Angular Leaf Spot Of Strawberry Research Articles

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Evaluation of a Viable-Cell Detection Assay for Xanthomonas fragariae with Latent Class Analysis

Most molecular diagnostic assays are designed to detect the simple presence of a protein or nucleic acid without regard to whether that protein or nucleic acid originated from a viable and presumably pathogenic organism at the time of isolation. We recently developed a viable-cell detection assay (propidium monoazide [PMA]-qPCR) for specific detection of viable (living) cells of Xanthomonas fragariae, the pathogen causing angular leaf spot of strawberry, and herein describe a unique set of statistical analyses for validation of this assay. For any detection assay or test, calculation of its sensitivity and specificity is essential for determining its diagnostic capabilities, particularly when evaluating competing assays or tests. This is often achieved by running competing tests concurrently on a set of samples with known pathogen density or disease status and cross-tabulating results. However, the PMA-qPCR assay is unique among PCR-based diagnostics because it is designed specifically to detect DNA from living cells only, whereas most traditional PCR assays are designed to detect DNA from the target organism regardless of its state of viability. Thus, one cannot directly compare the results from a cell-viability assay with those from a general assay to gauge the performance of either assay because the assays target two different but overlapping populations (i.e., the viable-cell population and the viable- plus nonviable-cell population). To address this challenge, two standard statistical approaches to diagnostic test evaluation were used jointly to estimate the test performance of the PMA-qPCR assay relative to two common assays for detection of X. fragariae. In both analyses, the PMA-qPCR outperformed the qPCR for detection of viable cells under a range of conditions. Viability testing is extremely useful in certification and disease management applications, and with the information on test performance generated here, the test can be put to practical use to design sampling strategies to account for the errors in testing. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

First Report of Bacterial Angular Leaf Spot of Strawberry Caused by Xanthomonas fragariae in Yunnan Province, China.

First Report of Bacterial Angular Leaf Spot of Strawberry Caused by Xanthomonas fragariae in Yunnan Province, China.

Propolis and Lentinula edodes extracts can control the angular leaf spot of strawberry by different mechanisms

Propolis and Lentinula edodes extracts can control the angular leaf spot of strawberry by different mechanisms

Draft Genome Sequences of Two Xanthomonas fragariae Strains.

Xanthomonas fragariae is the causal agent of angular leaf spot of strawberry. Short-read sequences were generated for two X. fragariae strains with different virulence phenotypes on the Illumina HiSeq 2000 platform. These genome sequences will contribute to a better understanding of pathogen evolution and the genes contributing to virulence in X. fragariae.

First Report of Xanthomonas fragariae Causing Angular Leaf Spot on Strawberry (Fragaria x ananassa) in Taiwan.

