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.

Caliciopsis moriondi, a new species for a fungus long confused with the pine pathogen C. pinea.

The genus Caliciopsis (Eurotiomycetes, Coryneliales) includes saprobic and plant pathogenic species. Caliciopsis canker is caused by Caliciopsis pinea Peck, a species first reported in the 19th century in North America. In recent years, increasing numbers of outbreaks of Caliciopsis canker have been reported on different Pinus spp. in the eastern USA. In Europe, the disease has only occasionally been reported causing cankers, mostly on Pinus radiata in stressed plantations. The aim of this study was to clarify the taxonomy of Caliciopsis specimens collected from infected Pinus spp. in Europe and North America using an integrative approach, combining morphology and phylogenetic analyses of three loci. The pathogenicity of the fungus was also considered. Two distinct groups were evident, based on morphology and multilocus phylogenetic analyses. These represent the known pathogen Caliciopsis pinea that occurs in North America and a morphologically similar, but phylogenetically distinct, species described here as Caliciopsis moriondi sp. nov., found in Europe and at least one location in eastern North America. Caliciopsis moriondi differs from C. pinea in various morphological features including the length of the ascomata, as well as their distribution on the stromata.

Real-time loop-mediated isothermal amplification assay for rapid detection of Fusarium circinatum.

Fusarium circinatum is the causal agent of pitch canker, a lethal disease of pine and other conifers. Since F. circinatum is a quarantine organism, its timely detection could efficiently prevent its introduction into new areas or facilitate spread management in already infected sites. In this study, we developed a sequence-specific probe loop-mediated isothermal amplification (LAMP) assay for F. circinatum using a field-deployable portable instrument. The assay was able to recognize the pathogen in host tissues in just 30 min, and the sensitivity of the assay made it possible to detect even small amounts of F. circinatum DNA (as low as 0.5 pg/µl). The high efficiency of this method suggests its use as a standard diagnostic tool during phytosanitary controls.

Real-time loop-mediated isothermal amplification: an early-warning tool for quarantine plant pathogen detection.

An effective framework for early warning and rapid response is a crucial element to prevent or mitigate the impact of biological invasions of plant pathogens, especially at ports of entry. Molecular detection of pathogens by using PCR-based methods usually requires a well-equipped laboratory. Rapid detection tools that can be applied as point-of-care diagnostics are highly desirable, especially to intercept quarantine plant pathogens such as Xylella fastidiosa, Ceratocystis platani and Phytophthora ramorum, three of the most devastating pathogens of trees and ornamental plants in Europe and North America. To this aim, in this study we developed three different loop mediated isothermal amplification (LAMP) assays able to detect each target pathogen both in DNA extracted from axenic culture and in infected plant tissues. By using the portable instrument Genie® II, the LAMP assay was able to recognize X. fastidiosa, C. platani and P. ramorum DNA within 30 min of isothermal amplification reaction, with high levels of specificity and sensitivity (up to 0.02 pg µL-1 of DNA). These new LAMP-based tools, allowing an on-site rapid detection of pathogens, are especially suited for being used at ports of entry, but they can be also profitably used to monitor and prevent the possible spread of invasive pathogens in natural ecosystems.

Comparative transcriptional and metabolic responses of Pinus pinea to a native and a non-native Heterobasidion species.

Heterobasidion irregulare is a causal agent of root and butt-rot disease in conifers, and is native to North America. In 1944 it was introduced in central Italy in a Pinus pinea stand, where it shares the same niche with the native species Heterobasidion annosum. The introduction of a non-native pathogen may have significant negative effects on a naïve host tree and the ecosystem in which it resides, requiring a better understanding of the system. We compared the spatio-temporal phenotypic, transcriptional and metabolic host responses to inoculation with the two Heterobasidion species in a large experiment with P. pinea seedlings. Differences in length of lesions at the inoculation site (IS), expression of host genes involved in lignin pathway and in cell rescue and defence, and analysis of terpenes at both IS and 12 cm above the IS (distal site, DS), were assessed at 3, 14 and 35 days post inoculation (dpi). Results clearly showed that both species elicit similar physiological and biochemical responses in P. pinea seedlings. The analysis of host transcripts and total terpenes showed differences between inoculation sites and between pathogen and mock inoculated plants. Both pathogen and mock inoculations induced antimicrobial peptide and phenylalanine ammonia-lyase overexpression at IS beginning at 3 dpi; while at DS all the analysed genes, except for peroxidase, were overexpressed at 14 dpi. A significantly higher accumulation of terpenoids was observed at 14 dpi at IS, and at 35 dpi at DS. The terpene blend at IS showed significant variation among treatments and sampling times, while no significant differences were ever observed in DS tissues. Based on our results, H. irregulare does not seem to have competitive advantages over the native species H. annosum in terms of pathogenicity towards P. pinea trees; this may explain why the non-native species has not widely spread over the 73 years since its putative year of introduction into central Italy.

Duplex real-time PCR assay for the simultaneous detection of Caliciopsis pinea and Fusarium circinatum in pine samples.

Fusarium circinatum and Caliciopsis pinea are the causal agents of Pitch canker and Caliciopsis canker, respectively. These diseases affect pines and other conifers both in Europe and North America. The two pathogens cause similar bleeding cankers, especially at the early stage of colonization. Symptoms closely resembling those due to F. circinatum can be instead associated with C. pinea. Since F. circinatum is a quarantine organism, subjected to provisional emergency measures, its report immediately causes serious economic implications, while C. pinea, even if now emerging, is not regulated in the EU nor in the USA. For this reason, a reliable and accurate diagnostic tool able to distinguish between the two organisms was considered a priority. In this study, we developed and standardized a duplex real-time PCR assay allowing the simultaneous recognition of C. pinea and F. circinatum DNA in pine tissue in a reasonably short time and for amounts as small as 0.06 pg/µl. The molecular assay is, therefore, able to detect the infection even before symptoms have fully developed. The test was challenged with a very large set of strains (110 different isolates) collected in different regions of the world and host trees, and gave reliable results. The high efficiency of this method suggests its use as a standard diagnostic tool during phytosanitary controls. In addition, the duplex real-time PCR assay presented here is the first DNA-based method designed to detect C. pinea, which is becoming an increasing threat to pine stands both in North America and in Europe.