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

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

Structural and functional leaf diversity lead to variability in photosynthetic capacity across a range of Juglans regia genotypes.

Similar to other cropping systems, few walnut cultivars are used as scion in commercial production. Germplasm collections can be used to diversify cultivar options and hold potential for improving crop productivity, disease resistance and stress tolerance. In this study, we explored the anatomical and biochemical bases of photosynthetic capacity and response to water stress in 11 Juglans regia accessions in the U.S. department of agriculture, agricultural research service (USDA-ARS) National Clonal Germplasm. Net assimilation rate (An ) differed significantly among accessions and was greater in lower latitudes coincident with higher stomatal and mesophyll conductances, leaf thickness, mesophyll porosity, gas-phase diffusion, leaf nitrogen and lower leaf mass and stomatal density. High CO2 -saturated assimilation rates led to increases in An under diffusional and biochemical limitations. Greater An was found in lower-latitude accessions native to climates with more frost-free days, greater precipitation seasonality and lower temperature seasonality. As expected, water stress consistently impaired photosynthesis with the highest % reductions in lower-latitude accessions (A3, A5 and A9), which had the highest An under well-watered conditions. However, An for A3 and A5 remained among the highest under dehydration. J. regia accessions, which have leaf structural traits and biochemistry that enhance photosynthesis, could be used as commercial scions or breeding parents to enhance productivity.

Desiccation of the leaf mesophyll and its implications for CO2 diffusion and light processing.

Leaves balance CO2 and radiative absorption while maintaining water transport to maximise photosynthesis. Related species with contrasting leaf anatomy can provide insights into inherent and stress-induced links between structure and function for commonly measured leaf traits for important crops. We used two walnut species with contrasting mesophyll anatomy to evaluate these integrated exchange processes under non-stressed and drought conditions using a combination of light microscopy, X-ray microCT, gas exchange, hydraulic conductance, and chlorophyll distribution profiles through leaves. Juglans regia had thicker palisade mesophyll, higher fluorescence in the palisade, and greater low-mesophyll porosity that were associated with greater gas-phase diffusion (gIAS ), stomatal and mesophyll (gm ) conductances and carboxylation capacity. More and highly-packed mesophyll cells and bundle sheath extensions (BSEs) in Juglans microcarpa led to higher fluorescence in the spongy and in proximity to the BSEs. Both species exhibited drought-induced reductions in mesophyll cell volume, yet the associated increases in porosity and gIAS were obscured by declines in biochemical activity that decreased gm . Inherent differences in leaf anatomy between the species were linked to differences in gas exchange, light absorption and photosynthetic capacity, and drought-induced changes in leaf structure impacted performance via imposing species-specific limitations to light absorption, gas exchange and hydraulics.

Co-located quantitative trait loci mediate resistance to Agrobacterium tumefaciens, Phytophthora cinnamomi, and P. pini in Juglans microcarpa × J. regia hybrids.

Soil-borne plant pathogens represent a serious threat that undermines commercial walnut (Juglans regia) production worldwide. Crown gall, caused by Agrobacterium tumefaciens, and Phytophthora root and crown rots, caused by various Phytophthora spp., are among the most devastating walnut soil-borne diseases. A recognized strategy to combat soil-borne diseases is adoption of resistant rootstocks. Here, resistance to A. tumefaciens, P. cinnamomi, and P. pini is mapped in the genome of Juglans microcarpa, a North American wild relative of cultivated walnut. Half-sib J. microcarpa mother trees DJUG 31.01 and DJUG 31.09 were crossed with J. regia cv. Serr, producing 353 and 400 hybrids, respectively. Clonally propagated hybrids were genotyped by sequencing to construct genetic maps for the two populations and challenged with the three pathogens. Resistance to each of the three pathogens was mapped as a major QTL on the long arm of J. microcarpa chromosome 4D and was associated with the same haplotype, designated as haplotype b, raising the possibility that the two mother trees were heterozygous for a single Mendelian gene conferring resistance to all three pathogens. The deployment of this haplotype in rootstock breeding will facilitate breeding of a walnut rootstock resistant to both crown gall and Phytophthora root and crown rots.

Predicting Stomatal Closure and Turgor Loss in Woody Plants Using Predawn and Midday Water Potential.