Angular leaf spot of strawberry, considered an A2 quarantine pest by the European and Mediterranean Plant Protection Organization (EPPO 2019), is an important bacterial disease in many regions. Since 2017, symptoms similar to angular leaf spot were observed in several strawberry cultivars including 'Taoyuan No. 1' and 'Xiang-Shui'. Early symptoms were angular, water-soaked lesions on the abaxial leaf surface, and later, reddish-brown irregular spots and coalesced lesions developed on the adaxial surface. In the humid conditions, sticky bacterial ooze exuding from lesions was observed. To isolate the causal agent, leaves showing water-soaked lesions were surface sterilized, cut into small pieces and soaked in 5 ml sterile water for at least 15 min. The supernatant from the cut-up pieces was serially diluted followed by spreading on sucrose peptone agar (SPA) (Hayward 1960). After incubating at 20°C for 4-5 days, single colonies grown on SPA were transferred to a new SPA plate and cultured at 20°C until colonies appeared. The yellow, glossy and mucoid colonies, which resembled the colony morphology of Xanthomonas fragariae, were selected as candidates for further confirmation. First, bacterial DNA of four candidate isolates, B001, B003 and B005 from Miaoli County and B004 from Taoyuan City, was PCR amplified with X. fragariae-specific primers: XF9/XF12 (Roberts et al. 1996) and 245A/B and 295A/B (Pooler et al. 1996). All four isolates could be detected by XF9/XF12 primer. Furthermore, isolates B003 and B004 could be detected by both 245A/B and 295A/B primers, while B001 and B005 could be detected by 295A/B only. Next, DNA gyrase subunit B (gyrB) was PCR amplified with the primers XgyrB1F/XgyrB1R (Young et al. 2008). The gyrB sequences of these four isolates were deposited in GenBank with accession numbers MT754942 to MT754945. The gyrB phylogenetic tree was constructed based on Bayesian inference analysis and maximum likelihood analysis. The gyrB sequences of the four isolates from Taiwan clustered in the clade containing the type strain of X. fragariae ICMP5715, indicating that they belong to X. fragariae. B001 and B005 formed a sub-group separated from B003 and B004, suggesting genetic differences between these isolates. To fulfill Koch's postulates, the abaxial surface of strawberry leaves were syringe infiltrated (KJP Silva et al., 2017) or wounded inoculated (Wang et al., 2017) with bacterial suspensions (final OD600 = 1.0-2.0) prepared from colonies of B001 and B003 washed from SPA plates. Inoculated plants were enclosed in a plastic bag (> 90% RH) at 25/20°C (day/night) under a 12-h/12-h photoperiod. After 7-14 days, water-soaked lesions similar to those observed in the field were developed on all inoculated leaves. The bacteria were successfully re-isolated from lesions of inoculated leaves and confirmed by specific primers XF9/XF12, 245A/B and 295A/B. We also found that the disease commonly occurs in the strawberry fields/nurseries with sprinkler irrigation during winter or early spring, and was particularly serious in the windward side or near riverside. To our knowledge, this is the first report of X. fragariae causing angular leaf spot on strawberry in Taiwan. Currently, the disease only occurs severely in certain regions, but establishment of effective management strategies will be needed to prevent spreading of this disease and potential economic loss in the future.

Risks for infection of strawberry plants with an aerosolized inoculum of Xanthomonas fragariae

Xanthomonas fragariae is the causative agent of angular leaf spot of strawberry, a quarantine organism in plant propagation material in the European Union. Field experiments were conducted to assess the risks for infection of strawberry plants through dispersal of an aerosolized inoculum. In practice, pathogen aerosols can be formed during mowing of an infected crop or by water splashing on symptomatic plants during overhead irrigation or rain. In our experiments, aerosols were generated by spraying suspensions of X. fragariae with a density of 108 cfu ml−1 or water under pressure vertically up into the air. In strawberry plants (cv Elsanta) placed at 1.3, 5 and 10 m distance downwind from the spray boom, infections were found, as evidenced with a combination of dilution–plating and molecular techniques, but more frequently in plants wetted prior to inoculation than in plants kept dry. A logarithmic decrease in infection incidence was found with the distance to the inoculum source. Symptomatic plants were found up to 5 m distance from the inoculum source. No infected plants were found in plants placed 4 m upwind or treated with water. In glasshouse studies, it was shown that under conditions favorable for disease development, spray-inoculation of strawberry plants with estimated densities of X. fragariae as low as 2000 cfu per plant were able to cause symptoms both in cv Elsanta and cv Sonata. Results indicate that there is a considerable risk on infections of strawberry plants exposed to aerosolized inoculum.

Colonization and Movement of Xanthomonas fragariae in Strawberry Tissues.

Xanthomonas fragariae causes angular leaf spot of strawberry, an important disease in strawberry growing regions worldwide. To better understand how X. fragariae multiplies and moves in strawberry plants, a green fluorescent protein (GFP)-labeled strain was constructed and used to monitor the pathogen's presence in leaf, petiole, and crown tissue with fluorescence microscopy following natural and wound inoculation in three strawberry cultivars. Taqman PCR was used to quantify bacterial densities in these same tissues regardless of the presence of GFP signal. Results showed X. fragariae colonized leaf mesophyll, the top 1 cm portion of the petiole adjacent to the leaf blade, and was occasionally found colonizing xylem vessels down to the middle of the petioles. The colonization of vascular bundles and the limited systemic movement that was observed appeared to be a passive process, of which the frequency increased with wounding and direct infiltration of bacteria into leaf veins. X. fragariae was able to directly enter petioles and colonize the space under the epidermis. Systemic movement of the bacteria into crown and other uninoculated tissues was not detected visually by GFP. However, X. fragariae was occasionally detected in these tissues by qPCR, but at quantities very near the qPCR detection limit. Petiole tissue harboring bacteria introduced either by direct entry through natural openings or wounds, or by systemic movement from infected foliar tissue, likely serves as a main source of initial inoculum in field plantings.

