First report of grapefruit rot caused by Colletotrichum gloeosporioides and C. karsti in France.

The grapefruit (Citrus paradisi) is a citrus hybrid tree (C. maxima & C. sinensis). Due to nutritional value and its bioactive compounds, the fruits are recognized as a functional food, valued as promoting health. French grapefruit production is low (7.5 Kt/year) but is confined to a restricted area in Corsica and benefits from a quality label, the economic impact of its cultivation being therefore locally significant. Since 2015 previously unreported symptoms have been repeatedly observed on grapefruits in more than half of the orchards in Corsica, with an incidence of 30% of fruits altered. Brown to black circular spots were observed on fruits and leaves, surrounded by chlorotic halos on the latter. On the mature fruit, lesions were round, 4 to 10 mm in diameter, brown and dry (e-Xtra 1). Although the lesions are superficial, the fruits cannot be marketed due to constraints linked to the quality label. 75 fungal isolates were obtained from symptomatic fruits or leaves collected in Corsica (in 2016, 2017, and 2021). Cultures obtained after 7 days on PDA at 25°C, were white to light grey in colour, forming concentric rings or dark spots on the agar surface. We did not observe any notable difference among the isolates except some evolved towards a more marked grey. Colonies tend to form a cottony aerial mycelium and orange conidial masses appear with age. The conidia were hyaline, aseptate, cylindrical with ends rounded, and measured 14.9 ± 0.95 µm length and 5.1 ± 0.45 µm width (n = 50). Cultural and morphological characteristics were similar to those described for C. gloeosporioides s. lat. or C. boninense s. lat. (Weir et al. 2012 ; Damm et al. 2012). Total genomic DNA was extracted from all isolates, and the ITS region of rDNA was amplified with ITS 5 & 4 primers, then sequenced (GenBank Accession Nos. OQ509805-808). For 90% of isolates GenBank BLASTn results were 100% identical to C. gloeosporioides isolates sequences, whereas for other isolates the resulting sequences were 100% identical to C. karsti or C. boninense isolates sequences. Four strains (three C. gloeosporioides with light colour differences, in order to see if there was diversity among isolates of C. gloeosporioides s. lato ; and one C. karsti) were further characterized by sequencing partial actin [ACT], calmodulin [CAL], chitin synthase [CHS-1], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], ß-tubulin 2 [TUB2], for all strains ; glutamine synthetase [GS], Apn2-Mat1-2-1 intergenic spacer and partial mating type (Mat1-2) gene [ApMAT] for C. gloeosporioides s. lat., and HIS3 for C. boninense s. lat. (Weir et al. 2012 ; Silva et al, 2012) (GenBank Accession Nos. OQ509805-808 & OQ507698-724). Multilocus phylogenetic analyses carried out with the obtained and Genbank available sequences confirmed that 3 isolates (UBOCC-A-116036, -116038, & -116039) clustered within C. gloeosporioides s. s., while the other (UBOCC-A-116037) clustered within C. karsti (e-Xtra 2) 'Star ruby' grapefruits were surface sterilized then wound-inoculated with 20 µl of a conidial suspension (105 conidia ml-1) of UBOCC-A-116036 & 116037 isolates or 20 µl sterile water for control (ten fruits for each isolate or control). After 10 days incubation at 20°C, symptoms, identical to those initially observed, developed around the inoculation point, while controls inoculated with water remained symptomless. Fungal colonies re-isolated from the lesions were morphologically like the original isolates. Recently, various infections caused by some Colletotrichum sp. have strongly compromised citrus production in different Mediterranean countries: ie Italy (Aiello et al. 2015), Portugal (Ramos et al. 2016), Tunisia (Ben Hadj Daoud et al. 2019), Turkey (Uysal et al. 2022). In these studies, C. gloeosporioides s. s. and C. karsti were identified as the causal agents. These two species were the predominant Colletotrichum sp. associated with Citrus and allied genera in Europe (Guarnaccia et al. 2017). To our knowledge, our study is the first report of C. gloeosporioides and C. karsti causing anthracnose on grapefruit in France, which confirms the incidence of these two pathogens on the Mediterranean rim. Given the economic importance of citrus cultivation in the Mediterranean region, the presence of Colletotrichum spp. should deserves to be monitored, and a control strategy should be considered.

