Functional Genomics Insights Into the Pathogenicity, Habitat Fitness, and Mechanisms Modifying Plant Development of Rhodococcus sp. PBTS1 and PBTS2.

Pistachio Bushy Top Syndrome (PBTS) is a recently emerged disease that has strongly impacted the pistachio industry in California, Arizona, and New Mexico. The disease is caused by two bacteria, designated PBTS1 that is related to Rhodococcus corynebacterioides and PBTS2 that belongs to the species R. fascians. Here, we assessed the pathogenic character of the causative agents and examined their chromosomal sequences to predict the presence of particular functions that might contribute to the observed co-occurrence and their effect on plant hosts. In diverse assays, we confirmed the pathogenicity of the strains on "UCB-1" pistachio rootstock and showed that they can also impact the development of tobacco species, but concurrently inconsistencies in the ability to induce symptoms were revealed. We additionally evidence that fas genes are present only in a subpopulation of pure PBTS1 and PBTS2 cultures after growth on synthetic media, that these genes are easily lost upon cultivation in rich media, and that they are enriched for in an in planta environment. Analysis of the chromosomal sequences indicated that PBTS1 and PBTS2 might have complementary activities that would support niche partitioning. Growth experiments showed that the nutrient utilization pattern of both PBTS bacteria was not identical, thus avoiding co-inhabitant competition. PBTS2 appeared to have the potential to positively affect the habitat fitness of PBTS1 by improving its resistance against increased concentrations of copper and penicillins. Finally, mining the chromosomes of PBTS1 and PBTS2 suggested that the bacteria could produce cytokinins, auxins, and plant growth-stimulating volatiles and that PBTS2 might interfere with ethylene levels, in support of their impact on plant development. Subsequent experimentation supported these in silico predictions. Altogether, our data provide an explanation for the observed pathogenic behavior and unveil part of the strategies used by PBTS1 and PBTS2 to interact with plants.

Characterization of Lilium longiflorum cv. 'Nellie White' Infection with Root-lesion Nematode Pratylenchus penetrans by Bright-field and Transmission Electron Microscopy.

Lilium longiflorum cv. Nellie White, commonly known as Easter lily, is an important floral crop with an annual wholesale value of over $26 million in the United States. The root-lesion nematode, Pratylenchus penetrans, is a major pest of lily due to the significant root damage it causes. In this study, we investigated the cytological aspects of this plant-nematode interaction using bright-field and transmission electron microscopy. We took advantage of an in vitro culture method to multiply lilies and follow the nematode infection over time. Phenotypic reactions of roots inoculated with P. penetrans were evaluated from 0 to 60 d after nematode infection. Symptom development progressed from initial randomly distributed discrete necrotic areas to advanced necrosis along entire roots of each inoculated plant. A major feature characterizing this susceptible host response to nematode infection was the formation of necrosis, browning, and tissue death involving both root epidermis and cortical cells. Degradation of consecutive cell walls resulted in loss of cell pressure, lack of cytoplasmic integrity, followed by cell death along the intracellular path of the nematode's migration. Pratylenchus penetrans was never seen in the vascular cylinder as the layer of collapsed endodermal cells presumably blocked the progression of nematodes into this area of the roots. This study presents the first detailed cytological characterization of P. penetrans infection of Easter lily plants.

High-Indexed Pt3Ni Alloy Tetrahexahedral Nanoframes Evolved through Preferential CO Etching.

