Assessment and comparison of rhizosphere communities in cultivated Vaccinium spp. provide a baseline for study of causative agents in decline.

It has long been recognized that the community of organisms associated with plant roots is a critical component of the phytobiome and can directly or indirectly contribute to the overall health of the plant. The rhizosphere microbial community is influenced by a number of factors including the soil type, the species of plants growing in those soils, and in the case of cultivated plants, the management practices associated with crop production. Vaccinium species, such as highbush blueberry and American cranberry, are woody perennials that grow in sandy, acidic soils with low to moderate levels of organic matter and a paucity of nutrients. When properly maintained, fields planted with these crops remain productive for many years. In some cases, however, yields and fruit quality decline over time, and it is suspected that degenerating soil health and/or changes in the rhizosphere microbiome are contributing factors. Determining the assemblage of bacterial and fungal microorganisms typically associated with the rhizosphere of these crops is a critical first step toward addressing the complex issue of soil health. We hypothesized that since blueberry and cranberry are in the same genus and grow in similar soils, that their associated rhizosphere microbial communities would be similar to each other. We analyzed the eukaryotic (primarily fungal) and bacterial communities from the rhizosphere of representative blueberry and cranberry plants growing in commercial fields in New Jersey. The data presented herein show that while the bacterial communities between the crops is very similar, the fungal communities associated with each crop are quite different. These results provide a framework for examining microbial components that might contribute to the health of Vaccinium spp. crops in New Jersey and other parts of the northeastern U.S.

Molecular and behavioral studies reveal differences in olfaction between winter and summer morphs of Drosophila suzukii.

Spotted-wing drosophila, Drosophila suzukii (Matsumura), is a major economic pest of several fruit crops in Europe, North and South America, and other parts of the world because it oviposits in ripening thin-skinned fruits. This vinegar fly exhibits two distinct morphotypes: a summer and a winter morph. Although adaptations associated with the winter morph enhance this invasive pest's capacity to survive in cold climates, winter is still a natural population bottleneck. Since monitoring early spring populations is important for accurate population forecasts, understanding the winter morph's response to olfactory cues may improve current D. suzukii management programs. In this study, a comparative transcriptome analysis was conducted to assess gene expression differences between the female heads of the two D. suzukii morphs, which showed significant differences in 738 genes (p ≤ 0.0001). Out of twelve genes related to olfaction determined to be differentially expressed in the transcriptome, i.e., those related to location of food sources, chemosensory abilities, and mating behavior, nine genes were upregulated in the winter morph while three were downregulated. Three candidate olfactory-related genes that were most upregulated or downregulated in the winter morph were further validated using RT-qPCR. In addition, behavioral assays were performed at a range of temperatures to confirm a differing behavioral response of the two morphs to food odors. Our behavioral assays showed that, although winter morphs were more active at lower temperatures, the summer morphs were generally more attracted to food odors. This study provides new insights into the molecular and behavioral differences in response to olfactory cues between the two D. suzukii morphs that will assist in formulating more effective monitoring and physiological-based control tools.

Contrasting a reference cranberry genome to a crop wild relative provides insights into adaptation, domestication, and breeding.

Cranberry (Vaccinium macrocarpon) is a member of the Heath family (Ericaceae) and is a temperate low-growing woody perennial native to North America that is both economically important and has significant health benefits. While some native varieties are still grown today, breeding programs over the past 50 years have made significant contributions to improving disease resistance, fruit quality and yield. An initial genome sequence of an inbred line of the wild selection 'Ben Lear,' which is parent to multiple breeding programs, provided insight into the gene repertoire as well as a platform for molecular breeding. Recent breeding efforts have focused on leveraging the circumboreal V. oxycoccos, which forms interspecific hybrids with V. macrocarpon, offering to bring in novel fruit chemistry and other desirable traits. Here we present an updated, chromosome-resolved V. macrocarpon reference genome, and compare it to a high-quality draft genome of V. oxycoccos. Leveraging the chromosome resolved cranberry reference genome, we confirmed that the Ericaceae has undergone two whole genome duplications that are shared with blueberry and rhododendron. Leveraging resequencing data for 'Ben Lear' inbred lines, as well as several wild and elite selections, we identified common regions that are targets of improvement. These same syntenic regions in V. oxycoccos, were identified and represent environmental response and plant architecture genes. These data provide insight into early genomic selection in the domestication of a native North American berry crop.

A ribosomal operon database and MegaBLAST settings for strain-level resolution of microbiomes.

