YDV Resistance in Barley: Phenotyping, Remote Imagery, and Virus-Vector Characterization.

Yellow Dwarf Viruses (YDV) spread by aphids are some of the most economically important barley (Hordeum vulgare L.) virus-vector complexes worldwide. Detection and control of these viruses are critical components in the production of barley, wheat, and numerous other grasses of agricultural importance. Genetic control of plant diseases is often preferable to chemical control to reduce the epidemiological, environmental, and economic cost of foliar insecticides. Accordingly, the objectives of this work were to I) screen a barley population for resistance to YDV under natural infection using phenotypic assessment of disease symptoms, II) implement drone imagery to further assess resistance and test its utility as a disease screening tool, III) identify the prevailing virus and vector types in the experimental environment, and IV) perform a genome-wide association study to identify genomic regions associated with measured traits. Significant genetic differences were found in a population of 192 barley inbred lines regarding their YDV symptom severity and symptoms were moderately to highly correlated with grain yield. The severity of YDV measured with aerial imaging was highly correlated with on-the-ground estimates (r=0.65). Three aphid species vectoring three YDV species were identified with no apparent genotypic influence on their distribution. A QTL impacting YDV resistance was detected on chromosome 2H, albeit undetected using aerial imaging. However, QTL for canopy cover and mean NDVI were successfully mapped using the drone. This work provides a framework for utilizing drone imagery in future resistance breeding efforts for YDV in cereals and grasses, and in other virus-vector disease complexes.

First report of Spiroplasma citri associated with disease symptoms in field-grown hemp (Cannabis sativa L.) in the Pacific Northwest.

Hemp (Cannabis sativa L.) is grown for cannabinoid oil production in Oregon. During the 2021 and 2022 growing seasons, plants with leaf curling, puckering, chlorotic mosaic, fasciation, and vein clearing were observed in disease surveys of Oregon hemp fields (Fig. 1). Symptoms were present on 1-10% of 2-4-month-old plants in fields located in Benton, Clackamas, Deschutes, Jackson, Josephine, Lane, Linn, Marion, Morrow, and Polk counties between July and September. Leaf and stem samples were collected from 38 symptomatic plants. Symptoms resembled those caused by beet leafhopper-vectored (BLH, Circulifer tenellus Baker) curtovirus or phytoplasma infection. Therefore, total nucleic acids were extracted (Dellaporta et al. 1983) from leaf material, and PCR conducted on all 38 samples to detect curtovirus coat proteins (BCTV-1/2 primers; Rondon et al. 2016) and 16S rRNA of phytoplasmas (nested primers P1/P7 followed by FU/RU; Lorenz et al. 1995). No curtoviruses or phytoplasmas were detected in any samples. Spiroplasma citri is also transmitted by BLH, so PCR was conducted with primers targeting the putative P89 adhesin gene and spiralin gene of S. citri (Yokomi et al. 2008). For all samples, PCR using P89F/R primers resulted in a 707 bp amplicon, and a 675 bp amplicon with Spiralin-f/r primers. PCR products were purified with ExoSapIT (Applied Biosystems, Waltham, MA), and two representative samples (ScH1; ScH2) were Sanger sequenced (EuroFins, Lancaster, PA) in the forward and reverse direction. Pairwise aligned P89 sequences were found to be 99 to 100% (ScH1: 633/637 bp; ScH2: 654/654 bp) identical to S. citri accession KT377386. Aligned/span>spiralin sequences were found to be 99 to 100% (ScH1: 661/664 bp; ScH2: 647/647 bp) identical to S. citri accession CP013197 in the NCBI GenBank Database. All sequences were deposited into GenBank (accession no. OQ969983, OQ992766, OQ969984, OQ969986). Frozen leaf material from one sample was used to culture S. citri according to Lee and Davis (1984). Leaf tissue was surface sterilized for 60 s in 1% NaOCl in 70% ethanol, cut into pieces in LD8 broth, and incubated for 25 min at 25˚C. The solution was passed through a 0.45um filter and incubated for 14 days at 30°C with constant shaking (150 rpm). Then, cultures were centrifuged for 2 min at 12,000 rpm to pelletize, resuspended in 100µL sterile distilled water and passed through a 0.2um filter. From this culture, 2 µL of broth was used as template for both the P89F/R and Spiralin-f/r primer sets. Amplicons were purified and sequenced as above (accession nos. OQ969982, OQ969985). Leaf tissue from both representative samples (ScH1 and ScH2) were positive for S. citri using double antibody sandwich ELISA (Agdia, Inc., Elkhart, IN) following manufacturer instructions. These results from sequencing, culturing, and ELISA testing indicate the hemp samples were infected with S. citri. To our knowledge this is the first report of disease symptoms in hemp associated with S. citri in Oregon and the Pacific Northwest. Infection by S. citri can limit yield by reducing photosynthetic capability of the plant and distortion of plant growth. Other pathogens like curtoviruses and phytoplasmas have also been detected in hemp in Oregon and the U.S. (Hu 2021; Rivedal et al. 2022), and the addition of S. citri associated with disease symptoms indicates a need for BLH management research. This discovery has implications for arid regions with other S. citri hosts including cruciferous plants, carrots, and tree fruit crops, all of which were grown near the sampled hemp fields in this study.

