A dual RNA-seq analyses revealed dynamic arms race during the invasion of walnut by Colletotrichum gloeosporioides.

BACKGROUND: Walnut anthracnose caused by Colletotrichum gloeosporioides seriously endangers the yield and quality of walnut, and has now become a catastrophic disease in the walnut industry. Therefore, understanding both pathogen invasion mechanisms and host response processes is crucial to defense against C. gloeosporioides infection. RESULTS: Here, we investigated the mechanisms of interaction between walnut fruits (anthracnose-resistant F26 fruit bracts and anthracnose-susceptible F423 fruit bracts) and C. gloeosporioides at three infection time points (24hpi, 48hpi, and 72hpi) using a high-resolution time series dual transcriptomic analysis, characterizing the arms race between walnut and C. gloeosporioides. A total of 20,780 and 6670 differentially expressed genes (DEGs) were identified in walnut and C. gloeosporioides against 24hpi, respectively. Generous DEGs in walnut exhibited opposite expression patterns between F26 and F423, which indicated that different resistant materials exhibited different transcriptional responses to C. gloeosporioides during the infection process. KEGG functional enrichment analysis indicated that F26 displayed a broader response to C. gloeosporioides than F423. Meanwhile, the functional analysis of the C. gloeosporioides transcriptome was conducted and found that PHI, SignalP, CAZy, TCDB genes, the Fungal Zn (2)-Cys (6) binuclear cluster domain (PF00172.19) and the Cytochrome P450 (PF00067.23) were largely prominent in F26 fruit. These results suggested that C. gloeosporioides secreted some type of effector proteins in walnut fruit and appeared a different behavior based on the developmental stage of the walnut. CONCLUSIONS: Our present results shed light on the arms race process by which C. gloeosporioides attacked host and walnut against pathogen infection, laying the foundation for the green prevention of walnut anthracnose.

Topography- and depth-dependent rhizosphere microbial community characteristics drive ecosystem multifunctionality in Juglans mandshurica forest.

Rhizosphere microbial community characteristics and ecosystem multifunctionality (EMF), both affected by topographic factors, are closely correlated. However, more targeted exploration is yet required to fully understand the variations of rhizosphere microbial communities along topographic gradients in different soil layers, as well as whether and how they regulate EMF under specific site conditions. Here, we conducted relevant research on Juglans mandshurica forests at six elevation gradients and two slope positions ranging from 310 to 750 m in Tianjin Baxian Mountain. Results demonstrated that rhizosphere soil physicochemical properties and enzyme activities of both layers (0-20 cm and 20-40 cm) varied significantly with elevation, while only at top layer did slope position have significant impacts on most indicators. Bacterial richness and diversity were higher in the top layer at slope bottom and middle-high elevation, the difference in fungi was not as noticeable. Both topographic factors and soil depth significantly impacted microbial community structure, with Candidatus_Udaeobacter of bacteria, Mortierella, Sebacina, and Hygrocybe of fungi mainly contributing to the dissimilarity between communities. EMF rose with increasing elevation, bacteria were more critical drivers of this process than fungi, and topographic factors could affect EMF by altering bacterial diversity and dominant taxa abundance. For evaluating EMF, the aggregate structure of sub layer and the carbon cycle-related indicators of top layer were of higher importance. Our results revealed the depth-dependent characteristics of the rhizosphere microbial community along topographic gradients in studied stands, as well as the pivotal regulatory role of bacteria on EMF, while also highlighting depth as an important variable for analyzing soil properties and EMF. This work helps us better understand the response of individuals and communities of J. mandshurica to changing environmental conditions, further providing a scientific reference for the management and protection of secondary forests locally and in North China.

Arbuscular mycorrhizal symbiosis with Rhizophagus irregularis DAOM197198 modifies the root transcriptome of walnut trees.