Current methods to characterize microbial communities generally employ sequencing of the 16S rRNA gene (<500 bp) with high accuracy (∼99%) but limited phylogenetic resolution. However, long-read sequencing now allows for the profiling of near-full-length ribosomal operons (16S-ITS-23S rRNA genes) on platforms such as the Oxford Nanopore MinION. Here, we describe an rRNA operon database with >300 ,000 entries, representing >10 ,000 prokaryotic species and ∼ 150, 000 strains. Additionally, BLAST parameters were identified for strain-level resolution using in silico mutated, mock rRNA operon sequences (70-95% identity) from four bacterial phyla and two members of the Euryarchaeota, mimicking MinION reads. MegaBLAST settings were determined that required <3 s per read on a Mac Mini with strain-level resolution for sequences with >84% identity. These settings were tested on rRNA operon libraries from the human respiratory tract, farm/forest soils and marine sponges ( n = 1, 322, 818 reads for all sample sets). Most rRNA operon reads in this data set yielded best BLAST hits (95 ± 8%). However, only 38-82% of library reads were compatible with strain-level resolution, reflecting the dominance of human/biomedical-associated prokaryotic entries in the database. Since the MinION and the Mac Mini are both portable, this study demonstrates the possibility of rapid strain-level microbiome analysis in the field.

Application of Plant Defense Elicitors Fails to Enhance Herbivore Resistance or Mitigate Phytoplasma Infection in Cranberries.

Synthetic elicitors of the salicylic acid (SA) and jasmonic acid (JA) plant defense pathways can be used to increase crop protection against herbivores and pathogens. In this study, we tested the hypothesis that elicitors of plant defenses interact with pathogen infection to influence crop resistance against vector and nonvector herbivores. To do so, we employed a trophic system comprising of cranberries (Vaccinium macrocarpon), the phytoplasma that causes false blossom disease, and two herbivores-the blunt-nosed leafhopper (Limotettix vaccinii), the vector of false blossom disease, and the nonvector gypsy moth (Lymantria dispar). We tested four commercial elicitors, including three that activate mainly SA-related plant defenses (Actigard, LifeGard, and Regalia) and one activator of JA-related defenses (Blush). A greenhouse experiment in which phytoplasma-infected and uninfected plants received repeated exposure to elicitors revealed that both phytoplasma infection and elicitor treatment individually improved L. vaccinii and L. dispar mass compared to uninfected, untreated controls; however, SA-based elicitor treatments reduced L. vaccinii mass on infected plants. Regalia also improved L. vaccinii survival. Phytoplasma infection reduced plant size and mass, increased levels of nitrogen (N) and SA, and lowered carbon/nitrogen (C/N) ratios compared to uninfected plants, irrespective of elicitor treatment. Although none of our elicitor treatments influenced transcript levels of a phytoplasma-specific marker gene, all of them increased N and reduced C/N levels; the three SA activators also reduced JA levels. Taken together, our findings reveal positive effects of both phytoplasma infection and elicitor treatment on the performance of L. vaccinii and L. dispar in cranberries, likely via enhancement of plant nutrition and changes in phytohormone profiles, specifically increases in SA levels and corresponding decreases in levels of JA. Thus, we found no evidence that the tested elicitors of plant defenses increase resistance to insect herbivores or reduce disease incidence in cranberries.

Transcriptome analysis identifies genes related to the waxy coating on blueberry fruit in two northern-adapted rabbiteye breeding populations.

BACKGROUND: Blueberry is of high economic value. Most blueberry varieties selected for the fresh market have an appealing light blue coating or "bloom" on the fruit due to the presence of a visible heavy epicuticular wax layer. This waxy layer also serves as natural defense against fruit desiccation and deterioration. RESULTS: In this study, we attempted to identify gene(s) whose expression is related to the protective waxy coating on blueberry fruit utilizing two unique germplasm populations that segregate for the waxy layer. We bulked RNA from waxy and non-waxy blueberry progenies from the two northern-adapted rabbiteye hybrid breeding populations ('Nocturne' x T 300 and 'Nocturne' x US 1212), and generated 316.85 million RNA-seq reads. We de novo assembled this data set integrated with other publicly available RNA-seq data and trimmed the assembly into a 91,861 blueberry unigene collection. All unigenes were functionally annotated, resulting in 79 genes potentially related to wax accumulation. We compared the expression pattern of waxy and non-waxy progenies using edgeR and identified overall 1125 genes in the T 300 population and 2864 genes in the US 1212 population with at least a two-fold expression difference. After validating differential expression of several genes by RT-qPCR experiments, a candidate gene, FatB, which encodes acyl-[acyl-carrier-protein] hydrolase, emerged whose expression was closely linked to the segregation of the waxy coating in our populations. This gene was expressed at more than a five-fold higher level in waxy than non-waxy plants of both populations. We amplified and sequenced the cDNA for this gene from three waxy plants of each population, but were unable to amplify the cDNA from three non-waxy plants that were tested from each population. We aligned the Vaccinium deduced FATB protein sequence to FATB protein sequences from other plant species. Within the PF01643 domain, which gives FATB its catalytic function, 80.08% of the amino acids were identical or had conservative replacements between the blueberry and the Cucumis melo sequence (XP_008467164). We then amplified and sequenced a large portion of the FatB gene itself from waxy and non-waxy individuals of both populations. Alignment of the cDNA and gDNA sequences revealed that the blueberry FatB gene consists of six exons and five introns. Although we did not sequence through two very large introns, a comparison of the exon sequences found no significant sequence differences between the waxy and non-waxy plants. This suggests that another gene, which regulates or somehow affects FatB expression, must be segregating in the populations. CONCLUSIONS: This study is helping to achieve a greater understanding of epicuticular wax biosynthesis in blueberry. In addition, the blueberry unigene collection should facilitate functional annotation of the coming chromosomal level blueberry genome.