A diagnostic tool for improved detection of Xanthomonas fragariae using a rapid and highly specific LAMP assay designed with comparative genomics

Molecular diagnostics of plant pathogens are crucial to prevent disease spread and to enhance food quality and security. A comparative genomics approach using genomes of different Xanthomonas species and pathovars was applied to identify highly specific targets in the genome of Xanthomonas fragariae, the causal agent of angular leaf spot of strawberry, listed under quarantine regulations in Europe. A reliable and sensitive loop‐mediated isothermal amplification (LAMP) assay was designed using a unique marker, providing a highly specific and rapid detection technique, convenient for on‐site detection. Specificity of the designed assay was tested on 37 strains from a culture collection of X. fragariae, 82 strains of other Xanthomonas species and pathovars and 11 strains of other bacterial genera isolated from strawberry leaves. A detection limit of 102 fg was achieved, approximating to 20 genome copies per reaction. When performing analyses with crude plant material, a consistent lower detection efficiency of 102 CFU mL−1 was achieved. The LAMP assay designed in this study was adapted to work on crude plant material without any prior extensive extraction steps or incubation period; moreover, it does not require advanced analytical knowledge or a fully equipped laboratory. Results were produced within 7–20 min, depending on the pathogen concentration, thus providing a high‐throughput and user‐friendly method for detection and screening of plant material in support of quarantine regulations.

Finished Genome Sequences of Xanthomonas fragariae, the Cause of Bacterial Angular Leaf Spot of Strawberry.

Xanthomonas fragariae is a foliar pathogen of strawberry that is of significant concern to nursery production of strawberry transplants and field production of strawberry fruit. Long-read sequencing was employed to generate finished genomes for two isolates (each with one chromosome and two plasmids) from symptomatic plants in northern California.

Inhibition of Xanthomonas fragariae, Causative Agent of Angular Leaf Spot of Strawberry, through Iron Deprivation.

In commercial production settings, few options exist to prevent or treat angular leaf spot (ALS) of strawberry, a disease of economic importance and caused by the bacterial pathogen Xanthomonas fragariae. In the process of isolating and identifying X. fragariae bacteria from symptomatic plants, we observed growth inhibition of X. fragariae by bacterial isolates from the same leaf macerates. Identified as species of Pseudomonas and Rhizobium, these isolates were confirmed to suppress growth of X. fragariae in agar overlay plates and in microtiter plate cultures, as did our reference strain Pseudomonas putida KT2440. Screening of a transposon mutant library of KT2440 revealed that disruption of the biosynthetic pathway for the siderophore pyoverdine resulted in complete loss of X. fragariae antagonism, suggesting iron competition as a mode of action. Antagonism could be replicated on plate and in culture by addition of purified pyoverdine or by addition of the chelating agents tannic acid and dipyridyl, while supplementing the medium with iron negated the inhibitory effects of pyoverdine, tannic acid and dipyridyl. When co-inoculated with tannic acid onto strawberry plants, X. fragariae’s ability to cause foliar symptoms was greatly reduced, suggesting a possible opportunity for iron-based management of ALS. We discuss our findings in the context of ‘nutritional immunity,’ the idea that plant hosts restrict pathogen access to iron, either directly, or indirectly through their associated microbiota.

A Loop-Mediated Isothermal Amplification Assay and Sample Preparation Procedure for Sensitive Detection of Xanthomonas fragariae in Strawberry.

