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.

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.

An roGFP2-Based Bacterial Bioreporter for Redox Sensing of Plant Surfaces.

The reduction-oxidation (redox) environment of the phytobiome (i.e., the plant-microbe interface) can strongly influence the outcome of the interaction between microbial pathogens, commensals, and their host. We describe a noninvasive method using a bacterial bioreporter that responds to reactive oxygen species and redox-active chemicals to compare microenvironments perceived by microbes during their initial encounter of the plant surface. A redox-sensitive variant of green fluorescent protein (roGFP2), responsive to changes in intracellular levels of reduced and oxidized glutathione, was expressed under the constitutive SP6 and fruR promoters in the epiphytic bacterium Pantoea eucalypti 299R (Pe299R/roGFP2). Analyses of Pe299R/roGFP2 cells by ratiometric fluorometry showed concentration-dependent responses to several redox active chemicals, including hydrogen peroxide (H2O2), dithiothreitol (DTT), and menadione. Changes in intracellular redox were detected within 5 min of addition of the chemical to Pe299R/roGFP2 cells, with approximate detection limits of 25 and 6 µM for oxidation by H2O2 and menadione, respectively, and 10 µM for reduction by DTT. Caffeic acid, chlorogenic acid, and ascorbic acid mitigated the H2O2-induced oxidation of the roGFP2 bioreporter. Aqueous washes of peach and rose flower petals from young blossoms created a lower redox state in the roGFP2 bioreporter than washes from fully mature blossoms. The bioreporter also detected differences in surface washes from peach fruit at different stages of maturity and between wounded and nonwounded sites. The Pe299R/roGFP2 reporter rapidly assesses differences in redox microenvironments and provides a noninvasive tool that may complement traditional redox-sensitive chromophores and chemical analyses of cell extracts.

First Report of the Peach Root-Knot Nematode, Meloidogyne floridensis Infecting Almond on Root-Knot Nematode Resistant 'Hansen 536' and 'Bright's Hybrid 5' Rootstocks in California, USA.

In April-August 2018, samples of galled roots with rhizosphere soil were collected from almond orchards in Atwater, Merced County and Bakersfield, Kern County, California. Almond trees (Prunus dulcis) grafted on 'Hansen 536' and 'Brights Hybrid®5' (peach-almond hybrid) rootstocks showed strong symptoms of growth decline. Extracted root-knot nematodes were identified by both morphological and molecular methods as M. floridensis. Meloidogyne floridensis was initially found in Florida, USA, and has not been reported from any other states and countries. This is a first report of M. floridensis in California and outside of Florida.In April-August 2018, samples of galled roots with rhizosphere soil were collected from almond orchards in Atwater, Merced County and Bakersfield, Kern County, California. Almond trees (Prunus dulcis) grafted on 'Hansen 536' and 'Brights Hybrid®5' (peach-almond hybrid) rootstocks showed strong symptoms of growth decline. Extracted root-knot nematodes were identified by both morphological and molecular methods as M. floridensis. Meloidogyne floridensis was initially found in Florida, USA, and has not been reported from any other states and countries. This is a first report of M. floridensis in California and outside of Florida.

Neoscytalidium dimidiatum Causing Canker, Shoot Blight and Fruit Rot of Almond in California.

Almond trees with trunk and branch cankers were observed in several orchards across almond-producing counties in California. Symptoms of cankers included bark lesions, discoloration of xylem tissues, longitudinal wood necrosis, and extensive gumming. Spur and shoot blight associated with rotted fruit were detected in two orchards in Kern County. The fungus Neoscytalidium dimidiatum was consistently recovered from the various cankers, infected fruit, and blighted shoots and its identity was confirmed based on phylogenetic and morphological studies. Phylogenetic analyses of the internal transcribed spacer, translation elongation factor 1-α, and ß-tubulin genes comparing 47 strains from California with reference specimens within the family Botryosphaeriaceae and coupled with detailed morphological observations validated the identity of the pathogenic fungus. Pathogenicity tests conducted in the field using 1- to 2-year-old branches inoculated with mycelium plugs or conidial suspensions and attached fruit inoculated with conidial suspensions fulfilled Koch's postulates. N. dimidiatum appeared highly virulent in almond-producing cankers of up to 22 cm in length within 4 weeks using mycelium plug inoculations as well as severe fruit rot combined with spur blight on the fruit-bearing spurs. This study reports, for the first time, the fungus N. dimidiatum as a pathogen of almond in California causing canker, shoot blight, and fruit rot. Disease symptoms are described and illustrated.

Susceptibility of Walnut and Hickory Species to Geosmithia morbida.