Interactions between cranberries and fungi: the proposed function of organic acids in virulence suppression of fruit rot fungi.

Cranberry fruit are a rich source of bioactive compounds that may function as constitutive or inducible barriers against rot-inducing fungi. The content and composition of these compounds change as the season progresses. Several necrotrophic fungi cause cranberry fruit rot disease complex. These fungi remain mostly asymptomatic until the fruit begins to mature in late August. Temporal fluctuations and quantitative differences in selected organic acid profiles between fruit of six cranberry genotypes during the growing season were observed. The concentration of benzoic acid in fruit increased while quinic acid decreased throughout fruit development. In general, more rot-resistant genotypes (RR) showed higher levels of benzoic acid early in fruit development and more gradual decline in quinic acid levels than that observed in the more rot-susceptible genotypes. We evaluated antifungal activities of selected cranberry constituents and found that most bioactive compounds either had no effects or stimulated growth or reactive oxygen species (ROS) secretion of four tested cranberry fruit rot fungi, while benzoic acid and quinic acid reduced growth and suppressed secretion of ROS by these fungi. We propose that variation in the levels of ROS suppressive compounds, such as benzoic and quinic acids, may influence virulence by the fruit rot fungi. Selection for crops that maintain high levels of virulence suppressive compounds could yield new disease resistant varieties. This could represent a new strategy for control of disease caused by necrotrophic pathogens that exhibit a latent or endophytic phase.

Population structure of blueberry mosaic associated virus: Evidence of reassortment in geographically distinct isolates.

The population structure of blueberry mosaic associated virus (BlMaV), a putative member of the family Ophioviridae, was examined using 61 isolates collected from North America and Slovenia. The studied isolates displayed low diversity in the movement and nucleocapsid proteins and low ratios of non-synonymous to synonymous nucleotide substitutions, indicative of strong purifying selection. Phylogenetic analyses revealed grouping primarily based on geography with some isolates deviating from this rule. Phylogenetic incongruence in the two regions, coupled with detection of reassortment events, indicated the possible role of genetic exchange in the evolution of BlMaV.

New and emerging viruses of blueberry and cranberry.

Blueberry and cranberry are fruit crops native to North America and they are well known for containing bioactive compounds that can benefit human health. Cultivation is expanding within North America and other parts of the world raising concern regarding distribution of existing viruses as well as the appearance of new viruses. Many of the known viruses of these crops are latent or asymptomatic in at least some cultivars. Diagnosis and detection procedures are often non-existent or unreliable. Whereas new viruses can move into cultivated fields from the wild, there is also the threat that devastating viruses can move into native stands of Vaccinium spp. or other native plants from cultivated fields. The aim of this paper is to highlight the importance of blueberry and cranberry viruses, focusing not only on those that are new but also those that are emerging as serious threats for production in North America and around the world.

Generation and analysis of blueberry transcriptome sequences from leaves, developing fruit, and flower buds from cold acclimation through deacclimation.

