Field Evaluation of Reduced Rate Brassicaceae Seed Meal Amendment and Rootstock Genotype on the Microbiome and Control of Apple Replant Disease.

An orchard field trial was conducted to assess the utility of reduced rate Brassicaceae seed meal (SM) amendment in concert with specific rootstock genotypes for effective control of apple replant disease. Three amendment rates of a 1:1 formulation of Brassica juncea-Sinapis alba SM were compared with preplant 1,3-dichloropropene/chloropicrin soil fumigation for disease control efficacy. When applied at the highest rate (6.6 t ha-1) in the spring of planting, SM caused significant phytotoxicity and tree mortality, which was higher for Gala/M.26 than for Gala/G.41 but was not observed at SM application rates of 2.2 or 4.4 t ha-1. SM treatment resulted in growth and yield increases of Gala/M.26 and Gala/G.41 trees in a manner similar to the fumigation treatment and significantly greater than the no treatment control. Tree growth in soils treated with SM at 4.4 t ha-1 was similar or superior to that obtained with SM at 6.6 t ha-1 and superior to that attained at an SM application rate of 2.2 t ha-1. Soil fumigation and all SM treatments reduced Pratylenchus penetrans root infestation relative to the control treatment at the end of the initial growing season. Lesion nematode root densities in the fumigation treatment, but not SM treatments, rapidly recovered and were indistinguishable from the control at the end of the second growing season. Soil fumigation and all SM treatments significantly suppressed Pythium spp. root infection relative to the control. Trees grafted to rootstock G.41 possessed lower P. penetrans root densities relative to trees grafted to rootstock M.26. One year after planting, composition of microbial communities from SM-amended soils was distinct from those detected in control and fumigated soils, and the differences were amplified with increasing SM application rate. Specific fungal and bacterial phyla associated with suppression of plant pathogens were more abundant in SM-treated soil relative to the control, and they were similar in abundance in 4.4- and 6.6-t ha-1 SM treatments. Findings from this study demonstrated that use of the appropriate apple rootstock genotype will allow for effective replant disease control at SM application rates significantly less than that utilized previously (6.6 t ha-1).

Interaction of Brassicaceae Seed Meal Soil Amendment and Apple Rootstock Genotype on Microbiome Structure and Replant Disease Suppression.

Preplant soil application of a Brassica juncea-Sinapis alba seed meal formulation (SM) at a rate of 6.6 t ha-1 alters composition of the orchard soil microbiome in a manner that yields sustainable long-term suppression of soilborne pathogens in apple production systems. However, the cost of SM amendment has hindered the adoption of this tactic to manage apple replant disease in commercial orchards. Greenhouse trials were conducted to assess the effect of reduced SM application rates in concert with apple rootstock genotype on structure of the rhizosphere microbiome and associated disease control outcomes. At all application rates assessed, SM treatment increased tree growth and reduced disease development relative to the control. In general, total tree biomass and leader shoot length were similar in soils treated with SM at 4.4 or 6.6 t ha-1 regardless of rootstock genotype. Equivalent increase in tree biomass when cultivated in soil treated at the lowest and highest SM amendment rate was attained when used in conjunction with G.41 or G.210 apple rootstocks. Suppression of Pythium spp. or Pratylenchus penetrans root densities was similar at all SM application rates. When cultivated in nontreated replant orchard soil, Geneva rootstocks (G.41 and G.210) exhibited lower levels of Pythium spp. and P. penetrans root colonization relative to Malling rootstocks (M.9 and MM.106). For a given rootstock, structure of the rhizosphere microbiome was similar in soils treated with SM at 4.4 and 6.6 t ha-1. G.41 and G.210 rootstocks but not M.9 or MM.106 cultivated in soil treated with SM at 2.2 t ha-1 possessed a rhizosphere bacterial community structure that differed significantly from the control. Findings indicate that effective control of apple replant disease may be attained at lower SM amendment rates than employed previously, with lower effective rates possible when integrated with tolerant rootstock genotypes such as G.41 or G.210.

Effect of Soil Physical Conditions on Emission of Allyl Isothiocyanate and Subsequent Microbial Inhibition in Response to Brassicaceae Seed Meal Amendment.