Walnut trees are cultivated and exploited worldwide for commercial timber and nut production. They are heterografted plants, with the rootstock selected to grow in different soil types and conditions and to provide the best anchorage, vigor, and resistance or tolerance to soil borne pests and diseases. However, no individual rootstock is tolerant of all factors that impact walnut production. In Europe, Juglans regia is mainly used as a rootstock. Like most terrestrial plants, walnut trees form arbuscular mycorrhizal symbioses, improving water and nutrient uptake and providing additional ecosystem services. Effects of arbuscular mycorrhizal symbiosis on root gene regulation, however, has never been assessed. We analyzed the response of one rootstock of J. regia to colonization by the arbuscular mycorrhizal fungus Rhizophagus irregularis DAOM197198. Plant growth as well as the nitrogen and phosphorus concentrations in roots and shoots were significantly increased in mycorrhizal plants versus non-colonized plants. In addition, we have shown that 1,549 genes were differentially expressed, with 832 and 717 genes up- and down-regulated, respectively. The analysis also revealed that some rootstock genes involved in plant nutrition through the mycorrhizal pathway, are regulated similarly as in other mycorrhizal woody species: Vitis vinifera and Populus trichocarpa. In addition, an enrichment analysis performed on GO and KEGG pathways revealed some regulation specific to J. regia (i.e., the juglone pathway). This analysis reinforces the role of arbuscular mycorrhizal symbiosis on root gene regulation and on the need to finely study the effects of diverse arbuscular mycorrhizal fungi on root gene regulation, but also of the scion on the functioning of an arbuscular mycorrhizal fungus in heterografted plants such as walnut tree.

Genome-wide identification of walnut (Juglans regia) PME gene family members and expression analysis during infection with Cryptosphaeria pullmanensis pathogens.

Walnuts exhibit a higher resistance to diseases, though they are not completely immune. This study focuses on the Pectin methylesterase (PME) gene family to investigate whether it is involved in disease resistance in walnuts. These 21 genes are distributed across 12 chromosomes, with four pairs demonstrating homology. Variations in conserved motifs and gene structures suggest diverse functions within the gene family. Phylogenetic and collinear gene pairs of the PME family indicate that the gene family has evolved in a relatively stable way. The cis-acting elements and gene ontology enrichment of these genes, underscores their potential role in bolstering walnuts' defense mechanisms. Transcriptomic analyses were conducted under conditions of Cryptosphaeria pullmanensis infestation and verified by RT-qPCR. The results showed that certain JrPME family genes were activated in response, leading to the hypothesis that some members may confer resistance to the disease.

Heart Rot Disease of Walnut Caused by Nothophoma juglandis sp. nov. and Its Endophytic Biocontrol Agent.

English walnut (Juglans regia L.) is an economically important hardwood tree species cultivated worldwide. Walnut heart rot disease leading to heartwood decay of trees has been frequently observed in a number of plantations in China. To identify the causal agent, 29 diseased stem samples were collected from walnut plantations in Beijing, and 54 fungal isolates were obtained. Koch's postulates were developed, and the results showed that Nothophoma juglandis, a species new to science, was the causal agent of walnut heart rot disease. Granulobasidium vellereum, a notable biocontrol agent, was coisolated with N. juglandis. An antagonistic assay on dual culture and walnut stems (both in the field and detached branches) proved that G. vellereum acted as a potential biocontrol agent against N. juglandis, as it could significantly inhibit the expansion of N. juglandis. The optimal temperature for mycelial growth and pathogenicity of N. juglandis was 26.6 and 27.0°C, respectively, which frequently occur in the summer of the walnut-growing regions in China.

Characterization of Xanthomonas arboricola pv. juglandis Bacteriophages against Bacterial Walnut Blight and Field Evaluation.

