Transcriptome profile of pecan scab resistant and susceptible trees from a pecan provenance collection.

Pecan scab is a devastating disease that causes damage to pecan (Carya illinoinensis (Wangenh.) K. Koch) fruit and leaves. The disease is caused by the fungus Venturia effusa (G. Winter) and the main management practice for controlling the disease is by application of fungicides at 2-to-3-week intervals throughout the growing season. Besides disease-related yield loss, application of fungicides can result in considerable cost and increases the likelihood of fungicide resistance developing in the pathogen. Resistant cultivars are available for pecan growers; although, in several cases resistance has been overcome as the pathogen adapts to infect resistant hosts. Despite the importance of host resistance in scab management, there is little information regarding the molecular basis of genetic resistance to pecan scab.The purpose of this study was to elucidate mechanisms of natural pecan scab resistance by analyzing transcripts that are differentially expressed in pecan leaf samples from scab resistant and susceptible trees. The leaf samples were collected from trees in a provenance collection orchard that represents the natural range of pecan in the US and Mexico. Trees in the orchard have been exposed to natural scab infections since planting in 1989, and scab ratings were collected over three seasons. Based on this data, ten susceptible trees and ten resistant trees were selected for analysis. RNA-seq data was collected and analyzed for diseased and non-diseased parts of susceptible trees as well as for resistant trees. A total of 313 genes were found to be differentially expressed when comparing resistant and susceptible trees without disease. For susceptible samples showing scab symptoms, 1,454 genes were identified as differentially expressed compared to non-diseased susceptible samples. Many genes involved in pathogen recognition, defense responses, and signal transduction were up-regulated in diseased samples of susceptible trees, whereas differentially expressed genes in pecan scab resistant samples were generally down-regulated compared to non-diseased susceptible samples.Our results provide the first account of candidate genes involved in resistance/susceptibility to pecan scab under natural conditions in a pecan orchard. This information can be used to aid pecan breeding programs and development of biotechnology-based approaches for generating pecan cultivars with more durable scab resistance.

Proteomic Analysis of Pecan (Carya illinoinensis) Nut Development.

Pecan (Carya illinoinensis) nuts are an economically valuable crop native to the United States and Mexico. A proteomic summary from two pecan cultivars at multiple time points was used to compare protein accumulation during pecan kernel development. Patterns of soluble protein accumulation were elucidated using qualitative gel-free and label-free mass-spectrometric proteomic analyses and quantitative (label-free) 2-D gel electrophoresis. Two-dimensional (2-D) gel electrophoresis distinguished a total of 1267 protein spots and shotgun proteomics identified 556 proteins. Rapid overall protein accumulation occurred in mid-September during the transition to the dough stage as the cotyledons enlarge within the kernel. Pecan allergens Car i 1 and Car i 2 were first observed to accumulate during the dough stage in late September. While overall protein accumulation increased, the presence of histones diminished during development. Twelve protein spots accumulated differentially based on 2-D gel analysis in the weeklong interval between the dough stage and the transition into a mature kernel, while eleven protein spots were differentially accumulated between the two cultivars. These results provide a foundation for more focused proteomic analyses of pecans that may be used in the future to identify proteins that are important for desirable traits, such as reduced allergen content, improved polyphenol or lipid content, increased tolerance to salinity, biotic stress, seed hardiness, and seed viability.

Comparative transcriptome analyses reveal insights into catkin bloom patterns in pecan protogynous and protandrous cultivars.