Acidifying Spray Suspensions of Oxytetracycline and Kasugamycin Enhances Their Effectiveness for Fire Blight Control in Apple and Pear.

The stability of the fire blight control material, oxytetracycline, in water is strongly affected by pH, increasing with increasing acidity. From 2017 to 2021, pear and apple orchard trials were conducted to evaluate if acidic amendments to oxytetracycline sprays improve fire blight control. Compared with the water-treated control, infection suppression after two bloom applications of an acidified commercial oxytetracycline formulation averaged 85.9 ± 0.4% compared with 72.2 ± 1.7% without an acidifier, but individual trials frequently had insufficient statistical power to separate among acidified and non-acidified antibiotic treatments. Across trials, a significant linear relationship was observed for regression of relative infection suppression from oxytetracycline (hydrochloride formulation) on spray tank pH. Similar relationships were observed for oxytetracycline (calcium complex formulation) and kasugamycin (P values were 0.055 and 0.069, respectively). Also based on regression, acidified oxytetracycline and kasugamycin suppressed epiphytic populations of Erwinia amylovora on flowers to a greater degree than the antibiotic only. As spray suspensions, commercial oxytetracycline formulations at label rate and amended with citric acid (1.2 g/liter) in well water had pH values near 3.4, but after spraying, the pH of flowers washed in deionized water (1 ml/flower) measured in a range of 5.2 to 5.5 compared with a pH range of 5.8 to 6.0 after a treatment of oxytetracycline only. In pear fruit finish trials, sprays acidified with citric acid-based materials had negligible effects on fruit russeting. Based on a serological assay, the detectable residual of oxytetracycline on apple foliage was increased by co-application with citric acid compared with a non-acidified control.

Refinement of Nonantibiotic Spray Programs for Fire Blight Control in Organic Pome Fruit.