Xanthomonas arboricola pv. juglandis (hereafter X. juglandis) is the etiological agent of walnut blight, the most important bacterial disease affecting walnut production worldwide. Currently, the disease is treated mainly with copper-derived compounds (e.g., CuSO4) despite the evidence of genetic resistance in these strains. Regarding the effectiveness and sustainability, the use of a bacteriophage appears to be a biocontrol alternative to reduce X. juglandis load and symptomatology of walnut blight. Here, the phages f20-Xaj, f29-Xaj, and f30-Xaj were characterized, and their effectiveness in walnut orchards against walnut blight was determined. These bacteriophages showed a specific lytic infection in X. juglandis strains isolated from Chile and France. Phylogenetic analysis of the complete genome of f20-Xaj and f30-Xaj indicates that these phages belong to the Pradovirus genus. In the field, the cocktail of these bacteriophages showed similar effectivity to CuSO4 in the reduction of incidence and severity in walnut tissue. Moreover, the bacterial load of X. juglandis was significantly reduced in the presence of bacteriophages in contrast to a CuSO4 treatment. These results show that the use of bacteriophages can be an alternative to combat the symptoms of walnut blight caused by X. juglandis.

Characterization and microsatellite marker development for a common bark and ambrosia beetle associate, Geosmithia obscura.

Symbioses between Geosmithia fungi and wood-boring and bark beetles seldom result in disease induction within the plant host. Yet, exceptions exist such as Geosmithia morbida, the causal agent of Thousand Cankers Disease (TCD) of walnuts and wingnuts, and Geosmithia sp. 41, the causal agent of Foamy Bark Canker disease of oaks. Isolates of G. obscura were recovered from black walnut trees in eastern Tennessee and at least one isolate induced cankers following artificial inoculation. Due to the putative pathogenicity and lack of recovery of G. obscura from natural lesions, a molecular diagnostic screening tool was developed using microsatellite markers mined from the G. obscura genome. A total of 3256 candidate microsatellite markers were identified (2236, 789, 137 di-, tri-, and tetranucleotide motifs, respectively), with 2011, 703, 101 di-, tri-, and tetranucleotide motifs, respectively, containing markers with primers. From these, 75 microsatellite markers were randomly selected, screened, and optimized, resulting in 28 polymorphic markers that yielded single, consistently recovered bands, which were used in downstream analyses. Five of these microsatellite markers were found to be specific to G. obscura and did not cross-amplify into other, closely related species. Although the remaining tested markers could be useful, they cross-amplified within different Geosmithia species, making them not reliable for G. obscura detection. Five novel microsatellite markers (GOBS9, GOBS10, GOBS41, GOBS43, and GOBS50) were developed based on the G. obscura genome. These species-specific microsatellite markers are available as a tool for use in molecular diagnostics and can assist future surveillance studies.

Inactivation of Foodborne Pathogens on Inshell Walnuts by UV-C Radiation.

ABSTRACT: Inshell walnuts can be contaminated with pathogens through direct contact or cross-contamination during harvesting and postharvest hulling, drying, or storage. This study aimed to assess the efficacy of UV-C radiation in inactivating foodborne pathogens on inshell walnut surfaces. Intact inshell walnut surfaces were inoculated separately with Salmonella,Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus and then were subjected to UV-C radiation at doses of 29.4, 147.0, 294.0, 588.0, and 882.0 mJ/cm2. UV-C radiation inactivated the inoculated pathogens in a dose-dependent manner, and a tailing effect was observed for the inactivation of pathogens. UV-C radiation at 29.4 and 882.0 mJ/cm2 reduced the populations of Salmonella Enteritidis PT 30, Salmonella Typhimurium, E. coli O157:H7, L. monocytogenes, and S. aureus on inshell walnut surfaces by 0.82 to 1.25 and 1.76 to 2.41 log CFU per walnut, respectively. Scanning electron photomicrographs showed pathogenic bacterial cells in the cracks and crevices of the inshell walnut surface, and the shielding of microorganisms by the cracks and crevices may have contributed to the tailing effect observed during UV-C inactivation. No significant changes (P > 0.05) were found in walnut lipid oxidation following UV-C radiation at doses up to 882.0 mJ/cm2. Together, the results indicate that UV-C radiation could be a potential technology for reducing the populations of various foodborne pathogens on inshell walnut surfaces while maintaining the quality of walnuts.

Comparative Profiling of Wood Canker Pathogens from Spore Traps and Symptomatic Plant Samples Within California Almond and Walnut Orchards.