Generation of allyl isothiocyanate (AITC) in soil treated with residues of specific Brassicaceae species yields direct and indirect suppression of soilborne plant pathogens. Soil physical conditions demonstrably affected the quantity of AITC generated in response to soil incorporation of a Brassica juncea/Sinapis alba seed meal (SM) formulation. The concentration of AITC generated in SM-amended soil increased with an increase in temperature from 10 to 30°C. AITC emission was also elevated with an increase in soil water potential from -1,000 kPa through -40 kPa; however, a significant decrease in AITC emission was observed in a saturated soil environment (0 kPa). Peak AITC emission was obtained 2 to 3 h after SM amendment under optimal conditions but the peak was delayed in soils incubated at low temperature or in extreme moisture environments. Although AITC production varied significantly across different orchard soils, all three orchard soils yielded the same pattern of AITC release in response to SM amendment over the spectrum of soil water potentials examined in this study. Mycelial growth inhibition in fungi and oomycetes isolated from apple roots was dependent on both AITC concentration and exposure time. Pythium ultimum exhibited sensitivity to AITC at concentrations ranging from 0.01 to 0.22 µg g-1 of soil, whereas Hypocrea lixii was insensitive to AITC. Exposure to AITC at a concentration of 0.22 µg g-1 of soil for a period of 2 h restricted hyphal growth of Rhizoctonia solani AG-5, Ilyonectria destructans, and Mortierella alpina. R. solani AG-5 exhibited significant growth inhibition when incubated at AITC concentrations of 0.008 to 0.011 µg g-1 of soil for 10 h. These findings provide information that will be useful in the management of appropriate soil variables to obtain optimal yields of AITC in response to SM soil amendments and indicate that a standard soil moisture prescription may be suitable for use when applying this SM formulation for soilborne disease control.

Timing of Perennial Canker Development in Apple Trees Caused by Neofabraea perennans and Neofabraea kienholzii.

Members of the genera Neofabraea and Phlyctema have been reported to incite canker diseases of apple trees and a postharvest decay of apple fruit referred to as "bull's-eye rot." Neofabraea kienholzii was recently identified as participating in the bull's-eye rot disease complex of apple and other pome fruit. In this study, apple twigs inoculated with N. kienholzii were shown to develop symptoms of a canker disease closely resembling perennial canker of pome fruit trees caused by N. perennans. Cankers resulting from infection by either Neofabraea spp. were more likely to be induced when twig inoculations occurred in October, and to a lesser degree in April, compared with all other inoculation dates evaluated in this study. Although N. kienholzii tended to induce cankers that were smaller in size compared with N. perennans, both pathogens shared similar seasonal trends in the initiation and expansion of tree cankers. N. perennans and N. kienholzii were recovered from inoculated twigs 6 months postinoculation regardless of when inoculations were conducted, indicating that both pathogens can survive on diseased twigs year-round. In addition, acervuli were observed more often on twigs inoculated in September and April compared with those inoculated in other months. Data from this work should help further our understanding of the epidemiology of N. kienholzii. This information also highlights the importance of proper branch pruning, canker removal, and aphid control. Such management activities should be conducted in a manner that helps minimize further spread of the pathogen.

Prospects for Biological Soilborne Disease Control: Application of Indigenous Versus Synthetic Microbiomes.

Biological disease control of soilborne plant diseases has traditionally employed the biopesticide approach whereby single strains or strain mixtures are introduced into production systems through inundative/inoculative release. The approach has significant barriers that have long been recognized, including a generally limited spectrum of target pathogens for any given biocontrol agent and inadequate colonization of the host rhizosphere, which can plague progress in the utilization of this resource in commercial field-based crop production systems. Thus, although potential exists, this model has continued to lag in its application. New omics' tools have enabled more rapid screening of microbial populations allowing for the identification of strains with multiple functional attributes that may contribute to pathogen suppression. Similarly, these technologies also enable the characterization of consortia in natural systems which provide the framework for construction of synthetic microbiomes for disease control. Harnessing the potential of the microbiome indigenous to agricultural soils for disease suppression through application of specific management strategies has long been a goal of plant pathologists. Although this tactic also possesses limitation, our enhanced understanding of functional attributes of suppressive soil systems through application of community and metagenomic analysis methods provide opportunity to devise effective resource management schemes. As these microbial communities in large part are fostered by the resources endemic to soil and the rhizosphere, substrate mediated recruitment of disease-suppressive microbiomes constitutes a practical means to foster their establishment in crop production systems.