Fire blight-susceptible, certified organic pome fruit is produced on 9,000 ha in the Pacific Northwest region of the United States with acreage continuing to expand despite a 2014 prohibition on antibiotics as allowable materials for infection suppression. Nonantibiotic practices for fire blight pathogen suppression mirror conventional management, but the full-bloom-to-petal-fall period when antibiotics are typically sprayed for fire blight control continues to receive research scrutiny owing to drawbacks and weaknesses of alternative materials. As solitary treatments, effective nonantibiotic materials (e.g., a yeast biocontrol, soluble coppers, and potassium aluminum sulfate) raise the risk of a crop-value-reducing, phytotoxic response termed "fruit russeting." Conversely, materials with less russeting risk (e.g., Bacillus-based biorationals) are less effective for fire blight control. Spray programs using a sequence of materials applied from midbloom to petal fall have the potential to provide high levels of protection with reduced russeting risk. In orchard trials, the effects of nonantibiotic spray programs on the epiphytic population size of Erwinia amylovora in flowers, yeast biocontrol population size, floral pH, infection suppression, and fruit russeting revealed strategies for sequencing sprays of nonantibiotic materials. The yeast biocontrol, Blossom Protect (Aureobasidium pullulans), sprayed at 70% bloom, was an important contributor to fire blight pathogen suppression as was the soluble copper material, Previsto, when applied at full bloom. Choice of material for the petal-fall spray timing was important to fruit russeting risk but apparently less important to overall infection incidence. Consequently, treatment programs of Blossom Protect at 70% bloom, a soluble copper at full bloom, and a Bacillus-based biorational at petal fall, best balance the quality of infection suppression with the risk of fruit russeting.

Induction of Systemic Acquired Resistance Aids Restoration of Tree Health in Field-Grown Pear and Apple Diseased with Fire Blight.

Induction of systemic acquired resistance as a therapeutic aid to restoration of tree health was evaluated in 3- to 14-year-old pear and apple trees diseased with fire blight. Acibenzolar-S-methyl (ASM) was applied to diseased trees in late spring near the time of removal of primary fire blight cankers, which had originated from floral infection. Suspensions of ASM (7.5 to 22.5 g of active ingredient per liter plus silicone surfactant) were painted onto a 30- to 45-cm length of branch tissue immediately below primary pruning cuts or sprayed onto an 80- to 100-cm length of central trunk. In some experiments, a second ASM treatment was made in late June to early July in conjunction with secondary pruning of redeveloped cankers. After pruning primary cankers, effects of ASM were measured by assessing weight and length of secondary cankers that were the result of fire blight redevelopment. Over 5 years of field experiments, trees that received an ASM treatments yielded 62% less diseased wood at the time of secondary and tertiary canker removal compared with non-ASM-treated trees. Moreover, tree mortality and proportion of pruning cuts where fire blight redeveloped were reduced by ASM. Induction of systemic acquired resistance could prove practical as an aid to pruning therapy in young, fire-blight-susceptible pear and apple trees where, after canker removal, disease symptoms frequently redevelop owing to residual cells of the pathogen distributed within symptomless portions of the tree.

Comparison of Methods of Acibenzolar-S-Methyl Application for Post-Infection Fire Blight Suppression in Pear and Apple.

Greenhouse-grown, 1-year-old potted 'Bosc' pear and apple rootstock cultivars 'M.9' and 'M.26' were inoculated with the fire blight pathogen, Erwinia amylovora, and subjected to trunk paint, root drench, or foliar spray treatments with acibenzolar-S-methyl (ASM, 4 to 30 mg a.i./tree) to induce systemic acquired resistance. Each method of ASM treatment suppressed fire blight canker expansion by 22 to 25%. Furthermore, ASM application method and ASM treatment timing (at or ±3 weeks relative to inoculation) interacted significantly (P ≤ 0.02) in each experiment. A root drench was most effective when applied 3 weeks before inoculation (36% suppression) whereas trunk paints and foliar sprays were more effective at inoculation (43 and 34%, suppression, respectively). Sizes of fire blight cankers in potted apple rootstocks M.9 and M.26 (under scions 'Gala' or 'Cameo') inoculated directly with the pathogen were reduced by 82 and 87% after two pretreatments of ASM applied as a trunk paint or root drench, respectively. Expression of pathogenesis-related (PR) genes PR-1 and -2 in apple leaves sampled after an ASM trunk paint were elevated significantly (P ≤ 0.05) relative to control trees for at least 9 weeks after treatment. Results of this study are being used to guide field research on postinfection therapy with ASM in 1- to 10-year-old pear and apple trees where fire blight has proven difficult to manage with therapeutic pruning only.