Fungi causing wood canker diseases are major factors limiting productivity and longevity of almond and walnut orchards. The goal of this study was to compare pathogen profiles from spore traps with those of plant samples collected from symptomatic almond and walnut trees and assess if profiles could be influenced by orchard type and age, rainfall amount and frequency, and/or neighboring trees. Three almond orchards and one walnut orchard with different characteristics were selected for this study. Fungal inoculum was captured weekly from nine trees per orchard using a passive spore-trapping device, during a 30-week period in the rainy season (October to April) and for two consecutive years. Fungal taxa identified from spore traps were compared with a collection of fungal isolates obtained from 61 symptomatic wood samples collected from the orchards. Using a culture-dependent approach coupled with molecular identification, we identified 18 known pathogenic species from 10 fungal genera (Ceratocystis destructans, Collophorina hispanica, Cytospora eucalypti, Diaporthe ampelina, Diaporthe chamaeropis/rhusicola, Diaporthe eres, Diaporthe novem, Diplodia corticola, Diplodia mutila, Diplodia seriata, Dothiorella iberica, Dothiorella sarmentorum, Dothiorella viticola, Eutypa lata, Neofusicoccum mediterraneum, Neofusicoccum parvum, Neoscytalidium dimidiatum, and Pleurostoma richardsiae), plus two unidentified Cytospora and Diaporthe species. However, only four species were identified with both methods (Diplodia mutila, Diplodia seriata, Dothiorella Iberica, and E. lata), albeit not consistently across orchards. Our results demonstrate a clear disparity between the two diagnostic methods and caution against using passive spore traps to predict disease risks. In particular, the spore trap approach failed to capture: insect-vectored pathogens such as Ceratocystis destructans that were often recovered from almond trunk and scaffold; Diaporthe chamaeropis/rhusicola commonly isolated from wood samples likely because Diaporthe species have a spatially restricted dispersal mechanism, as spores are exuded in a cirrus; and pathogenic species with low incidence in wood samples such as P. richardsiae and Collophorina hispanica. We propose that orchard inoculum is composed of both endemic taxa that are characterized by frequent and repeated trapping events from the same trees and isolated from plant samples, as well as immigrant taxa characterized by rare trapping events. We hypothesize that host type, orchard age, precipitation, and alternative hosts at the periphery of orchards are factors that could affect pathogen profile. We discuss the limitations and benefits of our methodology and experimental design to develop guidelines and prediction tools for fungal wood canker diseases in California orchards.

De Novo Arginine Synthesis Is Required for Full Virulence of Xanthomonas arboricola pv. juglandis During Walnut Bacterial Blight Disease.

Walnut blight (WB) disease caused by Xanthomonas arboricola pv. juglandis (Xaj) threatens orchards worldwide. Nitrogen metabolism in this bacterial pathogen is dependent on arginine, a nitrogen-enriched amino acid that can either be synthesized or provided by the plant host. The arginine biosynthetic pathway uses argininosuccinate synthase (argG), associated with increased bacterial virulence. We examined the effects of bacterial arginine and nitrogen metabolism on the plant response during WB by proteomic analysis of the mutant strain Xaj argG-. Phenotypically, the mutant strain produced 42% fewer symptoms and survived in the plant tissue with 2.5-fold reduced growth compared with wild type, while showing itself to be auxotrophic for arginine in vitro. Proteomic analysis of infected tissue enabled the profiling of 676 Xaj proteins and 3,296 walnut proteins using isobaric labeling in a data-dependent acquisition approach. Comparative analysis of differentially expressed proteins revealed distinct plant responses. Xaj wild type (WT) triggered processes of catabolism and oxidative stress in the host under observed disease symptoms, while most of the host biosynthetic processes triggered by Xaj WT were inhibited during Xaj argG- infection. Overall, the Xaj proteins revealed a drastic shift in carbon and energy management induced by disruption of nitrogen metabolism while the top differentially expressed proteins included a Fis transcriptional regulator and a peptidyl-prolyl isomerase. Our results show the critical role of de novo arginine biosynthesis to sustain virulence and minimal growth during WB. This study is timely and critical as copper-based control methods are losing their effectiveness, and new sustainable methods are urgently needed in orchard environments.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A Secreted Chorismate Mutase from Xanthomonas arboricola pv. juglandis Attenuates Virulence and Walnut Blight Symptoms.

