Shifts in Soil Bacterial Communities as a Function of Carbon Source Used During Anaerobic Soil Disinfestation

Anaerobic soil disinfestation (ASD) is an organic amendment-based management practice for controlling soil-borne plant pathogens. Pathogen suppression appears to be carbon source-dependent and mediated by bacteria that proliferate and produce volatile organic compounds, as well as physico-chemical changes (i.e., elevated temperature, lowered redox potential and pH, release of metal ions) in soil. ASD is under study for adoption in tree crops as a replacement for chemical-fumigation, but its widespread use is limited by incomplete understanding of its suppression mechanisms and high economic costs. The carbon substrate is one component of the ASD process that can be optimized to enhance effectiveness and affordability. While rice bran is currently the standard carbon source used for ASD, we identified three alternative substrates (molasses, mustard seed meal, and tomato pomace) that are similar in efficacy to rice bran at generating and sustaining soil anoxia and reducing populations of introduced plant pathogens. Here, we used replicated ASD field trials to determine if rice bran and the alternative carbon substrates would elicit similar soil bacterial communities (characterized via amplicon sequencing of the 16S rRNA gene v4 region) and to assess if any observed community shifts were consistent across repeated trials. We found significant, but minimal differences in community composition between ASD carbon treatments (F4, 30 = 2.80, P < 0.001, R2 = 0.22) and trials (F1, 30 = 5.24, P < 0.001, R2 = 0.10). In both trials, the abundances of Bacteroidales, Clostridiales, Selenomonadales, and Enterobacteriales increased significantly (>5 log2 fold change) in all ASD treatments compared to untreated controls. A group of shared core genera belonging to the Clostridiales and Selenomonadales were identified in both trials and constituted 22.6 and 21.5% of the communities. Bacterial taxa that were most responsive to ASD treatments had the genomic potential for denitrification, nitrogen fixation, and fermentation reactions that produce organic acids (such as acetate and butyrate) known to inhibit in vitro growth of plant pathogens based on predicted metagenomes. Together, these results indicate that reproducible and effective implementation of ASD is achievable with alternative carbon substrates to rice bran

Comparative Genomics of Plant-Associated Pseudomonas spp.: Insights into Diversity and Inheritance of Traits Involved in Multitrophic Interactions

We provide here a comparative genome analysis of ten strains within the Pseudomonas fluorescens group including seven new genomic sequences. These strains exhibit a diverse spectrum of traits involved in biological control and other multitrophic interactions with plants, microbes, and insects. Multilocus sequence analysis placed the strains in three sub-clades, which was reinforced by high levels of synteny, size of core genomes, and relatedness of orthologous genes between strains within a sub-clade. The heterogeneity of the P. fluorescens group was reflected in the large size of its pan-genome, which makes up approximately 54% of the pan-genome of the genus as a whole, and a core genome representing only 45–52% of the genome of any individual strain. We discovered genes for traits that were not known previously in the strains, including genes for the biosynthesis of the siderophores achromobactin and pseudomonine and the antibiotic 2-hexyl-5-propyl-alkylresorcinol; novel bacteriocins; type II, III, and VI secretion systems; and insect toxins. Certain gene clusters, such as those for two type III secretion systems, are present only in specific sub-clades, suggesting vertical inheritance. Almost all of the genes associated with multitrophic interactions map to genomic regions present in only a subset of the strains or unique to a specific strain. To explore the evolutionary origin of these genes, we mapped their distributions relative to the locations of mobile genetic elements and repetitive extragenic palindromic (REP) elements in each genome. The mobile genetic elements and many strain-specific genes fall into regions devoid of REP elements (i.e., REP deserts) and regions displaying atypical tri-nucleotide composition, possibly indicating relatively recent acquisition of these loci. Collectively, the results of this study highlight the enormous heterogeneity of the P. fluorescens group and the importance of the variable genome in tailoring individual strains to their specific lifestyles and functional repertoire

Comparative Genomics of Plant-Associated Pseudomonas spp.: Insights into Diversity and Inheritance of Traits Involved in Multitrophic Interactions