Quantitative Real-Time PCR Based on SYBR Green Technology for the Identification of Philaenus italosignus Drosopoulos & Remane (Hemiptera Aphrophoridae).

The use of molecular tools to identify insect pests is a critical issue, especially when rapid and reliable tests are required. We proposed a protocol based on qPCR with SYBR Green technology to identify Philaenus italosignus (Hemiptera, Aphrophoridae). The species is one of the three spittlebugs able to transmit Xylella fastidiosa subsp. pauca ST53 in Italy, together with Philaenus spumarius and Neophilaenus campestris. Although less common than the other two species, its identification is key to verifying which role it can play when locally abundant. The proposed assay shows analytical specificity being inclusive with different populations of the target species and exclusive with non-target taxa, either taxonomically related or not. Moreover, it shows analytical sensibility, repeatability, and reproducibility, resulting in an excellent candidate for an official diagnostic method. The molecular test can discriminate P. italosignus from all non-target species, including the congeneric P. spumarius.

DNA Extraction Methods to Obtain High DNA Quality from Different Plant Tissues.

DNA extraction from plant samples is very important for a good performance of diagnostic molecular assays in phytopathology. The variety of matrices (such as leaves, roots, and twigs) requires a differentiated approach to DNA extraction. Here we describe three categories of matrices: (a) symptomatic bark/wood tissue; (b) residues of frass resulting from insect woody trophic activities, portions of the galleries produced in the wood, and tissues surrounding exit holes; and (c) leaves of different plant species. To improve the performances of diagnostic assays, we here describe DNA extraction procedures that have been optimized for each matrix type.

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

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.

Complete Genome Sequence of the Plant-Pathogenic Fungus Colletotrichum lupini.

Colletotrichum is a fungal genus (Ascomycota, Sordariomycetes, Glomerellaceae) that includes many economically important plant pathogens that cause devastating diseases of a wide range of plants. In this work, using a combination of long- and short-read sequencing technologies, we sequenced the genome of Colletotrichum lupini RB221, isolated from white lupin (Lupinus albus) in France during a survey in 2014. The genome was assembled into 11 nuclear chromosomes and a mitochondrial genome with a total assembly size of 63.41 Mb and 36.55 kb, respectively. In total, 18,324 protein-encoding genes have been predicted, of which only 39 are specific to C. lupini. This resource will provide insight into pathogenicity factors and will help provide a better understanding of the evolution and genome structure of this important plant pathogen.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Rapid Identification of Anoplophora chinensis (Coleoptera: Cerambycidae) From Adult, Larval, and Frass Samples Using TaqMan Probe Assay.

A molecular diagnostic method using TaqMan probe qPCR is presented for the identification of Anoplophora chinensis (Förster) (Coleoptera: Cerambycidae) from whole body insects (adults and larvae) and frass samples stored under different conditions. The results showed a perfect amplification of DNA from all samples; the repeatability and reproducibility of the protocol were very good, with standard deviations of inter-run and intra-run variability less than or equal to 0.5. The assay allowed to discern all A. chinensis samples from those of the other non-target wood-borer species, with 100% correspondence to the homologous sequences. No amplification or cross reactions were observed with A. glabripennis (Motschulsky) (Coleoptera: Cerambycidae), which is the most related species among those tested. The protocol was validated by an internal blind panel test which showed a good correspondence between the results obtained by different operators in the same lab. The analytical sensitivity for the lab frass with the Probe qPCR, namely the lowest amount of A. chinensis DNA that can be detected (LoD), was 0.64 pg/µl with a Cq of 34.87. The use of indirect evidence for the identification of a pest is an important feature of the method, which could be crucial to detect the presence of wood-boring insects. This diagnostic tool can help prevent the introduction of A. chinensis into new environments or delimit existing outbreak areas thanks to indirect frass diagnosis.

Rapid Detection of Pityophthorus juglandis (Blackman) (Coleoptera, Curculionidae) with the Loop-Mediated Isothermal Amplification (LAMP) Method.

