine regulatory network. The differential regulatory functions for RpeA and RpeB also affected the capacity of 30-84 for fungal inhibition. Based on these results, a model is proposed to illustrate the relationship of RpeA/RpeB to other regulatory genes controlling phenazine biosynthesis in P. chlororaphis 30-84, a regulatory hierarchy that may be conserved in other pseudomonads and may play a role in stress response.

Volatile organic compounds emitted by Burkholderia pyrrocinia CNUC9 trigger induced systemic salt tolerance in Arabidopsis thaliana.

Salinity is among the most significant abiotic stresses that negatively affects plant growth and agricultural productivity worldwide. One ecofriendly tool for broadly improving plant tolerance to salt stress is the use of bio-inoculum with plant growth-promoting rhizobacteria (PGPR). In this study, a bacterium strain CNUC9, which was isolated from maize rhizosphere, showed several plant growth-promoting characteristics including the production of 1-aminocyclopropane-1-carboxylate deaminase, indole acetic acid, siderophore, and phosphate solubilization. Based on 16S rRNA and recA gene sequence analysis, we identified strain CNUC9 as Burkholderia pyrrocinia. Out of bacterial determinants to elicit plant physiological changes, we investigated the effects of volatile organic compounds (VOCs) produced by B. pyrrocinia CNUC9 on growth promotion and salinity tolerance in Arabidopsis thaliana. Higher germination and survival rates were observed after CNUC9 VOCs exposure under 100 mM NaCl stress. CNUC9 VOCs altered the root system architecture and total leaf area of A. thaliana compared to the control. A. thaliana exposed to VOCs induced salt tolerance by increasing its total soluble sugar and chlorophyll content. In addition, lower levels of reactive oxygen species, proline, and malondialdehyde were detected in CNUC9 VOCs-treated A. thaliana seedlings under stress conditions, indicating that VOCs emitted by CNUC9 protected the plant from oxidative damage induced by salt stress. VOC profiles were obtained through solid-phase microextraction and analyzed by gas chromatography coupled with mass spectrometry. Dimethyl disulfide (DMDS), methyl thioacetate, and 2-undecanone were identified as products of CNUC9. Our results indicate that optimal concentrations of DMDS and 2-undecanone promoted growth in A. thaliana seedlings. Our findings provide greater insight into the salt stress alleviation of VOCs produced by B. pyrrocinia CNUC9, as well as potential sustainable agriculture applications.

Drought-Stress Tolerance in Wheat Seedlings Conferred by Phenazine-Producing Rhizobacteria.

The specific role of phenazines produced by rhizosphere-colonizing Pseudomonas in mediating wheat seedling drought-stress tolerance and recovery from water deficit was investigated using Pseudomonas chlororaphis 30-84 and isogenic derivatives deficient or enhanced in phenazine production compared to wild type. Following a 7-day water deficit, seedlings that received no-inoculum or were colonized by the phenazine mutant wilted to collapse, whereas seedlings colonized by phenazine producers displayed less severe symptoms. After a 7-day recovery period, survival of seedlings colonized by phenazine-producing strains exceeded 80%, but was less than 60% for no-inoculum controls. A second 7-day water deficit reduced overall survival rates to less than 10% for no-inoculum control seedlings, whereas survival was ∼50% for seedlings colonized by phenazine-producers. The relative water content of seedlings colonized by phenazine-producers was 10-20% greater than for the no-inoculum controls at every stage of water deficit and recovery, resulting in higher recovery indices than observed for the no-inoculum controls. For 10-day water deficits causing the collapse of all seedlings, survival rates remained high for plants colonized by phenazine-producers, especially the enhanced phenazine producer (∼74%), relative to the no-inoculum control (∼25%). These observations indicate that seedlings colonized by the phenazine-producing strains suffered less from dehydration during water deficit and recovered better, potentially contributing to better resilience from a second drought/recovery cycle. Seedlings colonized by phenazine-producing strains invested more in root systems and produced 1.5 to 2 fold more root tips than seedlings colonized by the phenazine mutant or the no-inoculum controls when grown with or without water deficit. The results suggest that the presence of phenazine-producing bacteria in the rhizosphere provides wheat seedlings with a longer adjustment period resulting in greater drought-stress avoidance and resilience.

