Insights Regarding Resistance of 'Nemaguard' Rootstock to the Bacterium Xylella fastidiosa.

'Nemaguard' is a commonly used rootstock for almond and stone fruits due to resistance to nematodes and enhanced scion vigor. Nemaguard also happens to be resistant to strains of Xylella fastidiosa that cause almond leaf scorch disease. Previous research showed that prior to June-budding, this rootstock can prevent infection of almond nursery stock by X. fastidiosa. Further, the rootstock also promotes recovery from infection in susceptible almond scions. Objectives of this study were to 1) compare movement and bacterial populations of X. fastidiosa in almond and Nemaguard, 2) determine whether the metabolic profile of infected versus noninfected plants of each species correspond with differences in pathogen distribution, and 3) evaluate the impact of feeding on Nemaguard on transmission efficiency and pathogen populations in insects. Results showed limited or no movement of X. fastidiosa beyond the point of mechanical inoculation in Nemaguard, whereas X. fastidiosa was detected in susceptible almond and isolated from plant samples distal to the point of inoculation. Large differences in the concentration of phenolic compounds between Nemaguard and almond were also found, although this was not impacted by infection status. After acquiring X. fastidiosa from infected plants, vector access periods of up to 14 days on Nemaguard neither reduced pathogen populations in vectors nor reduced transmission efficiency of X. fastidiosa to susceptible plants when compared with similar vector-access periods on susceptible grapevines. Results suggest Nemaguard, in spite of having high phenolic concentrations in its xylem, does not directly impact X. fastidiosa survival and that future research should focus on identification of potential physical traits that prevent bacterial attachment, multiplication, or movement within the plant.

Lessons from a Phenotyping Center Revealed by the Genome-Guided Mapping of Powdery Mildew Resistance Loci.

The genomics era brought unprecedented opportunities for genetic analysis of host resistance, but it came with the challenge that accurate and reproducible phenotypes are needed so that genomic results appropriately reflect biology. Phenotyping host resistance by natural infection in the field can produce variable results due to the uncontrolled environment, uneven distribution and genetics of the pathogen, and developmentally regulated resistance among other factors. To address these challenges, we developed highly controlled, standardized methodologies for phenotyping powdery mildew resistance in the context of a phenotyping center, receiving samples of up to 140 grapevine progeny per F1 family. We applied these methodologies to F1 families segregating for REN1- or REN2-mediated resistance and validated that some but not all bioassays identified the REN1 or REN2 locus. A point-intercept method (hyphal transects) to quantify colony density objectively at 8 or 9 days postinoculation proved to be the phenotypic response most reproducibly predicted by these resistance loci. Quantitative trait locus (QTL) mapping with genotyping-by-sequencing maps defined the REN1 and REN2 loci at relatively high resolution. In the reference PN40024 genome under each QTL, nucleotide-binding site-leucine-rich repeat candidate resistance genes were identified-one gene for REN1 and two genes for REN2. The methods described here for centralized resistance phenotyping and high-resolution genetic mapping can inform strategies for breeding resistance to powdery mildews and other pathogens on diverse, highly heterozygous hosts.

Discovery and genome-guided mapping of REN12 from Vitis amurensis, conferring strong, rapid resistance to grapevine powdery mildew.

