Multilocus Characterization, Gene Expression Analysis of Putative Immunodominant Protein Coding Regions, and Development of Recombinase Polymerase Amplification Assay for Detection of 'Candidatus Phytoplasma Pruni' in Prunus avium.

Western X (WX) disease, caused by 'Candidatus Phytoplasma pruni', is a devastating disease of sweet cherry resulting in the production of small, bitter-flavored fruits that are unmarketable. Escalation of WX disease in Washington State prompted the development of a rapid detection assay based on recombinase polymerase amplification (RPA) to facilitate timely removal and replacement of diseased trees. Here, we report on a reliable RPA assay targeting putative immunodominant protein coding regions that showed comparable sensitivity to polymerase chain reaction (PCR) in detecting 'Ca. Phytoplasma pruni' from crude sap of sweet cherry tissues. Apart from the predominant strain of 'Ca. Phytoplasma pruni', the RPA assay also detected a novel strain of phytoplasma from several WX-affected trees. Multilocus sequence analyses using the immunodominant protein A (idpA), imp, rpoE, secY, and 16S ribosomal RNA regions from several 'Ca. Phytoplasma pruni' isolates from WX-affected trees showed that this novel phytoplasma strain represents a new subgroup within the 16SrIII group. Examination of high-throughput sequencing data from total RNA of WX-affected trees revealed that the imp coding region is highly expressed, and as supported by quantitative reverse transcription PCR data, it showed higher RNA transcript levels than the previously proposed idpA coding region of 'Ca. Phytoplasma pruni'.

Hop stunt viroid: Effect on Host (Humulus lupulus) Transcriptome and Its Interactions With Hop Powdery Mildew (Podospheara macularis).

Viroids are the smallest known plant pathogens that exploit host systems for their replication and cause diseases in many hosts. In this study, the host response of hop plants to Hop stunt viroid (HSVd) infection was studied through transcriptome analysis. RNA sequence analysis of hop leaves infected with HSVd revealed dynamic changes in hop gene expression. Defense-related genes and genes involved in lipid and terpenoid metabolism are the major categories that showed differential expression due to HSVd infection. Additionally, the effect of HSVd on development of hop powdery mildew (Podospheara macularis) (HPM) was studied. Transcriptome analysis followed by quantitative reverse transcription-polymerase chain reaction analysis showed that transcript levels of pathogenesis-related (PR) genes such as PR protein 1, chitinase, and thaumatin-like protein genes are induced in leaves infected with HPM alone. The response in these genes to HPM is significantly down-regulated in leaves with HSVd-HPM mixed infection. These results confirm that HSVd alters host metabolism, physiology, and plant defense responses. Nevertheless, in detached leaf assays, HPM consistently expanded faster on HSVd-negative leaves relative to HSVd-positive leaves. Although HSVd infection suppresses elements associated with the host immunity response, infection by HSVd is antagonistic to HPM infection of hops.

A rapid isothermal assay for the detection of Hop stunt viroid in hop plants (Humulus lupulus), and its application in disease surveys.

Hop stunt disease caused by Hop stunt viroid (HSVd) is a growing threat to hop cultivation globally. HSVd spreads mainly by use of contaminated planting material and by mechanical means. Thorough testing of hop yards and removal of infected bines are critical components of efforts to control the spread of the disease. Reverse transcription-polymerase chain reaction (RT-PCR) has become the primary technique used for HSVd detection; however, sample handling and analysis are technically challenging. In this study, a robust reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed to facilitate analysis of multiple samples. The assay was optimized with all major variants of HSVd from other host species in addition to hop variants. Used in conjunction with sample collection cards, RT-RPA accommodates large sample numbers. Greenhouse and farm samples tested with RT-RPA were also tested with RT-PCR and a 100% correlation between the two techniques was found.

Hop Stunt Viroid: Effects on Vegetative Growth and Yield of Hop Cultivars, and Its Distribution in Central Washington State.

Hop stunt viroid (HSVd) is an economically important pathogen that reduces growth and yield of hops. Visual symptoms of infected hop are highly dependent on cultivar. A study was conducted using six cultivars of hop to determine the impact on yield. Average dry cone yields of infected 'Glacier', 'Cascade', and 'Willamette' were reduced by 62, 14, and 34%, respectively, relative to noninoculated healthy plants. No significant yield reduction was observed for 'Nugget', 'Columbus', and 'Galena'. The α-acid and ß-acid contents showed a parallel pattern. Horticultural parameters of Willamette and Nugget were measured in the final year of the study. Internode length, shoot length, and side-arm length were reduced by 29, 26, and 73%, respectively, for infected Willamette bines relative to noninfected bines; no effects were observed resulting from infection of Nugget. To understand the current potential impact of HSVd, a survey was conducted to determine its distribution in central Washington. The survey revealed that 17% of hop plants tested are infected. Hop yield and hop plant longevity will be significantly affected by this level of infection. Copyright © 2017 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Safeguarding Fruit Crops in the Age of Agricultural Globalization.

