Experimental evidence of microbial inheritance in plants and transmission routes from seed to phyllosphere and root.

While the environment is considered the primary origin of the plant microbiome, the potential role of seeds as a source of transmitting microorganisms has not received much attention. Here we tested the hypothesis that the plant microbiome is partially inherited through vertical transmission. An experimental culturing device was constructed to grow oak seedlings in a microbe-free environment while keeping belowground and aboveground tissues separated. The microbial communities associated with the acorn's embryo and pericarp and the developing seeding's phyllosphere and root systems were analysed using amplicon sequencing of fungal ITS and bacterial 16S rDNA. Results showed that the seed microbiome is diverse and non-randomly distributed within an acorn. The microbial composition of the phyllosphere was diverse and strongly resembled the composition found in the embryo, whereas the roots and pericarp each had a less diverse and distinct microbial community. Our findings demonstrate a high level of microbial diversity and spatial partitioning of the fungal and bacterial community within both seed and seedling, indicating inheritance, niche differentiation and divergent transmission routes for the establishment of root and phyllosphere communities.

Preharvest and Postharvest Applications of a Pomegranate Peel Extract to Control Citrus Fruit Decay During Storage and Shelf Life.

Green and blue molds are the most important postharvest diseases affecting citrus in storage. These diseases are commonly controlled with fungicides, but legislative restrictions, consumer concerns, and the development of resistant strains of the pathogens have increasingly led to the search for alternative methods of control. A pomegranate peel extract (PGE) was very effective in controlling Valencia orange and clementine postharvest rot under commercial conditions. After cold storage and 7 days of shelf life, the incidence of decay on oranges sprayed before harvest with PGE at 12, 6, and 3 g/liter was reduced by 78.9, 76.0, and 64.6%, respectively. Similarly, postharvest dipping treatments with PGE reduced rot by 90.2, 84.3, and 77.6%, respectively. Comparable levels of protection were also achieved on clementines. On both oranges and clementines, the extract provided a significantly higher level of protection compared with imazalil, a fungicide commonly used for postharvest treatments. The high level of efficacy and the consistent results on different fruit species (clementines and oranges) and with different application methods (preharvest and postharvest) were evidence of reliability and flexibility. PGE also showed a strong antimicrobial activity against fungi and bacteria, suggesting its possible use in sanitizers to reduce the microbial contamination of recirculated water in packinghouses. The results of the present study encourage the integration of conventional chemical fungicides and sanitizers with PGE to control citrus postharvest rot.

Development and Application of a Quantitative PCR Detection Method to Quantify Venturia oleaginea in Asymptomatic Olive (Olea europaea) Leaves.

Olive leaf spot (OLS), caused by Venturia oleaginea, is one of the most common and serious diseases of olive trees in the Mediterranean region. Understanding the pathogen life cycle is important for the development of effective control strategies. Current knowledge is incomplete owing to a lack of effective detection methods. It is extremely difficult to culture V. oleaginea in vitro, so primers were designed to amplify and sequence the internal transcribed spacer ITS1-5.8S-ITS2 region of the fungus directly from infected olive leaves. Sanger sequencing indicated a unique ITS region present in the European strains screened, confirming the appropriateness of the target region for developing a quantitative PCR (qPCR) assay. Furthermore, high-throughput sequencing of the same region excluded the presence of other Venturia species in the olive phyllosphere. The qPCR assay proved very specific and sensitive, enabling the detection of approximately 26 copies of target DNA. The analysis of symptomless leaves during early stages of the epidemic from the end of winter through spring revealed a similar quantity of pathogen DNA regardless of the leaf growth stage. In contrast, the pathogen titer changed significantly during the season. Data indicated that leaf infections start earlier than expected over the season and very young leaves are as susceptible as adult leaves. These findings have important practical implications and suggest the need for improved scheduling of fungicide treatments. The qPCR assay represents a valuable tool providing quantitative results and enables detection of V. oleaginea in all olive organs, including those in which OLS cannot be studied using previously available methods.

Analysis of the Fungal Diversity in Citrus Leaves with Greasy Spot Disease Symptoms.