Knowledge about physiological stress thresholds provides crucial information about plant performance and survival under drought. In this study, we report on the triphasic nature of the relationship between plant water potential (Ψ) at predawn and midday and describe a method that predicts Ψ at stomatal closure and turgor loss exclusively from this water potential curve (WP curve). The method is based on a piecewise linear regression model that was developed to predict the boundaries (termed Θ1 and Θ2) separating the three phases of the curve and corresponding slope values. The method was tested for three economically important woody species. For all species, midday Ψ was much more negative than predawn Ψ during phase I (mild drought), reductions in midday Ψ were minor while predawn Ψ continued to decline during phase II (moderate drought), and midday and predawn Ψ reached similar values during phase III (severe drought). Corresponding measurement of leaf gas exchange indicated that boundary Θ1 between phases I and II coincided with Ψ at stomatal closure. Data from pressure-volume curves demonstrated that boundary Θ2 between phases II and III predicted Ψ at leaf turgor loss. The WP curve method described here is an advanced application of the Scholander-type pressure chamber to categorize plant dehydration under drought into three distinct phases and to predict Ψ thresholds of stomatal closure and turgor loss.

Temporal Responses of Microbial Communities to Anaerobic Soil Disinfestation.

Anaerobic soil disinfestation (ASD) is an organic amendment-based management tool for controlling soil-borne plant diseases and is increasingly used in a variety of crops. ASD results in a marked decrease in soil redox potential and other physicochemical changes, and a turnover in the composition of the soil microbiome. Mechanisms of ASD-mediated pathogen control are not fully understood, but appear to depend on the carbon source used to initiate the process and involve a combination of biological (i.e., release of volatile organic compounds) and abiotic (i.e., lowered pH, release of metal ions) factors. In this study, we examined how the soil microbiome changes over time in response to ASD initiated with rice bran, tomato pomace, or red grape pomace as amendments using growth chamber mesocosms that replicate ASD-induced field soil redox conditions. Within 2 days, the soil microbiome rapidly shifted from a diverse assemblage of taxa to being dominated by members of the Firmicutes for all ASD treatments, whereas control mesocosms maintained diverse and more evenly distributed communities. Rice bran and tomato pomace amendments resulted in microbial communities with similar compositions and trajectories that were different from red grape pomace communities. Quantitative PCR showed nitrogenase gene abundances were higher in ASD communities and tended to increase over time, suggesting the potential for altering soil nitrogen availability. These results highlight the need for temporal and functional studies to understand how pathogen suppressive microbial communities assemble and function in ASD-treated soils.

Complete Genome Sequences of Brenneria rubrifaciens Strain 6D370 and Brenneria nigrifluens Strain ATCC 13028, Causative Agents of Bark Cankers in Walnut.

Brenneria rubrifaciens and Brenneria nigrifluens, respectively, cause deep and shallow bark canker disease in walnut. B. rubrifaciens exhibits quorum sensing-controlled virulence and rubrifacine pigment production. The complete genome sequences of these species will be useful for studying the role of genes regulated by quorum sensing, including pathways mediating pathogenesis.

Variations in xylem embolism susceptibility under drought between intact saplings of three walnut species.

A germplasm collection containing varied Juglans genotypes holds potential to improve drought resistance of plant materials for commercial production. We used X-ray computed microtomography to evaluate stem xylem embolism susceptibility/repair in relation to vessel anatomical features (size, arrangement, connectivity and pit characteristics) in 2-year-old saplings of three Juglans species. In vivo analysis revealed interspecific variations in embolism susceptibility among Juglans microcarpa, J. hindsii (both native to arid habitats) and J. ailantifolia (native to mesic habitats). Stem xylem of J. microcarpa was more resistant to drought-induced embolism as compared with J. hindsii and J. ailantifolia (differences in embolism susceptibility among older and current year xylem were not detected in any species). Variations in most vessel anatomical traits were negligible among the three species; however, we detected substantial interspecific differences in intervessel pit characteristics. As compared with J. hindsii and J. ailantifolia, low embolism susceptibility in J. microcarpa was associated with smaller pit size in larger diameter vessels, a smaller area of the shared vessel wall occupied by pits, lower pit frequency and no changes in pit characteristics as vessel diameters increased. Changes in amount of embolized vessels following 40 days of re-watering were minor in intact saplings of all three species highlighting that an embolism repair mechanism did not contribute to drought recovery. In conclusion, our data indicate that interspecific variations in drought-induced embolism susceptibility are associated with species-specific pit characteristics, and these traits may provide a future target for breeding efforts aimed at selecting walnut germplasm with improved drought resistance.