Powdery mildew resistance genes restrict infection attempts at different stages of pathogenesis. Here, a strong and rapid powdery mildew resistance phenotype was discovered from Vitis amurensis 'PI 588631' that rapidly stopped over 97% of Erysiphe necator conidia, before or immediately after emergence of a secondary hypha from appressoria. This resistance was effective across multiple years of vineyard evaluation on leaves, stems, rachises, and fruit and against a diverse array of E. necator laboratory isolates. Using core genome rhAmpSeq markers, resistance mapped to a single dominant locus (here named REN12) on chromosome 13 near 22.8-27.0 Mb, irrespective of tissue type, explaining up to 86.9% of the phenotypic variation observed on leaves. Shotgun sequencing of recombinant vines using skim-seq technology enabled the locus to be further resolved to a 780 kb region, from 25.15 to 25.93 Mb. RNASeq analysis indicated the allele-specific expression of four resistance genes (NLRs) from the resistant parent. REN12 is one of the strongest powdery mildew resistance loci in grapevine yet documented, and the rhAmpSeq sequences presented here can be directly used for marker-assisted selection or converted to other genotyping platforms. While no virulent isolates were identified among the genetically diverse isolates and wild populations of E. necator tested here, NLR loci like REN12 are often race-specific. Thus, stacking of multiple resistance genes and minimal use of fungicides should enhance the durability of resistance and could enable a 90% reduction in fungicides in low-rainfall climates where few other pathogens attack the foliage or fruit.

Management of Almond Leaf Scorch Disease: Long-Term Data on Yield, Tree Vitality, and Disease Progress.

Almond leaf scorch disease (ALSD) has been a chronic problem for California almond growers. This disease is caused by the bacterial pathogen Xylella fastidiosa and is transmitted by xylem-feeding insects. Previous research suggested that retaining, rather than roguing, ALSD-affected trees may be more economically beneficial because ALSD-affected trees produced a reasonable yield and did not die over a 3-year period. Because almond orchards are kept in production for approximately 25 years, longer-term data are needed to fully evaluate the merits of retaining ALSD-affected trees. Extension of yield evaluations from 3 to 5 years demonstrated that yield loss due to ALSD was consistent over 5 years, with yields of ALSD-affected trees reduced by 20 and 40% compared with unaffected trees for 'Nonpareil' and 'Sonora', respectively. To assess risk of ALSD-affected trees serving as a source of inocula for secondary (tree-to-tree) spread and to evaluate vitality of ALSD-affected trees, previous surveys of two orchards were extended from 3 to 6 or 7 years. The relationship between disease incidence (percentage of trees infected) and survey year was linear for all cultivars examined at both orchards. Furthermore, at each orchard, the spatial location of infections detected after the first survey was random with respect to the spatial location of infections identified during the first survey, suggesting that ALSD-affected trees retained in orchards did not serve as a source for secondary spread. Over the 6- to 7-year study period, death of ALSD-affected trees was rare, with only 9% of ALSD-affected trees dying. Because orchards used in this study had relatively high disease incidence, 61 orchards containing Sonora were surveyed to determine typical levels of ALSD incidence. ALSD was widespread, with at least one infected tree in 56% of orchards surveyed, but incidence was typically low (mean incidence = 0.47%). Collectively, the results suggest that retaining ALSD-affected trees may be economically beneficial in older orchards.

Complete Genome Sequence of Curtobacterium sp. Strain TXMA1, Isolated from a Grapevine in Texas, USA.

The genome of Curtobacterium sp. strain TXMA1, isolated from a grapevine in Texas showing leaf marginal necrosis symptoms, was sequenced. The TXMA1 genome has a 3,454,876-bp, circular chromosome with a GC content of 71.74%, 3,213 open reading frames (ORFs), 47 tRNAs, and 4 complete rRNA operons (5S, 16S, and 23S).

Discovery of the REN11 Locus From Vitis aestivalis for Stable Resistance to Grapevine Powdery Mildew in a Family Segregating for Several Unstable and Tissue-Specific Quantitative Resistance Loci.

