Globalization has a dramatic effect on the trade and movement of seeds, fruits and vegetables, with a corresponding increase in economic losses caused by the introduction of transboundary plant pathogens. Current diagnostic techniques provide a useful and precise tool to enact surveillance protocols regarding specific organisms, but this approach is strictly targeted, while metabarcoding and shotgun metagenomics could be used to simultaneously detect all known pathogens and potentially new ones. This review aims to present the current status of high-throughput sequencing (HTS) diagnostics of fungal and bacterial plant pathogens, discuss the challenges that need to be addressed, and provide direction for the development of methods for the detection of a restricted number of related taxa (specific surveillance) or all of the microorganisms present in a sample (general surveillance). HTS techniques, particularly metabarcoding, could be useful for the surveillance of soilborne, seedborne and airborne pathogens, as well as for identifying new pathogens and determining the origin of outbreaks. Metabarcoding and shotgun metagenomics still suffer from low precision, but this issue can be limited by carefully choosing primers and bioinformatic algorithms. Advances in bioinformatics will greatly accelerate the use of metagenomics to address critical aspects related to the detection and surveillance of plant pathogens in plant material and foodstuffs.

The yeast Metschnikowia fructicola was reported as an efficient biological control agent of postharvest diseases of fruits and vegetables, and it is the bases of the commercial formulated product “Shemer.” Several mechanisms of action by which M. fructicola inhibits postharvest pathogens were suggested including iron-binding compounds, induction of defense signaling genes, production of fungal cell wall degrading enzymes and relatively high amounts of superoxide anions. We assembled the whole genome sequence of two strains of M. fructicola using PacBio and Illumina shotgun sequencing technologies. Using the PacBio, a high-quality draft genome consisting of 93 contigs, with an estimated genome size of approximately 26 Mb, was obtained. Comparative analysis of M. fructicola proteins with the other three available closely related genomes revealed a shared core of homologous proteins coded by 5,776 genes. Comparing the genomes of the two M. fructicola strains using a SNP calling approach resulted in the identification of 564,302 homologous SNPs with 2,004 predicted high impact mutations. The size of the genome is exceptionally high when compared with those of available closely related organisms, and the high rate of homology among M. fructicola genes points toward a recent whole-genome duplication event as the cause of this large genome. Based on the assembled genome, sequences were annotated with a gene description and gene ontology (GO term) and clustered in functional groups. Analysis of CAZymes family genes revealed 1,145 putative genes, and transcriptomic analysis of CAZyme expression levels in M. fructicola during its interaction with either grapefruit peel tissue or Penicillium digitatum revealed a high level of CAZyme gene expression when the yeast was placed in wounded fruit tissue.

Candida oleophila is an effective biocontrol agent used to control post-harvest diseases of fruits and vegetables. C. oleophila I-182 was the active agent used in the first-generation yeast-based commercial product, Aspire^®, for post-harvest disease management. Several action modes, like competition for nutrients and space, induction of pathogenesis-related genes in host tissues, and production of extracellular lytic enzymes, have been demonstrated for the biological control activity exhibited by C. oleophila through which it inhibits post-harvest pathogens. In the present study, the whole genome of C. oleophila I-182 was sequenced using PacBio and Illumina shotgun sequencing technologies, yielding an estimated genome size of 14.73 Mb. The genome size is similar in length to that of the model yeast strain Saccharomyces cerevisiae S288c. Based on the assembled genome, protein-coding sequences were identified and annotated. The predicted genes were further assigned with gene ontology terms and clustered in special functional groups. A comparative analysis of C. oleophila proteome with the proteomes of 11 representative yeasts revealed 2 unique and 124 expanded families of proteins in C. oleophila. Availability of the genome sequence will facilitate a better understanding the properties of biocontrol yeasts at the molecular level.

Date palm (Phoenix dactylifera) is considered to be a highly important food crop in several African and Middle Eastern countries due to its nutritional value and health-promoting properties. Microbial contamination of dates has been of concern to consumers, but very few works have analyzed in detail the microbial load of the different parts of date fruit. In the present work, we characterized the fungal communities of date fruit using a metagenomic approach, analyzing the data for differences between microbial populations residing in the pulp and peel of “Medjool” dates at the different stages of fruit development. The results revealed that Penicillium, Cladosporium, Aspergillus, and Alternaria were the most abundant genera in both parts of the fruit, however, the distribution of taxa among the time points and tissue types (peel vs. pulp) was very diverse. Penicillium was more abundant in the pulp at the green developmental stage (Kimri), while Aspergillus was more frequent in the peel at the brown developmental stage (Tamer). The highest abundance of Alternaria was detected at the earliest sampled stage of fruit development (Hababauk stage). Cladosporium had a high level of abundance in peel tissues at the Hababauk and yellow (Khalal) stages. Regarding the yeast community, the abundance of Candida remained stable up until the Khalal stage, but exhibited a dramatic increase in abundance at the Tamer stage in peel tissues, while the level of Metschnikowia, a genus containing several species with postharvest biocontrol activity, exhibited no significant differences between the two tissue types or stages of fruit development. This work constitutes a comprehensive metagenomic analysis of the fungal microbiome of date fruits, and has identified changes in the composition of the fungal microbiome in peel and pulp tissues at the different stages of fruit development. Notably, this study has also characterized the endophytic fungal microbiome present in pulp tissues of dates.