Loop-Mediated Isothermal Amplification (LAMP) and SYBR Green qPCR for Fast and Reliable Detection of Geosmithia morbida (Kolařik) in Infected Walnut

Abstract

Walnut species (Juglans spp.) are multipurpose trees, widely employed in plantation forestry for high-quality timber and nut production, as well as in urban greening as ornamental plants. These species are currently threatened by the thousand cankers disease (TCD) complex, an insect–fungus association which involves the ascomycete Geosmithia morbida (GM) and its vector, the bark beetle Pityophthorus juglandis. While TCD has been studied extensively where it originated in North America, little research has been carried out in Europe, where it was more recently introduced. A key step in research to cope with this new phytosanitary emergency is the development of effective molecular detection tools. In this work, we report two accurate molecular methods for the diagnosis of GM, based on LAMP (real-time and visual) and SYBR Green qPCR, which are complimentary to and integrated with similar recently developed assays. Our protocols detected GM DNA from pure mycelium and from infected woody tissue with high accuracy, sensitivity, and specificity, without cross-reactivity to a large panel of taxonomically related species. The precision and robustness of our tests guarantee high diagnostic standards and could be used to support field diagnostic end-users in TCD monitoring and surveillance campaigns.

Stilbocrea banihashemiana sp. nov. a New Fungal Pathogen Causing Stem Cankers and Twig Dieback of Fruit Trees

“…Members of Bionectriaceae are less known as canker pathogens. However, also this family encompasses species that were reported as causal agents of cankers on woody plants, such as Nectriella pironii Alfieri & Samuels [ 11 ], which causes gall and stem cankers on fig, and Geosmithia morbida M. Kolark, E. Freeland, C. Utley & Tissera, the causal agent of Thousand Cankers Disease (TCD) of walnut ( Juglans regia L.) [ 12 , 13 , 14 ]. Moreover, some species of Stilbocrea , a genus within this family, cause wood discoloration and trunk diseases, e.g., S. colubrensis Lechat & J. Fourn on Bambusa vulgaris (Schrad.…”

Thousand Cankers Disease in Walnut Trees in Europe: Current Status and Management

“…Various protocols for the diagnosis of TCD have been developed in recent years [ 34 , 48 , 49 , 50 , 51 , 52 ]. Firstly, Lamarche et al [ 48 ] have developed an efficient and sensitive real-time PCR assay based on TaqMan chemistry that is highly specific, targeting a discriminant part of the GM β-tubulin gene.…”

“…Since the loop-mediated isothermal AMPlification (LAMP) reaction offers a wide range of possibilities for point-of-care application, its use is generally preferred as a field-deployable molecular approach [ 56 ]. Recently, LAMP-based assays able to detect GM and WTB DNA in a short time (10-15 min) have been developed [ 51 , 52 ]. In particular, these two assays were highly specific, with a sensitivity as low as 3.2 pg/µL and 0.64 pg/µL, respectively, allowing for the rapid, field-deployable screening of environmental samples to identify both the pathogen and the vector (even when only larvae and insect frass are available).…”

Rapid and cost‐effective detection of Fusarium euwallaceae from woody tissues

“…Using PCR to identify F . euwallaceae within plant material without prior DNA purification and using species‐specific primers has not yet been attempted (Rizzo et al, 2021, 2022).…”

“…Vázquez-Rosas-Landa et al ( 2021) also used a comparative genomic approach to develop accurate diagnostic systems for various ascomycete fungi, one of which included a diagnostic PCR marker for F. kuroshium by amplification of a specific fragment from DNA extracted directly from plant tissues with symptoms, without needing to culture the fungus. Using PCR to identify F. euwallaceae within plant material without prior DNA purification and using speciesspecific primers has not yet been attempted (Rizzo et al, 2021(Rizzo et al, , 2022.…”