Walnut blight is a significant above-ground disease of walnuts caused by Xanthomonas arboricola pv. juglandis (Xaj). The secreted form of chorismate mutase (CM), a key enzyme of the shikimate pathway regulating plant immunity, is highly conserved between plant-associated beta and gamma proteobacteria including phytopathogens belonging to the Xanthomonadaceae family. To define its role in walnut blight disease, a dysfunctional mutant of chorismate mutase was created in a copper resistant strain Xaj417 (XajCM). Infections of immature walnut Juglans regia (Jr) fruit with XajCM were hypervirulent compared with infections with the wildtype Xaj417 strain. The in vitro growth rate, size and cellular morphology were similar between the wild-type and XajCM mutant strains, however the quantification of bacterial cells by dPCR within walnut hull tissues showed a 27% increase in XajCM seven days post-infection. To define the mechanism of hypervirulence, proteome analysis was conducted to compare walnut hull tissues inoculated with the wild type to those inoculated with the XajCM mutant strain. Proteome analysis revealed 3296 Jr proteins (five decreased and ten increased with FDR ≤ 0.05) and 676 Xaj417 proteins (235 increased in XajCM with FDR ≤ 0.05). Interestingly, the most abundant protein in Xaj was a polygalacturonase, while in Jr it was a polygalacturonase inhibitor. These results suggest that this secreted chorismate mutase may be an important virulence suppressor gene that regulates Xaj417 virulence response, allowing for improved bacterial survival in the plant tissues.

Antifungal Activity of Bacillus velezensis CE 100 against Anthracnose Disease (Colletotrichum gloeosporioides) and Growth Promotion of Walnut (Juglans regia L.) Trees.

Walnut anthracnose caused by Colletotrichum gloeosporioides is a deleterious disease that severely affects the production of walnut (Juglans regia L.). The aim of this study was to assess the antifungal and growth promotion activities of Bacillus velezensis CE 100 as an alternative to chemical use in walnut production. The crude enzyme from B. velezensis CE 100 exhibited chitinase, protease, and ß-l,3-glucanase activity and degraded the cell wall of C. gloeosporioides, causing the inhibition of spore germination and mycelial growth by 99.3% and 33.6% at 100 µL/mL, respectively. The field application of B. velezensis CE 100 culture broth resulted in a 1.3-fold and 6.9-fold decrease in anthracnose disease severity compared to the conventional and control groups, respectively. Moreover, B. velezensis CE 100 produced indole-3-acetic acid (up to 1.4 µg/mL) and exhibited the potential for ammonium production and phosphate solubilization to enhance the availability of essential nutrients. Thus, field inoculation of B. velezensis CE 100 improved walnut root development, increased nutrient uptake, enhanced chlorophyll content, and consequently improved total biomass by 1.5-fold and 2.0-fold compared to the conventional and control groups, respectively. These results demonstrate that B. velezensis CE 100 is an effective biocontrol agent against anthracnose disease and a potential plant growth-promoting bacteria in walnut tree production.

Genome-Wide Identification and Transcriptional Expression Profiles of Transcription Factor WRKY in Common Walnut (Juglans regia L.).

