David M. Weller

Induced Systemic Resistance by Beneficial Microbes

Beneficial microbes in the microbiome of plant roots improve plant health. Induced systemic resistance (ISR) emerged as an important mechanism by which selected plant growth-promoting bacteria and fungi in the rhizosphere prime the whole plant body for enhanced defense against a broad range of pathogens and insect herbivores. A wide variety of root-associated mutualists, including Pseudomonas, Bacillus, Trichoderma, and mycorrhiza species sensitize the plant immune system for enhanced defense without directly activating costly defenses. This review focuses on molecular processes at the interface between plant roots and ISR-eliciting mutualists, and on the progress in our understanding of ISR signaling and systemic defense priming. The central role of the root-specific transcription factor MYB72 in the onset of ISR and the role of phytohormones and defense regulatory proteins in the expression of ISR in aboveground plant parts are highlighted. Finally, the ecological function of ISR-inducing microbes in the root microbiome is discussed.

Pseudomonas Biocontrol Agents of Soilborne Pathogens: Looking Back Over 30 Years

ABSTRACT Pseudomonas spp. are ubiquitous bacteria in agricultural soils and have many traits that make them well suited as biocontrol agents of soilborne pathogens. Tremendous progress has been made in characterizing the process of root colonization by pseudomonads, the biotic and abiotic factors affecting colonization, bacterial traits and genes contributing to rhizosphere competence, and the mechanisms of pathogen suppression. This review looks back over the last 30 years of Pseudomonas biocontrol research and highlights key studies, strains, and findings that have had significant impact on shaping our current understanding of biological control by bacteria and the direction of future research.

NATURAL PLANT PROTECTION BY 2, 4- DIACETYLPHLOROGLUCINOL PRODUCING PSEUDOMONAS SPP. IN TAKE ALL DECLINE SOILS

Take-all decline (TAD) is a natural biological control of the wheat root disease “take-all” that develops in response to the disease during extended monoculture of wheat. The research to date on TAD has been mostly descriptive and no particular occurrence is yet fully understood. We demonstrate that root-associated fluorescent Pseudomonas spp. producing the antibiotic 2,4-diacetylphloroglucinol (Phl) are key components of the natural biological control that operates in TAD soils in Washington State (U.S.A.). Phl-producing Pseudomonas spp. were present on roots of wheat grown in TAD soils at or above the threshold population density required for significant suppression of take-all of wheat. The specific suppression that operates in TAD soils was lost when Phl-producing fluorescent Pseudomonas spp. were eliminated, and conducive soils gained suppressiveness to take-all when Phl-producing Pseudomonas strains were introduced via mixing in small amounts of TAD soil. Introduction of selected Phl-producing strai...

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.

Current Concepts in the Use of Introduced Bacteria for Biological Disease Control: Mechanisms and Antifungal Metabolites

The resurgence of interest in the use of introduced microorganisms for biological control of plant pathogens during the past 10 years has been driven in part by trends in agriculture toward greater sustainability and increased public concern for hazards associated with the use of synthetic pesticides. Rapidly evolving technologies from molecular biology and genetics have provided new insights into the underlying mechanisms by which biocontrol agents function and have allowed evaluation of the behavior of microbial inoculants in natural environments to a degree not previously possible. The results from these advances bear directly on two fundamental sources of inconsistency in the performance of microorganisms introduced for biological control that until now have retarded their commercial development and widespread use, namely, inadequate colonization of the target site and variability in the expression or level of activity of the mechanism(s) responsible for pathogen suppression.

Exploiting genotypic diversity of 2,4-diacetylphloroglucinol-producing Pseudomonas spp.: characterization of superior root-colonizing P. fluorescens strain Q8r1-96.