Citrus greasy spot (CGS) is a disease of citrus with worldwide distribution and recent surveys have revealed a high level of incidence and severity of symptoms of the disease in Sicily, southern Italy. Although Mycosphaerel la citri (anamorph Zasmidium citri-griseum) and other related species are generally considered as causal agents, the etiology of CGS is still unclear. Here, we report the use of an amplicon metagenomic approach to investigate the fungal communities on citrus leaves symptomatic or asymptomatic for CGS from an orchard in Sicily showing typical CGS symptoms. A total of 35,537 high-quality chimeric free reads were obtained and assigned to 176 operational taxonomic units (OTUs), clustered at 99 % similarity threshold. Data revealed a dominating presence of the phylum Ascomycota (92.6 %) over other fungal phyla. No significant difference was observed between symptomatic and asymptomatic leaves according to both alpha and beta diversity analyses. The family Mycosphaerellaceae was the most abundant and was represented by the genera Ramularia, Mycosphaerella, and Septoria with 44.8, 2.4, and 1.7 % of the total detected sequences, respectively. However, none of the species currently reported as causal agents of CGS was detected in the present study. The most abundant sequence type (ST) was associated to Ramularia brunnea, a species originally described to cause leaf spot in a perennial herbaceous plant of the family Asteraceae. Results exclude that CGS symptoms observed in Sicily are caused by Z. citri-griseum and, moreover, they indicate that a considerable part of the fungal diversity in citrus leaves is still unknown.

A Metabarcoding Survey on the Fungal Microbiota Associated to the Olive Fruit Fly.

The occurrence of interaction between insects and fungi is interesting from an ecological point of view, particularly when these interactions involve insect pests and plant pathogens within an agroecosystem. In this study, we aimed to perform an accurate analysis on the fungal microbiota associated to Bactrocera oleae (Rossi) through a metabarcoding approach based on 454 pyrosequencing. From this analysis, we retrieved 43,549 reads that clustered into 128 operational taxonomic units (OTUs), of which 29 resulted in the "core" associate fungi of B. oleae. This fungal community was mainly represented by sooty mould fungi, such as Cladosporium spp., Alternaria spp. and Aureobasidium spp., by plant pathogens like Colletotrichum spp. and Pseudocercospora spp., along with several other less abundant taxa whose ecology is unclear in most of the cases. Our findings lead to new insights into the microbial ecology of this specific ecological niche, enabling the understanding of a complex network of interactions within the olive agroecosystem.

Evaluation of a Pomegranate Peel Extract as an Alternative Means to Control Olive Anthracnose.

Olive anthracnose is caused by different species of Colletotrichum spp. and may be regarded as the most damaging disease of olive fruit worldwide, greatly affecting quality and quantity of the productions. A pomegranate peel extract (PGE) proved very effective in controlling the disease. The extract had a strong in vitro fungicidal activity against Colletotrichum acutatum sensu stricto, was very effective in both preventive and curative trials with artificially inoculated fruit, and induced resistance in treated olive tissues. In field trials, PGE was significantly more effective than copper, which is traditionally used to control the disease. The highest level of protection was achieved by applying the extract in the early ascending phase of the disease outbreaks because natural rots were completely inhibited with PGE at 12 g/liter and were reduced by 98.6 and by 93.0% on plants treated with PGE at 6 and 3 g/liter, respectively. Two treatments carried out 30 and 15 days before the expected epidemic outbreak reduced the incidence of the disease by 77.6, 57.0, and 51.8%, depending on the PGE concentration. The analysis of epiphytic populations showed a strong antimicrobial activity of PGE, which sharply reduced both fungal and bacterial populations. Because PGE was obtained from a natural matrix using safe chemicals and did not have any apparent phytotoxic effect on treated olive fruit, it may be regarded as a safe and effective natural antifungal preparation to control olive anthracnose and improve olive productions.

Genetic Analysis of Phytophthora nicotianae Populations from Different Hosts Using Microsatellite Markers.