Thousand cankers disease (TCD) of walnut is a result of feeding in the phloem by the walnut twig beetle (WTB), Pityophthorus juglandis, and subsequent canker formation caused by Geosmithia morbida around galleries. TCD has caused extensive morbidity and mortality to Juglans nigra in the western United States and, in 2010, was discovered in the eastern United States, where the tree is a highly valuable timber resource. WTB and G. morbida also have been found in J. regia orchards throughout major production areas in California, and the numbers of damaged trees are increasing. We tested the susceptibility of walnut and hickory species to G. morbida in greenhouse and field studies. Carya illinoinensis, C. aquatica, and C. ovata were immune. All walnut species tested, including J. ailantifolia, J. californica, J. cinerea, J. hindsii, J. major, J. mandshurica, J. microcarpa, J. nigra, and J. regia, developed cankers following inoculation with G. morbida. J. nigra was the most susceptible, whereas J. major, a native host of the WTB and, presumably, G. morbida, had smaller and more superficial cankers. Canker formation differed among maternal half-sibling families of J. nigra and J. cinerea, indicating genetic variability in resistance to G. morbida. Our inoculation studies with G. morbida have corroborated many of the field observations on susceptibility of walnut and hickory species to TCD, although the ability of the WTB to successfully attack and breed in walnut is also an important component in TCD resistance.

Fusarium spp., Cylindrocarpon spp., and Environmental Stress in the Etiology of a Canker Disease of Cold-Stored Fruit and Nut Tree Seedlings in California.

The principal objective of this study was to determine the etiology of a canker disease in dormant stone fruit and apple tree seedlings maintained in refrigerated storage that has significantly impacted California fruit and nut tree nurseries. Signs and symptoms of the disease develop during storage or soon after planting, with subsequent decline and death of young trees. Isolations from both diseased and healthy almond and apple trees and Koch's postulates using stem segments of desiccation-stressed almond trees as hosts implicated Fusarium avenaceum and F. acuminatum as the primary causal agents. F. solani, Ilyonectria robusta, and Cylindrocarpon obtusiusculum were also capable of causing similar symptoms but were less frequently encountered in isolations of diseased tissue. Loss of bark turgidity in excised almond stem segments, as can occur in cold-stored seedlings, correlated with increased susceptibility to F. acuminatum, with maximum canker development occurring after relative bark turgidity dropped below a threshold of approximately 86%. Healthy almond trees, almond scion budwood, and a wheat cover crop used in fields where tree seedlings were grown and maintained until cold storage all possessed asymptomatic infections of F. acuminatum, F. avenaceum, and C. obtusiusculum as determined by activation following overnight freezing, cold storage, or desiccation.

Propiconazole Sensitivity in Populations of Geotrichum candidum, the Cause of Sour Rot of Peach and Nectarine, in California.

The sour rot pathogen of peach (Prunus persica var. persica) and nectarine (P. persica var. nectarina) fruit, Geotrichum candidum, can cause significant postharvest losses in California fruit production. Harvested peach and nectarine fruit, treated with fungicide at the packinghouse but culled after inspection because of disease and defects, were collected for further assessment and pathogen isolation. The incidence of fruit with sour rot was 3.4 ± 1.0 to 26.1 ± 2.3%. Culled fruit that had been treated with postharvest fungicides from five different orchards had a significantly higher incidence of sour rot when compared with nontreated fruit. Since August 2006, propiconazole has been used as a postharvest treatment to protect peach and nectarine fruit against sour rot. The mean effective concentration that inhibits 50% of mycelial growth (EC50) value of 57 isolates of G. candidum to propiconazole collected before and during 2006 was 0.072 µg/ml. However, 61 isolates from propiconazole-treated, diseased fruit collected from 2007 to 2009 had a mean EC50 value for mycelial growth of 0.378 µg/ml, a fivefold shift in mean sensitivity. Propiconazole applied as either a protective or curative treatment significantly reduced disease severity and decay development in fruit inoculated with a propiconazole-sensitive isolate. The fungicide was significantly less effective when applied as a preventive or a curative application to fruit that were inoculated with a less-sensitive isolate of G. candidum. Under laboratory conditions, isolates of the pathogen less sensitive to propiconazole were stable over multiple transfers on fungicide-free medium. The potential for the development of G. candidum populations with reduced sensitivity to propiconazole and the increased risk of crop loss due to the practice of returning culled fruit to production orchards are discussed.

Biology and Sources of Inoculum of Geotrichum candidum Causing Sour Rot of Peach and Nectarine Fruit in California.

Geotrichum candidum causes sour rot of fresh-market stone fruit such as peach and nectarine. Since 2001, the incidence of sour rot has increased in California, a semi-arid production area, which is considered atypical for the occurrence of the disease. In this study, sour rot developed at significantly higher incidence on wounded fruit compared with unwounded fruit, and disease severity increased as fruit matured. In packinghouse surveys, sour rot was found on up to 4% of non-fungicide- treated peach and nectarine fruit. In laboratory assays, sour rot developed when fruit were inoculated with a minimum of 20 conidia per inoculation site. Inoculum of G. candidum could be detected in California orchard soils at depths of up to 10 cm. The amount of inoculum in the soil was positively correlated with that on leaf and fruit surfaces of trees growing at a specific site. Moreover, inoculum of G. candidum was detected at different areas of packing lines in seven packinghouses. There were significant differences among the packinghouses evaluated, and these differences could be attributed, in part, to different sanitation practices used. Nitidulid beetles and fruit flies were found to play a role in disease transmission. These insects acquired sour rot inoculum in the orchard, and 25% of nitidulid beetles and 26% of fruit flies collected were positive for the pathogen. Spore survival in the soil over a 12-month period decreased significantly when soil depth increased from 10 to 20 cm. This study identified sources of inoculum of G. candidum in orchards and packinghouses, and provides information to guide development of disease management programs under the semi-arid conditions of California.