Variations in fecal Schistosoma japonicum egg counts were studied in ZhuXi administrative village, JiangXi Province, China. Population stool examinations were collected with duplicate, standard, 41.5-mg Kato-Katz thick smears on seven consecutive days for 570 individuals from two natural (individual) villages: village I with high endemicity and village II with low endemicity. The proportion of individuals with at least one positive count increased from 42.4% after a single measurement to 68.3% after seven measurements in village I (n = 356), and from 17.0% to 36.0% in village II (n = 214), respectively. This demonstrates a very high variation in repeated S. japonicum egg counts and a considerable lack of sensitivity of the Kato-Katz technique; light and moderate infections are especially missed with a single or a few measurements. The observed day-to-day variation in individual egg counts is highly aggregated (variance higher than the mean) and suggestive of a negative binomial distribution. For five individuals on three days, repeated sampling from different locations of a stool specimen shows a clear trend with egg counts decreasing from the beginning of the stool to the end and from the outside layer to the center. Ten multiple samples from a particular subsection (10-30 g) of a stool specimen for 44 positive individuals still showed aggregation in egg counts, particularly for high intensities of infection. This means that the aggregation in repeated daily S. japonicum egg counts cannot be explained alone by a specific day-to-day component and variation in the concentration of eggs at different locations in the stool. There also exists clustering of eggs within parts of the stool.

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.

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.

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.

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.

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.