In all, 231 isolates of Phytophthora nicotianae representing 14 populations from different host genera, including agricultural crops (Citrus, Nicotiana, and Lycopersicon), potted ornamental species in nurseries (Lavandula, Convolvulus, Myrtus, Correa, and Ruta), and other plant genera were characterized using simple-sequence repeat markers. In total, 99 multilocus genotypes (MLG) were identified, revealing a strong association between genetic grouping and host of recovery, with most MLG being associated with a single host genus. Significant differences in the structure of populations were revealed but clonality prevailed in all populations. Isolates from Citrus were found to be genetically related regardless of their geographic origin and were characterized by high genetic uniformity and high inbreeding coefficients. Higher variability was observed for other populations and a significant geographical structuring was determined for isolates from Nicotiana. Detected differences were related to the propagation and cultivation systems of different crops. Isolates obtained from Citrus spp. are more likely to be distributed worldwide with infected plant material whereas Nicotiana and Lycopersicon spp. are propagated by seed, which would not contribute to the spread of the pathogen and result in a greater chance for geographic isolation of lineages. With regard to ornamental species in nurseries, the high genetic variation is likely the result of the admixture of diverse pathogen genotypes through the trade of infected plant material from various geographic origins, the presence of several hosts in the same nursery, and genetic recombination through sexual reproduction of this heterothallic species.

Chemical Characterization of Different Sumac and Pomegranate Extracts Effective against Botrytis cinerea Rots.

Pomegranate (Punica granatum L.) peel and sumac (Rhus coriaria L.) fruit and leaf extracts were chemically characterized and their ability to inhibit table grape (cv. Italia) rots caused by Botrytis cinerea was evaluated on artificially inoculated berries. Different extraction methods were applied and extracts were characterized through Ultra Fast High Performance Liquid Chromatography coupled to Photodiode array detector and Electrospray ionization Mass spectrometer (UPLC-PDA-ESI/MSn) for their phenol and anthocyanin contents. The concentrated pomegranate peel extract (PGE-C) was the richest in phenols (66.97 g gallic acid equivalents/kg) while the concentrated sumac extract from fruits (SUF-C) showed the highest anthocyanin amount (171.96 mg cyanidin 3-glucoside equivalents/kg). Both phenolic and anthocyanin profile of pomegranate and sumac extracts were quite different: pomegranate extract was rich in cyanidin 3-glucoside, pelargonidin 3-glucoside and ellagic acid derivatives, while sumac extract was characterized by 7-methyl-cyanidin 3-galactoside and gallic acid derivatives. The concentrated extracts from both pomegranate peel and sumac leaves significantly reduced the development of Botrytis rots. In particular, the extract from pomegranate peel completely inhibited the pathogen at different intervals of time (0, 12, and 24 h) between treatment and pathogen inoculation on fruits maintained at 22-24 °C and high relative humidity (RH). This extract may represent a valuable alternative to control postharvest fungal rots in view of its high efficacy because of the low cost of pomegranate peel, which is a waste product of processing factories.

Characterization of Basidiomycetes associated with wood rot of citrus in southern Italy.

The characterization of Basidiomycetes associated with wood rots in commercial citrus orchards in southern Italy revealed that both white and brown rot fungi are implicated in this disease. Fomitiporia mediterranea was the most prevalent species causing a white rot, followed by Fomitopsis sp. which, by contrast, was associated with brown rot wood decay. Furthermore, Phellinus spp. and other nonidentified basidiomycetous fungi showing genetic affinity with the genera Phellinus and Coniophora were occasionally isolated. Artificial inoculations on lemon (Citrus limon) branches showed a faster wood colonization by Fomitopsis sp. compared with F. mediterranea, indicating that the former species as a potentially serious pathogen of citrus trees. The analysis of F. mediterranea internal transcribed spacer (ITS) sequences revealed a high level of genetic variability, with 13 genotypes which were both homozygous (6 genotypes) and heterozygous (7 genotypes). The presence of heterozygous genomes based on ITS sequences has never been reported before for F. mediterranea. This, together with the high frequency of basidiomata on infected wood, unambiguously confirms the outcrossing nature of reproduction in F. mediterranea and the primary role of basidiospores in the dissemination of inoculum. Similarly, high genetic variability was observed analyzing Fomitopsis sp. Because basidiomata of this fungus have not been observed on citrus trees, it can be hypothesized that basidiospores are produced on alternative host plants.

Effectiveness of phenolic compounds against citrus green mould.