Carbon Source-Dependent Effects of Anaerobic Soil Disinfestation on Soil Microbiome and Suppression of Rhizoctonia solani AG-5 and Pratylenchus penetrans.

The effect of carbon source on efficacy of anaerobic soil disinfestation (ASD) toward suppression of apple root infection by Rhizoctonia solani AG-5 and Pratylenchus penetrans was examined. Orchard grass (GR), rice bran (RB), ethanol (ET), composted steer manure (CM), and Brassica juncea seed meal (SM) were used as ASD carbon inputs, with plant assays conducted in natural and pasteurized orchard soils. Subsequent studies investigated the effect of GR application rate used in ASD on control of these pathogens. In general, apple root infection by R. solani AG-5 was significantly lower in ET, GR, RB, and SM ASD treatments compared with the control. Among different ASD treatments, apple seedling growth was significantly greater when GR or SM was used as the carbon input relative to all other ASD treatments. R. solani AG-5 DNA abundance was significantly reduced in all ASD treatments, regardless of amendment type, compared with the control. In independent experiments, ASD-GR was consistently superior to ASD-CM for limiting pathogen activity in soils. ASD treatment with a grass input rate of 20 t ha(-1) provided superior suppression of P. penetrans but grass application rate did not affect ASD efficacy in control of R. solani AG-5. The soil microbiome from ASD-GR-treated soils was clearly distinct from the control and ASD-CM-treated soils. In contrast, composition of the microbiome from control and ASD-CM-treated soils could not be differentiated. Comparative results from pasteurized and nonpasteurized soils suggest that there is potential for GR based ASD treatment to recruit microbial elements that persist over the anaerobic phase of soil incubation, which may functionally contribute to disease suppression. When ASD was conducted with GR, microbial diversity was markedly reduced relative to the control or ASD-CM soil suggesting that this parameter, typically associated with system resilience, was not instrumental to the function of ASD-induced soil suppressiveness.

Brassica seed meal soil amendments transform the rhizosphere microbiome and improve apple production through resistance to pathogen reinfestation.

Brassicaceae seed meal (SM) formulations were compared with preplant 1,3-dichloropropene/chloropicrin (Telone-C17) soil fumigation for the ability to control apple replant disease and to suppress pathogen or parasite reinfestation of organic orchard soils at two sites in Washington State. Preplant soil fumigation and an SM formulation consisting of either Brassica juncea-Sinapis alba or B. juncea-B. napus each provided similar levels of disease control during the initial growing season. Although tree growth was similar in fumigated and SM-amended soil during the initial growing season, tree performance in terms of growth and yield was commonly superior in B. juncea-S. alba SM-amended soil relative to that in fumigated soil at the end of four growing seasons. SM-amended soils were resistant to reinfestation by Pratylenchus penetrans and Pythium spp. relative to fumigated soils and corresponded with enhanced tree performance. Phytotoxic symptoms were observed in response to SM amendment at one of two orchard sites, were dependent upon season of application, and occurred in an SM formulation-specific manner. After 2 years, the rhizosphere microbiome in fumigated soils had reverted to one that was indistinguishable from the no-treatment control. In contrast, rhizosphere soils from the SM treatment possessed unique bacterial and fungal profiles, including specific microbial elements previously associated with suppression of plant-pathogenic fungi, oomycetes, and nematodes. Overall diversity of the microbiome was reduced in the SM treatment rhizosphere, suggesting that enhanced "biodiversity" was not instrumental in achieving system resistance or pathogen suppression.

Effect of Rootstock Genotype and Arbuscular Mycorrhizal Fungal (AMF) Species on Early Colonization of Apple.