ABSTRACT The genotypic diversity that occurs in natural populations of antagonistic microorganisms provides an enormous resource for improving biological control of plant diseases. In this study, we determined the diversity of indigenous 2,4-diacetylphloroglucinol (DAPG)-producingPseudomonas spp. occurring on roots of wheat grown in a soil naturally suppressive to take-all disease of wheat. Among 101 isolates, 16 different groups were identified by random amplified polymorphic DNA (RAPD) analysis. One RAPD group made up 50% of the total population of DAPG-producing Pseudomonas spp. Both short- and long-term studies indicated that this dominant genotype, exemplified by P. fluorescens Q8r1-96, is highly adapted to the wheat rhizosphere. Q8r1-96 requires a much lower dose (only 10 to 100 CFU seed−1 or soil−1) to establish high rhizosphere population densities (107 CFU g of root−1) than Q2-87 and 1M1-96, two genotypically different, DAPG-producing P. fluorescens strains. Q8r1-96 maintained a rhizosphere population density of approximately 105 CFU g of root−1 after eight successive growth cycles of wheat in three different, raw virgin soils, whereas populations of Q2-87 and 1M1-96 dropped relatively quickly after five cycles and were not detectable after seven cycles. In short-term studies, strains Q8r1-96, Q2-87, and 1M1-96 did not differ in their ability to suppress take-all. After eight successive growth cycles, however, Q8r1-96 still provided control of take-all to the same level as obtained in the take-all suppressive soil, whereas Q2-87 and 1M1-96 gave no control anymore. Biochemical analyses indicated that the superior rhizosphere competence of Q8r1-96 is not related to in situ DAPG production levels. We postulate that certain rhizobacterial genotypes have evolved a preference for colonization of specific crops. By exploiting diversity of antagonistic rhizobacteria that share a common trait, biological control can be improved significantly.

Effect of Population Density of Pseudomonas fluorescens on Production of 2,4-Diacetylphloroglucinol in the Rhizosphere of Wheat

ABSTRACT The role of antibiotics in biological control of soilborne pathogens, and more generally in microbial antagonism in natural disease-suppressive soils, often has been questioned because of the indirect nature of the supporting evidence. In this study, a protocol for high pressure liquid chromatography/mass spectrometry is described that allowed specific identification and quantitation of the antibiotic 2,4-diacetylphloroglucinol (Phl) produced by naturally occurring fluorescent Pseudomonas spp. on roots of wheat grown in a soil suppressive to take-all of wheat. These results provide, for the first time, biochemical support for the conclusion of previous work that Phl-producing fluorescent Pseudomonas spp. are key components of the natural biological control that operates in take-all-suppressive soils in Washington State. This study also demonstrates that the total amount of Phl produced on roots of wheat by P. fluorescens strain Q2-87, at densities ranging from approximately 10(5) to 10(7) CFU/g of root, is proportional to its rhizosphere population density and that Phl production per population unit is a constant (0.62 ng/10(5) CFU). Thus, Phl production in the rhizosphere of wheat is strongly related to the ability of the introduced strain to colonize the roots.

Bacillus sp. L324-92 for biological control of three root diseases of wheat grown with reduced tillage.

ABSTRACT Strain L324-92 is a novel Bacillus sp. with biological activity against three root diseases of wheat, namely take-all caused by Gaeumannomyces graminis var. tritici, Rhizoctonia root rot caused by Rhizoctonia solani AG8, and Pythium root rot caused mainly by Pythium irregulare and P. ultimum, that exhibits broad-spectrum inhibitory activity and grows at temperatures from 4 to 40 degrees C. These three root diseases are major yieldlimiting factors for wheat in the U.S. Inland Pacific Northwest, especially wheat direct-drilled into the residue of a previous cereal crop. Strain L324-92 was selected from among approximately 2,000 rhizosphere/rhizoplane isolates of Bacillus species isolated from roots of wheat collected from two eastern Washington wheat fields that had long histories of wheat. Roots were washed, heat-treated (80 degrees C for 30 min), macerated, and dilution-plated on (1)/(10)-strength tryptic soy agar. Strain L324-92 inhibited all isolates of G. graminis var. tritici, Rhizoctonia species and anastomosis groups, and Pythium species tested on agar at 15 degrees C; provided significant suppression of all three root diseases at 15 degrees C in growth chamber assays; controlled either Rhizoctonia root rot, takeall, or both; and increased yields in field tests in which one or more of the three root diseases of wheats were yield-limiting factors. The ability of L324-92 to grow at 4 degrees C probably contributes to its biocontrol activity on direct-drilled winter and spring wheat because, under Inland Northwest conditions, leaving harvest residues of the previous crop on the soil surface keeps soils cooler compared with tilled soils. These results suggest that Bacillus species with desired traits for biological control of wheat root diseases are present within the community of wheat rhizosphere microorganisms and can be recovered by protocols developed earlier for isolation of fluorescent Pseudomonas species effective against take-all.