Race-specific resistance loci, whether having qualitative or quantitative effects, present plant-breeding challenges for phenotypic selection and deciding which loci to select or stack with other resistance loci for improved durability. Previously, resistance to grapevine powdery mildew (GPM, caused by Erysiphe necator) was predicted to be conferred by at least three race-specific loci in the mapping family B37-28 × C56-11 segregating for GPM resistance from Vitis aestivalis. In this study, 9 years of vineyard GPM disease severity ratings plus a greenhouse and laboratory assays were genetically mapped, using a rhAmpSeq core genome marker platform with 2,000 local haplotype markers. A new qualitative resistance locus, named REN11, on the chromosome (Chr) 15 was found to be effective in nearly all (11 of 12) vineyard environments on leaves, rachis, berries, and most of the time (7 of 12) stems. REN11 was independently validated in a pseudo-testcross with the grandparent source of resistance, "Tamiami." Five other loci significantly predicted GPM severity on leaves in only one or two environments, which could indicate race-specific resistance or their roles in different timepoints in epidemic progress. Loci on Chr 8 and 9 reproducibly predicted disease severity on stems but not on other tissues and had additive effects with REN11 on the stems. The rhAmpSeq local haplotype sequences published in this study for REN11 and Chr 8 and 9 stem quantitative trait locus (QTL) can be used directly for marker-assisted selection or converted to SNP assays. In screening for REN11 in a diversity panel of 20,651 vines representing the diversity of Vitis, this rhAmpSeq haplotype had a false positive rate of 0.034% or less. The effects of the other foliar resistance loci detected in this study seem too unstable for genetic improvement regardless of quantitative effect size, whether due to race specificity or other environmental variables.

Variable development rate and survival of navel orangeworm (Lepidoptera: Pyralidae) on wheat bran diet and almonds.

A series of laboratory and field studies were conducted using three lines of navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae), reared on wheat bran diet and almonds, Prunus dulcis (Mill.) D.A.Webb, at constant and fluctuating temperature. The duration of development on wheat bran diet at constant temperature differed significantly among the three lines. Development was as much as 40% faster at constant temperature than at fluctuating temperatures, consequently the developmental duration determined at constant temperature was not an absolute measure. When the maximum temperature in fluctuating regimes exceeded 43 degrees C, survival decreased by 50% compared with the constant temperature control. In almonds held at constant temperature, the developmental rate on new-crop nuts was variety-dependent and was fastest on 'Nonpareil' almonds and slowest on the experimental selection '23-122'. Development and survival were also variety-dependent on unharvested (mummy) almonds, and navel orangeworm average emergence was earliest from Nonpareil and latest from 'Carmel' nuts, differing by 529 degree-days, whereas survival was the highest on 'Butte', 35.7%, and the lowest on Carmel nuts, 7.2%. In our trials, both the speed of development and survival depended on host age, variety and quality, indicating that almonds were a dynamic rather than a static nutrient source for navel orangeworm. Identifying the factors responsible for variation in development and survival will give insight into improving control strategies.

Genetic Structure of a Worldwide Germplasm Collection of Prunus armeniaca L. Reveals Three Major Diffusion Routes for Varieties Coming From the Species' Center of Origin.

The characterization of the largest worldwide representative data set of apricot (Prunus armeniaca L.) germplasm was performed using molecular markers. Genetic diversity and structure of the cultivated apricot genetic resources were analyzed to decipher the history of diffusion of this species around the world. A common set of 25 microsatellite markers was used for genotyping a total of 890 apricot accessions in different collections from the center of origin to the more recent regions of apricot culture. Using a Bayesian model-based clustering approach, the apricot genotypes can be structured into five different genetic clusters (FST = 0.174), correlated with the geographical regions of origin of the accessions. Accessions from China and Central Asia were clustered together and exhibited the highest levels of diversity, confirming an origin in this region. A loss of genetic diversity was observed from the center of origin to both western and eastern zones of recent apricot culture. Altogether, our results revealed that apricot spread from China and Central Asia, defined as the center of origin, following three major diffusion routes with a decreasing gradient of genetic variation in each geographical group. The identification of specific alleles outside the center of origin confirmed the existence of different secondary apricot diversification centers. The present work provides more understanding of the worldwide history of apricot species diffusion as well as the field of conservation of the available genetic resources. Data have been used to define an apricot core collection based on molecular marker diversity which will be useful for further identification of genomic regions associated with commercially important horticultural traits through genome-wide association studies to sustain apricot breeding programs.

