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.

Effects of whole-orchard recycling on nitrate leaching potential in almond production systems.

Inefficient nitrogen (N) fertilization and irrigation have led to unhealthy nitrate levels in groundwater bodies of agricultural areas in California. Simultaneously, high commodity prices and drought have encouraged perennial crop growers to turnover less-productive orchards, providing opportunities to recycle tree biomass in situ and to use high-carbon (C) residues to conserve soil and water resources. Although climate change adaptation and mitigation benefits of high-C soil amendments have been shown, uncertainties remain regarding the benefits and trade-offs of this practice for N cycling and retention. We used established almond [Prunus dulcis (Mill.) D. A. Webb] orchard trials on Hanford fine sandy loam with short-term and long-term biomass recycling legacies to better understand the changes in N dynamics and retention capacity associated with this practice. In a soil column experiment, labeled N fertilizer was added and traced into various N pools, including microbial biomass and inorganic fractions in soil and leachate. Shifts in microbial communities were characterized using the abundance of key N cycling functional genes regulating nitrification and denitrification processes. Our findings showed that, in the short term, biomass recycling led to N immobilization within the orchard biomass incorporation depth zone (0-15 cm) without impacts on N leaching potential. However, this practice drastically reduced nitrate leaching potential by 52%, 10 yr after biomass incorporation without an increase in N immobilization. Although the timing of these potential benefits as a function of microbial population and C and N biogeochemical cycles still needs to be clarified, our results highlight the potential of this practice to meaningfully mitigate nitrate discharges into groundwater while conserving soil resources.

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.

Identification and Pathogenicity of Fungal Species Associated with Canker Diseases of Pistachio in California.

A survey was conducted during 2015 and 2016 in pistachio orchards throughout the San Joaquin Valley of California to investigate the occurrence of canker diseases and identify the pathogens involved. Cankers and dieback symptoms were observed mainly in orchards aged >15 years. Symptoms of canker diseases included brown to dark brown discoloration of vascular tissues, wood necrosis, and branch dieback. In total, 58 fungal isolates were obtained from cankers and identified based on multilocus phylogenetic analyses (internal transcribed spacer, glyceraldehyde 3-phosphate dehydrogenase, ß-tubulin, calmodulin, actin 1, and translation elongation factor 1α) representing 11 fungal species: Colletotrichum karstii, Cytospora californica, Cytospora joaquinensis, Cytospora parapistaciae, Cytospora pistaciae, Diaporthe ambigua, Didymella glomerata, Diplodia mutila, Neofusicoccum mediterraneum, Phaeoacremonium canadense, and Schizophyllum commune. Pathogenicity tests conducted in the main pistachio cultivars Kerman, Golden Hills, and Lost Hills using the mycelium-plug method indicated that all fungal species were pathogenic to Pistacia vera. All species tested caused cankers in pistachio branches, although virulence among species varied from high to moderate. Overall, N. mediterraneum and Cytospora spp. were the most widespread and virulent species associated with canker diseases of pistachio in California.