The transcription factor WRKY is widely distributed in the plant kingdom, playing a significant role in plant growth, development and response to stresses. Walnut is an economically important temperate tree species valued for both its edible nuts and high-quality wood, and its response to various stresses is an important factor that determines the quality of its fruit. However, in walnut trees themselves, information about the WRKY gene family remains scarce. In this paper, we perform a comprehensive study of the WRKY gene family in walnut. In total, we identified 103 WRKY genes in the common walnut that are clustered into 4 groups and distributed on 14 chromosomes. The conserved domains all contained a WRKY domain, and motif 2 was observed in most WRKYs, suggesting a high degree of conservation and similar functions within each subfamily. However, gene structure was significantly differentiated between different subfamilies. Synteny analysis indicates that there were 56 gene pairs in J. regia and A. thaliana, 76 in J. regia and J. mandshurica, 75 in J. regia and J. microcarpa, 76 in J. regia and P. trichocarpa, and 33 in J. regia and Q. robur, indicating that the WRKY gene family may come from a common ancestor. GO and KEGG enrichment analysis showed that the WRKY gene family was involved in resistance traits and the plant-pathogen interaction pathway. In anthracnose-resistant F26 fruits (AR) and anthracnose-susceptible F423 fruits (AS), transcriptome and qPCR analysis results showed that JrWRKY83, JrWRKY73 and JrWRKY74 were expressed significantly more highly in resistant cultivars, indicating that these three genes may be important contributors to stress resistance in walnut trees. Furthermore, we investigate how these three genes potentially target miRNAs and interact with proteins. JrWRKY73 was target by the miR156 family, including 12 miRNAs; this miRNA family targets WRKY genes to enhance plant defense. JrWRKY73 also interacted with the resistance gene AtMPK6, showing that it may play a crucial role in walnut defense.

Metabolic pathway analysis of walnut endophytic bacterium Bacillus subtilis HB1310 related to lipid production from fermentation of cotton stalk hydrolysate based on genome sequencing.

OBJECTIVES: In this study, genome sequencing and metabolic analysis were used to identify and verify the key metabolic pathways for glucose and xylose utilization and fatty acid synthesis in the walnut endophytic bacterium (WEB) Bacillus subtilis HB1310. RESULTS: The genome sequence of WEB HB1310 was generated with a size of 4.1 Mb and GC content of 43.5%. Genome annotation indicated that the Embden-Meyerhof-Parnas, pentose phosphate, and fatty acid synthesis pathways were mainly involved in mixed sugar utilization and lipid production. In particular, diverse and abundant fatty acid synthesis genes were observed in a higher number than in other Bacillus strains. The tricarboxylic acid cycle competitively shared the carbon flux flowing before 48 h, and the acetic acid fermentation competed after 72 h. Moreover, fatty acid synthase activity was highly correlated with lipid titer with a correlation coefficient of 0.9626, and NADPH might be more utilized for the lipid synthesis within 48 h. CONCLUSIONS: This study is the first attempt to explain the metabolic mechanism of mixed sugar utilization and lipid production based on genomic information, which provides a theoretical basis for the metabolic regulation of bacterial lipid production from lignocellulosic hydrolysates.

Transcriptome and proteome analysis of walnut (Juglans regia L.) fruit in response to infection by Colletotrichum gloeosporioides.

BACKGROUND: Walnut anthracnose induced by Colletotrichum gloeosporioides is a disastrous disease affecting walnut production. The resistance of walnut fruit to C. gloeosporioides is a highly complicated and genetically programmed process. However, the underlying mechanisms have not yet been elucidated. RESULTS: To understand the molecular mechanism underlying the defense of walnut to C. gloeosporioides, we used RNA sequencing and label-free quantitation technologies to generate transcriptomic and proteomic profiles of tissues at various lifestyle transitions of C. gloeosporioides, including 0 hpi, pathological tissues at 24 hpi, 48 hpi, and 72 hpi, and distal uninoculated tissues at 120 hpi, in anthracnose-resistant F26 fruit bracts and anthracnose-susceptible F423 fruit bracts, which were defined through scanning electron microscopy. A total of 21,798 differentially expressed genes (DEGs) and 1929 differentially expressed proteins (DEPs) were identified in F26 vs. F423 at five time points, and the numbers of DEGs and DEPs were significantly higher in the early infection stage. Using pairwise comparisons and weighted gene co-expression network analysis of the transcriptome, we identified two modules significantly related to disease resistance and nine hub genes in the transcription expression gene networks. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of the DEGs and DEPs revealed that many genes were mainly related to immune response, plant hormone signal transduction, and secondary metabolites, and many DEPs were involved in carbon metabolism and photosynthesis. Correlation analysis between the transcriptome data and proteome data also showed that the consistency of the differential expression of the mRNA and corresponding proteins was relatively higher in the early stage of infection. CONCLUSIONS: Collectively, these results help elucidate the molecular response of walnut fruit to C. gloeosporioides and provide a basis for the genetic improvement of walnut disease resistance.