In perennial plants such as pecan, once reproductive maturity is attained, there are genetic switches that are regulated and required for flower development year after year. Pecan trees are heterodichogamous with both pistillate and staminate flowers produced on the same tree. Therefore, defining genes exclusively responsible for pistillate inflorescence and staminate inflorescence (catkin) initiation is challenging at best. To understand these genetic switches and their timing, this study analyzed catkin bloom and gene expression of lateral buds collected from a protogynous (Wichita) and a protandrous (Western) pecan cultivar in summer, autumn and spring. Our data showed that pistillate flowers in the current season on the same shoot negatively impacted catkin production on the protogynous 'Wichita' cultivar. Whereas fruit production the previous year on 'Wichita' had a positive effect on catkin production on the same shoot the following year. However, fruiting the previous year nor current year pistillate flower production had no significant effect on catkin production on 'Western' (protandrous cultivar) cultivar. The RNA-Seq results present more significant differences between the fruiting and non-fruiting shoots of the 'Wichita' cultivar compared to the 'Western' cultivar, revealing the genetic signals likely responsible for catkin production. Our data presented here, indicates the genes showing expression for the initiation of both types of flowers the season before bloom.

Influence of Geographical Orchard Location on the Microbiome from the Progeny of a Pecan Controlled Cross.

Carya illinoinensis (Wangenh.) K.Koch production has expanded beyond the native distribution as the genetic diversity of the species, in part, has allowed the trees to grow under broad geographic and climatic ranges. Research in other plant species has demonstrated that the phytobiome enhances their ability to survive and thrive in specific environments and, conversely, is influenced by the prevailing environment and plant genetics, among other factors. We sought to analyze the microbiota of pecan seedlings from the controlled cross 'Lakota' × 'Oaxaca' that were made in Georgia and Texas, respectively, to determine if the maternal geographical origin influences the microbiome of the resulting progeny. No significant differences in bacterial communities were observed between the seeds obtained from the two different states (p = 0.081). However, seed origin did induce significant differences in leaf fungal composition (p = 0.012). Results suggest that, in addition to some environmental, epigenetics, or host genetic components, ecological processes, such as dispersal mechanisms of the host, differentially impact the pecan microbiome, which may have ramifications for the health of trees grown in different environments. Future studies on the role of the microbiome in plant health and productivity will aid in the development of sustainable agriculture for improved food security.

Population Genetic Characteristics and Mating Type Frequency of Venturia effusa from Pecan in South America.

Scab, caused by the plant-pathogenic fungus Venturia effusa, is a major disease of pecan in South America, resulting in loss of quantity and quality of nut yield. Characteristics of the populations of V. effusa in South America are unknown. We used microsatellites to describe the genetic diversity and population structure of V. effusa in South America, and determined the mating type status of the pathogen. The four hierarchically sampled orchard populations from Argentina (AR), Brazil (BRC and BRS), and Uruguay (UR) had moderate to high genotypic and gene diversity. There was evidence of population differentiation (Fst = 0.196) but the correlation between geographic distance and genetic distance was not statistically significant. Genetic differentiation was minimal between the UR, BRC, and BRS populations, and these populations were more clearly differentiated from the AR population. The MAT1-1 and MAT1-2 mating types occurred in all four orchards and their frequencies did not deviate from the 1:1 ratio expected under random mating; however, multilocus linkage equilibrium was rejected in three of the four populations. The population genetics of South American populations of V. effusa has many similarities to the population genetics of V. effusa previously described in the United States. Characterizing the populations genetics and reproductive systems of V. effusa are important to establish the evolutionary potential of the pathogen and, thus, its adaptability-and can provide a basis for informed approaches to utilizing available host resistance and determining phytosanitary needs.

Evidence for Seed Transmission of Xylella fastidiosa in Pecan (Carya illinoinensis).

