Redefining IPM for Strawberry Production under the Emerging Threat of Anthracnose and Strawberry Sap Beetle

A sampling survey of strawberry acreage in New York was conducted to determine the distribution of two pests of increasing concern to strawberry growers in New York: strawberry sap beetle, Stelidota geminata (Say), and anthracnose, Colletotrichum acutatum. The 2002 sampling for both pests was conducted in a total of 37 strawberry fields at 14 farms, with farms distributed throughout four agricultural regions of New York

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 2×10(3) 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

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. [Graphic: 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

Youden’s index for the LAMP assay.

<p>The sensitivities and specificities used to calculate the index were estimated through latent class analyses for each combination of LAMP reaction times and qPCR C_t threshold values.</p

Reaction curves for qPCR and real-time LAMP.

<p>(A) qPCR and (B) LAMP amplification curves of pure bacterial DNA. (C) DNA melting curves for the LAMP assay in (B). (D) Gel electrophoresis results of the same samples processed in (B). Samples were run in 1.5% agarose gel with 100 bp DNA ladder and stained with ethidium bromide. (E) End product-detection with HNB dye of the same samples processed in (D). Samples 1–10 in (D) and (E) had the same concentration of <i>X</i>. <i>fragariae</i> as shown in the legend of (A) from top to bottom. (F) qPCR and (G) LAMP amplification curve of <i>X</i>. <i>fragariae-</i>spiked leaf samples processed through the sample preparation procedure.</p

qPCR sensitivities (A, B) and specificities (C, D) relative to LAMP assay results.

<p>The sensitivities and specificities were estimated through latent class analyses for each combination of qPCR C_t and LAMP T_t threshold values. Error bars are standard errors of the means and are shown for a select series in each plot to avoid clutter. The series with the largest error was typically chosen as the representative series. Data points lacking error bars for that series were either too small or were not estimable by the MLE procedure.</p

LAMP sensitivity (A, B) and specificity (C, D) relative to qPCR assay results.

<p>The sensitivities and specificities were estimated through latent class analyses for each combination of LAMP T_t and qPCR C_t threshold values. Error bars are standard errors of the means and are shown for a select series in each plot to avoid clutter. The series with the largest error was typically chosen as the representative series. Data points lacking error bars for that series were either too small or were not estimable by the MLE procedure.</p

Detection rates of LAMP and qPCR.

<p>(A) DNA extracted from pure <i>Xanthomonas fragariae</i> cells. (B) DNA extracted from mixtures of <i>X</i>. <i>fragariae</i> cells and strawberry tissues after 21 h of incubation. Mean detection rates were calculated based on the percentage of positive samples at selected thresholds in 9 (3 samples per experiment) and 18 samples (6 samples per experiment) per bacterial concentration in (A) and (B), respectively. Error bars are standard errors of the means.</p

The Use of Aerated Steam as a Heat Treatment for Managing Angular Leaf Spot in Strawberry Nursery Production and Its Effect on Plant Yield

Xanthomonas fragariae, the bacterium causing angular leaf spot (ALS) of strawberry, is found routinely on strawberry nursery stock. Although ALS can be a serious disease in production fields, it can be particularly problematic in the sale and trade of commercial nursery stock because of international trade regulations. Heat treatment has been shown to be an effective treatment for managing ALS on nursery stock in small-scale experimental trials. The objective of this research was to design, build, and trial precision thermotherapy units for managing ALS for both research and commercial applications and to test a new thermotherapeutic protocol on strawberry nursery stock that combines a conditioning thermal treatment with an eradicative thermal treatment. Small-plot trials were conducted to evaluate the efficacy of precision thermotherapy on control of ALS using known sources of infected nursery stock. Additionally, trials were conducted in cooperation with commercial nurseries to determine the impact of thermotherapy on plant health and yield and on the natural development of ALS. In the small-plot trials, ALS incidence was significantly lower in plots treated with precision thermotherapy. In the commercial trials, precision thermotherapy had a variable, but negligible, effect on plant growth and yield. ALS, when it occurred, was always lower in thermotherapy-treated plots. Heat is a near-universal biocide. Thus, in addition to managing ALS, the commercial application of precision thermal therapy to strawberry nursery stock may be effective for managing a wide range of pest and disease threats to strawberry while simultaneously reducing pesticide usage.[Graphic: 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. 202

Diversity and impact of herbivorous insects on Brazilian peppertree in Florida prior to release of exotic biological control agents

© 2017 Informa UK Limited, trading as Taylor & Francis Group. Brazilian peppertree, Schinus terebinthifolia Raddi (Anacardiaceae), is a South American plant that is highly invasive in Florida. The impact of insect herbivores on the performance of Brazilian peppertree was evaluated at two locations in Florida using an insecticide exclusion method. Although 38 species of insect herbivores were collected on the invasive tree, there were no differences in growth or reproductive output of insecticide protected and unprotected trees, providing evidence that insect feeding had no measurable impact on tree performance. The majority of insects collected on Brazilian peppertree were generalists, and several were serious agricultural pests