Stored citrus fruit suffer huge losses because of the development of green mould caused by Penicillium digitatum. Usually synthetic fungicides are employed to control this disease, but their use is facing some obstacles, such public concern about possible adverse effects on human and environmental health and the development of resistant pathogen populations. In the present study quercetin, scopoletin and scoparone--phenolic compounds present in several agricultural commodities and associated with response to stresses--were firstly tested in vitro against P. digitatum and then applied in vivo on oranges cv. Navelina. Fruits were wound-treated (100 µg), pathogen-inoculated, stored and surveyed for disease incidence and severity. Although only a minor (≤13%) control effect on P. digitatum growth was recorded in vitro, the in vivo trial results were encouraging. In fact, on phenolic-treated oranges, symptoms appeared at 6 days post-inoculation (DPI), i.e., with a 2 day-delay as compared to the untreated control. Moreover, at 8 DPI, quercetin, scopoletin, and scoparone significantly reduced disease incidence and severity by 69%-40% and 85%-70%, respectively, as compared to the control. At 14 DPI, scoparone was the most active molecule. Based on the results, these compounds might represent an interesting alternative to synthetic fungicides.

Comparative transcriptome profiling and co-expression network analysis uncover the key genes associated with pear petal defense responses against Monilinia laxa infection.

Pear brown rot and blossom blight caused by Monilinia laxa seriously affect pear production worldwide. Here, we compared the transcriptomic profiles of petals after inoculation with M. laxa using two pear cultivars with different levels of sensitivity to disease (Sissy, a relatively tolerant cultivar, and Kristalli, a highly susceptible cultivar). Physiological indexes were also monitored in the petals of both cultivars at 2 h and 48 h after infection (2 HAI and 48 HAI). RNA-seq data and weighted gene co-expression network analysis (WGCNA) allowed the identification of key genes and pathways involved in immune- and defense-related responses that were specific for each cultivar in a time-dependent manner. In particular, in the Kristalli cultivar, a significant transcriptome reprogramming occurred early at 2 HAI and was accompanied either by suppression of key differentially expressed genes (DEGs) involved in the modulation of any defense responses or by activation of DEGs acting as sensitivity factors promoting susceptibility. In contrast to the considerably high number of DEGs induced early in the Kristalli cultivar, upregulation of specific DEGs involved in pathogen perception and signal transduction, biosynthesis of secondary and primary metabolism, and other defense-related responses was delayed in the Sissy cultivar, occurring at 48 HAI. The WGCNA highlighted one module that was significantly and highly correlated to the relatively tolerant cultivar. Six hub genes were identified within this module, including three WRKY transcription factor-encoding genes: WRKY 65 (pycom05g27470), WRKY 71 (pycom10g22220), and WRKY28 (pycom17g13130), which may play a crucial role in enhancing the tolerance of pear petals to M. laxa. Our results will provide insights into the interplay of the molecular mechanisms underlying immune responses of petals at the pear-M. laxa pathosystem.

Analyses of the population structure in a global collection of Phytophthora nicotianae isolates inferred from mitochondrial and nuclear DNA sequences.

Genetic variation within the heterothallic cosmopolitan plant pathogen Phytophthora nicotianae was determined in 96 isolates from a wide range of hosts and geographic locations by characterizing four mitochondrial (10% of the genome) and three nuclear loci. In all, 52 single-nucleotide polymorphisms (SNPs) (an average of 1 every 58 bp) and 313 sites with gaps representing 5,450 bases enabled the identification of 50 different multilocus mitochondrial haplotypes. Similarly, 24 SNPs (an average of 1 every 69 bp), with heterozygosity observed at each locus, were observed in three nuclear regions (hyp, scp, and ß-tub) differentiating 40 multilocus nuclear genotypes. Both mitochondrial and nuclear markers revealed a high level of dispersal of isolates and an inconsistent geographic structuring of populations. However, a specific association was observed for host of origin and genetic grouping with both nuclear and mitochondrial sequences. In particular, the majority of citrus isolates from Italy, California, Florida, Syria, Albania, and the Philippines clustered in the same mitochondrial group and shared at least one nuclear allele. A similar association was also observed for isolates recovered from Nicotiana and Solanum spp. The present study suggests an important role of nursery populations in increasing genetic recombination within the species and the existence of extensive phenomena of migration of isolates that have been likely spread worldwide with infected plant material.