Pecan bacterial leaf scorch, caused by Xylella fastidiosa subsp. multiplex, is an economically significant disease of pecan with known detrimental effects on the yield of susceptible cultivars. In this study, endosperm was harvested from developing pecan seeds, and direct qPCR and sequencing were used to detect and confirm the presence of X. fastidiosa. DNA was isolated from mature seeds originating from seven trees, revealing a positivity rate up to 90%, and transmission of X. fastidiosa from infected seed to the germinated seedlings was found to be over 80%. Further epidemiological analyses were performed to determine where X. fastidiosa localizes in mature seed and seedlings. The highest concentrations of X. fastidiosa DNA were found in the hilum and outer integument of the seeds and the petioles, respectively. High-, medium-, and low-density seeds were harvested to determine the impact of the bacterium on seed density and seedling growth rate. The growth rate of seedlings originating from low-density seeds was significantly reduced compared to the medium- and high-density seeds. Despite the increased growth and germination rates, the high-density seed group had a greater proportion of samples that tested positive for the presence of X. fastidiosa by qPCR. The results demonstrate the ability of X. fastidiosa to colonize developing seeds and be efficiently transmitted from well-developed seeds to germinated seedlings. Continued research is needed to understand the plant-microbe interactions involved in the colonization of pecan seeds by X. fastidiosa and to develop effective phytosanitary approaches to reduce the risks posed by seed transmission.

Four chromosome scale genomes and a pan-genome annotation to accelerate pecan tree breeding.

Genome-enabled biotechnologies have the potential to accelerate breeding efforts in long-lived perennial crop species. Despite the transformative potential of molecular tools in pecan and other outcrossing tree species, highly heterozygous genomes, significant presence-absence gene content variation, and histories of interspecific hybridization have constrained breeding efforts. To overcome these challenges, here, we present diploid genome assemblies and annotations of four outbred pecan genotypes, including a PacBio HiFi chromosome-scale assembly of both haplotypes of the 'Pawnee' cultivar. Comparative analysis and pan-genome integration reveal substantial and likely adaptive interspecific genomic introgressions, including an over-retained haplotype introgressed from bitternut hickory into pecan breeding pedigrees. Further, by leveraging our pan-genome presence-absence and functional annotation database among genomes and within the two outbred haplotypes of the 'Lakota' genome, we identify candidate genes for pest and pathogen resistance. Combined, these analyses and resources highlight significant progress towards functional and quantitative genomics in highly diverse and outbred crops.

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.

Comment on "Evolutionary transitions between beneficial and phytopathogenic Rhodococcus challenge disease management".

We would like to address a number of concerns regarding this paper (Savory et al., 2017).

Complete Genome and Plasmid Sequences for Rhodococcus fascians D188 and Draft Sequences for Rhodococcus Isolates PBTS 1 and PBTS 2.

Rhodococcus fascians, a phytopathogen that alters plant development, inflicts significant losses in plant production around the world. We report here the complete genome sequence of R. fascians D188, a well-characterized model isolate, and Rhodococcus species PBTS (pistachio bushy top syndrome) 1 and 2, which were shown to be responsible for a disease outbreak in pistachios.

Phytophthora Species in Rivers and Streams of the Southwestern United States.

UNLABELLED: Phytophthora species were isolated from rivers and streams in the southwestern United States by leaf baiting and identified by sequence analysis of internal transcribed spacer (ITS) ribosomal DNA (rDNA). The major waterways examined included the Rio Grande River, Gila River, Colorado River, and San Juan River. The most prevalent species identified in rivers and streams were Phytophthora lacustris and P. riparia, both members of Phytophthora ITS clade 6. P. gonapodyides, P. cinnamomi, and an uncharacterized Phytophthora species in clade 9 were also recovered. In addition, six isolates recovered from the Rio Grande River were shown to be hybrids of P. lacustris × P. riparia Pathogenicity assays using P. riparia and P. lacustris failed to produce any disease symptoms on commonly grown crops in the southwestern United States. Inoculation of Capsicum annuum with P. riparia was shown to inhibit disease symptom development when subsequently challenged with P. capsici, a pathogenic Phytophthora species. IMPORTANCE: Many Phytophthora species are significant plant pathogens causing disease on a large variety of crops worldwide. Closer examinations of streams, rivers, and forest soils have also identified numerous Phytophthora species that do not appear to be phytopathogens and likely act as early saprophytes in aquatic and saturated environments. To date, the Phytophthora species composition in rivers and streams of the southwestern United States has not been evaluated. This article details a study to determine the identity and prevalence of Phytophthora species in rivers and streams located in New Mexico, Arizona, Colorado, Utah, and Texas. Isolated species were evaluated for pathogenicity on crop plants and for their potential to act as biological control agents.