Xanthomonas fragariae is a bacterium that causes angular leaf spot of strawberry. Asymptomatic infection is common and contributes to the difficulties in disease management. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay as an efficient method for detection of asymptomatic infections of X. fragariae. In addition, a new method of sample preparation was developed that allows sampling of a larger amount of plant tissue, hence increasing the detection rate in real-life samples. The sample preparation procedure includes an overnight incubation of strawberry tissues in phosphate-buffered saline (PBS), followed by a quick sample concentration and a boiling step to extract DNA for amplification. The detection limit of the LAMP assay was approximately 2×103 CFU/mL for pure bacteria culture and 300 CFU/mL for bacteria spiked strawberry leaf and petiole samples. LAMP provided a 2–3 fold lower detection limit than the standard qPCR assay but was faster, and more user-friendly. The LAMP assay should serve as a rapid, sensitive and cost-effective tool for detecting asymptomatic infections of X. fragariae in strawberry nursery stock and contribute to improved disease management.

Effect of sugar alcohols, antioxidants and activators of systemically acquired resistance on severity of bacterial angular leaf spot (Xanthomonas fragariae) of strawberry in controlled environment conditions

Bacterial angular leaf spot of strawberry caused by Xanthomonas fragariae is an increasingly important disease for which few management strategies are available. The resistance status of most cultivars is unknown and of those cultivars tested, few have significant levels of resistance. Copper formulations are available and have limited effectiveness but phytotoxicity is a problem with repeated applications. This controlled environment study examined the efficacy of foliar applications of various antioxidants, such as α-tocopherol and mannitol, along with the plant activators acetylsalicylic acid and acibenzolar-S-methyl, and the fungicide fosetyl-Al, for disease control. Mannitol and acibenzolar-S-methyl consistently provided excellent control of disease, while fosetyl-Al and α-tocopherol were significantly less effective. Other antioxidants and sugar alcohols similar to mannitol did not provide the same level of angular leaf spot control. Interestingly, interactions were observed between various sugar alcohols and strawberry cultivars in suppressing disease. The mode of action of sugar alcohols against angular leaf spot is likely not related to their antioxidant activity. Sugar alcohols, especially mannitol, may represent a new and non-toxic approach to bacterial angular leaf spot control.

Xanthomonas fragariae에 의한 딸기 세균성모무늬병

Angular leaf spot of strawberry caused by Xanthomonas fragariae occurred sporadically in the farmers open field at Sugok-myon, Jinju city, and greenhouse at Okjong-myon, Hadong-gun, Gyeongnam province, Korea. Typical symptoms of angular leaf spot initially appeared as a minute, water-soaked, rotted lesion on the lower leaf surface and the minute spots expanded to form angular leaf spots, usually delimited by small veins. The lesion was translucent under the transmitted light and dark green under reflected light. The slow growing pathogen was a gram-negative, motile bacterium with single polar flagellum, and producing yellowish orange pigment on YDC medium. It showed very strong pathogenicity on the leaves of strawberry. On the basis of biological characteristics and pathogenicity test on host plants, the bacteria was identified as a Xanthomonas fragariae. This is the first report on angular leaf spot of strawberry caused by X. fragariae in Korea.

Development and Optimization of a Real-Time Detection Assay for Xanthomonas fragariae in Strawberry Crown Tissue with Receiver Operating Characteristic Curve Analysis

Angular leaf spot of strawberry is caused by the bacterium Xanthomonas fragariae. The disease is transmitted primarily through systemically infected nursery stock. This creates problems for nurseries wishing to export plants to Europe because of quarantine restrictions. Currently, field inspections for symptoms are used to certify plants free of X. fragariae, but visual inspection is not useful for detecting plants infected systemically. To detect systemic infections, polymerase chain reaction (PCR) is the desired tool because of its sensitivity, specificity, and ease of use. In this study, we developed three sets of real-time PCR primers and probes and determined optimal reaction conditions for use of these primers for the detection of the bacterium X. fragariae in strawberry crown tissue. Real time detection proved to be both more sensitive and specific than standard PCR. Moreover, the detection of X. fragariae in crown tissue extract was possible with real-time PCR but not with standard PCR which is a significant improvement over standard PCR. The information on sensitivity and specificity of the primer sets was used to evaluate the performance of these primers with receiver operating characteristic (ROC) curve analysis under different tolerances. The results of this analysis can be used to provide guidance on threshold selection to manage disease below unacceptable levels. The results of this research may be useful to regulators and inspectors who must certify that plants meet European and Mediterranean Plant Protection Organization standards.