Haplotyping the Vitis collinear core genome with rhAmpSeq improves marker transferability in a diverse genus.

Transferable DNA markers are essential for breeding and genetics. Grapevine (Vitis) breeders utilize disease resistance alleles from congeneric species ~20 million years divergent, but existing Vitis marker platforms have cross-species transfer rates as low as 2%. Here, we apply a marker strategy targeting the inferred Vitis core genome. Incorporating seven linked-read de novo assemblies and three existing assemblies, the Vitis collinear core genome is estimated to converge at 39.8 Mb (8.67% of the genome). Adding shotgun genome sequences from 40 accessions enables identification of conserved core PCR primer binding sites flanking polymorphic haplotypes with high information content. From these target regions, we develop 2,000 rhAmpSeq markers as a PCR multiplex and validate the panel in four biparental populations spanning the diversity of the Vitis genus, showing transferability increases to 91.9%. This marker development strategy should be widely applicable for genetic studies in many taxa, particularly those ~20 million years divergent.

Effects of Almond Leaf Scorch Disease on Almond Yield: Implications for Management.

Almond leaf scorch (ALS) disease has been present in California's almond-growing regions for over 60 years. This disease is caused by the bacterium Xylella fastidiosa and the pathogen is vectored by xylem-feeding sharpshooters and spittlebugs. Currently, there are no effective management techniques that prevent trees from becoming infected. Within affected orchards throughout California's Central Valley, disease incidence and the risk of tree-to-tree spread appears to be low. Consequently, the decision to remove or keep infected trees depends on lost productivity. We compared yield and vitality between infected and uninfected almond for cvs. Sonora and Nonpareil. Sonora was examined at three sites over 3 years and Nonpareil was examined at one site over 2 years. Yields of ALS-affected trees were significantly lower for both cultivars, although yield losses of Sonora were proportionally greater than those of Nonpareil. Yields of infected trees did not decline incrementally over years; rather, they fluctuated similarly to those of uninfected trees. In addition, no infected trees died during the course of the study. These results are in direct contrast to previous anecdotal reports which suggest that yields of infected trees incrementally decline and infected trees eventually die. A simple economic model was developed to determine conditions under which rouging infected trees would increase returns. Based on the model, orchard age, yield loss due to infection, and the value of a maximally producing almond tree should be considered when deciding to remove ALS-affected trees.

Distinguishing Nonpareil marketing group almond cultivars through multivariate analyses.

More than 80% of the world's almonds are grown in California with several dozen almond cultivars available commercially. To facilitate promotion and sale, almond cultivars are categorized into marketing groups based on kernel shape and appearance. Several marketing groups are recognized, with the Nonpareil Marketing Group (NMG) demanding the highest prices. Placement of cultivars into the NMG is historical and no objective standards exist for deciding whether newly developed cultivars belong in the NMG. Principal component analyses (PCA) were used to identify nut and kernel characteristics best separating the 4 NMG cultivars (Nonpareil, Jeffries, Kapareil, and Milow) from a representative of the California Marketing Group (cultivar Carmel) and the Mission Marketing Group (cultivar Padre). In addition, discriminant analyses were used to determine cultivar misclassification rates between and within the marketing groups. All 19 evaluated carpological characters differed significantly among the 6 cultivars and during 2 harvest seasons. A clear distinction of NMG cultivars from representatives of the California and Mission Marketing Groups was evident from a PCA involving the 6 cultivars. Further, NMG kernels were successfully discriminated from kernels representing the California and Mission Marketing Groups with overall kernel misclassification of only 2% using 16 of the 19 evaluated characters. Pellicle luminosity was the most discriminating character, regardless of the character set used in analyses. Results provide an objective classification of NMG almond kernels, clearly distinguishing them from kernels of cultivars representing the California and Mission Marketing Groups.