BACKGROUND: There has been increased consumption of blueberries in recent years fueled in part because of their many recognized health benefits. Blueberry fruit is very high in anthocyanins, which have been linked to improved night vision, prevention of macular degeneration, anti-cancer activity, and reduced risk of heart disease. Very few genomic resources have been available for blueberry, however. Further development of genomic resources like expressed sequence tags (ESTs), molecular markers, and genetic linkage maps could lead to more rapid genetic improvement. Marker-assisted selection could be used to combine traits for climatic adaptation with fruit and nutritional quality traits. RESULTS: Efforts to sequence the transcriptome of the commercial highbush blueberry (Vaccinium corymbosum) cultivar Bluecrop and use the sequences to identify genes associated with cold acclimation and fruit development and develop SSR markers for mapping studies are presented here. Transcriptome sequences were generated from blueberry fruit at different stages of development, flower buds at different stages of cold acclimation, and leaves by next-generation Roche 454 sequencing. Over 600,000 reads were assembled into approximately 15,000 contigs and 124,000 singletons. The assembled sequences were annotated and functionally mapped to Gene Ontology (GO) terms. Frequency of the most abundant sequences in each of the libraries was compared across all libraries to identify genes that are potentially differentially expressed during cold acclimation and fruit development. Real-time PCR was performed to confirm their differential expression patterns. Overall, 14 out of 17 of the genes examined had differential expression patterns similar to what was predicted from their reads alone. The assembled sequences were also mined for SSRs. From these sequences, 15,886 blueberry EST-SSR loci were identified. Primers were designed from 7,705 of the SSR-containing sequences with adequate flanking sequence. One hundred primer pairs were tested for amplification and polymorphism among parents of two blueberry populations currently being used for genetic linkage map construction. The tetraploid mapping population was based on a cross between the highbush cultivars Draper and Jewel (V. darrowii is also in the background of 'Jewel'). The diploid mapping population was based on a cross between an F1 hybrid of V. darrowii and diploid V. corymbosum and another diploid V. corymbosum. The overall amplification rate of the SSR primers was 68% and the polymorphism rate was 43%. CONCLUSIONS: These results indicate that this large collection of 454 ESTs will be a valuable resource for identifying genes that are potentially differentially expressed and play important roles in flower bud development, cold acclimation, chilling unit accumulation, and fruit development in blueberry and related species. In addition, the ESTs have already proved useful for the development of SSR and EST-PCR markers, and are currently being used for construction of genetic linkage maps in blueberry.

In planta production of the highly potent resveratrol analogue pterostilbene via stilbene synthase and O-methyltransferase co-expression.

Resveratrol and related stilbenes are thought to play important roles in defence responses in several plant species and have also generated considerable interest as nutraceuticals owing to their diverse health-promoting properties. Pterostilbene, a 3,5-dimethylether derivative of resveratrol, possesses properties similar to its parent compound and, additionally, exhibits significantly higher fungicidal activity in vitro and superior pharmacokinetic properties in vivo. Recombinant enzyme studies carried out using a previously characterized O-methyltransferase sequence from Sorghum bicolor (SbOMT3) demonstrated its ability to catalyse the A ring-specific 3,5-bis-O-methylation of resveratrol, yielding pterostilbene. A binary vector was constructed for the constitutive co-expression of SbOMT3 with a stilbene synthase sequence from peanut (AhSTS3) and used for the generation of stably transformed tobacco and Arabidopsis plants, resulting in the accumulation of pterostilbene in both species. A reduced floral pigmentation phenotype observed in multiple tobacco transformants was further investigated by reversed-phase HPLC analysis, revealing substantial decreases in both dihydroquercetin-derived flavonoids and phenylpropanoid-conjugated polyamines in pterostilbene-producing SbOMT3/AhSTS3 events. These results demonstrate the potential utility of this strategy for the generation of pterostilbene-producing crops and also underscore the need for the development of additional approaches for minimizing concomitant reductions in key phenylpropanoid-derived metabolites.

Molecular Detection and Discrimination of Blueberry red ringspot virus Strains Causing Disease in Cultivated Blueberry and Cranberry.

Blueberry red ringspot virus (BRRV) causes red ringspots on the stems, leaves, and ripening fruit of infected highbush blueberry (Vaccinium corymbosum) plants. The disease was originally observed in New Jersey and has now been reported in other blueberry growing regions in the United States, as well as several locations in Europe. A disease with similar symptoms occurs in American cranberry (V. macrocarpon), but BRRV has never been confirmed as the causal agent. Serological detection of BRRV in infected plants has been unsatisfactory. Using a primer set designed for routine detection (RRSV3/RRSV4), we successfully amplified a fragment of the virus from all tissues of infected highbush blueberry plants. Using the same primer set, we confirmed natural infection of BRRV in rabbiteye (V. virgatum) blueberry cultivars and the rabbiteye × V. constablaei hybrid cultivar Little Giant. These species have not been previously reported as hosts for this virus. Viral fragments were cloned from representative blueberry and cranberry plants exhibiting ringspot symptoms. Phylogenetic analysis of sequence data showed that cranberry strains of BRRV are precursors to the more derived blueberry strains. The techniques reported in this paper are being used to evaluate strain variation in Vaccinium species and to identify the as yet unknown vector(s) of this virus.

