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.

Key chili and maize growing areas of Pakistan were selected for a focused baseline study of the levels of Aspergillus spp. Investigations were undertaken using a combination of molecular and culture-based techniques. Samples investigated included soil samples, one-year-old corn cobs, and fresh chili from selected locations. Aspergillus strains obtained from corn cobs were screened using coconut milk agar, resulting in one strain that was positive for aflatoxin production. Whole genome sequencing (WGS) with low coverage techniques were employed to screen the isolates for differences in the ribosomal RNA gene cluster and mitochondrial genome, with the aflatoxigenic strain proving to have a distinctive profile. Finally, strains were subjected to matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry (MALDI-ToF-MS) in order to obtain a proteomic ‘fingerprint’ which was used to distinguish the aflatoxigenic strain from the other isolates. The next generation sequencing (NGS) study was broadened to incorporate metabarcoding with ITS rRNA for determining the microbial biodiversity of the soil samples and presumptive screening for the presence of aflatoxigenic strains. Using information gleaned from the WGS results, a putative aflatoxigenic operational taxonomic unit (OTU) was observed in four of the 15 soil samples screened by metabarcoding. This method may have beneficial applications in early detection and surveillance programs in agricultural soils and commodities.

During the summer of 2016, severe dieback was observed on young potted Prunus lusitanica (Portugal laurel) plants in a nursery in the Pistoia province (Tuscany, Italy). Cylindrocarpon-like isolates were consistently recovered from diseased plant tissues. The combination of morphological and molecular traits, including sequence data of histone 3 and β-tubulin genes (HIS3, TUB2) and internal transcribed spacers (ITS), allowed the identification of Dactylonectria macrodidyma (Halleen, Schroers & Crous) L. Lombard & Crous (asexual form Cylindrocarpon macrodidymum) as the causal agent of the disease. Pathogenicity tests reproduced disease symptoms observed in the nursery after six months fulfilling Koch’s postulates. D. macrodidyma is a soilborne plant pathogen and is to be considered of great economic importance on P. lusitanica, especially under favorable conditions such as stress and/or reduction of plant vitality. The increasingly frequent reports of the disease caused by the pathogen in various nurseries suggest that pot cultivation, together with prolonged drought periods, may play a role in favoring infections. To the best of our knowledge, this is the first report worldwide of the occurrence of dieback on Prunus lusitanica caused by D. macrodidyma.