We provide here a comparative genome analysis of ten strains within the Pseudomonas fluorescens group including seven new genomic sequences. These strains exhibit a diverse spectrum of traits involved in biological control and other multitrophic interactions with plants, microbes, and insects. Multilocus sequence analysis placed the strains in three sub-clades, which was reinforced by high levels of synteny, size of core genomes, and relatedness of orthologous genes between strains within a sub-clade. The heterogeneity of the P. fluorescens group was reflected in the large size of its pan-genome, which makes up approximately 54% of the pan-genome of the genus as a whole, and a core genome representing only 45-52% of the genome of any individual strain. We discovered genes for traits that were not known previously in the strains, including genes for the biosynthesis of the siderophores achromobactin and pseudomonine and the antibiotic 2-hexyl-5-propyl-alkylresorcinol; novel bacteriocins; type II, III, and VI secretion systems; and insect toxins. Certain gene clusters, such as those for two type III secretion systems, are present only in specific sub-clades, suggesting vertical inheritance. Almost all of the genes associated with multitrophic interactions map to genomic regions present in only a subset of the strains or unique to a specific strain. To explore the evolutionary origin of these genes, we mapped their distributions relative to the locations of mobile genetic elements and repetitive extragenic palindromic (REP) elements in each genome. The mobile genetic elements and many strain-specific genes fall into regions devoid of REP elements (i.e., REP deserts) and regions displaying atypical tri-nucleotide composition, possibly indicating relatively recent acquisition of these loci. Collectively, the results of this study highlight the enormous heterogeneity of the P. fluorescens group and the importance of the variable genome in tailoring individual strains to their specific lifestyles and functional repertoire

Insect-Mediated Dispersal of the Rhizobacterium Pseudomonas chlororaphis

The southern corn rootworm, Diabrotica undecimpunctata subsp. howardi, a common and mobile insect pest, was shown to transmit the rhizobacte-rium Pseudomonas chlororaphis strain L11 between corn plants. Strain L11 has been genetically modified to contain the lacZY genes from Escherichia coli. It can reach high densities on roots and invade the roots and move into the foliage. D. undecimpunctata subsp. howardi became infested with L11 as larvae while feeding on roots of seed-inoculated corn and retained the bacteria through pupation, molting to the adult stage, and emergence from the soil. Bacterial densities on or in the insects increased 100-fold after they fed again as adults on L11-infested foliage. Adults retained the bacteria for at least 2 weeks after last exposure and could transmit L11 to new plants. The likelihood of transmission decreased with time since last exposure to L11, but increased with time spent on the new plants. This research demonstrates that rhizobacteria can escape the rhizosphere by moving in or onto foliage, where they can then be acquired and transmitted by insects. This transmission route may be common among naturally occurring rhizobacteria and facilitate the dispersal of both beneficial and harmful soilborne microorganisms

Transcriptomic Evidence of a Link between Cell Wall Biogenesis, Pathogenesis, and Vigor in Walnut Root and Trunk Diseases.

Crown gall disease (Agrobacterium tumefaciens), crown/root rot disease (Phytophthora spp.), root lesion disease (Pratylenchus vulnus) and tree vigor are key traits affecting the productivity and quality of walnuts in California. Unchallenged hybrid rootstocks were analyzed by RNA-seq to examine pre-formed factors affecting these traits. Enrichment analysis of the differentially expressed genes revealed that the increased expression of cell wall biogenesis-related genes plays a key role in susceptibility to A. tumefaciens, susceptibility to Phytophthora spp. and increased vigor. Analysis of the predicted subcellular loci of the encoded proteins revealed that many gene products associated with vigor and susceptibility were targeted to the plasma membrane and extracellular space, connecting these traits to sustaining barrier function. We observed that RNA processing and splicing, along with predicted nuclear targeting, were associated with resistance to A. tumefaciens, resistance to Phytophthora spp. and low vigor. Four genes within the J. microcarpa QTL region for resistance to A. tumefaciens and Phytophthora spp. were represented among our transcripts, with two of the genes being differentially expressed in association with resistance to A. tumefaciens and decreased vigor. No differential expression related to Phytophthora spp. or P. vulnus resistance was observed in this region. Additionally, the J. microcarpa haplotype expressed more transcripts associated with resistance to A. tumefaciens, Phytophthora spp. and low vigor, but not P. vulnus, than the J. regia haplotype. We also report unique and shared hormone and defense responses associated with each trait. This research suggests a link between cell wall biogenesis, vigor and critical root diseases of walnut