BABA and Phytophthora nicotianae Induce Resistance to Phytophthora capsici in Chile Pepper (Capsicum annuum).

Induced resistance in plants is a systemic response to certain microorganisms or chemicals that enhances basal defense responses during subsequent plant infection by pathogens. Inoculation of chile pepper with zoospores of non-host Phytophthora nicotianae or the chemical elicitor beta-aminobutyric acid (BABA) significantly inhibited foliar blight caused by Phytophthora capsici. Tissue extract analyses by GC/MS identified conserved change in certain metabolite concentrations following P. nicotianae or BABA treatment. Induced chile pepper plants had reduced concentrations of sucrose and TCA cycle intermediates and increased concentrations of specific hexose-phosphates, hexose-disaccharides and amino acids. Galactose, which increased significantly in induced chile pepper plants, was shown to inhibit growth of P. capsici in a plate assay.

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.

Genetic analysis of a novel Xylella fastidiosa subspecies found in the southwestern United States.

Xylella fastidiosa, the causal agent of several scorch diseases, is associated with leaf scorch symptoms in Chitalpa tashkentensis, a common ornamental landscape plant used throughout the southwestern United States. For a number of years, many chitalpa trees in southern New Mexico and Arizona exhibited leaf scorch symptoms, and the results from a regional survey show that chitalpa trees from New Mexico, Arizona, and California are frequently infected with X. fastidiosa. Phylogenetic analysis of multiple loci was used to compare the X. fastidiosa infecting chitalpa strains from New Mexico, Arizona, and trees imported into New Mexico nurseries with previously reported X. fastidiosa strains. Loci analyzed included the 16S ribosome, 16S-23S ribosomal intergenic spacer region, gyrase-B, simple sequence repeat sequences, X. fastidiosa-specific sequences, and the virulence-associated protein (VapD). This analysis indicates that the X. fastidiosa isolates associated with infected chitalpa trees in the Southwest are a highly related group that is distinct from the four previously defined taxons X. fastidiosa subsp. fastidiosa (piercei), X. fastidiosa subsp. multiplex, X. fastidiosa subsp. sandyi, and X. fastidiosa subsp. pauca. Therefore, the classification proposed for this new subspecies is X. fastidiosa subsp. tashke.

BiP and zein binding domains within the delta zein protein.

Zeins are alcohol soluble seed storage proteins synthesized within the endosperm of maize and subsequently deposited into endoplasmic reticulum (ER) derived protein bodies. The genes encoding the beta and delta zeins were previously introduced into tobacco with the expectation of improving the nutritional quality of plants (Bagga et al. in Plant Physiol 107:13, 1997). Novel protein bodies are produced in the leaves of transgenic plants accumulating the beta or delta zein proteins. The mechanism of protein body formation within leaves is unknown. It is also unknown how zeins are retained in the ER since they do not contain known ER retention motifs. Retention may be due to an interaction of zeins with an ER chaperone such as binding luminal protein (BiP). We have demonstrated protein-protein interactions with the delta zeins, beta zeins, and BiP proteins using an E. coli two-hybrid system. In this study, four putative BiP binding motifs were identified within the delta zein protein using a BiP scoring program (Blond-Elguindi et al. in Cell 75:717, 1993). These putative binding motifs were mutated and their effects on protein interactions were analyzed in both a prokaryotic two-hybrid system and in plants. These mutations resulted in reduced BiP-zein protein interaction and also altered zein-zein interactions. Our results indicate that specific motifs are necessary for BiP-delta zein protein interactions and that there are specific motifs which are necessary for zein-zein interactions. Furthermore, our data demonstrates that zein proteins must be able to interact with BiP and zeins for their stability and ability to form protein bodies.