Evaluation of Dormant-Stage Inoculum Sanitation as a Component of a Fire Blight Management Program for Fresh-Market Bartlett Pear.

From 2010 to 2013, the efficacy of copper-based inoculum sanitation as a component of fire blight management programs was evaluated in commercial pear orchards located in northern California. Forty-one 4-ha sections of orchard were split into two equal-sized plots, with the orchardist applying horticultural oil alone to one plot and horticultural oil plus a fixed copper bactericide to the other plot. These treatments were timed to begin just prior to and finish at the "green tip" phenological stage, which occurs about 5 weeks before full bloom. During bloom, flower samples were collected from the plots and subjected to a loop-mediated isothermal DNA amplification (LAMP) assay for specific detection of Erwinia amylovora. Overall, epiphytic populations of E. amylovora on flowers were detected rarely at midbloom (6% of samples) but commonly at petal fall (44% of samples). In three of four seasons, E. amylovora detection in flower samples at a given bloom stage was significantly suppressed in copper-plus-oil-treated plots compared with oil-only plots. All orchards also received antibiotic treatments during the bloom period and, perhaps as a consequence, the development of fire blight was sporadic and not affected significantly by the copper treatment in any season. The pathogen detection data indicate that copper sanitation may add value to a fire blight management program by delaying the increase of epiphytic populations of E. amylovora in flowers to the late stages of the bloom period, at which time the number of susceptible flowers declines rapidly.

Evaluation of Strategies for Fire Blight Control in Organic Pome Fruit Without Antibiotics.

Apple and pear produced organically under the U.S. National Organic Program (NOP) standard can be treated with antibiotics for suppression of fire blight caused by Erwinia amylovora. Recent regulatory actions by the NOP, however, have lessened the likelihood of antibiotic use after the 2014 season. In response, western U.S. organic apple and pear stakeholders identified two immediate-need research objectives related to fire blight control: development of effective non-antibiotic control programs based on combinations of registered biological products; and, in apple, integration of these products with lime sulfur, which is sprayed at early bloom to reduce fruit load. In orchard trials in Oregon, increasing the frequency of treatment with biological products improved suppression of floral infection. In apple, fruit load thinning with 2% lime sulfur plus 2% fish oil (LS+FO) at 30 and 70% bloom significantly (P ≤ 0.05) reduced the proportion of blighted flower clusters in four of five orchard trials. Moreover, lime sulfur significantly (P ≤ 0.05) suppressed epiphytic populations of E. amylovora after their establishment on apple flowers. Over four trials, treatment with Aureobasidium pullulans (Blossom Protect) after LS+FO reduced the incidence of fire blight by an average of 92% compared with water only; this level of control was similar to treatment with streptomycin. In three seasons, a spray of a Pantoea agglomerans product after the 70% bloom treatment of LS+FO established the antagonist on a significantly (P ≤ 0.05) higher proportion of flowers compared with a spray of this bacterium before the thinning treatment. Consequently, in apple, biological treatments for fire blight control are not advised until after lime sulfur treatments for fruit load thinning are completed.

Quantitative Molecular Detection of Xanthomonas hortorum pv. carotae in Carrot Seed Before and After Hot-Water Treatment.