Fairy Ring Disease of Cranberry: Assessment of Crop Losses and Impact on Cultivar Genotype.

Fairy ring is a disease of cultivated cranberry common in the eastern growing regions of the United States, especially New Jersey and Massachusetts. Rings may persist for many years, and current control recommendations are costly and largely ineffective. The goal of this study was to accurately assess the impact of this disease on cranberry, a long-lived, high-value, perennial crop. The rate of fairy ring expansion, rate of formation of new rings, and distribution of rings across three cultivars were determined using a geographical information system (GIS) database that incorporated aerial and satellite imagery. Ring growth rates, estimated from imagery collected over a 10-year period in cv. Ben Lear, averaged 0.455 m in radius per year. Rings were observed in 'Ben Lear' three times more frequently than in either 'Early Black' or 'Stevens' cultivars. Direct sampling showed that estimates for yield within rings were 22 to 68% less than unaffected areas of the field for cv. Ben Lear. These estimates included the effects of fruit rot, which was elevated within rings to 18 to 29% of the total harvest. The impact on yield of 'Stevens' and 'Early Black' was lower than in 'Ben Lear'. Most cranberry cultivars are clonal and variation in fruit morphology within rings, particularly in 'Ben Lear', prompted an analysis of vine genotype. Areas affected by fairy ring in 'Ben Lear' showed an increase in genetic diversity at least 0.4 to 4 times that of unaffected areas. Therefore, it appears that fairy ring not only directly reduces yield but also can increase the host genetic diversity. This likely is due to increased seedling establishment resulting from seed drop when fruit decompose. Because seedlings typically yield less than the parental cultivar, the increase in genetic diversity also may contribute to long-term reduction of productivity in a cranberry field.

Identification of a new phytophthora species causing root and runner rot of cranberry in new jersey.

ABSTRACT In New Jersey, Phytophthora cinnamomi is the pathogen most commonly isolated from diseased roots and runners of the cultivated cranberry (Vaccinium macrocarpon). A second distinct species of Phytophthora has been isolated from dying cranberry plants and surface irrigation water. This species is homothallic with paragynous antheridia and ellipsoid-limoniform, nonpapillate sporangia. It was tentatively identified as P. megasperma in an earlier report. Laboratory experiments demonstrate that the cardinal temperatures for vegetative growth are between 5 and 30 degrees C with an optimum near 25 degrees C. Sporangia are produced at temperatures between 10 and 20 degrees C with the majority of sporangia produced at 10 and 15 degrees C. In pathogenicity tests, no growth effect was observed on cranberry plants (cv. Early Black) when tests were conducted at 25 degrees C; however, significant reductions in plant growth occurred when tests were conducted at 15 degrees C. This species was insensitive to metalaxyl but was sensitive to buffered phosphorous acid. Sequence analysis of the internal transcribed spacer 1 (ITS1), 5.8S rDNA, and ITS2 regions place these isolates in Phytophthora clade 6 with greatest similarity to Phytophthora taxon raspberry. To our knowledge, this is the first report of isolates of this affiliation in North America. However, the observation of low temperature preferences makes this species unique in an otherwise high temperature clade. The isolates described in this study are tentatively classified as Phytophthora taxon cranberry.

Anthracnose Fruit Rot Resistance in Blueberry Cultivars.

Anthracnose fruit rot (causal agent, Colletotrichum acutatum) is an important disease in most blueberry growing regions of North America. Losses caused by the disease are usually seen as a postharvest rot with orange spore masses appearing on the surface of affected fruit. One hundred cultivars/selections of blueberry were screened for resistance to fruit rot between 1993 and 2003 by inoculating container-grown plants bearing green fruit. Visible rot symptoms on ripe fruits were evaluated after a 1-week incubation at room temperature. Our analyses revealed that infection levels were affected by mean May temperatures in New Jersey, generally increasing as temperatures increased; however, this effect was not consistent among all cultivars. A generalized linear mixed model was developed to predict resistance at the historic mean May temperature, conservatively explaining 59% of the variance in resistance. Percent infection ranged from 9 to 91% with a mean of 51% across all cultivars. Results for common cultivars corresponded well with field reports of their relative susceptibilities. An estimate of narrow-sense heritability of 0.32 suggested additive inheritance of resistance. Since very high inoculum loads were used in this study, cultivars exhibiting a low percentage of fruit rot are predicted to show superior field resistance to the disease and will be incorporated into an ongoing breeding program.