Effect of Producing Different Phenazines on Bacterial Fitness and Biological Control in Pseudomonas chlororaphis 30-84.

Pseudomonas chlororaphis 30-84 is a biological control agent selected for its ability to suppress diseases caused by fungal pathogens. P. chlororaphis 30-84 produces three phenazines: phenazine-1-carboxylic acid (PCA), 2-hydroxy-phenazine-1-carboxylic acid (2OHPCA) and a small amount of 2-hydroxy-phenazine (2OHPHZ), and these are required for fungal pathogen inhibition and wheat rhizosphere competence. The two, 2-hydroxy derivatives are produced from PCA via the activity of a phenazine-modifying enzyme encoded by phzO. In addition to the seven biosynthetic genes responsible for the production of PCA, many other Pseudomonas strains possess one or more modifying genes, which encode enzymes that act independently or together to convert PCA into other phenazine derivatives. In order to understand the fitness effects of producing different phenazines, we constructed isogenic derivatives of P. chlororaphis 30-84 that differed only in the type of phenazines produced. Altering the type of phenazines produced by P. chlororaphis 30-84 enhanced the spectrum of fungal pathogens inhibited and altered the degree of take-all disease suppression. These strains also differed in their ability to promote extracellular DNA release, which may contribute to the observed differences in the amount of biofilm produced. All derivatives were equally important for survival over repeated plant/harvest cycles, indicating that the type of phenazines produced is less important for persistence in the wheat rhizosphere than whether or not cells produce phenazines. These findings provide a better understanding of the effects of different phenazines on functions important for biological control activity with implications for applications that rely on introduced or native phenazine producing populations.

An upstream sequence modulates phenazine production at the level of transcription and translation in the biological control strain Pseudomonas chlororaphis 30-84.

Phenazines are bacterial secondary metabolites and play important roles in the antagonistic activity of the biological control strain P. chlororaphis 30-84 against take-all disease of wheat. The expression of the P. chlororaphis 30-84 phenazine biosynthetic operon (phzXYFABCD) is dependent on the PhzR/PhzI quorum sensing system located immediately upstream of the biosynthetic operon as well as other regulatory systems including Gac/Rsm. Bioinformatic analysis of the sequence between the divergently oriented phzR and phzX promoters identified features within the 5'-untranslated region (5'-UTR) of phzX that are conserved only among 2OHPCA producing Pseudomonas. The conserved sequence features are potentially capable of producing secondary structures that negatively modulate one or both promoters. Transcriptional and translational fusion assays revealed that deletion of 90-bp of sequence at the 5'-UTR of phzX led to up to 4-fold greater expression of the reporters with the deletion compared to the controls, which indicated this sequence negatively modulates phenazine gene expression both transcriptionally and translationally. This 90-bp sequence was deleted from the P. chlororaphis 30-84 chromosome, resulting in 30-84Enh, which produces significantly more phenazine than the wild-type while retaining quorum sensing control. The transcriptional expression of phzR/phzI and amount of AHL signal produced by 30-84Enh also were significantly greater than for the wild-type, suggesting this 90-bp sequence also negatively affects expression of the quorum sensing genes. In addition, deletion of the 90-bp partially relieved RsmE-mediated translational repression, indicating a role for Gac/RsmE interaction. Compared to the wild-type, enhanced phenazine production by 30-84Enh resulted in improvement in fungal inhibition, biofilm formation, extracellular DNA release and suppression of take-all disease of wheat in soil without negative consequences on growth or rhizosphere persistence. This work provides greater insight into the regulation of phenazine biosynthesis with potential applications for improved biological control.

Soybean mosaic virus Helper Component-Protease Alters Leaf Morphology and Reduces Seed Production in Transgenic Soybean Plants.