The Metabolomic-Gut-Clinical Axis of Mankai Plant-Derived Dietary Polyphenols.

BACKGROUND: Polyphenols are secondary metabolites produced by plants to defend themselves from environmental stressors. We explored the effect of Wolffia globosa 'Mankai', a novel cultivated strain of a polyphenol-rich aquatic plant, on the metabolomic-gut clinical axis in vitro, in-vivo and in a clinical trial. METHODS: We used mass-spectrometry-based metabolomics methods from three laboratories to detect Mankai phenolic metabolites and examined predicted functional pathways in a Mankai artificial-gut bioreactor. Plasma and urine polyphenols were assessed among the 294 DIRECT-PLUS 18-month trial participants, comparing the effect of a polyphenol-rich green-Mediterranean diet (+1240 mg/polyphenols/day, provided by Mankai, green tea and walnuts) to a walnuts-enriched (+440 mg/polyphenols/day) Mediterranean diet and a healthy controlled diet. RESULTS: Approximately 200 different phenolic compounds were specifically detected in the Mankai plant. The Mankai-supplemented bioreactor artificial gut displayed a significantly higher relative-abundance of 16S-rRNA bacterial gene sequences encoding for enzymes involved in phenolic compound degradation. In humans, several Mankai-related plasma and urine polyphenols were differentially elevated in the green Mediterranean group compared with the other groups (p < 0.05) after six and 18 months of intervention (e.g., urine hydroxy-phenyl-acetic-acid and urolithin-A; plasma Naringenin and 2,5-diOH-benzoic-acid). Specific polyphenols, such as urolithin-A and 4-ethylphenol, were directly involved with clinical weight-related changes. CONCLUSIONS: The Mankai new plant is rich in various unique potent polyphenols, potentially affecting the metabolomic-gut-clinical axis.

The hierarchy of root branching order determines bacterial composition, microbial carrying capacity and microbial filtering.

Fine roots vary dramatically in their functions, which range from resource absorption to within-plant resource transport. These differences should alter resource availability to root-associated microorganisms, yet most root microbiome studies involve fine root homogenization. We hypothesized that microbial filtering would be greatest in the most distal roots. To test this, we sampled roots of six temperate tree species from a 23-year-old common garden planting, separating by branching order. Rhizoplane bacterial composition was characterized with 16S rRNA gene sequencing, while bacterial abundance was determined on a subset of trees through flow cytometry. Root order strongly impacted composition across tree species, with absorptive lower order roots exerting the greatest selective pressure. Microbial carrying capacity was higher in absorptive roots in two of three tested tree species. This study indicates lower order roots as the main point of microbial interaction with fine roots, suggesting that root homogenization could mask microbial recruitment signatures.

Survey of Salmonella in raw tree nuts at retail in the United States.

The objective of this survey was to estimate the prevalence, contamination level, and genetic diversity of Salmonella in selected raw, shelled tree nuts (Brazil nuts, cashews, hazelnuts, macadamia nuts, pecans, pine nuts, pistachios, and walnuts) at retail markets in the United States. A total of 3,374 samples of eight tree nuts were collected from different types of retail stores and markets nationwide between September 2015 and March 2017. These samples (375 g) were analyzed using a modified FDA's BAM Salmonella culture method. Of the 3,374 samples, 15 (0.44%) (95% confidence interval [CI] [0.25, 0.73]) were culturally confirmed as containing Salmonella; 17 isolates were obtained. Among these isolates, there were 11 serotypes. Salmonella was not detected in Brazil nuts (296), hazelnuts (487), pecans (510), pine nuts (500), and walnuts (498). Salmonella prevalence estimates in cashews (510), macadamia (278), and pistachios (295) were 0.20% (95% CI [<0.01, 1.09]), 2.52% (95% CI [1.02, 5.12]), and 2.37% (95% CI [0.96, 4.83]), respectively. The rates of Salmonella isolation from major/big-chain supermarkets (1381), small-chain supermarkets (328), discount/variety/drug stores (1329), and online (336) were 0.29% (95% CI [0.08, 0.74]), 0.30% (95% CI [0.01, 1.69]), 0.45% (95% CI [0.17, 0.98]), and 1.19% (95% CI [0.33, 3.02]), respectively. Salmonella prevalence in organic (530) and conventional (2,844) nuts was not different statistically (P = 0.0601). Of the enumerated samples (15), 80% had Salmonella levels ≤0.0092 most probable number (MPN)/g. The highest contamination level observed was 0.75 MPN/g. The prevalence and contamination levels of Salmonella in the tree nuts analyzed were generally comparable to previous reports. Pulsed-field gel electrophoresis, serotype, and sequencing data all demonstrated that Salmonella population in nuts is very diverse genetically. PRACTICAL APPLICATION: The prevalence, contamination level, and genetic diversity of Salmonella in eight types of tree nuts (3,374 samples collected nationwide) revealed in this survey could help the development of mitigation strategies to reduce public health risks associated with consumption of these nuts.

