Phylogenomic analyses and comparative genomics of Pseudomonas syringae associated with almond (Prunus dulcis) in California.

We sequenced and comprehensively analysed the genomic architecture of 98 fluorescent pseudomonads isolated from different symptomatic and asymptomatic tissues of almond and a few other Prunus spp. Phylogenomic analyses, genome mining, field pathogenicity tests, and in vitro ice nucleation and antibiotic sensitivity tests were integrated to improve knowledge of the biology and management of bacterial blast and bacterial canker of almond. We identified Pseudomonas syringae pv. syringae, P. cerasi, and P. viridiflava as almond canker pathogens. P. syringae pv. syringae caused both canker and foliar (blast) symptoms. In contrast, P. cerasi and P. viridiflava only caused cankers, and P. viridiflava appeared to be a weak pathogen of almond. Isolates belonging to P. syringae pv. syringae were the most frequently isolated among the pathogenic species/pathovars, composing 75% of all pathogenic isolates. P. cerasi and P. viridiflava isolates composed 8.3 and 16.7% of the pathogenic isolates, respectively. Laboratory leaf infiltration bioassays produced results distinct from experiments in the field with both P. cerasi and P. syringae pv. syringae, causing significant necrosis and browning of detached leaves, whereas P. viridiflava conferred moderate effects. Genome mining revealed the absence of key epiphytic fitness-related genes in P. cerasi and P. viridiflava genomic sequences, which could explain the contrasting field and laboratory bioassay results. P. syringae pv. syringae and P. cerasi isolates harboured the ice nucleation protein, which correlated with the ice nucleation phenotype. Results of sensitivity tests to copper and kasugamycin showed a strong linkage to putative resistance genes. Isolates harbouring the ctpV gene showed resistance to copper up to 600 µg/ml. In contrast, isolates without the ctpV gene could not grow on nutrient agar amended with 200 µg/ml copper, suggesting ctpV can be used to phenotype copper resistance. All isolates were sensitive to kasugamycin at the label-recommended rate of 100µg/ml.

Limited Evidence for Accumulation of Latent Infections of Canker-Causing Pathogens in Shoots of Stone Fruit and Nut Crops in California.

Prevalence of latent infections of the canker-causing fungi Botryosphaeria dothidea and species of Cytospora, Diplodia, Lasiodiplodia, Neofusicoccum, and Phomopsis in young shoots of almond, prune, and walnut trees in California was studied to test the hypotheses that latent infections accumulate from current-season shoots to 1-year-old shoots in the orchard and there are distinct associations among pathogen taxa present as latent infections in the same shoot. Samples of newly emerged and 1-year-old shoots were periodically collected in each almond, prune, and walnut orchard for two growing seasons. A real-time quantitative PCR assay was used to quantify latent infection with three parameters: incidence, molecular severity, and latent infection index. Diplodia spp. were absent from most samples. For almond, Lasiodiplodia spp. and Cytospora spp. were detected with a maximum incidence >90%, while B. dothidea and Neofusicoccum spp. incidence was <20% in most cases. In prune orchards, the incidence levels of B. dothidea were >50% in most cases, while those of Cytospora spp. and Lasiodiplodia spp. were 30 to 60% and 30 to 100%, respectively. For walnut, many samplings showed higher incidence in 1-year-old (30 to 80%) than in newly emerged shoots (10 to 50%). Accumulation of latent infection between the two shoot age classes was detected in only a few cases. The percentages of samples showing coexistence of two, three, and four pathogen taxa in the same shoot were 20 to 25, <10, and <5%, respectively. Pairwise associations among pathogen taxa in the same shoot were significant in many cases.

Fungal Pathogens Associated With Canker Diseases of Almond in California.

Almond canker diseases are destructive and can reduce the yield as well as the lifespan of almond orchards. These diseases may affect the trunk and branches of both young and mature trees and can result in tree death soon after orchard establishment in severe cases. Between 2015 and 2018, 70 almond orchards were visited throughout the Central Valley of California upon requests from farm advisors for canker disease diagnosis. Two major canker diseases were identified, including Botryosphaeriaceae cankers and Ceratocystis canker. In addition, five less prevalent canker diseases were identified, including Cytospora, Eutypa, Diaporthe, Collophorina, and Pallidophorina canker. Seventy-four fungal isolates were selected for multilocus phylogenetic analyses of internal transcribed spacer region ITS1-5.8S-ITS2 and part of the translation elongation factor 1-α, ß-tubulin, and glyceraldehyde 3-phosphate dehydrogenase gene sequences; 27 species were identified, including 12 Botryosphaeriaceae species, Ceratocystis destructans, five Cytospora species, Collophorina hispanica, four Diaporthe species, two Diatrype species, Eutypa lata, and Pallidophorina paarla. The most frequently isolated species were Ceratocystis destructans, Neoscytalidium dimidiatum, and Cytospora californica. Pathogenicity experiments on almond cultivar Nonpareil revealed that Neofusicoccum parvum, Neofusicoccum arbuti, and Neofusicoccum mediterraneum were the most virulent. Botryosphaeriaceae cankers were predominantly found in young orchards and symptoms were most prevalent on the trunks of trees. Ceratocystis canker was most commonly found in mature orchards and associated with symptoms found on trunks or large scaffold branches. This study provides a thorough examination of the diversity and pathogenicity of fungal pathogens associated with branch and trunk cankers of almond in California.