Phytophthora × pelgrandis Causes Root and Collar Rot of Lavandula stoechas in Italy.

In 2007, Phytophthora isolates with atypical morphological and biological characteristics were found associated with root and collar rot of potted plants of Stoechas lavender (Lavandula stoechas) in an ornamental nursery in Italy. A polyphasic approach, including morphological and cultural observations, sequencing the ITS-rDNA region, the Pheca and the mitochondrial coxI genes, multiplex PCRs with primers specific for P. nicotianae or P. cactorum, as well as random amplified polymorphic DNA-polymerase chain reaction, was used to characterize these isolates. On the basis of morpho-cultural and molecular analyses, the isolates from Stoechas lavender were identified as Phytophthora × pelgrandis, a natural hybrid of P. nicotianae × P. cactorum previously reported in other European countries, the Americas, and Taiwan, as a pathogen of ornamentals and loquat plants. In pathogenicity tests using potted plants of Stoechas lavender, the P. × pelgrandis isolates, similarly to the parental species P. nicotianae, induced the symptoms observed on plants with natural infections and were reisolated only from artificially inoculated plants. Dispersal of P. × pelgrandis on this host could exacerbate the damage caused by Phytophthora root and collar rot, of which the main causal agent presently is P. nicotianae on lavender in Europe. Application of hygienic measures is important to reduce the proliferation and spread of the Phytophthora hybrids.

Characterization of Phytopythium Species Involved in the Establishment and Development of Kiwifruit Vine Decline Syndrome.

Since 2012, the kiwifruit vine decline syndrome (KVDS) has progressively compromised Italian kiwifruit orchards. Different abiotic and biotic factors have been associated with the establishment and development of KVDS. During monitoring of orchards affected by KVDS in north-western Italy during 2016-2019, 71 Phytopythium spp. were isolated. Based on maximum likelihood concatenated phylogeny on the ITS1-5.8S-ITS2 region of the rDNA, large subunit rDNA, and cytochrome oxidase I, isolates were identified as P. vexans (52), P. litorale (10), P. chamaehyphon (7) and P. helicoides (2). Phytopythium litorale and P. helicoides are reported for the first time as agents of KVDS in Italy. To demonstrate pathogenicity and fulfil Koch's postulates, representative isolates of P. vexans, P. litorale, P. chamaehyphon and P. helicoides were inoculated in potted plants. In these trials, waterlogging was applied to stress plant with a temporary anoxia and to favour the production of infective zoospores by the oomycetes. In experiments in vitro, the four species showed the highest growth at 25-30 °C, depending on the media used. P. helicoides was able to grow also at 40 °C. The four species were able to grow in vitro at a pH ranging from 5.0 to 8.0, showing that pH had less effect on growth than temperature. The present study suggests a strong role of different species of Phytopythium in the establishment and development of KVDS. Phytopythium spp. could be favoured by the average increase in soil temperatures during summer, associated with global warming.

Plant genotype influence the structure of cereal seed fungal microbiome.

Plant genotype is a crucial factor for the assembly of the plant-associated microbial communities. However, we still know little about the variation of diversity and structure of plant microbiomes across host species and genotypes. Here, we used six species of cereals (Avena sativa, Hordeum vulgare, Secale cereale, Triticum aestivum, Triticum polonicum, and Triticum turgidum) to test whether the plant fungal microbiome varies across species, and whether plant species use different mechanisms for microbiome assembly focusing on the plant ears. Using ITS2 amplicon metagenomics, we found that host species influences the diversity and structure of the seed-associated fungal communities. Then, we tested whether plant genotype influences the structure of seed fungal communities across different cultivars of T. aestivum (Aristato, Bologna, Rosia, and Vernia) and T. turgidum (Capeiti, Cappelli, Mazzancoio, Trinakria, and Timilia). We found that cultivar influences the seed fungal microbiome in both species. We found that in T. aestivum the seed fungal microbiota is more influenced by stochastic processes, while in T. turgidum selection plays a major role. Collectively, our results contribute to fill the knowledge gap on the wheat seed microbiome assembly and, together with other studies, might contribute to understand how we can manipulate this process to improve agriculture sustainability.

Plant Genotype Shapes the Bacterial Microbiome of Fruits, Leaves, and Soil in Olive Plants.