Chemically controlling crystal structures in nanoscale is challenging, yet provides an effective way to improve catalytic performances. Pt-based nanoframes are a new class of nanomaterials that have great potential as high-performance catalysts. To date, these nanoframes are formed through acid etching in aqueous solutions, which demands long reaction time and often yields ill-defined surface structures. Herein we demonstrate a robust and unprecedented protocol for facile development of high-performance nanoframe catalysts using size and crystallographic facet-controlled PtNi4 tetrahexahedral nanocrystals prepared through a colloidal synthesis approach as precursors. This new protocol employs the Mond process to preferentially dealloy nickel component in the ⟨100⟩ direction through carbon monoxide etching of carbon-supported PtNi4 tetrahexahedral nanocrystals at an elevated temperature. The resultant Pt3Ni alloy tetrahexahedral nanoframes possess an open, stable, and high-indexed microstructure, containing a segregated Pt thin layer strained to the Pt-Ni alloy surfaces and featuring a down-shift d-band center as revealed by the density functional theory calculations. These nanoframes exhibit much improved catalytic performance, such as high stability under prolonged electrochemical potential cycles, promoting direct electro-oxidation of formic acid to carbon dioxide and enhancing oxygen reduction reaction activities. Because carbon monoxide can be generated from the carbon support through thermal annealing in air, a common process for pretreating supported catalysts, the developed approach can be easily adopted for preparing industrial scale catalysts that are made of Pt-Ni and other alloy nanoframes.

Independent origins of diploidy in Metarhizium.

In fungi, stable diploid genome arrangements are rare. Here we present evidence from nuclear intergenic DNA sequencing, microsatellite genotyping, and configuration of the mating-type locus to demonstrate two independent origins of persistent diploid genome organization in the Metarhizium majus species complex. Most taxa in the complex are genotypically haploid, with individual isolates consistently displaying a single allele across all nuclear loci, as well as having a single mating-type locus. In contrast, individuals of M. majus and the clade designated here MGT1 are shown to be diploid, based on a consistent finding of heterozygosity and the presence of both MAT1 and MAT2 mating-type loci. In single locus phylogenies, nuclear intergenic alleles of M. majus and MGT1 each form monophyletic groups, indicating that diploidy in both taxa likely originated by the union of conspecific individuals. Sequence divergence in the APN2/MAT1-1-3 and APN2/MAT2-1 intergenic spacers indicate the two MAT loci are physically separated in the genomes of both diploid taxa, although the linkage relationship of the MAT loci to one another is unknown. The presence of both mating genes in a single nucleus suggests these diploid genomes may represent a mating event that failed to complete meiosis. Whether or not these isolates are able to complete the sexual cycle under any conditions and form ascospores remains an open question.

Comparison of helper component-protease RNA silencing suppression activity, subcellular localization, and aggregation of three Korean isolates of Turnip mosaic virus.

In 2014, we performed a nationwide survey in Korean radish fields to investigate the distribution and variability of Turnip mosaic virus (TuMV). Brassica rapa ssp. pekinensis sap-inoculated with three isolates of TuMV from infected radish tissue showed different symptom severities, whereas symptoms in Raphanus sativus were similar for each isolate. The helper component-protease (HC-Pro) genes of each isolate were sequenced, and phylogenetic analysis showed that the three Korean isolates were clustered into the basal-BR group. The HC-Pro proteins of these isolates were tested for their RNA silencing suppressor (VSR) activity and subcellular localization in Nicotiana benthamiana. A VSR assay by co-agroinfiltration of HC-Pro with soluble-modified GFP (smGFP) showed that HC-Pro of isolate R007 and R041 showed stronger VSR activity than R065. The HC-Pros showed 98.25 % amino acid identity, and weak VSR isolate (R065) has a single variant residue in the C-terminal domain associated with protease activity and self-interaction compared to isolates with strong VSR activity. Formation of large subcellular aggregates of GFP:HC-Pro fusion proteins in N. benthamiana was only observed for HC-Pro from isolates with strong VSR activity, suggesting that R065 'weak' HC-Pro may have diminished self-association; substitution of the variant C-terminal residue largely reversed the HC-Pro aggregation and silencing suppressor characteristics. The lack of correlation between VSR efficiency and induction of systemic necrosis (SN) suggests that differences in viral accumulation due to HC-Pro are not responsible for SN.

Correlations of carotenoid content and transcript abundances for fibrillin and carotenogenic enzymes in Capsicum annum fruit pericarp.