ABSTRACT Transgenic soybean (Glycine max) plants expressing Soybean mosaic virus (SMV) helper component-protease (HC-Pro) showed altered vegetative and reproductive phenotypes and responses to SMV infection. When inoculated with SMV, transgenic plants expressing the lowest level of HC-Pro mRNA and those transformed with the vector alone initially showed mild SMV symptoms. Plants that accumulated the highest level of SMV HC-Pro mRNA showed very severe SMV symptoms initially, but after 2 weeks symptoms disappeared, and SMV titers were greatly reduced. Analysis of SMV RNA abundance over time with region-specific probes showed that the HC-Pro region of the SMV genome was degraded before the coat protein region. Transgenic soybean plants that expressed SMV HC-Pro showed dose-dependent alterations in unifoliate leaf morphologies and seed production where plants expressing the highest levels of HC-Pro had the most deformed leaves and the lowest seed production. Accumulation of microRNAs (miRNAs) and mRNAs putatively targeted by miRNAs was analyzed in leaves and flowers of healthy, HC-Pro-transgenic, and SMV-infected plants. Neither expression of SMV HC-Pro nor SMV infection produced greater than twofold changes in accumulation of six miRNAs. In contrast, SMV infection was associated with twofold or greater increases in the accumulation of four of seven miRNA-targeted mRNAs tested.

The Phenazine 2-Hydroxy-Phenazine-1-Carboxylic Acid Promotes Extracellular DNA Release and Has Broad Transcriptomic Consequences in Pseudomonas chlororaphis 30-84.

Enhanced production of 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) by the biological control strain Pseudomonas chlororaphis 30-84 derivative 30-84O* was shown previously to promote cell adhesion and alter the three-dimensional structure of surface-attached biofilms compared to the wild type. The current study demonstrates that production of 2-OH-PCA promotes the release of extracellular DNA, which is correlated with the production of structured biofilm matrix. Moreover, the essential role of the extracellular DNA in maintaining the mass and structure of the 30-84 biofilm matrix is demonstrated. To better understand the role of different phenazines in biofilm matrix production and gene expression, transcriptomic analyses were conducted comparing gene expression patterns of populations of wild type, 30-84O* and a derivative of 30-84 producing only PCA (30-84PCA) to a phenazine defective mutant (30-84ZN) when grown in static cultures. RNA-Seq analyses identified a group of 802 genes that were differentially expressed by the phenazine producing derivatives compared to 30-84ZN, including 240 genes shared by the two 2-OH-PCA producing derivatives, the wild type and 30-84O*. A gene cluster encoding a bacteriophage-derived pyocin and its lysis cassette was upregulated in 2-OH-PCA producing derivatives. A holin encoded in this gene cluster was found to contribute to the release of eDNA in 30-84 biofilm matrices, demonstrating that the influence of 2-OH-PCA on eDNA production is due in part to cell autolysis as a result of pyocin production and release. The results expand the current understanding of the functions different phenazines play in the survival of bacteria in biofilm-forming communities.

Roles of the Gac-Rsm pathway in the regulation of phenazine biosynthesis in Pseudomonas chlororaphis 30-84.

The GacS/GacA two-component regulatory system activates the production of secondary metabolites including phenazines crucial for biological control activity in Pseudomonas chlororaphis 30-84. To better understand the role of the Gac system on phenazine regulation, transcriptomic analyses were conducted by comparing the wild-type strain to a gacA mutant. RNA-seq analysis identified 771 genes under GacA control, including many novel genes. Consistent with previous findings, phenazine biosynthetic genes were significantly downregulated in a gacA mutant. The transcript abundances of phenazine regulatory genes such as phzI, phzR, iopA, iopB, rpoS, and pip also were reduced. Moreover, the transcript abundance of three noncoding RNAs (ncRNAs) including rsmX, rsmY, and rsmZ was significantly decreased by gacA mutation consistent with the presence of consensus GacA-binding sites associated with their promoters. Our results also demonstrated that constitutive expression of rsmZ from a non-gac regulated promoter resulted in complete restoration of N-acyl-homoserine lactone (AHL) and phenazine production as well as the expression of other gac-dependent secondary metabolites in gac mutants. The role of RsmA and RsmE in phenazine production also was investigated. Overexpression of rsmE, but not rsmA, resulted in decreased AHL and phenazine production in P. chlororaphis, and only a mutation in rsmE bypassed the requirement for GacA in phenazine gene expression. In contrast, constitutive expression of the phzI/phzR quorum sensing system did not rescue phenazine production in the gacA mutant, indicating the direct posttranscriptional control by Gac on the phenazine biosynthetic genes. On the basis of these results, we propose a model to illustrate the hierarchic role of phenazine regulators modulated by Gac in the control of phenazine production. The transcriptomic analysis also was used to identify additional genes regulated by GacA that may contribute to the biological control capability of strain 30-84.