Genetic Diversity of phlD from 2,4-Diacetylphloroglucinol-Producing Fluorescent Pseudomonas spp.

ABSTRACT Fluorescent Pseudomonas spp. that produce 2,4-diacetylphloroglucinol (2,4-DAPG) have biocontrol activity against damping-off, root rot, and wilt diseases caused by soilborne fungal pathogens, and play a key role in the natural suppression of Gaeumannomyces graminis var. tritici, known as take-all decline. Diversity within phlD, an essential gene in the biosynthesis of 2,4-DAPG, was studied by restriction fragment length polymorphism (RFLP) analysis of 123 2,4-DAPG-producing isolates from six states in the United States and six other locations worldwide. Clusters defined by RFLP analysis of phlD correlated closely with clusters defined previously by BOX-polymerase chain reaction (PCR) genomic fingerprinting, indicating the usefulness of phlD as a marker of genetic diversity and population structure among 2,4-DAPG producers. Genotypes defined by RFLP analysis of phlD were conserved among isolates from the same site and cropping history. Random amplified polymorphic DNA analyses of genomic DNA revealed a higher degree of polymorphism than RFLP and BOX-PCR analyses. Genotypic diversity in a subset of 30 strains representing all the phlD RFLP groups did not correlate with production in vitro of monoacetylphloroglucinol, 2,4-DAPG, or total phloroglucinol compounds. Twenty-seven of the 30 representative strains lacked pyrrolnitrin and pyoluteorin biosynthetic genes as determined by the use of specific primers and probes.

Changes in Populations of Rhizosphere Bacteria Associated with Take-All Disease of Wheat

ABSTRACT Take-all, caused by Gaeumannomycesgraminis var. tritici, is one of the most important fungal diseases of wheat worldwide. Knowing that microbe-based suppression of the disease occurs in monoculture wheat fields following severe outbreaks of take-all, we analyzed the changes in rhizosphere bacterial communities following infection by the take-all pathogen. Several bacterial populations were more abundant on diseased plants than on healthy plants, as indicated by higher counts on a Pseudomonas-selective medium and a higher fluorescence signal in terminal restriction fragment length polymorphism analyses of amplified 16S ribosomal DNA (rDNA). Amplified rDNA restriction analysis (ARDRA) of the most abundant cultured populations showed a shift in dominance from Pseudomonasto Chryseobacterium species in the rhizosphere of diseased plants. Fluorescence-tagged ARDRA of uncultured rhizosphere washes revealed an increase in ribotypes corresponding to several bacterial genera, including those subsequently identified by partial 16S sequencing as belonging to species of alpha-, beta-, and gamma-proteobacteria, sphingobacteria, and flavobacteria. The functional significance of some of these populations was investigated in vitro. Of those isolated, only a small subset of the most abundantPseudomonas spp. and aphlD+Pseudomonas sp. showed any significant ability to inhibit G.graminis var. tritici directly. When cultured strains were mixed with the inhibitoryphlD+Pseudomonasstrain, the Chryseobacterium isolates showed the least capacity to inhibit this antagonist of the pathogen, indicating that increases in Chryseobacterium populations may facilitate the suppression of take-all by 2,4-diacetylphloroglucinol-producingphlD+ pseudomonads.