Draft Genome Sequence of Agrobacterium tumefaciens Biovar 1 Strain 186, Isolated from Walnut.

Agrobacterium tumefaciens biovar 1 strain 186 was isolated from a walnut tree expressing crown gall symptoms. The draft genome sequence of this strain harbored genes for crown gall formation and will be useful for understanding its virulence on Paradox, the predominant hybrid rootstock used for the cultivation of English walnut in California.

Patterns of drought-induced embolism formation and spread in living walnut saplings visualized using X-ray microtomography.

Embolism formation and spread are dependent on conduit structure and xylem network connectivity. Detailed spatial analysis has been limited due to a lack of non-destructive methods to visualize these processes in living plants. We used synchrotron X-ray computed tomography (microCT) to visualize these processes in vivo for Juglans microcarpa Berl. saplings subjected to drought, and also evaluated embolism repair capability after re-watering. Cavitation was not detected in vivo until stem water potentials (Ψ(stem)) reached -2.2 MPa, and loss of stem hydraulic conductivity as derived from microCT images predicted that 50% of conductivity was lost at Ψ(stem) of ∼ -3.5 MPa; xylem vulnerability as determined with the centrifuge method was comparable only in the range of Ψ(stem) from -2.5 to -3.5 MPa. MicroCT images showed that cavitation appeared initially in isolated vessels not connected to other air-filled conduits. Once embolized vessels were present, multiple vessels in close proximity cavitated, and 3-D analysis along the stem axis revealed some connections between cavitated vessels. A tomography-derived automated xylem network analysis found that only 36% of vessels had one or more connections to other vessels. Cavitation susceptibility was related to vessel diameter, with large diameter vessels (>40 µm, mean diameter 25-30 µm) cavitating mainly under moderate stress (Ψ(stem) > -3 MPa) and small diameter vessels (<30 µm) under severe stress. After re-watering there was no evidence for short or longer term vessel refilling over 2 weeks despite a rapid recovery of plant water status. The low embolism susceptibility in 1-year-old J. microcarpa may aid sapling survival during establishment.

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.

AHL signals induce rubrifacine production in a bruI mutant of Brenneria rubrifaciens.

Several members of the bacterial genus Brenneria are pathogenic on different tree species. Cell-free extracts from the bacterial phytopathogens Brenneria rubrifaciens, B. salicis, and B. nigrifluens induced production of the red pigment rubrifacine in the B. rubrifaciens bruI insertional mutant Br-212. Analysis of the bruI locus identified an adjacent open reading frame, designated bruR, with homology to luxR. High-performance liquid chromatography and mass spectrometry analysis of ethyl acetate extracts from wild-type B. rubrifaciens and Escherichia coli expressing the bruI gene identified two acyl homoserine lactone (AHL) peaks, N-(3-oxohexanoyl)-homoserine lactone (3OC6HSL) and N-hexanoyl-homoserine lactone (C6HSL). Addition of synthetic 3OC6HSL and C6HSL at 10 µM to the bruI mutant, strain Br-212, induced rubrifacine production and the ability to elicit a hypersensitive reaction (HR) in tobacco leaves. Synthetic C6HSL was less effective at inducing pigment production than 3OC6HSL at 10 µM. The bruI mutant Br-212 did not produce detectable AHLs, indicating that C6HSL and 3OC6HSL are the major AHLs produced by this species. The AHLs N-heptanoyl-DL-homoserine lactone (C7HSL), N-octanoyl-DL-homoserine lactone (C8HSL), and N-(3-oxooctanoyl)-DL-homoserine lactone (3OC8HSL) also induced pigment production in Br-212 and restored its ability to elicit an HR in tobacco, suggesting that cross-talk with other bacterial species may be possible.

The role of tyloses in crown hydraulic failure of mature walnut trees afflicted by apoplexy disorder.