Biocontrol and growth-promoting effect of Trichoderma asperellum TaspHu1 isolate from Juglans mandshurica rhizosphere soil.

To better apply the biocontrol agent Trichoderma spp. in Northeast China, collecting and screening more suitable native Trichoderma strains is necessary. In the present study, 10 isolates were obtained from Juglans mandshurica rhizosphere soils in Heilongjiang Province, and were identified as T. asperellum (four isolates), T. harzianum (four), T. hamatum (one), T. atroviride (one). The fastest-growing isolate per species on potato dextrose agar medium were further evaluated in stress tolerance tests (salt, alkali, nutritional stress, and low temperature) and confrontation assays (eight pathogens), which showed that T. asperellum TaspHu1 possessed the best adaptation and biological control ability. Then, Solanum lycopersicum (tomato) seeds were sown and treated with a series of concentrations of TaspHu1 spore suspension, as was unsown soil. Tomato seedlings treated by TaspHu1 had a significantly greater height, stem diameter, soluble protein content and soluble sugar content. Furthermore, their nitrate reductase activity and catalase activity were significantly increased, and these promoting effects depended on the concentration of the spore suspension. Meanwhile, a decrease in chlorophyll content was observed in the tomato seedlings treated with TaspHu1. In addition, strain TaspHu1 enhanced the tomato seedlings' absorption of available nitrogen, but did not influence the soil available nitrogen content. Furthermore, the resistance of tomato seedlings against Alternaria alternata was enhanced by TaspHu1 (smaller, fewer leaf spots), the seedlings' hormone signal transduction genes JAR1, MYC2, NPR1, PR1, and GH3.2 were highly expressed. Thus, TaspHu1 is a promising biocontrol candidate for use in agriculture and forestry.

Proteome Analysis of Walnut Bacterial Blight Disease.

The interaction between the plant host, walnut (Juglans regia; Jr), and a deadly pathogen (Xanthomonas arboricola pv. juglandis 417; Xaj) can lead to walnut bacterial blight (WB), which depletes walnut productivity by degrading the nut quality. Here, we dissect this pathosystem using tandem mass tag quantitative proteomics. Walnut hull tissues inoculated with Xaj were compared to mock-inoculated tissues, and 3972 proteins were identified, of which 3296 are from Jr and 676 from Xaj. Proteins with differential abundance include oxidoreductases, proteases, and enzymes involved in energy metabolism and amino acid interconversion pathways. Defense responses and plant hormone biosynthesis were also increased. Xaj proteins detected in infected tissues demonstrate its ability to adapt to the host microenvironment, limiting iron availability, coping with copper toxicity, and maintaining energy and intermediary metabolism. Secreted proteases and extracellular secretion apparatus such as type IV pilus for twitching motility and type III secretion effectors indicate putative factors recognized by the host. Taken together, these results suggest intense degradation processes, oxidative stress, and general arrest of the biosynthetic metabolism in infected nuts. Our results provide insights into molecular mechanisms and highlight potential molecular tools for early detection and disease control strategies.