The plant microbiome plays an important role in plant biology, ecology, and evolution. While recent technological developments enabled the characterization of plant-associated microbiota, we still know little about the impact of different biotic and abiotic factors on the diversity and structures of these microbial communities. Here, we characterized the structure of bacterial microbiomes of fruits, leaves, and soil collected from two olive genotypes (Sinopolese and Ottobratica), testing the hypothesis that plant genotype would impact each compartment with a different magnitude. Results show that plant genotype differently influenced the diversity, structure, composition, and co-occurence network at each compartment (fruits, leaves, soil), with a stronger effect on fruits compared to leaves and soil. Thus, plant genotype seems to be an important factor in shaping the structure of plant microbiomes in our system, and can be further explored to gain functional insights leading to improvements in plant productivity, nutrition, and defenses.

The Fungal Microbiome of Wheat Flour Includes Potential Mycotoxin Producers.

Consumers are increasingly demanding higher quality and safety standards for the products they consume, and one of this is wheat flour, the basis of a wide variety of processed products. This major component in the diet of many communities can be contaminated by microorganisms before the grain harvest, or during the grain storage right before processing. These microorganisms include several fungal species, many of which produce mycotoxins, secondary metabolites that can cause severe acute and chronic disorders. Yet, we still know little about the overall composition of fungal communities associated with wheat flour. In this study, we contribute to fill this gap by characterizing the fungal microbiome of different types of wheat flour using culture-dependent and -independent techniques. Qualitatively, these approaches suggested similar results, highlighting the presence of several fungal taxa able to produce mycotoxins. In-vitro isolation of fungal species suggest a higher frequency of Penicillium, while metabarcoding suggest a higher abundance of Alternaria. This discrepancy might reside on the targeted portion of the community (alive vs. overall) or in the specific features of each technique. Thus, this study shows that commercial wheat flour hosts a wide fungal diversity with several taxa potentially representing concerns for consumers, aspects that need more attention throughout the food production chain.

Olive leaf spot caused by Venturia oleaginea: An updated review.

Olive leaf spot (OLS) caused by Venturia oleaginea is widespread in all olive-growing areas and continents, where can cause severe yield losses. The disease is often underestimated for the difficulty to reveal early leaf symptoms and for the pathogen-induced phylloptosis, which creates the illusion of healthy and restored plants. The present review provide updated information on taxonomy, pathogen life style and cycle, epidemiology, diagnosis, and control. Application of copper-based fungicides is the main method to control OLS. However, the regulation 2009/1107 of the European Commission include these fungicides in the list of substances candidates for substitution. It is therefore urgent to find alternative control strategies especially for organic agriculture. Among new approaches/strategies for controlling OLS, promising results have been obtained using nanotechnology, endophytic microbes, and biostimulants.

Extracts from Environmental Strains of Pseudomonas spp. Effectively Control Fungal Plant Diseases.

The use of synthetic chemical products in agriculture is causing severe damage to the environment and human health, but agrochemicals are still widely used to protect our crops. To counteract this trend, we have been looking for alternative strategies to control plant diseases without causing harm to the environment or damage to our health. However, these alternatives are still far from completely replacing chemical products. Microorganisms have been widely known as a biological tool to control plant diseases, but their use is still limited due to the high variability in their efficacy, together with issues in product registration. However, the metabolites produced by these microorganisms can represent a novel tool for the environment-friendly management of plant diseases, while reducing the issues mentioned above. In this study, we explore the soil microbial diversity in natural systems to look for microorganisms with the potential to be used in pre- and post-harvest protection against fungal plant pathogens. Using a simple workflow, we isolated 22 bacterial strains that were tested both in vitro and in vivo for their ability to counteract the growth of common plant pathogens. The three best isolates, identified as members of the bacterial genus Pseudomonas, were used to produce a series of alcoholic extracts, which were then tested for their action against plant pathogens in simulated real-world applications. Results show that extracts from these isolates have an exceptional biocontrol activity and can be successfully used to control plant pathogens in operational setups. Thus, this study shows that the environmental microbiome is an important source of microorganisms producing metabolites that might provide an alternative strategy to synthetic chemical products.

Metagenomics Approaches for the Detection and Surveillance of Emerging and Recurrent Plant Pathogens.

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.