In the Central Valley of California, mature walnut trees afflicted with apoplexy disorder exhibit rapid and complete crown defoliation within a few weeks of symptom initiation. Symptoms are typically found throughout the entire crown and are initially expressed as wilting and chlorosis followed by scorching of leaves. Since the cause of apoplexy disorder is unknown, we set out to elucidate the water relations physiology underlying this condition. Stem water potential (Psi(s)) of healthy, asymptomatic trees remained high throughout the 2007 growing season, while that of apoplexy-afflicted trees decreased significantly with the onset of symptoms for both healthy-appearing and symptomatic portions of these trees. Psi(s)s were significantly reduced by at least 0.7 MPa in the lower, middle and upper portions of the symptomatic canopies compared with those from asymptomatic trees. Heat pulse velocities measured in the main trunk at three radial depths consistently decreased prior to the onset of symptoms and eventually reached zero with complete crown defoliation. Comparison of sap flow with predicted water use based on walnut evapotranspiration suggests that stomata of symptomatic trees were closing at higher evaporative demand prior to symptom formation. Specific hydraulic conductivity (K(s)) of symptomatic stems was significantly lower than that of asymptomatic stems, and no detectable K(s) could be measured on several of the symptomatic stem samples. However, shallow root K(s) did not significantly differ between symptomatic and asymptomatic trees, suggesting that hydraulic failure was isolated to the crown of these grafted trees. Light and scanning electron microscopy of stem and trunk sapwood revealed extensive tylose development in vessels throughout the crown of symptomatic trees. Analysis of the formation of tyloses on multiple dates revealed rapid development of these vessel occlusions in conjunction with visual symptom formation and dramatic decreases in sap flow. In 2008, tylose development was associated with elevated ethylene production in the active sapwood of symptomatic trees. The cause of elevated ethylene associated with tylose production and symptom formation is yet to be determined.

Genetic loci involved in rubrifacine production in the walnut pathogen Brenneria rubrifaciens.

Brenneria rubrifaciens produces a unique red pigment known as rubrifacine that has been hypothesized to play a role in pathogenesis on walnut. Analysis of DNA flanking the Tn5 insertion site in 20 rubrifacine minus (pig(-)) mutants identified three regions required for rubrifacine production. The first region was homologous to nonribosomal peptide synthetases (NRPS), the second was homologous to autoinducer synthase genes (expI homologs), and the third region was homologous to the slyA gene of Candidatus blochmania and Escherichia coli. Pigment production was not necessary for elicitation of the hypersensitive response (HR) in tobacco and had little impact on virulence in tissue-cultured walnut plants. The expI-interrupted mutants exhibited reduced virulence on walnut and were HR negative on tobacco. Pigment production was restored in Br-212 when grown in the presence of wild-type B. rubrifaciens, E. coli carrying the cloned expI-like gene, or introduction of the cloned wild-type copy of the expI-like gene. Two Brenneria spp., B. nigrifluens and B. salicis, also restored pigment production in Br-212. These results demonstrate that rubrifacine production and virulence of B. rubrifaciens on walnut are under the control of a quorum-sensing system and are sensitive to signal molecules from other Brenneria spp.

Relationship between avirulence gene (avrA) diversity in Ralstonia solanacearum and bacterial wilt incidence.

Bacterial wilt, caused by Ralstonia solanacearum, is a serious disease of tobacco in North and South Carolina. In contrast, the disease rarely occurs on tobacco in Georgia and Florida, although bacterial wilt is a common problem on tomato. We investigated whether this difference in disease incidence could be explained by qualitative characteristics of avirulence gene avrA in the R. solanacearum population in the southeastern United States. Sequence analysis established that wild-type avrA has a 792-bp open reading frame. Polymerase chain reaction (PCR) amplification of avrA from 139 R. solanacearum strains generated either 792-bp or approximately 960-bp DNA fragments. Strains that elicited a hypersensitive reaction (HR) on tobacco contained the 792-bp allele, and were pathogenic on tomato and avirulent on tobacco. All HR-negative strains generated a approximately 960-bp DNA fragment, and wilted both tomato and tobacco. The DNA sequence of avrA in six HR-negative strains revealed the presence of one of two putative miniature inverted-repeat transposable elements (MITEs): a 152-bp MITE between nucleotides 542 and 543, or a 170-bp MITE between nucleotides 461 and 462 or 574 and 575. Southern analysis suggested that the 170-bp MITE is unique to strains from the southeastern United States and the Caribbean. Mutated avrA alleles were present in strains from 96 and 75% of North and South Carolina sites, respectively, and only in 13 and 0% of the sites in Georgia and Florida, respectively. Introduction of the wildtype allele on a plasmid into four HR-negative strains reduced their virulence on both tobacco and tomato. Inactivation of avrA in an HR-positive, avirulent strain, resulted in a mutant that was weakly virulent on tobacco. Thus, the incidence of bacterial wilt of tobacco in the southeastern United States is partially explained by which avrA allele dominates the local R. solanacearum population.