The walnut twig beetle Pityophthorus juglandis is a phloem-boring bark beetle responsible, in association with the ascomycete Geosmithia morbida, for the Thousand Cankers Disease (TCD) of walnut trees. The recent finding of TCD in Europe prompted the development of effective diagnostic protocols for the early detection of members of this insect/fungus complex. Here we report the development of a highly efficient, low-cost, and rapid method for detecting the beetle, or even just its biological traces, from environmental samples: the loop-mediated isothermal amplification (LAMP) assay. The method, designed on the 28S ribosomal RNA gene, showed high specificity and sensitivity, with no cross reactivity to other bark beetles and wood-boring insects. The test was successful even with very small amounts of the target insect's nucleic acid, with limit values of 0.64 pg/µL and 3.2 pg/µL for WTB adults and frass, respectively. A comparison of the method (both in real time and visual) with conventional PCR did not display significant differences in terms of LoD. This LAMP protocol will enable quick, low-cost, and early detection of P. juglandis in areas with new infestations and for phytosanitary inspections at vulnerable sites (e.g., seaports, airports, loading stations, storage facilities, and wood processing companies).

TaqMan probe assays on different biological samples for the identification of three ambrosia beetle species, Xylosandrus compactus (Eichoff), X. crassiusculus (Motschulsky) and X. germanus (Blandford) (Coleoptera Curculionidae Scolytinae).

Molecular assays based on qPCR TaqMan Probes were developed to identify three species of the genus Xylosandrus, X. compactus, X. crassiusculus and X. germanus (Coleoptera Curculionidae Scolytinae). These ambrosia beetles are xylophagous species alien to Europe, causing damages to many ornamental and fruiting trees as well as shrubs. DNA extraction was carried out from adults, larvae and biological samples derived from insect damages on infested plants. For X. compactus, segments of galleries in thin infested twigs were cut and processed; in the case of X. crassiusculus, raw frass extruded from exit holes was used, while DNA of X. germanus was extracted from small wood chips removed around insect exit holes. The assays were inclusive for the target species and exclusive for all the non-target species tested. The LoD was 3.2 pg/µL for the frass of X. crassiusculus and 0.016 ng/µL for the woody matrices of the other two species. Both repeatability and reproducibility were estimated on adults and woody samples, showing very low values ranging between 0.00 and 4.11. Thus, the proposed diagnostic assays resulted to be very efficient also on the woody matrices used for DNA extraction, demonstrating the applicability of the protocol in the absence of dead specimens or living stages.

Rapid and Sensitive Detection of Tomato Brown Rugose Fruit Virus in Tomato and Pepper Seeds by Reverse Transcription Loop-Mediated Isothermal Amplification Assays (Real Time and Visual) and Comparison With RT-PCR End-Point and RT-qPCR Methods.

Tomato brown rugose fruit virus (ToBRFV) represents an emerging viral threat to the productivity of tomato and pepper protected cultivation worldwide. This virus has got the status of quarantine organism in the European Union (EU) countries. In particular, tomato and pepper seeds will need to be free of ToBRFV before entering the EU and before coming on the market. Thus, lab tests are needed. Here, we develop and validate a one-step reverse transcription LAMP platform for the detection of ToBRFV in tomato and pepper leaves, by real-time assay [reverse transcription loop-mediated isothermal amplification (RT-LAMP)] and visual screening (visual RT-LAMP). Moreover, these methods can also be applied successfully for ToBRFV detection in tomato and pepper seeds. The diagnostic specificity and sensitivity of both RT-LAMP and visual RT-LAMP are both 100%, with a detection limit of nearly 2.25 fg/µl, showing the same sensitivity as RT-qPCR Sybr Green, but 100 times more sensitive than end-point RT-PCR diagnostic methods. In artificially contaminated seeds, the proposed LAMP assays detected ToBRFV in 100% of contaminated seed lots, for up to 0.025-0.033% contamination rates in tomato and pepper, respectively. Our results demonstrate that the proposed LAMP assays are simple, inexpensive, and sensitive enough for the detection of ToBRFV, especially in seed health testing. Hence, these methods have great potential application in the routine detection of ToBRFV, both in seeds and plants, reducing the risk of epidemics.

Development of a loop-mediated isothermal amplification (LAMP) assay for the identification of the invasive wood borer Aromia bungii (Coleoptera: Cerambycidae) from frass.