The effect of plant cultivar on the degree of mycorrhization and the benefits mediated by arbuscular mycorrhizal fungi (AMF) have been documented in many crops. In apple, a wide variety of rootstocks are commercially available; however, it is not clear whether some rootstock genotypes are more susceptible to mycorrhization than others and/or whether AMF species identity influences rootstock compatibility. This study addresses these questions by directly testing the ability/efficacy of four different AMF species (Rhizophagus irregularis, Septoglomus deserticola, Claroideoglomus claroideum or Claroideoglomus etunicatum) to colonize a variety of commercially available Geneva apple rootstock genotypes (G.11, G.41, G.210, G.969, and G.890). Briefly, micropropagated plantlets were inoculated with individual species of AMF or were not inoculated. The effects of the rootstock genotype/AMF interaction on mycorrhization, plant growth, and/or leaf nutrient concentrations were assessed. We found that both rootstock genotype and the identity of the AMF are significant sources of variation affecting the percentage of colonization. However, these factors largely operate independently in terms of the extent of root colonization. Among the AMF tested, C. etunicatum and R. irregularis represented the most compatible fungal partners, regardless of apple rootstock genotype. Among the rootstocks tested, semi-dwarfing rootstocks appeared to have an advantage over dwarfing rootstocks in regard to establishing and maintaining associations with AMF. Nutrient uptake and plant growth outcomes were also influenced in a rootstock genotype/AMF species-specific manner. Our findings suggest that matching host genetics with compatible AMF species has the potential to enhance agricultural practices in nursery and orchard systems.

A framework for the targeted recruitment of crop-beneficial soil taxa based on network analysis of metagenomics data.

BACKGROUND: The design of ecologically sustainable and plant-beneficial soil systems is a key goal in actively manipulating root-associated microbiomes. Community engineering efforts commonly seek to harness the potential of the indigenous microbiome through substrate-mediated recruitment of beneficial members. In most sustainable practices, microbial recruitment mechanisms rely on the application of complex organic mixtures where the resources/metabolites that act as direct stimulants of beneficial groups are not characterized. Outcomes of such indirect amendments are unpredictable regarding engineering the microbiome and achieving a plant-beneficial environment. RESULTS: This study applied network analysis of metagenomics data to explore amendment-derived transformations in the soil microbiome, which lead to the suppression of pathogens affecting apple root systems. Shotgun metagenomic analysis was conducted with data from 'sick' vs 'healthy/recovered' rhizosphere soil microbiomes. The data was then converted into community-level metabolic networks. Simulations examined the functional contribution of treatment-associated taxonomic groups and linked them with specific amendment-induced metabolites. This analysis enabled the selection of specific metabolites that were predicted to amplify or diminish the abundance of targeted microbes functional in the healthy soil system. Many of these predictions were corroborated by experimental evidence from the literature. The potential of two of these metabolites (dopamine and vitamin B12) to either stimulate or suppress targeted microbial groups was evaluated in a follow-up set of soil microcosm experiments. The results corroborated the stimulant's potential (but not the suppressor) to act as a modulator of plant beneficial bacteria, paving the way for future development of knowledge-based (rather than trial and error) metabolic-defined amendments. Our pipeline for generating predictions for the selective targeting of microbial groups based on processing assembled and annotated metagenomics data is available at https://github.com/ot483/NetCom2 . CONCLUSIONS: This research demonstrates how genomic-based algorithms can be used to formulate testable hypotheses for strategically engineering the rhizosphere microbiome by identifying specific compounds, which may act as selective modulators of microbial communities. Applying this framework to reduce unpredictable elements in amendment-based solutions promotes the development of ecologically-sound methods for re-establishing a functional microbiome in agro and other ecosystems. Video Abstract.

Toward a holistic view of orchard ecosystem dynamics: A comprehensive review of the multiple factors governing development or suppression of apple replant disease.

Replant diseases are a common occurrence in perennial cropping systems. In apple, progress toward the development of a universally effective disease management strategy, beyond the use of broad-spectrum soil fumigants, is impeded by inconsistencies in defining replant disease etiology. A preponderance of evidence attributes apple replant disease to plant-induced changes in the soil microbiome including the proliferation of soilborne plant pathogens. Findings from alternative studies suggest that the contribution of abiotic factors, such as the accumulation of phenolic detritus from previous orchard plantings, may play a part as well. Engineering of the resident soil microbiome using resource-based strategies is demonstrating potential to limit activity of replant pathogens and improve productivity in newly established orchards. An understanding of factors promoting the assembly of a disease-suppressive soil microbiome along with consideration of host factors that confer disease tolerance or resistance is imperative to the developing a more holistic view of orchard ecosystem dynamics. Here, we review the literature concerning the transition of orchard soil from a healthy state to a replant disease-conducive state. Included in the scope of this review are studies on the influence of soil type and geography on the apple replant pathogen complex. Furthermore, several tolerance and innate resistance mechanisms that have been described in apple to date, including the role of root chemistry/exudates are discussed. Finally, the interplay between apple rootstock genotype and key resource-based strategies which have been shown to "reshape" the plant holobiont in favor of a more prophylactic or disease-suppressive state is highlighted.

