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.

First Report of Cytospora azerbaijanica Causing Cytospora Canker and Shoot Dieback on Peach (Prunus persica) in California, U.S.A.

Peaches (Prunus persica L.) are an important crop in the United States with California leading the nation in peach production, with approximately 505,000 tons valued at $378.3 million (USDA National Agricultural Statistics Service, 2021, https://www.nass.usda.gov/). From April to July 2022, symptoms of branch and scaffold canker as well as shoot dieback were observed in three peach (cvs. Loadel, Late Ross and Starn) orchards located in San Joaquin County, California. Samples were collected from about 12 trees for each cultivar. Fast-growing, white, flat colonies were consistently isolated from active cankers on acidified potato dextrose agar (APDA) following the method described by (Lawrence et al. 2017). Pure fungal cultures were obtained by transferring single hyphal tips onto new APDA Petri plates. A total of 22 isolates were obtained. Each fungal isolate was recovered from a single diseased branch (40 to 55% recovery). All isolates in this study shared similar morphological characteristics. Fungal colonies were fast-growing with relatively even but slightly dentate margin, flat with white to off-white mycelium that turned vinaceous buff to pale greyish sepia (Rayner 1970) with age. Black, globose, ostiolated pycnidia, 0.8-(1.3)-2.2 mm diameter, with brownish surface hyphae formed on peach wood embedded in PDA after approximately three weeks and exudated buff-colored mucilage. Pycnidia were both solitary and aggregated and had multiple internal locules sharing invaginated walls. Conidiogenous cells were hyaline, smooth-walled, septate, tapering towards the apex, 13-(18.2)-25.1 × 0.8-(1.3)-1.9 µm (n = 40). Conidia were hyaline, allantoid, smooth, aseptate, 5.5-(6.3)-7.1 × 1.4-(1.9)-2.3 µm (n = 40). Genomic DNA was extracted and sequences of the internal transcribed spacer region (ITS) using ITS5/ITS4 universal primers, translation elongation factor 1α gene (TEF) using primers EF1-728F/EF1-986R, second largest subunit of RNA polymerase II (RPB2) using primers RPB2-5F2/fRPB2-7cR, and actin gene region (ACT) using primers ACT-512F/ACT-783R were obtained and compared with sequences available in GenBank (Lawrence et al. 2018; Hanifeh et al. 2022). Isolates were identified as Cytospora azerbaijanica following DNA sequencing and morphological identification. Consensus sequences of the four genes of two representative isolates (SJC-66 and SJC-69) were deposited into GenBank database (ITS: OQ060581 and OQ060582; ACT: OQ082292, OQ082295; TEF: OQ082290 and OQ082293; RPB2: OQ082291 and OQ082294). The Basic Local Alignment Search Tool (BLAST) indicated that the sequenced RPB2 genes of isolates (SJC-66 and SJC-69) were at least 99% identical to that of Cytospora sp. strain shd47 (Accession: MW824360) covering at least 85% of the sequences. The actin genes from our isolates were at least 97.85% identical to that of Cytospora sp. strain shd47 (Accession: MZ014513), covering 100% of the sequences. The translation elongation factor gene from isolates (SJC-66 and SJC-69) was at least 96.4% identical to that of Cytospora sp. strain shd166 (Accession: OM372512), covering 100% of the query. Those top hit strains belong to C. azerbaijanica, recently reported by Hanifeh et al. (2022). Pathogenicity tests were performed by inoculating eight wounded, 2- to 3-year-old healthy branches on each of eight 7-year-old peach trees, cvs. Loadel, Late Ross and Starn, using 5-mm-diameter mycelium plugs collected from the margin of an actively growing fungal colony on APDA. Controls were mock-inoculated with sterile agar plugs. Inoculation sites were covered with petroleum jelly and wrapped with Parafilm to keep moisture. The experiment was performed twice. After four months, inoculation tests resulted in vascular discoloration (canker) above and below the inoculation sites (average necrosis length of 114.1 mm). Cytospora azerbaijanica was re-isolated from all infected branches (70 to 100% recovery) completing Koch's postulates. Controls remained symptomless and no fungi were isolated from the slightly discolored tissue. Cytospora species are destructive canker and dieback pathogens of numerous woody hosts worldwide. Recently, C. azerbaijanica was reported in causing canker disease of apple trees in Iran (Hanifeh et al. 2022). To our knowledge, this is the first report of C. azerbaijanica causing canker and shoot dieback of peach trees in the United States and worldwide. These findings will aid towards a better understanding of genetic diversity and host range of C. azerbaijanica.