Molecular assays to detect and quantify DNA from viable cells of the seedborne pathogen Xanthomonas hortorum pv. carotae in carrot seed were developed and evaluated for use on nontreated and hot-water-treated seed lots. Both a TaqMan real-time polymerase chain reaction (PCR) assay and a loop-mediated isothermal amplification (LAMP) dilution endpoint assay detected and quantified DNA from viable pathogen cells after treatment of carrot seed washes with the live-dead discriminating dye propidium monoazide (PMA). The detection limits of the assays were approximately 101 CFU for pure cultures of X. hortorum pv. carotae, and 102 to 103 CFU/g seed from naturally infested carrot seed lots. X. hortorum pv. carotae in and on carrot seed was killed by soaking the seed in hot water (52°C for 25 min), and a subsequent PMA treatment of these hot-water-treated seed washes suppressed detection of the pathogen with both the real-time PCR and LAMP assays. For 36 commercial seed lots treated with PMA but not hot water, regression of colony counts of X. hortorum pv. carotae measured by dilution plating on a semiselective agar medium versus estimates of pathogen CFU determined by the molecular assays resulted in significant (P ≤ 0.05) linear relationships (R2 = 0.68 for the real-time PCR assay and 0.79 for the LAMP assay). The molecular assays provided quantitative estimates of X. hortorum pv. carotae infestations in carrot seed lots in <24 h, which is a significant improvement over the 7 to 14 days required to obtain results from the traditional dilution-plating assay.

Genome sequencing and comparative analysis of the carrot bacterial blight pathogen, Xanthomonas hortorum pv. carotae M081, for insights into pathogenicity and applications in molecular diagnostics.

Xanthomonas hortorum pv. carotae (Xhc) is an economically important pathogen of carrots. Its ability to epiphytically colonize foliar surfaces and infect seeds can result in bacterial blight of carrots when grown in warm and humid regions. We used high-throughput sequencing to determine the genome sequence of isolate M081 of Xhc. The short reads were de novo assembled and the resulting contigs were ordered using a syntenic reference genome sequence from X. campestris pv. campestris ATCC 33913. The improved, high-quality draft genome sequence of Xhc M081 is the first for its species. Despite its distance from other sequenced xanthomonads, Xhc M081 still shared a large inventory of orthologous genes, including many clusters of virulence genes common to other foliar pathogenic species of Xanthomonas. We also mined the genome sequence and identified at least 21 candidate type III effector genes. Two were members of the avrBs2 and xopQ families that demonstrably elicit effector-triggered immunity. We showed that expression in planta of these two type III effectors from Xhc M081 was sufficient to elicit resistance gene-mediated hypersensitive responses in heterologous plants, indicating a possibility for resistance gene-mediated control of Xhc. Finally, we identified regions unique to the Xhc M081 genome sequence, and demonstrated their potential in the design of molecular diagnostics for this pathogen.

Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Erwinia amylovora on Pear and Apple Fruit Flowers.

Fire blight of pear and apple is frequently an inoculum-limited disease but weather-based forecasting models commonly assume that the pathogen is omnipresent. To improve fire blight risk assessment during flowering, we developed a rapid pathogen detection protocol that uses loop-mediated isothermal amplification (LAMP) to detect DNA of epiphytic Erwinia amylovora on samples of pear and apple flowers. LAMP detected a single flower colonized epiphytically by E. amylovora in a sample of 100 flower clusters (approximately 600 flowers). Samples of 100 flower clusters from orchards (approximately one sample per hectare) were washed and subjected to LAMP, which was completed in 2 h. In three experimental orchards inoculated with E. amylovora, positive LAMP reactions were attained from nine of nine 100-flower cluster samples; pathogen populations in the floral washes averaged 5.2 × 103 CFU per flower as determined by dilution plating. Samples of pear and apple flowers obtained from 60 commercial orchards located in Oregon, Washington, California, and Utah resulted in detection of E. amylovora by LAMP assay from 34 sites, 20 of which developed fire blight. Of samples at early bloom, 10% were positive for epiphytic E. amylovora compared with 28% at petal fall; pathogen density in washes of positive samples averaged 3.2 × 102 CFU per flower. In another 26 orchards, all floral washes were negative for E. amylovora by LAMP and by dilution plating; a light severity of fire blight was observed in 8 of these orchards. Overall, positive detection of epiphytic E. amylovora in commercial orchards by LAMP-based scouting generally occurred at later stages of bloom after heat (risk) units had begun to accumulate, an indication that weather-based forecasting models may be an adequate measure of fire blight risk for many orchardists. Nonetheless, several orchardists communicated that information from the LAMP-based rapid detection protocol resulted in modification of their fire blight management practices.