Contrasting effects of genotype and root size on the fungal and bacterial communities associated with apple rootstocks.

The endophytic microbiome of plants is believed to have a significant impact on its physiology and disease resistance, however, the role of host genotype in determining the composition of the endophytic microbiome of apple root systems remains an open question that has important implications for defining breeding objectives. In the current study, the bacterial and fungal microbiota associated with four different apple rootstocks planted in April, 2018 in the same soil environment and harvested in May, 2019 were evaluated to determine the role of genotype on the composition of both the bacterial and fungal communities. Results demonstrated a clear impact of genotype and root size on microbial composition and diversity. The fungal community was more affected by plant genotype whereas the bacterial community was shaped by root size. Fungal and bacterial abundance was equal between different-sized roots however, significantly higher microbial counts were detected in rhizosphere samples compared to root endosphere samples. This study provides information that can be used to develop a comprehensive and readily applicable understanding of the impact of genotype and environmental factors on the establishment of plant microbiome, as well as its potential function and impact on host physiology.

Analysis of Environmental Variables and Carbon Input on Soil Microbiome, Metabolome and Disease Control Efficacy in Strawberry Attributable to Anaerobic Soil Disinfestation.

Charcoal rot and Fusarium wilt, caused by Macrophomina phaseolina and Fusarium oxysporum f. sp. fragariae, respectively, are major soil-borne diseases of strawberry that have caused significant crop losses in California. Anaerobic soil disinfestation has been studied as an industry-level option to replace soil fumigants to manage these serious diseases. Studies were conducted to discern whether Gramineae carbon input type, incubation temperature, or incubation duration influences the efficacy of this disease control tactic. In experiments conducted using 'low rate' amendment applications at moderate day/night temperatures (24/18 °C), and carbon inputs (orchard grass, wheat, and rice bran) induced an initial proliferation and subsequent decline in soil density of the Fusarium wilt pathogen. This trend coincided with the onset of anaerobic conditions and a corresponding generation of various anti-fungal compounds, including volatile organic acids, hydrocarbons, and sulfur compounds. Generation of these metabolites was associated with increases in populations of Clostridium spp. Overall, carbon input and incubation temperature, but not incubation duration, significantly influenced disease suppression. All Gramineae carbon inputs altered the soil microbiome and metabolome in a similar fashion, though the timing and maximum yield of specific metabolites varied with input type. Fusarium wilt and charcoal rot suppression were superior when anaerobic soil disinfestation was conducted using standard amendment rates of 20 t ha-1 at elevated temperatures combined with a 3-week incubation period. Findings indicate that anaerobic soil disinfestation can be further optimized by modulating carbon source and incubation temperature, allowing the maximum generation of antifungal toxic volatile compounds. Outcomes also indicate that carbon input and environmental variables may influence treatment efficacy in a target pathogen-dependent manner which will require pathogen-specific optimization of treatment protocols.

Comparative Analysis of the Apple Root Transcriptome as Affected by Rootstock Genotype and Brassicaceae Seed Meal Soil Amendment: Implications for Plant Health.