Understanding the Process of Latent Infection of Canker-Causing Pathogens in Stone Fruit and Nut Crops in California.

The Botryosphaeriaceae family is considered a fungal family that includes pathogens causing latent infection of woody plants, and a number of species were identified as causal pathogens of canker and shoot blight diseases. To better understand the process of latent infection of major canker-causing pathogens in woody tissues in different tree crops important in California, shoot and bud samples were randomly collected from four tree crops: almond, dried plum, pistachio, and walnut. The previously developed DNA primers and quantitative real-time PCR (qPCR) assay systems were applied to detect six canker-causing pathogen groups, including Botryosphaeria dothidea, and species of Cytospora, Diplodia, Lasiodiplodia, Neofusicoccum, and Phomopsis. The concepts of molecular severity (MS) and latent infection index (LII) were introduced and applied to quantify the latent infection levels for these samples. Variation in incidence of latent infection among pathogen groups was observed, whereas the incidences were relatively low among species of Phomopsis and Diplodia. High incidences of Cytospora spp. were observed in two dried plum (prune) orchards. Most orchards showed high incidences of B. dothidea and Lasiodiplodia spp. and moderate incidences of Neofusicoccum spp. Variations in MS were observed among samples of the studied orchards, ranging from 4 to 8. The overall results of LII demonstrated that species of Diplodia and Phomopsis were less important in population development of canker-causing pathogens at the latent phase. Lasiodiplodia spp. were the most aggressive and had been well developed in populations among the studied tree crops. Cytospora spp. became predominant in two of the three dried plum orchards, whereas B. dothidea and Neofusicoccum spp. showed trends of increase in incidence across various tree crops. This study also demonstrated the usefulness of this sensitive qPCR approach in providing evidence of the latent phase of major canker-causing pathogens of stone fruit and nut crops at an early stage of latent infection in woody plant tissues.

Timing Spring Insecticide Applications to Target both Amyelois transitella (Lepidoptera: Pyralidae) and Anarsia lineatella (Lepidoptera: Gelechiidae) in Almond Orchards.

Amyelois transitella (Walker) (Lepidoptera: Pyralidae) and Anarsia lineatella Zeller (Lepidoptera: Gelechiidae) are key Lepidoptera pests of almonds in California. Spring insecticide applications (early to mid-May) targeting either insect were not usually recommended because of the potential to disrupt natural enemies when broad-spectrum organophosphates and pyrethroids were applied. The registration of reduced risk compounds such as chlorantraniliprole, methoxyfenozide, and spinetoram, which have a higher margin of safety for natural enemies, makes spring (early to mid-May) application an acceptable control approach. We examined the efficacy of methoxyfenozide, spinetoram, and chlorantraniliprole at three spring application timings including the optimum spring timing for both A. lineatella and A. transitella in California almonds. Our study also examined the possibility of reducing larval populations of A. lineatella and A. transitella simultaneously with a single spring insecticide application. There were no significant differences in the field efficacy of insecticides targeting either A. lineatella or A. transitella, depending on application timing for the three spring timings examined in this study. In most years (2009-2011), all three timings for each compound resulted in significantly less A. transitella and A. lineatella damage when compared with an untreated control, though there was some variation in efficacy between the two species. Early to mid-May applications of the reduced-risk insecticides chlorantraniliprole and spinetoram can be used to simultaneously target A. transitella and A. lineatella with similar results across the potential timings.

Pear Scab: Components of Potential Ascospore Dose and Validation of an Ascospore Maturity Model.

Variables necessary to calculate potential ascospore dose (PAD) for Venturia pirina, the causal agent of pear scab, were measured: area of individual leaves averaged 20.0 cm2, number of lesions per infected leaf ranged from 1.8 to 2.2, 13.5 to 41.7% of lesions were fertile (contained pseudothecia), number of pseudothecia per fertile lesion ranged from 15.2 to 24.4, and number of asci per pseudothecium averaged 131.5. The proportions of orchard floor covered with leaves (leaf litter density) were 0.34 and 0.67 for mowed and nonmowed orchards, respectively. Incidence of infected leaves ranged from 0.1 to 32.3%. Calculated PAD ranged from 1,114 to 708,320 ascospores per m2 in a commercial and an unsprayed orchard, respectively. For validation of a previously published model relating ascospore maturity to degree-days with a 0°C base, a linear regression equation was developed using 11 data sets collected between 1981 and 1998. The slope of the line was not different (P = 0.951) from that of the original model.