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.

Orchard recycling improves climate change adaptation and mitigation potential of almond production systems.

There is an urgent need to develop climate smart agroecosystems capable of mitigating climate change and adapting to its effects. In California, high commodity prices and increased frequency of drought have encouraged orchard turnover, providing an opportunity to recycle tree biomass in situ prior to replanting an orchard. Whole orchard recycling (WOR) has potential as a carbon (C) negative cultural practice to build soil C storage, soil health, and orchard productivity. We tested the potential of this practice for long term C sequestration and hypothesized that associated co-benefits to soil health will enhance sustainability and resiliency of almond orchards to water-deficit conditions. We measured soil health metrics and productivity of an almond orchard following grinding and incorporation of woody biomass vs. burning of old orchard biomass 9 years after implementation. We also conducted a deficit irrigation trial with control and deficit irrigation (-20%) treatments to quantify shifts in tree water status and resilience. Biomass recycling led to higher yields and substantial improvement in soil functioning, including nutrient content, aggregation, porosity, and water retention. This practice also sequestered significantly higher levels of C in the topsoil (+5 t ha-1) compared to burning. We measured a 20% increase in irrigation water use efficiency and improved soil and tree water status under stress, suggesting that in situ biomass recycling can be considered as a climate smart practice in California irrigated almond systems.

Characterization and PCR-based detection of benzimidazole-resistant isolates of Monilinia laxa in California.

Monilinia laxa is a pathogen of brown rot of stone fruit and almond in California, causing blossom blights and fruit rots. In this study, low-level resistance to the benzimidazole fungicides benomyl and thiophanate-methyl was detected in field isolates of M laxa collected from stone fruits and almonds in California. Low-resistant (LR) isolates grew in potato dextrose agar (PDA) plates amended with benomyl and thiophanate-methyl at 1 and 5 microg ml(-1), respectively, but not in plates amended with benomyl at 5 microg ml(-1) or thiophanate-methyl at 50 microg ml(-1). The benzimidazole LR isolates were characterized by temperature sensitivity and the DNA sequence of the beta-tubulin gene. The LR isolates showed high-temperature sensitivity, being sensitive to 1 microg ml(-1) of benomyl at 28 degrees C but resistant at 8-24 degrees C. Analysis of the DNA sequence of the beta-tubulin gene showed that the LR isolates had a point mutation at the amino-acid position 240, causing substitution of leucine by phenylalanine. Based on the point mutation, a pair of allele-specific PCR primers was developed for rapid detection of LR isolates of M laxa. In addition, a pair of PCR primers specific to M laxa was developed on the basis of the differences in the DNA sequence of the intron 6 of beta-tubulin gene from M laxa, M fructicola and other fungal species. The primer pair amplified the expected 376-bp DNA fragment from all M laxa isolates tested, but not from 14 other fungal species isolated from stone fruit and almond crops. The restriction endonuclease BsmA I recognized the sequence GTCTCC in the PCR products from sensitive (S) isolates only, but not the GTTTCC sequence in the PCR products from LR isolates. The endonuclease digested the 376-bp PCR products from S isolates to produce two bands (111 and 265 bp) on agarose gels. Thus, both allele-specific PCR and the PCR-restriction fragment length polymorphism (PCR-RFLP) methods could be useful for rapidly detecting benzimidazole-resistant isolates of M laxa from stone fruit and almond crops in California.

Effects of Wounding, Inoculum Density, and Biological Control Agents on Postharvest Brown Rot of Stone Fruits.