Brassicaceae seed meal (SM) soil amendment has been utilized as an effective strategy to control the biological complex of organisms, which includes oomycetes, fungi, and parasitic nematodes, that incites the phenomenon termed apple replant disease. Soil-borne disease control attained in response to Brassicaceae SM amendment is reliant on multiple chemical and biological attributes, including specific SM-generated modifications to the soil/rhizosphere microbiome. In this study, we conducted a comparative analyses of apple root gene expression as influenced by rootstock genotype combined with a seed meal (SM) soil amendment. Apple replant disease (ARD) susceptible (M.26) and tolerant (G.210) rootstocks cultivated in SM-amended soil exhibited differential gene expression relative to corresponding non-treated control (NTC) orchard soil. The temporal dynamics of gene expression indicated that the SM-amended soil system altered the trajectory of the root transcriptome in a genotype-specific manner. In both genotypes, the expression of genes related to plant defense and hormone signaling were altered in SM-amended soil, suggesting SM-responsive phytohormone regulation. Altered gene expression was temporally associated with changes in rhizosphere microbiome density and composition in the SM-treated soil. Gene expression analysis across the two rootstocks cultivated in the pathogen-infested NTC soil showed genotype-specific responses indicative of different defensive strategies. These results are consistent with previously described resistance mechanisms of ARD "tolerant" rootstock cultivars and also add to our understanding of the multiple mechanisms by which SM soil amendment and the resulting rhizosphere microbiome affect apple rootstock physiology. Future studies which assess transcriptomic and metagenomic data in parallel will be important for illuminating important connections between specific rhizosphere microbiota, gene-regulation, and plant health.

Efficacy of Brassicaceous Seed Meal Formulations for the Control of Apple Replant Disease in Conventional and Organic Production Systems.

The efficacy of brassicaceous seed meals for the control of apple replant disease and the effects of such treatments on the causal pathogen complex were examined in conventional and organic production systems. When used in conjunction with a postplant application of mefenoxam, Brassica juncea and Sinapis alba seed meal soil amendments were as effective as preplant fumigation of soil with 1,3-dichloropropene-chloropicrin in terms of disease control, tree growth, and overall fruit yields of Gala/M26. Brassica napus seed meal amendment-mefenoxam soil drench also enhanced yields in a manner comparable to preplant fumigation, but vegetative growth was intermediate between the control and fumigation treatments. When applied alone, seed meal amendments failed to enhance tree growth or control disease to the level attained in response to soil fumigation. Postplant mefenoxam treatments revealed that failure of seed meal amendments to enhance tree growth and yield when used independently was due, at least in part, to increased apple root infection by Pythium spp. in B. napus and S. alba seed meal-amended soils, and by Phytophthora cambivora in B. juncea-amended soil. As mefenoxam treatment is not compatible with organic cropping systems, a seed meal blend was formulated which, based upon biological activity, was predicted to suppress known components of the target pathogen complex without need of additional treatment. Gala/M26 trees planted in soils treated with a 1:1 ratio of B. juncea:B. napus seed meal blend performed as well in terms of disease control and vegetative growth as trees cultivated in fumigated soil at an organic-certified orchard. Because these trials utilized the highly susceptible rootstock M26, the results demonstrate that these amendments are a viable alternative to soil fumigation for the control of apple replant disease in both conventional and organic systems.

Protozoan-induced regulation of cyclic lipopeptide biosynthesis is an effective predation defense mechanism for Pseudomonas fluorescens.

Environmental bacteria are exposed to a myriad of biotic interactions that influence their function and survival. The grazing activity of protozoan predators significantly impacts the dynamics, diversification, and evolution of bacterial communities in soil ecosystems. To evade protozoan predation, bacteria employ various defense strategies. Soil-dwelling Pseudomonas fluorescens strains SS101 and SBW25 produce the cyclic lipopeptide surfactants (CLPs) massetolide and viscosin, respectively, in a quorum-sensing-independent manner. In this study, CLP production was shown to protect these bacteria from protozoan predation as, compared to CLP-deficient mutants, strains SS101 and SBW25 exhibited resistance to grazing by Naegleria americana in vitro and superior persistence in soil in the presence of this bacterial predator. In the wheat rhizosphere, CLP-producing strains had a direct deleterious impact on the survival of N. americana. In vitro assays further showed that N. americana was three times more sensitive to viscosin than to massetolide and that exposure of strain SS101 or SBW25 to this protozoan resulted in upregulation of CLP biosynthesis genes. Enhanced expression of the massABC and viscABC genes did not require physical contact between the two organisms as gene expression levels were up to threefold higher in bacterial cells harvested 1 cm from feeding protozoans than in cells collected 4 cm from feeding protozoans. These findings document a new natural function of CLPs and highlight that bacterium-protozoan interactions can result in activation of an antipredator response in prey populations.

Epiphytic bacteria and yeasts on apple blossoms and their potential as antagonists of Erwinia amylovora.