The expansion of fruit production and markets into new geographic areas provides novel opportunities and challenges for the agricultural and marketing industries. Evidence that fruit consumption helps prevent nutrient deficiencies and reduces the risk of cardiovascular disease and cancer has assisted in the expansion of all aspects of the fruit industry. In today's competitive global market environment, producers need access to the best plant material available in terms of genetics and health if they are to maintain a competitive advantage in the market. An ever-increasing amount of plant material in the form of produce, nursery plants, and breeding stock moves vast distances, and this has resulted in an increased risk of pest and disease introductions into new areas. One of the primary concerns of the global fruit industry is a group of systemic pathogens for which there are no effective remedies once plants are infected. These pathogens and diseases require expensive management and control procedures at nurseries and by producers locally and nationally. Here, we review (i) the characteristics of some of these pathogens, (ii) the history and economic consequences of some notable disease epidemics caused by these pathogens, (iii) the changes in agricultural trade that have exacerbated the risk of pathogen introduction, (iv) the path to production of healthy plants through the U.S. National Clean Plant Network and state certification programs, (v) the economic value of clean stock to nurseries and fruit growers in the United States, and (vi) current efforts to develop and harmonize effective nursery certification programs within the United States as well as with global trading partners.

Rapid and sensitive detection of Little cherry virus 2 using isothermal reverse transcription-recombinase polymerase amplification.

Little cherry virus 2 (LChV2) (genus Ampelovirus) is the primary causal agent of little cherry disease (LCD) in sweet cherry (Prunus avium) in North America and other parts of the world. This mealybug-transmitted virus does not induce significant foliar symptoms in most sweet cherry cultivars, but does cause virus-infected trees to yield unevenly ripened small fruits with poor flavor. Most fruits from infected trees are unmarketable. In the present study, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) technique was developed using LChV2 coat protein specific primers and probe. Detection of terminally labeled amplicons was achieved with a high affinity lateral flow strip. The RT-RPA is confirmed to be simple, fast, and specific. In comparison, although it retains the sensitivity of RT-PCR, it is a more cost-effective procedure. RT-RPA will be a very useful tool for detecting LChV2 from crude extracts in any growth stage of sweet cherry from field samples.

Complete nucleotide sequence of a strain of cherry mottle leaf virus associated with peach wart disease in peach.

The complete nucleotide sequence and genome organization of a peach virus isolate from a naturally infected peach tree showing typical peach wart-like symptoms on the fruit surface was determined and compared to sequences of members of the family Betaflexiviridae. The genome consists of 7,987 nucleotides, excluding the poly-A tail, and has four open reading frames (ORFs). Analysis of the whole genome and putative proteins encoded by each ORF revealed greatest sequence similarity to a cherry isolate of cherry mottle leaf virus (CMLV). The two isolates have similar genome organizations and share 88 and 93 % homology in their corresponding products of the replicase and coat protein genes, respectively. CMLV has been reported from several Prunus spp. and may be associated with peach wart-like disease symptoms on peach fruit.

Complete genome organization of American hop latent virus and its relationship to carlaviruses.

The complete genomic sequence of American hop latent virus (AHLV; genus Carlavirus) was determined. The genome consists of 8,601 nucleotides plus a 3'-polyadenylate tail. The genome encompasses six potential open reading frames (ORF) in the positive sense, and their organization is typical of other carlaviruses. Analysis of the coat protein coding sequence at both the nucleic acid level and the amino acid level indicates that AHLV is only remotely related to the other carlaviruses known to infect common hop. Polyclonal antibodies were produced against the bacterially expressed coat protein of AHLV. These antibodies differentiated between AHLV and other carlaviruses of hop.

Complete nucleotide sequences and genome organization of a cherry isolate of cherry leaf roll virus.

The complete nucleotide sequence of cherry leaf roll virus (CLRV, genus Nepovirus) from a naturally infected cherry tree (Prunus avium cv. Bing) in North America was determined. RNA1 and RNA2 consist of 7,893 and 6,492 nucleotides, respectively, plus a poly-(A) tail. Each RNA encodes a single potential open reading frame. The first 657 nucleotides of RNA1 and RNA2 are 99% identical and include the 5'-UTR and the first 214 deduced amino acids of the polyproteins following the first of two in-frame start codons. Phylogenetic analysis reveals close relationships between CLRV and members of subgroup C of the genus Nepovirus.

A Strain of Clover yellow vein virus that Causes Severe Pod Necrosis Disease in Snap Bean.