Nested Pcr (polymerase chain reaction) for detection of Xanthomonas fragariae in symptomless strawberry plants

For rapid and sensitive detection of Xanthomonas fragariae, the causal agent of angular leaf spot of strawberry, a nested polymerase chain reaction (Pcr) was developed. A specific fragment was amplified from X fragariae Dna using primers 245A and 245B, described previously (Pooler et al. 1996). The fragment was sequenced and internal primers suitable for nested Pcr were selected. Using this internal pair of primers, a specific fragment was amplified from all of the 14 X. fragariae isolates tested but no fragment was amplified from X. campestris isolates or from unidentified bacteria, which were isolated from strawberry plants. Whereas detection limit of simple Pcr was 20 pg Dna per reaction, nested Pcr was up to a hundred times more sensitive and even from 200 fg Dna per reaction, a fragment was amplified. In addition, fragments amplified by nested Pcr were always clearly detectable on agarose gels, whereas fragments amplified by simple Pcr were only visible as faint bands when template concentrations decreased. Applicability of nested Pcr was tested on samples from naturally infected fields and from nursery plants showing no symptoms by visible inspection. In symptomatic plants, X. fragariae was regularly detected in leaves, especially in old leaves. In the crown, it was only occasionally detected. Simple Pcr was sufficient for confirmation of symptomatic infection and sometimes for detection of X. fragariae in symptomless parts of symptom-bearing plants and in latently infected nursery plants, respectively. However, using more sensitive nested Pcr, latent infections were more frequently detected and Pcr fragments were more clearly visible on agarose gels.

Bacterial leaf blight of strawberry (Fragaria (x) ananassa) caused by a pathovar of Xanthomonas arboricola, not similar to Xanthomonas fragariae Kennedy & King. Description of the causal organism as Xanthomonas arboricola pv. fragariae (pv. nov., comb. nov.)

A new bacterial disease of strawberry is described. This disease, called bacterial leaf blight of strawberry, is characterized by dry, brown necrotic leaf spots and large brown V‐shaped lesions along the leaf margin, midrib and major veins. Symptoms are different from angular leaf spot of strawberry caused by the bacterium Xanthomonas fragariae. Strains of the bacterial leaf blight pathogen were characterized in a polyphasic approach by biochemical tests, fatty acid analysis, protein electrophoresis, serology, PCR, pigment analysis, ice‐nucleation activity, AFLP analysis, DNA:DNA hybridization, pathogenicity and host range tests, and compared with a number of reference strains of X. fragariae and other Xanthomonas species. Bacterial leaf blight strains formed a homogeneous group in all tests, completely different from X. fragariae. They were the only strains causing leaf blight of strawberry upon artificial inoculation into strawberry. Fatty acid and protein electrophoretic analysis showed that the strains belong to the phenon X. campestris (sensu latu, including pathovars now classified as belonging to X. arboricola). AFLP analysis and DNA:DNA hybridization further clarified their taxonomic position as belonging to X. arboricola. The name X. arboricola pv. fragariae is proposed for the bacterium causing leaf blight of strawberry with strain PD2780 (LMG 19145) as pathovar type strain. Criteria for routine identification are given and the taxonomic status is discussed.

Structural Determination of theO-Specific Polysaccharide from theXanthomonas fragariaeLipopolysaccharide Fraction

The structure of the O-specific polysaccharide from the lipopolysaccharide fraction of the phytopathogenic bacterium Xanthomonas fragariae, the causal agent of the angular leaf spot of strawberry, is described. On the basis of the spectroscopic data, chemical analyses and degradation methods, a structure is suggested.

Structural Determination of the O-Specific Polysaccharide from the Xanthomonas fragariae Lipopolysaccharide Fraction

The structure of the O-specific polysaccharide from the lipopolysaccharide fraction of the phytopathogenic bacterium Xanthomonas fragariae, the causal agent of the angular leaf spot of strawberry, is described. On the basis of the spectroscopic data, chemical analyses and degradation methods, a structure is suggested.

Angular leaf spot of strawberry, a new disease in Argentina. Comparative control with antibiotics and fungicides.

Angular leaf spot of strawberry, a new disease in Argentina. Comparative control with antibiotics and fungicides.