Apple blossoms were sampled for indigenous epiphytic populations of culturable microorganisms during different stages of bloom at two locations in central Washington State and one site in Corvallis, OR. Frequencies and population sizes of bacteria on stigmas of apple were lower in Washington than at Corvallis, where average relative humidity was higher and possibly favored greater colonization; however, bacteria at Corvallis were mainly pseudomonads, whereas those in Washington were diverse, composed of several genera. In Washington, yeast as well as bacteria were isolated from both stigmatic and hypanthial surfaces. Sampled blossoms were processed immediately to assess microbial populations, or after a 24-h incubation at 28 degrees C and high relative humidity, which broadened the range of detectable taxa evaluated as potential antagonists. Identifications were based on fatty acid methyl ester profiles and rDNA sequence analyses. Yeasts or yeastlike organisms were detected at frequencies similar to or greater than bacteria, particularly in hypanthia. When microbial isolates were tested for their capacity to suppress Erwinia amylovora on stigmas of detached crab apple flowers, many were ineffective. The best antagonists were the bacteria Pantoea agglomerans and Pseudomonas spp. and a few yeasts identified as Cryptococcus spp. Further evaluation of these taxa on flowers could lead to the discovery of additional biocontrol agents for fire blight.

Hybridization of an ITS-based macroarray with ITS community probes for characterization of complex communities of fungi and fungal-like protists.

The ability to characterize fungal community structure and dynamics in the environment is constantly challenged by the high levels of diversity that must be confronted. Large-scale oligonucleotide arrays for use in such analytical studies are currently under development; however, the implementation of this approach generally requires substantial time and financial resources. To address the need for a more accessible tool for fungal community profiling and broad diagnostics, we evaluated the potential utility of a reverse dot blot approach utilizing macroarray targets and probes that each consisted of a PCR product of the entire fungal ITS1-5.8S-ITS2 gene region. Samples used to generate the array targets included both culturable and non-culturable fungi and fungal-like protists representing a range of ecological functions. Tests performed using single-species probes within the genus Pythium demonstrated that taxonomic lineages could generally be distinguished when ITS DNA sequence similarity differed by greater than 5-10%. An artificially constructed community probe of known composition successfully detected eight of the 10 lineages contained on the array with only one clear false positive in 95 targets. The approach was also successfully applied to environmental samples. Taxa resident in the soil of a local orchard were identified using the array and matched those documented in previous studies. Closely related taxa from a previously uncharacterized and geographically distant orchard soil were also identified by the array and had affinities to Leptodontium, Cadophora, Zalerion, and Geomyces. These taxa were further confirmed to be present in the sample by cloning and DNA sequencing. A minority of lineages had DNA targets with low melting temperatures which were not detected on the arrays except under conditions that compromised specificity. Membrane-based ITS macroarrays coupled with community ITS probes possessed sufficient power to detect multiple genus-level lineages of fungi in complex samples and should have broad applications in the study of fungal communities.

Interaction of Brassicaceous Seed Meal and Apple Rootstock on Recovery of Pythium spp. and Pratylenchus penetrans from Roots Grown in Replant Soils.

Pythium spp. and Pratylenchus penetrans are significant components of the diverse pathogen complex that incites apple replant disease in Washington State. The structure of the Pythium population differs among orchard soils but is composed of multiple pathogenic species. Studies were conducted to determine the effect of brassicaceous seed meals and apple rootstock on the activity and composition of these pathogen populations. Brassicaceous seed meals differed in capacity to suppress Pythium numbers and apple root infection, as well as differentially transformed composition of the population recovered from apple roots. Brassica juncea seed meal (SM) was the sole seed meal examined to suppress Pythium numbers and root infection; however, a persisting population was always detected in which Pythium irregulare existed as the dominant or co-dominant species. In general, the Geneva series rootstocks were less susceptible to root infection by native populations of Pythium, whereas M26, MM106, and MM111 were highly susceptible. Apple rootstocks from the Geneva series consistently supported lower populations of P. penetrans than did Malling or Malling-Merton rootstocks. B. juncea SM was superior to Brassica napus SM or Sinapis alba SM in suppressing lesion nematode populations. Significant rootstock × seed meal interaction was detected, and nematode suppression in response to B. napus or S. alba SM was only observed when used in concert with a tolerant rootstock, while B. juncea SM suppressed lesion nematode root populations irrespective of rootstock. These findings demonstrate that utilization of brassicaceous seed meal amendments for replant disease suppression must employ an appropriate rootstock in order to achieve optimal disease control.