Evaluation of Likelihood of Co-Occurrence of Erwinia amylovora with Mature Fruit of Winter Pear.

ABSTRACT Phytosanitary concerns about fire blight prohibit export of U.S.-grown pears to some countries without this disease. To examine these concerns, we evaluated the potential for co-occurrence of Erwinia amylovora with mature, symptomless winter pear fruit by inoculation experiments and by survey of commercial orchards. Immature pear and apple fruit were inoculated in orchards with E. amylovora strain 153N as resuspended lyophilized cells or as ooze from diseased tissues. Regardless of inoculum source, population size of Ea153N on fruit declined by an order of magnitude every 3 to 4 days during the first 2 weeks after inoculation; at 56 days after inoculation, Ea153N was not detected, except on 1 of 450 fruit with 4 colony forming units (CFU). After inoculation of flowers, calyx-end survival of Ea153N on pear and apple fruit declined from high populations at petal fall to a few cells at harvest, with no detection of the pathogen after a 7-week cold storage. Migration of Ea153N into symptomless pear fruit from diseased branches was evaluated by enrichment assay and nested polymerase chain reaction of internal fruit core tissues; these assays failed to detect the pathogen in healthy fruit from diseased trees. At harvest, E. amylovora could not be detected on 5,599 of 5,600 fruit of d'Anjou pear sampled from commercial orchards in major production areas of the Pacific Northwest; one fruit yielded 32 CFU of the pathogen. Postharvest, mature pear fruit contaminated with Ea153N and subsequently wounded required a dose of >10,000 cells at the wound site to allow for persistence of the pathogen through a 7-week-cold storage. We conclude that epiphytic E. amylovora shows similar survival characteristics on both pear and apple fruit, this pathogen is not an endophyte within mature symptomless pear fruit, its presence is exceptionally rare on commercially produced fruit, and that epiphytic survival of E. amylovora through a postharvest chilling period is unlikely given the unrealistically high population size required for persistence.

Bioavailability of Iron to Pseudomonas fluorescens Strain A506 on Flowers of Pear and Apple.

ABSTRACT The addition of 0.1 mM FeCl(3) to a defined culture medium induces the bacterial epiphyte Pseudomonas fluorescens strain A506 (A506) to produce an antibiotic toxic to the fire blight pathogen, Erwinia amylovora. Consequently, because A506 is registered and applied as a commercial product to suppress E. amylovora before floral infection of pear and apple, the relative availability of iron to A506 on surfaces of pear and apple flowers is of potential significance. An 'iron biosensor' construct of A506 was developed by transformation with an iron-regulated promoter (pvd) fused to a promoterless ice nucleation reporter gene (inaZ). This construct, A506 (pvd-inaZ), established high populations on pear and apple flowers, ranging from 10(4) to 10(6) CFU/flower. In seven trials on pear and apple trees, A506 (pvd-inaZ) expressed high ice nucleation activity (INA) on flowers, indicating limited iron bioavailability or a low-iron environment unlikely to induce antibiotic production by A506. A506 (pvd-inaZ) also colonized flowers when mixed with chemicals containing iron: FeSO(4) or the iron chelates ferric ethylenediaminedi-(o-hydroxyphenyl-acetic) acid (FeEDDHA) and ferric diethylenetriamine pentaacetate (FeDTPA). These compounds represent an array of commercial iron formulations applied to foliage to avert iron chlorosis. Treatment of flowers with a mixture of A506 (pvd-inaZ) and 3 mM FeEDDHA or FeDTPA significantly decreased INA compared with flowers treated with A506 (pvd-inaZ) in water. Lower concentrations (0.3 mM) of FeEDDHA, however, did not consistently suppress INA. These results indicate that apple and pear flowers represent an iron-limited environment to A506 and that treatment with 3 mM FeEDDHA is needed to increase significantly the level of iron available to this bacterium.