The effects of wounding, inoculum density, and three isolates (New, Ta291, and 23-E-6) of Trichoderma spp. and one isolate (BI-54) of Rhodotorula sp. on postharvest brown rot of stone fruits were determined at 20°C and 95% relative humidity (RH). Brown rot was observed frequently on wounded nectarine, peach, and plum fruits inoculated with two spores of Monilinia fructicola per wound, and occasionally on unwounded nectarine and peach fruits inoculated with the same spore load. Brown rot was observed on wounded plums only. A substantial increase in lesion diameter of brown rot was also recorded on wounded nectarines and peaches inoculated with suspensions of ≤20 spores and ≤200 spores per wound, respectively, compared with unwounded fruit. At concentrations of 107 and 108 spores per ml, all Trichoderma isolates substantially reduced brown rot on peaches (63 to 98%) and plums (67 to 100%) when fruits were inoculated with M. fructicola following the application of a biological control agent. Similarly, at 108 spores per ml, the yeast BI-54 also suppressed brown rot on peaches completely and on plums by 54%. Significant brown rot reduction was also achieved with the isolate New at a concentration of 108 spores per ml, even when the biocontrol agent was applied 12 h after inoculation with M. fructicola and under continuous conditions of 95% RH. The isolates Ta291 and 23-E-6 also reduced brown rot significantly under drier (50% RH) incubation conditions. These isolates provided the best control of brown rot on plums when they were applied 12 h earlier than inoculation with M. fructicola. Satisfactory control of brown rot on plums inoculated with M. fructicola at 8 × 104 spores per ml was achieved with New at 106 spores per ml and with Ta291 at 107 spores per ml. Measures taken to avoid injuring fruit will greatly reduce brown rot of stone fruit at any spore load for plum, but only at ≤50 spores per mm2 for peach, and at ≤5 spores per mm2 for nectarine. This study identifies two isolates (Ta291 and New) of Trichoderma atroviride, one isolate (23-E-6) of T. viride, and one of Rhodotorula sp. that show potential for further development as biocontrol agents of postharvest brown rot of stone fruits.

Development of Apothecia from Stone Fruit Infected and Stromatized by Monilinia fructicola in California.

Apothecia were produced in the orchard, lath house, and laboratory from peach and nectarine fruit infected and stromatized by Monilinia fructicola. Fully stromatized "mummies" and nonstromatized infected fruit were placed in the orchard either on the soil surface or buried to a depth of 2 to 3 cm. Mummies were placed in the orchard at monthly intervals from August to February in 1993-94 and 1994-95. Nonstromatized infected fruit, which were fleshy and decomposed rapidly, were soon unavailable and were only placed in the orchard in August and September. Apothecia developed in February and early March only from mummies that were placed in the orchard in either October, November, or December. Mummies placed in the field in August, September, January, and February did not produce apothecia. Leaving mummies on the soil surface versus burying them 2 to 3 cm did not affect the development of apothecia. Apothecia were never produced from nonstromatized or recently-infected (fleshy) fruit. In the laboratory, apothecia were only produced from mummies that were partially buried in moist sand and stored without light at 2°C and >97% relative humidity (RH) for more than 8 weeks prior to incubation for 2 weeks (12, 15, or 20°C) with a 12-h photoperiod. Mummies that were incubated at >97% RH for less than 8 weeks or incubated at <90% RH never produced apothe-cia when stored at 2°C and then transferred to warmer temperatures with light. In orchard experiments, apothecia were only observed in plots with nondisturbed orchard floor vegetation; whereas no apothecia were found in either herbicide-treated or rototilled plots. Apothecia in the San Joaquin Valley were only produced from mummies that were subject to an 8-week or greater cold-temperature incubation while in contact with soil.

Significance of Thinned Fruit as a Source of the Secondary Inoculum of Monilinia fructicola in California Nectarine Orchards.

The significance of thinned fruit as a source of secondary inoculum in the spread of brown rot, caused by Monilinia fructicola, under semi-arid weather conditions of the San Joaquin Valley in California, was investigated in seven nectarine orchards in 1995 and 1996. Between 6 and 60% (depending on the orchard) of thinned fruit showed sporulation by M. fructicola. Brown rot was significantly less severe at preharvest (five orchards) and postharvest (one orchard) on fruit harvested from trees in plots from which thinned fruit were completely removed than on those in plots from which thinned fruit were not removed. M. fructicola sporulated more frequently on thinned fruit placed into irrigation trenches than on those left on the dry berms in tree rows. The incidence of preharvest fruit brown rot increased exponentially as the density of thinned fruit increased on the orchard floor. These results suggest that thinned fruit left on the floor of nectarine orchards can be a significant inoculum source of secondary infections. Removal or destruction of thinned fruit should reduce brown rot in nectarine and possibly other stone fruit orchards under semi-arid California conditions.