Temporal Dynamics of the Soil Metabolome and Microbiome During Simulated Anaerobic Soil Disinfestation.

Significant interest exists in engineering the soil microbiome to attain suppression of soil-borne plant diseases. Anaerobic soil disinfestation (ASD) has potential as a biologically regulated disease control method; however, the role of specific metabolites and microbial community dynamics contributing to ASD mediated disease control is mostly uncharacterized. Understanding the trajectory of co-evolutionary processes leading to syntrophic generation of functional metabolites during ASD is a necessary prelude to the predictive utilization of this disease management approach. Consequently, metabolic and microbial community profiling were used to generate highly dimensional datasets and network analysis to identify sequential transformations through aerobic, facultatively anaerobic, and anaerobic soil phases of the ASD process and distinct groups of metabolites and microorganisms linked with those stages. Transient alterations in abundance of specific microbial groups, not consistently accounted for in previous studies of the ASD process, were documented in this time-course study. Such events initially were associated with increases and subsequent diminution in highly labile metabolites conferred by the carbon input. Proliferation and dynamic compositional changes in the Firmicutes community continued throughout the anaerobic phase and was linked to temporal changes in metabolite abundance including accumulation of small chain organic acids, methyl sulfide compounds, hydrocarbons, and p-cresol with antimicrobial properties. Novel potential modes of disease control during ASD were identified and the importance of the amendment and "community metabolism" for temporally supplying specific classes of labile compounds were revealed.

Targeted Metabolic Profiling Indicates Apple Rootstock Genotype-Specific Differences in Primary and Secondary Metabolite Production and Validate Quantitative Contribution From Vegetative Growth.

Previous reports regarding rhizodeposits from apple roots are limited, and complicated by microbes, which readily colonize root systems and contribute to modify rhizodeposit metabolite composition. This study delineates methods for collection of apple rhizodeposits under axenic conditions, indicates rootstock genotype-specific differences and validates the contributions of vegetative activity to rhizodeposit quantity. Primary and phenolic rhizodeposit metabolites collected from two apple rootstock genotypes, G935 and M26, were delineated 2 months after root initiation by utilizing gas chromatography/liquid chromatography-mass spectrometry (GC/LC-MS), respectively. Twenty-one identified phenolic compounds and 29 sugars, organic acids, and amino acids, as well as compounds tentatively identified as triterpenoids were present in the rhizodeposits. When adjusted for whole plant mass, hexose, erythrose, galactose, phloridzin, kaempferol-3-glucoside, as well as glycerol, and glyceric acid differed between the genotypes. Phloridzin, phloretin, epicatechin, 4-hydroxybenzoic acid, and chlorogenic acid were among the phenolic compounds found in higher relative concentration in rhizodeposits, as assessed by LC-MS. Among primary metabolites assessed by GC-MS, amino acids, organic acids, and sugar alcohols found in relatively higher concentration in the rhizodeposits included L-asparagine, L-cysteine, malic acid, succinic acid, and sorbitol. In addition, putative ursane triterprenoids, identified based on accurate mass comparison to previously reported triterpenoids from apple peel, were present in rhizodeposits in high abundance relative to phenolic compounds assessed via the same extraction/instrumental method. Validation of metabolite production to tree vegetative activity was conducted using a separate set of micropropagated trees (genotype MM106) which were treated with a toxic volatile compound (butyrolactone) to inhibit activity/kill leaves and vegetative growth. This treatment resulted in a reduction of total collected rhizodeposits relative to an untreated control, indicating active vegetative growth contributes to rhizodeposit metabolites. Culture-based assays indicated an absence of bacterial or fungal endophytes in roots of micropropagated G935 and M26 plants. However, the use of fungi-specific primers in qPCR indicated the presence of fungal DNA in 30% of the samples, thus the contribution of endophytes to rhizodeposits cannot be fully eliminated. This study provides fundamental information for continued research and application of rhizosphere ecology driven by apple rootstock genotype specific rhizodeposition.