The fruits of Capsicum spp. are especially rich sites for carotenoid synthesis and accumulation, with cultivar-specific carotenoid accumulation profiles. Differences in chromoplast structure as well as carotenoid biosynthesis are correlated with distinct carotenoid accumulations and fruit color. In the present study, the inheritance of chromoplast shape, carotenoid accumulation profiles, and transcript levels of four genes were measured. Comparisons of these traits were conducted using fruit from contrasting variants, Costeño Amarillo versus Costeño Red, and from F1 hybrids; crosses between parental lines with novel versions of these traits. Intermediate chromoplast shapes were observed in the F1, but no association between specific carotenoid accumulation and chromoplast shape was detected. Increased total carotenoid content was associated with increased ß-carotene and violaxanthin content. Transcript levels for phytoene synthase (Psy) and ß-carotene hydroxylase (CrtZ-2) were positively correlated with increased levels of specific carotenoids. No correlation was detected between transcript levels of capsanthin/capsorubin synthase (Ccs) and carotenoid composition or chromoplast shape. Transcript levels of fibrillin, were differentially correlated with specific carotenoids, negatively correlated with accumulation of capsanthin, and positively correlated with violaxanthin. The regulation of carotenoid accumulation in chromoplasts in Capsicum fruit continues to be a complex process with multiple steps for control.

First Report of Rhodococcus Isolates Causing Pistachio Bushy Top Syndrome on 'UCB-1' Rootstock in California and Arizona.

'UCB-1' (Pistacia atlantica × Pistacia integerrima) rootstock is a hybrid cultivar widely used by the U.S. pistachio industry. In the last three years, a large number of micropropagated UCB-1 pistachio rootstocks planted in California and Arizona orchards exhibited shortened internodes, stunted growth, swollen lateral buds, bushy/bunchy growth, stem galls with multiple buds, and twisted roots with minimal lateral branching. Field T-budding success in affected orchards was reduced to approximately 30% with unusual bark cracking often observed around the bud-union. The percentage of abnormal rootstocks within affected orchards varied from 10 to 90%. We have termed the cumulative symptoms "pistachio bushy top syndrome" (PBTS) to describe these affected trees. Two isolates, both containing virulence factors from the phytopathogen Rhodococcus fascians, were identified on symptomatic trees in field and nursery samples. Micropropagated UCB-1 trees inoculated with the Rhodococcus isolates exhibited stunted growth, shortened internode length, swollen lateral buds, sylleptic branching, and differences in root morphology, compared with control UCB-1 trees. To our knowledge, this is the first report of Rhodococcus isolates, related to Rhodococcus fascians, causing disease on a commercial tree crop and the results presented indicate that this organism is responsible at least in part for PBTS in California and Arizona.

Carotenoid Extraction and Quantification from Capsicum annuum.

Carotenoids are ubiquitous pigments that play key roles in photosynthesis and also accumulate to high levels in fruit and flowers. Specific carotenoids play essential roles in human health as these compounds are precursors for Vitamin A; other specific carotenoids are important sources of macular pigments and all carotenoids are important anti-oxidants. Accurate determination of the composition and concentration of this complex set of natural products is therefore important in many different scientific areas. One of the richest sources of these compounds is the fruit of Capsicum; these red, yellow and orange fruit accumulate multiple carotenes and xanthophylls. This report describes the detailed method for the extraction and quantification of specific carotenes and xanthophylls.

Multiple microscopic approaches demonstrate linkage between chromoplast architecture and carotenoid composition in diverse Capsicum annuum fruit.

Increased accumulation of specific carotenoids in plastids through plant breeding or genetic engineering requires an understanding of the limitations that storage sites for these compounds may impose on that accumulation. Here, using Capsicum annuum L. fruit, we demonstrate directly the unique sub-organellar accumulation sites of specific carotenoids using live cell hyperspectral confocal Raman microscopy. Further, we show that chromoplasts from specific cultivars vary in shape and size, and these structural variations are associated with carotenoid compositional differences. Live-cell imaging utilizing laser scanning confocal (LSCM) and confocal Raman microscopy, as well as fixed tissue imaging by scanning and transmission electron microscopy (SEM and TEM), all demonstrated morphological differences with high concordance for the measurements across the multiple imaging modalities. These results reveal additional opportunities for genetic controls on fruit color and carotenoid-based phenotypes.