The red-necked longhorn beetle Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae) is native to east Asia, where it is a major pest of cultivated and ornamental species of the genus Prunus. Morphological or molecular discrimination of adults or larval specimens is required to identify this invasive wood borer. However, recovering larval stages of the pest from trunks and branches causes extensive damage to plants and is timewasting. An alternative approach consists in applying non-invasive molecular diagnostic tools to biological traces (i.e., fecal pellets, frass). In this way, infestations in host plants can be detected without destructive methods. This paper presents a protocol based on both real-time and visual loop-mediated isothermal amplification (LAMP), using DNA of A. bungii extracted from fecal particles in larval frass. Laboratory validations demonstrated the robustness of the protocols adopted and their reliability was confirmed performing an inter-lab blind panel. The LAMP assay and the qPCR SYBR Green method using the F3/B3 LAMP external primers were equally sensitive, and both were more sensitive than the conventional PCR (sensitivity > 103 to the same starting matrix). The visual LAMP protocol, due to the relatively easy performance of the method, could be a useful tool to apply in rapid monitoring of A. bungii and in the management of its outbreaks.

A duplex real-time PCR with probe for simultaneous detection of Geosmithia morbida and its vector Pityophthorus juglandis.

The cultivation of walnuts (Juglans sp.) in Europe retains high economic, social, and environmental value. The recent reporting of the Thousand Cankers Disease (TCD) fungus, Geosmithia morbida, and of its vector, Pityophthorus juglandis, in walnut trees in Italy is alarming the whole of Europe. Although Italy is at present the only foothold of the disease outside North America, given the difficulties inherent in traditional identification of both members of this beetle/fungus complex, a rapid and effective protocol for the early detection and identification of TCD organisms is an absolute priority for Europe. Here we report the development of an effective and sensitive molecular tool based on simplex/duplex qPCR assays for the rapid, accurate and highly specific detection of both the bionectriaceous fungal pathogen and its bark-beetle vector. Our assay performed excellently, detecting minute amounts of target DNA without any non-specific amplification. Detection limits from various and heterogeneous matrices were lower than other reported assays. Our molecular protocol could assist in TCD organism interception at entry points, territory monitoring for the early identification and eradication of outbreaks, delineation of quarantine areas, and tracing back TCD entry and dispersal pathways.

Molecular Identification of Anoplophora glabripennis (Coleoptera: Cerambycidae) From Frass by Loop-Mediated Isothermal Amplification.

Anoplophora glabripennis (Motschulsky, 1853), native to eastern Asia, is a destructive woodborer of many ornamental species, leading to the decline and the death of the attacked trees. In outbreak areas as Europe or North America, this pest is usually identified using morphological or molecular analyses of adult or larval specimens. However, the procedures for collecting A. glabripennis specimens from infested plants are too expensive and time consuming for routine screening. A noninvasive diagnostic tool based on frass discrimination is therefore crucial for the rapid identification of A. glabripennis at different development stages in the host. This article describes a rapid diagnostic protocol based on loop-mediated isothermal amplification (LAMP). DNA extracted from A. glabripennis frass was amplified with both visual and real-time LAMP and compared with those of nontarget species. The results show that the method is reliable and accurate and therefore could be a promising diagnostic tool in phytosanitary surveys.

Genome Resources for the Endophytic Fungus Paraphaeosphaeria sporulosa.

Paraphaeosphaeria genus includes plant pathogens or biocontrol agents as well as bioremediators and endophytic fungi. Paraphaeosphaeria sporulosa 10515 was isolated in 2013 as an endophyte of Festuca spp. collected on Mount Etna at 1,832 meters above sea level. Here, we present the first-draft whole-genome sequence of a P. sporulosa endophytic isolate. This data will be useful for future research on understanding the genetic bases of endophytism.

Molecular Detection of the Seed-Borne Pathogen Colletotrichum lupini Targeting the Hyper-Variable IGS Region of the Ribosomal Cluster.