Soybean aphid (Aphis glycines) outbreaks occurring since 2000 have been associated with severe virus epidemics in snap bean (Phaseolus vulgaris) production in the Great Lakes region. Our objective was to identify specific viruses associated with the disease complex observed in the region and to survey bean germplasm for sources of resistance to the causal agents. The principle causal agent of the disease complex associated with extensive pod necrosis was identified as Clover yellow vein virus (ClYVV), designated ClYVV-WI. The virus alone caused severe mosaic, apical necrosis, and stunting. Putative coat protein amino acid sequence from clones of amplicons generated by reverse-transcription polymerase chain reaction was 98% identical to ClYVV strain no. 30 identified in Japan that has not been reported to cause pod necrosis. ClYVV-WI amplicons were 96% identical to a mild strain of ClYVV from Oregon. A distinguishing feature of this new strain is that it does not react with Potyvirus broad-spectrum monoclonal antibody PTY 1. A survey of common bean lines and cultivars revealed that, in addition to UI-31 and US1140 with known resistance to ClYVV, lines with the bc-3 gene for resistance to Bean common mosaic necrosis virus also were resistant to ClYVV-WI. An evaluation of 63 snap bean cultivars and breeding lines revealed just one, Roma 442, with a moderate level of tolerance to ClYVV-WI. Introgression of the bc-3 gene and resistances from UI-31 and US1140 into snap bean may offer a high level of resistance to extensive pod necrosis disease caused by ClYVV in the Great Lakes region.

Population Biology of Pseudoperonospora humuli in Oregon and Washington.

Pseudoperonospora humuli populations from Oregon and Washington were analyzed for genetic variation using random amplified polymorphic DNA (RAPD) and DNA amplification fingerprinting (DAF) markers. The genetic structure of the Oregon and Washington populations differed considerably. There was little genetic diversity in Washington, with only five RAPD and six DAF groups detected among 40 isolates tested. One genotype was predominant in Washing-ton. In contrast, 18 RAPD and 34 DAF groups were found among the 40 isolates tested from Oregon. No unique band profile associated with host cultivar was observed. It is suggested that the distinct difference in population structure between the two geographic regions might be due to climatic differences resulting in a higher frequency of sexual reproduction of P. humuli in Oregon than in Washington.

Biological control of apple blue mold with Pseudomonas fluorescens.

Pseudomonas fluorescens isolate 1100-6 was evaluated as a potential biological control agent for apple blue mold caused by Penicillium expansum or Penicillium solitum. Both the wild-type isolate 1100-6 and a genetically modified derivative labeled with the gene encoding the green fluorescent protein (GFP) were compared. The P. fluorescens isolates with or without GFP equally reduced the growth of Penicillium spp. and produced large zones of inhibition in dual culture plate assays. Cell-free metabolites produced by the bacterial antagonists reduced the colony area of Penicillium isolates by 17.3% to 78.5%. The effect of iron chelate on the antagonistic potential of P. fluorescens was also studied. The use of iron chelate did not have a major effect on the antagonistic activity of P. fluorescens. With or without GFP, P. fluorescens significantly reduced the severity and incidence of apple decay by 2 P. expansum isolates after 11 d at 20 degrees C and by P. expansum and P. solitum after 25 d at 5 degrees C when the biocontrol agents were applied in wounds 24 or 48 h before challenging with Penicillium spp. Populations of P. fluorescens labeled with the GFP were determined 1, 9, 14, and 20 d after inoculation at 5 degrees C. The log CFU/mL per wound increased from 6.95 at the time of inoculation to 9.12 CFU/mL (P < 0.05) 25 d after inoculation at 5 degrees C. The GFP strain did not appear to penetrate deeply into wounds based on digital photographs taken with an inverted fluorescence microscope. These results indicate that P. fluorescens isolate 1100-6 could be an important new biological control for apple blue mold.

Incidence and Spatial Distribution of Viruses in Hop Gardens of Washington State.

The incidences of Hop latent virus (HpLV), Hop mosaic virus (HpMV), and American hop latent virus (AHLV), members of the genus Carlavirus, and Prunus necrotic ringspot virus and Apple mosaic virus, members of the genus Ilarvirus, were assessed for two hop cultivars, Horizon and Nugget, in Washington State. The spatial distribution of plants infected by the carlaviruses was assessed in two Horizon gardens in 2000 and one Nugget garden in 1993, 1994, and 1995. In the first Horizon garden (garden 1) and the Nugget garden, plants were separated by 2.1 m within and between rows. In these gardens, cultivation and the wide plant spacing discouraged contact between plants in either direction. In the second Horizon garden (garden 2), plants were separated by 4.3 m between rows and 1.0 m within rows. In all gardens, mechanical operations operated predominantly along rows; however, the closer plant spacing within rows in garden 2 permitted contact between adjacent plants within rows. In both Horizon gardens, the distribution of plants infected with HpMV was aggregated within rows. However, the distribution of plants infected with HpLV and AHLV was strongly influenced by contact between plants. In the Nugget garden, the distribution of plants infected by all three carlaviruses was autocorrelated within rows by 1995.