Incidence of brown rot disease caused by Gnomoniopsis smithogilvyi on buds, flowers and chestnuts and rapid HRM-based detection of the disease.

Chestnut production is considered one of the most important economic resources of rural mountainous areas in Greece. Lately, producers report a steep rise in the incidence of brown rot disease caused by the fungus Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales), which results in severe chestnut rot. The pathogen is considered an emerging pathogen in many countries worldwide (Italy, France, Switzerland, Australia, New Zealand). This study aimed at (a) exploring the incidence of the brown rot disease in Vria (Regional Unit of Pieria, Region of Central Makedonia, Greece), (b) isolating and identifying the causal agent of the disease, (c) exploring the fungus presence at different phenological stages of the chestnut trees, and (d) implementing species-specific Bar- High Resolution Melting Analysis (HRM) for the early detection of G. smithogilvyi in chestnuts. G. smithogilvyi occurrence in chestnut tissues was more severe in June (59 %), nearly disappeared in July (19 %) and August (7 %) and increased again during harvesting time in September (57 %). This result could be attributed to a sum of different factors, including climate conditions. Moreover, it was demonstrated that G. smithogilvyi can be identified using a Bar-HRM analysis of chestnut tissues (buds, flowers and nuts). Results of this study clearly demonstrate that Bar-HRM can be used for the accurate, rapid and reliable identification of G. smithogilvyi universally on infected samples from different localities.

Rapid and Accurate Detection of Gnomoniopsis smithogilvyi the Causal Agent of Chestnut Rot, through an Internally Controlled Multiplex PCR Assay.

The fungus Gnomoniopsis smithogilvyi is a significant threat to the production of sweet chestnut (Castanea sativa) nuts in Australia and worldwide. The pathogen causes nut rot, which leads to substantial production losses. Early and accurate diagnosis of the disease is essential to delineate and implement control strategies. A specific and sensitive multiplex PCR was developed based on the amplification of three barcode sequences of G. smithogilvyi. The assay reliability was enhanced by including the amplification of a host gene as an internal control. Primers were thoroughly evaluated in silico before assessing them in vitro. Primer annealing temperature and concentration were optimised to enhance the assay sensitivity and specificity. The assay detection limit ranged between 0.1 and 1.0 pg (5 and 50 fg/µL) of genomic DNA per reaction. No cross-reactivity was observed with genomic DNA from closely and distantly related fungal species. We also characterised Australian G. smithogilvyi isolates phenotypically and genotypically and found significant differences in morphologic and virulence traits of the isolates. An understanding of the virulence of G. smithogilvyi and the availability of a reliable and accurate diagnostic technique will enable earlier detection of the pathogen, which will contribute to effective control strategies for the disease.

Rapid diagnostics for Gnomoniopsis smithogilvyi (syn. Gnomoniopsis castaneae) in chestnut nuts: new challenges by using LAMP and real-time PCR methods.

Nuts of the sweet chestnut (Castanea sativa) are a widely appreciated traditional food in Europe. In recent years producers and consumers reported a drop of nut quality due to the presence of rot diseases caused by Gnomoniopsis smithogilvyi. Early detection of this pathogen is fundamental to the economic viability of the chestnut industry. In the present study, we developed three molecular methods based on real-time portable LAMP, visual LAMP and qPCR assays for G. smithogilvyi. The molecular assays were specific for G. smithogilvyi and did not amplify the other 11 Gnomoniopsis species and 11 other fungal species commonly associated with chestnuts. The detection limit of both the qPCR and real-time portable LAMP (P-LAMP) assays was 0.128 pg/µL, while the visual LAMP (V-LAMP) assay enabled the detection up to 0.64 pg/µL. By using these newly developed molecular tools, the pathogen was detected in symptomatic and asymptomatic nuts, but not in leaves. The reliability of these molecular methods, including the P-LAMP assay, was particularly useful in detecting G. smithogilvyi of harvested nuts in field, even in the absence of rot symptoms.

Gnomoniopsis smithogilvyi causes chestnut canker symptoms in Castanea sativa shoots in Switzerland.

A screening of Castanea sativa scions for grafting for the presence of endophytes showed that the opportunistic fungal pathogen Gnomoniopsis smithogilvyi was the most abundant member of the endophytic flora. This fungus is known as a pathogen affecting chestnut fruits in Italy and Australia. Here, we present evidence that it causes cankers very similar to the ones due to Cryphonectria parasitica infection on twigs and scions of chestnut trees. We found natural infections of G. smithogilvyi in healthy grafted plants as well as in scions from chestnut trees. The identity of the fungus isolated from asymptomatic tissues was verified by applying Koch's postulates and corroborated by DNA sequencing of four different gene regions. In contrast to C. parasitica that appears on the bark as yellow to orange pycnidia, stromata and slimy twisted tendrils, G. smithogilvyi forms orange to red and black pycnidia, gray stromata and cream-colored to beige slimy twisted tendrils on the bark. These Swiss strains are closely related to G. smithogilvyi strains from Australia and from New Zealand, Gnomoniopsis sp. and Gnomoniopsis castanea from New Zealand, Italy, France and Switzerland. While the strains from Ticino are genetically very close to G. smithogilvyi and G. castanea from Italy, the differences between the strains from Ticino and Geneva suggest two different origins. The present study supports the hypothesis that a single species named G. smithogilvyi, which is known to be the agent of chestnut rot, also causes wood cankers on chestnut.