Lupins anthracnose is a destructive seed and airborne disease caused by Colletotrichum lupini, affecting stems and pods. Primary seed infections as low as 0.01-0.1% can cause very severe yield losses. One of the most effective management strategies is the development of a robust and sensitive seed detection assay to screen seed lots before planting. PCR-based detection systems exhibit higher levels of sensitivity than conventional techniques, but when applied to seed tests they require the extraction of PCR-quality DNA from target organisms in backgrounds of saprophytic organisms and inhibitory seed-derived compounds. To overcome these limitations, a new detection protocol for C. lupini based on a biological enrichment step followed by a PCR assay was developed. Several enrichment protocols were compared with Yeast Malt Broth amended with ampicillin, streptomycin, and lactic acid were the most efficient. A species-specific C. lupini primer pair was developed based on rDNA IGS sequences. The specificity was evaluated against 17 strains of C. lupini, 23 different Colletotrichum species, and 21 different organisms isolated from seeds of Lupinus albus cv. Multitalia, L. luteus cv. Mister, and L. angustifolius cv. Tango. The protocol described here enabled the detection of C. lupini in samples artificially infected with less than 1/10,000 infected seed.

Combined Metabarcoding and Multi-locus approach for Genetic characterization of Colletotrichum species associated with common walnut (Juglans regia) anthracnose in France.

Juglans regia (walnut) is a species belonging to the family Juglandaceae. Broadly spread in diverse temperate and subtropical regions, walnut is primarily cultivated for its nuts. In France, Colletotrichum sp. on walnut was detected for the first time in 2007; in 2011 the disease led to 50-70% losses in nut production. A combined approach of metabarcoding analysis and multi-locus genetic characterization of isolated strains has been used for taxonomic designation and to study the genetic variability of this pathogen in France. Evidence indicates that four Colletotrichum species are associated with walnut in France: 3 belong to the C. acutatum species complex and 1 to the C. gloeosporioides species complex. Results also show that C. godetiae is the most abundant species followed by C. fioriniae; while C. nymphaeae and another Colletotrichum sp. belonging to the C. gloeosporioides complex are found rarely. Representative isolates of detected species were also used to confirm pathogenicity on walnut fruits. The results show a high variability of lesion's dimensions among isolates tested. This study highlights the genetic and pathogenic heterogeneity of Colletotrichum species associated with walnut anthracnose in France providing useful information for targeted treatments or selection of resistant cultivars, in order to better control the disease.

Development of a rapid PCR-Nucleic Acid Lateral Flow Immunoassay (PCR-NALFIA) based on rDNA IGS sequence analysis for the detection of Macrophomina phaseolina in soil.

The 'Nucleic Acid Lateral Flow Immunoassay' (NALFIA) using a generic 'Lateral Flow Device' (LFD), combined with PCR employing labelled primers (PCR-NALFIA), enables to circumvent the use of electrophoresis, making the diagnostic procedure more rapid and easier. If the specific amplicon is present in the sample, a coloured band, with an intensity proportional to the amplicon concentration, will develop on the LFD strip in addition to the control band. Species-specific primers for M. phaseolina based on the rDNA intergenic spacer (IGS) were developed and their specificity was checked and confirmed using 20 isolates of M. phaseolina and other 16 non-target fungi. A DNA extraction protocol based on a bead-beating technique using silica beads, skimmed milk and PVP was also developed. The M. phaseolina specific primers MP102F/MP102R, 5' labelled with biotin and FITC respectively, were used in the PCR-NALFIA assay to identify the pathogen starting from mycelium or microsclerotia. Microsclerotia of M. phaseolina (1, 10, 100 and 200) were manipulated under a stereomicroscope and their DNA was extracted using microsclerotia alone or mixed with different types of soil. The resulting DNA, used for the PCR-NALFIA assay, provided positive results for all the samples tested. A semi-quantitative grey-scale reference card based on the PCR-NALFIA assay using intervals corresponding to microsclerotia soil number was developed. For this purpose, the normalized pixel grey volumes obtained after a densitometric analysis of the test line intensity generated by the LFD dipsticks were used.

Genome Sequence of Fusarium graminearum ITEM 124 (ATCC 56091), a Mycotoxigenic Plant Pathogen.

Fusarium graminearum is among the main causal agents of Fusarium head blight (FHB), or scab, of wheat and other cereals, caused by a complex of Fusarium species, worldwide. Besides causing economic losses in terms of crop yield and quality, F. graminearum poses a severe threat to animal and human health. Here, we present the first draft whole-genome sequence of the mycotoxigenic Fusarium graminearum strain ITEM 124, also providing useful information for comparative genomics studies.